JP2006316022A - gamma-POLYGLUTAMIC ACID (gamma-PGA, H FORM) AND gamma-POLYGLUTAMATE USED AS NUTRITION SUPPLEMENT IN DIETARY PRODUCT - Google Patents
gamma-POLYGLUTAMIC ACID (gamma-PGA, H FORM) AND gamma-POLYGLUTAMATE USED AS NUTRITION SUPPLEMENT IN DIETARY PRODUCT Download PDFInfo
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- JP2006316022A JP2006316022A JP2005142168A JP2005142168A JP2006316022A JP 2006316022 A JP2006316022 A JP 2006316022A JP 2005142168 A JP2005142168 A JP 2005142168A JP 2005142168 A JP2005142168 A JP 2005142168A JP 2006316022 A JP2006316022 A JP 2006316022A
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- polyglutamate
- pga
- polyglutamic acid
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Abstract
Description
本発明は、腸内でのカルシウム吸収や骨のカルシウム吸収を向上させるためのγ−ポリグルタミン酸（γ−ＰＧＡ、Ｈ体）、γ−ポリグルタメートＮａ+体、γ−ポリグルタメートＫ+体、γ−ポリグルタメートＮＨ4 +体、γ−ポリグルタメートＭｇ++体、γ−ポリグルタメートＣａ++体、又はこれらの混合物のダイエタリー製品における使用に関する。更に詳細には、本発明は、カルシウムの吸収促進や骨粗鬆症の予防、カルシウム損失の低減、骨強度の維持、成長や健康状態の改善のために、γ−ポリグルタミン酸（γ−ＰＧＡ、Ｈ体）、γ−ポリグルタメートＮａ+体、γ−ポリグルタメートＫ+体、γ−ポリグルタメートＮＨ4 +体、γ−ポリグルタメートＭｇ++体、γ−ポリグルタメートＣａ++体、又はこれらの混合物を、食品又は動物用飼料組成物中において錯形成吸着剤（complex adsorbent）又は栄養補助剤として使用することに関する。 The present invention relates to γ-polyglutamic acid (γ-PGA, H form), γ-polyglutamate Na + form, γ-polyglutamate K + form, γ for improving calcium absorption in the intestine and bone calcium absorption. -Polyglutamate NH 4 + form, γ-polyglutamate Mg ++ form, γ-polyglutamate Ca ++ form, or a mixture thereof in a dietary product. More specifically, the present invention relates to γ-polyglutamic acid (γ-PGA, H form) for promoting calcium absorption, preventing osteoporosis, reducing calcium loss, maintaining bone strength, and improving growth and health. Γ-polyglutamate Na + isomer, γ-polyglutamate K + isomer, γ-polyglutamate NH 4 + isomer, γ-polyglutamate Mg ++ isomer, γ-polyglutamate Ca ++ isomer, or a mixture thereof, It relates to use as a complex adsorbent or nutritional supplement in food or animal feed compositions.
「トランスフォーミング増殖因子−β」（ＴＧＦ−β）はタンパク質ファミリーの一種であり、進化過程において高度に保存されており、広範な種類の細胞に影響を及ぼす。当初、ＴＧＦ−βは初代細胞培養を足場非依存的に増殖させる因子として特定された。インビボにおいてＴＧＦ−βは、結合組織の形成（deposition）を促進し、間充織細胞の増殖を誘起する。ＴＧＦ−βは、造血幹細胞（非特許文献１参照）やＮＫ細胞（非特許文献２参照）等の免疫系細胞の増殖及び分化に影響を及ぼす。ＴＧＦ−βはまた、癌や白血病等の過増殖を抑制するために投与することができる（特許文献１参照）。 “Transforming growth factor-β” (TGF-β) is a member of the protein family that is highly conserved during evolution and affects a wide variety of cells. Initially, TGF-β was identified as a factor that allowed primary cell cultures to grow in an anchorage-independent manner. In vivo, TGF-β promotes connective tissue deposition and induces mesenchymal cell proliferation. TGF-β affects the proliferation and differentiation of immune system cells such as hematopoietic stem cells (see Non-patent Document 1) and NK cells (see Non-Patent Document 2). TGF-β can also be administered to suppress overgrowth such as cancer and leukemia (see Patent Document 1).
特許文献２及び３の開示に従って、脱石灰した骨から尿素又は塩酸グアニジンを用いて「骨形成因子（ＢＭＰ）」を抽出・再沈澱させた。セイデンとトーマス（Seyedin and Thomas）は特許文献４において、カオトロピック剤を用いた抽出と、ｐＨ４．８でＣＭＣに吸着したフラクションからの活性物質を回収することにより骨形成刺激骨由来タンパク質を部分精製したことを報告している。この新規なタンパク質フラクションは「骨形成因子（osteogenic factor）」と称され、約３００００ダルトン未満の分子量を有すると特定された。 According to the disclosures of Patent Documents 2 and 3, “bone forming factor (BMP)” was extracted and reprecipitated from decalcified bone using urea or guanidine hydrochloride. Seidin and Thomas partially purified bone-derived stimulating bone-derived protein in US Pat. No. 5,057,099 by extraction with a chaotropic agent and recovering active substances from fractions adsorbed to CMC at pH 4.8. It is reported that. This novel protein fraction is referred to as the “osteogenic factor” and has been identified as having a molecular weight of less than about 30000 daltons.
アクチビンは、二量体タンパク質であり、構造的にＴＧＦ−β１に類似するタンパク質ファミリーの一種で、インヒビンに類似している。インヒビンは、アクチビンサブユニットと他のアクチビンサブユニットとからなるヘテロ二量体である。アクチビンは、卵胞刺激ホルモンの放出（非特許文献３参照）やランゲルハンス島からのインスリン分泌（非特許文献４参照）、骨髄培養における赤芽球多能性前駆細胞（erythroid and multipotential progenitor cell）のコロニー形成（非特許文献５参照）を刺激し、また、インビボにおいて軟骨性骨の形成を誘起する（非特許文献６参照）ことが判明している。 Activin is a dimeric protein, a member of a protein family that is structurally similar to TGF-β1 and is similar to inhibin. Inhibin is a heterodimer composed of activin subunits and other activin subunits. Activin is a release of follicle-stimulating hormone (see Non-Patent Document 3), insulin secretion from Langerhans Island (see Non-Patent Document 4), and colonies of erythroid and multipotential progenitor cells in bone marrow culture. It has been found that it stimulates formation (see Non-Patent Document 5) and induces the formation of cartilage bone in vivo (see Non-Patent Document 6).
骨シアロタンパク質（ＢＳＰ）は高度にグリコシレート化及びサルフェート化されたリンタンパク質であり、石灰化した結合組織にのみ認められる。ポリグルタミン酸モチーフは、ヒドロキシアパタイトと細胞表面インテグリンとを結合する能力を有する。ＢＳＰは骨ニュークリエーターが有する生物物理学的及び化学的機能を有する。ヒドロキシアパタイト結合ポリグルタミン酸配列は二官能性の物質（entites）を提供するが、この物質により、ＢＳＰのターゲティングと骨表面への正常細胞及び転移細胞の付着が仲介される（非特許文献７参照）。 Bone sialoprotein (BSP) is a highly glycosylated and sulfated phosphoprotein found only in calcified connective tissue. The polyglutamic acid motif has the ability to bind hydroxyapatite and cell surface integrins. BSP has the biophysical and chemical functions of bone neucreators. Hydroxyapatite-linked polyglutamic acid sequences provide bifunctional entities, which mediate BSP targeting and adhesion of normal and metastatic cells to the bone surface (see Non-Patent Document 7). .
本技術分野において、可溶性、抗原性及び生物学的タンパク質クリアランスを変化させるためにポリマーと共有結合させて改変したタンパク質に関する文献がある（特許文献５、特許文献６、特許文献７及び特許文献８参照）。特許文献９（１９９２年１１月１１日公開）は、骨成長因子と標的組織への親和性を有するターゲティング分子とを含む組成物を開示しており、この骨成長因子及びターゲティング分子はクロスリンカーと化学的に結合している。クロスリンカーとしては合成親水性ポリマーが好ましい。これら分子は骨に対する親和性を有することが好ましい。骨成長因子としてはＴＧＦ−β、アクチビン、骨形成因子（ＢＭＰ）又は骨シアロタンパク質（ＢＳＰ）が好ましい。標的とする組織の例としては骨や、軟骨、骨成長因子が標的とするその他の各種組織や細胞が挙げられる。これら組成物は、骨形成の促進目的での使用、及び骨粗鬆症や骨関節炎、加齢による骨量損失においてよく見受けられる骨損失の修復や処置における使用を意図したものである。 In this technical field, there are documents related to proteins modified by covalent bonding with polymers in order to change solubility, antigenicity and biological protein clearance (see Patent Document 5, Patent Document 6, Patent Document 7 and Patent Document 8). ). Patent Document 9 (published on November 11, 1992) discloses a composition comprising a bone growth factor and a targeting molecule having an affinity for a target tissue, the bone growth factor and the targeting molecule comprising a crosslinker and It is chemically bonded. As the crosslinker, a synthetic hydrophilic polymer is preferable. These molecules preferably have affinity for bone. The bone growth factor is preferably TGF-β, activin, bone morphogenetic factor (BMP) or bone sialoprotein (BSP). Examples of target tissues include bones, cartilage, and other various tissues and cells targeted by bone growth factors. These compositions are intended for use in promoting bone formation and in the repair and treatment of bone loss often found in osteoporosis, osteoarthritis, and bone loss due to aging.
人体のカルシウム吸収は、基本的に二経路によることが知られている。即ち、能動輸送と受動輸送である。能動輸送経路は主としてビタミンＤ及び種々のホルモンの調節により制御されており、ここではカルシウムは小腸上部において濃度勾配とは逆勾配で吸収される。一方、受動輸送経路では、カルシウムは小腸下部で濃度勾配に従って吸収される。可溶性カルシウムが大量に存在する場合、小腸下部に対する受動輸送の比率が非常に高くなる。能動輸送経路では、可溶性カルシウムの濃度を増加させても一定量以上に吸収量を増加させることはできないが、受動輸送では腸内の可溶性カルシウムの濃度が増加するにつれ吸収が上昇する。腸内のカルシウム吸収率は１０〜５０％であると報告されている。腸における乳タンパク質であるカゼインの酵素分解物である、カゼインホスホペプチド（ＣＰＰ）は小腸の可溶性カルシウムの濃度を増加させ、これによりカルシウム吸収を促進させる（特許文献１０参照（１９９５年９月５日発行））。カルシウムは、ＣＰＰに含まれる酸性アミノ酸のカルボキシレート基とホスホセリンのホスフェート基に配位することにより可溶性の状態に維持される。 It is known that the absorption of calcium in the human body is basically by two pathways. That is, active transport and passive transport. The active transport pathway is primarily controlled by the regulation of vitamin D and various hormones, where calcium is absorbed in the upper small intestine with a reverse gradient to the concentration gradient. On the other hand, in the passive transport pathway, calcium is absorbed in the lower small intestine according to a concentration gradient. When soluble calcium is present in large quantities, the ratio of passive transport to the lower small intestine is very high. In the active transport pathway, even if the concentration of soluble calcium is increased, the amount of absorption cannot be increased beyond a certain amount, but in passive transport, the absorption increases as the concentration of soluble calcium in the intestine increases. Intestinal calcium absorption has been reported to be 10-50%. Casein phosphopeptide (CPP), which is an enzymatic degradation product of casein, a milk protein in the intestine, increases the concentration of soluble calcium in the small intestine, thereby promoting calcium absorption (see Patent Document 10 (September 5, 1995). Issue)). Calcium is maintained in a soluble state by coordinating to the carboxylate group of acidic amino acids contained in CPP and the phosphate group of phosphoserine.
骨粗鬆症や骨折は、高齢者や閉経後の女性によく見られる代謝性疾患である。ダイエタリーカルシウムは、高齢者の骨粗鬆症の予防のためカルシウム要求量を考慮する場合や、骨強度や骨のミネラル密度を増加する際に不可欠である。骨粗鬆症は、骨損失を特徴とする疾患であり、骨折の危険性の増加や背中痛や関節痛に関連している。骨粗鬆症の予防には十分なダイエタリー栄養物、特にカルシウム含有量の高い食品の摂取が重要である。ダイエタリーカルシウムは吸収性及び生体利用性が非常に高く、年齢とともにカルシウム吸収が低下する事実を考えると、高齢者のカルシウム要求量を考慮するうえで重要である。 Osteoporosis and fractures are metabolic diseases that are common in elderly people and postmenopausal women. Dietary calcium is indispensable when considering calcium requirements for the prevention of osteoporosis in the elderly and when increasing bone strength and bone mineral density. Osteoporosis is a disease characterized by bone loss and is associated with an increased risk of fracture, back pain and joint pain. In order to prevent osteoporosis, it is important to take sufficient dietary nutrients, particularly foods with a high calcium content. Dietary calcium has an extremely high absorbability and bioavailability, and considering the fact that calcium absorption decreases with age, it is important in considering the calcium requirements of the elderly.
本発明は、γ−ポリグルタミン酸（γ−ＰＧＡ、Ｈ体）、γ−ポリグルタメートＮａ+体、γ−ポリグルタメートＫ+体、γ−ポリグルタメートＮＨ4 +体、γ−ポリグルタメートＭｇ++体、γ−ポリグルタメートＣａ++体又はこれらの混合物を、ダイエタリーカルシウムの吸収と骨芽細胞の増殖とを促進する栄養補助剤として、食品や動物用飼料において使用することに関する。 The present invention relates to γ-polyglutamic acid (γ-PGA, H form), γ-polyglutamate Na + form, γ-polyglutamate K + form, γ-polyglutamate NH 4 + form, γ-polyglutamate Mg ++ form Γ-polyglutamate Ca ++ or a mixture thereof is used in foods and animal feeds as a nutritional supplement that promotes dietary calcium absorption and osteoblast proliferation.
本発明において、栄養補助剤としてγ−ポリグルタミン酸（γ−ＰＧＡ、Ｈ体）、γ−ポリグルタメートＮａ+体、γ−ポリグルタメートＫ+体、γ−ポリグルタメートＮＨ4 +体、γ−ポリグルタメートＭｇ++体、γ−ポリグルタメートＣａ++体又はこれらの混合物を使用すれば、錯体形成によってカルシウム、マグネシウムのいずれも効果的に溶解・安定化することができ、食品や飼料組成物に用いた場合、効果的にカルシウム及びマグネシウムの生体利用性を向上させることができる。 In the present invention, γ-polyglutamic acid (γ-PGA, H form), γ-polyglutamate Na + form, γ-polyglutamate K + form, γ-polyglutamate NH 4 + form, γ-polyglutamate as nutritional supplements If Mg ++ form, γ-polyglutamate Ca ++ form or a mixture thereof is used, both calcium and magnesium can be effectively dissolved and stabilized by complex formation, and can be used for food and feed compositions. If so, the bioavailability of calcium and magnesium can be improved effectively.
γ−ポリグルタミン酸（γ−ＰＧＡ、Ｈ体）及びγ−ポリグルタメート（Ｎａ+、Ｋ+、ＮＨ4 +、Ｃａ++及びＭｇ++体）は生分解性であって、Ｌ−グルタミン酸から液内発酵プロセス（Ｈ．クボタ（Kubota）ら、「枯草菌Ｆ−２−０１によるポリ（γ−グルタミン酸）の生産（Production of Poly((-Glutamic Acid) by Bacillus subtitlis F-2-01）」、Ｂｉｏｓｃｉ．Ｂｉｏｔｅｃｈ．Ｂｉｏｃｈｅｍ．５７（７）、１２１２−１２１３、１９９３及びＹ．オガワ（Ogawa）ら、「ジャー内発酵における枯草菌（ナットウ）によるγ−ポリグルタミン酸の効果的な生産（Efficient Production of (-Polyglutamic Acid by Bacillus subtilis (natto) in Jar Fermentation）」、６１（１０）、１６８４−１６８７、１９９７参照）によって生産される非毒性バイオポリマーである。γ−ポリグルタメート（Ｎａ+、Ｋ+、ＮＨ4 +、Ｃａ++及びＭｇ++体）は吸水性に優れ、金属イオンのＣａ++、Ｍｇ++、Ｚｎ++、Ｍｎ++、Ｓｅ++++及びＣｒ+++に対し良好な配位能を有し、錯体を形成する。また、γ−ポリグルタメートのポリアニオン性は水系において前記金属イオンを溶解・安定化するという用途に関し研究されている。図１にγ−ポリグルタミン酸（γ−ＰＧＡ）及びγ−ポリグルタメート（Ｎａ+、Ｋ+、ＮＨ4+、Ｃａ++、Ｍｇ++体）の分子構造を示す。図２、図３及び図４に、1Ｈ−ＮＭＲ、13Ｃ−ＮＭＲ及びＦＴ−ＩＲスペクトルをそれぞれ示す。これらスペクトル及び分析データを表１にまとめた。図５にｐＨ滴定曲線を示す。 γ-polyglutamic acid (γ-PGA, H form) and γ-polyglutamate (Na + , K + , NH 4 + , Ca ++ and Mg ++ forms) are biodegradable and are liquids from L-glutamic acid. Internal fermentation process (H. Kubota et al., "Production of Poly ((-Glutamic Acid) by Bacillus subtitlis F-2-01)", Biosci. Biotech. Biochem. 57 (7), 1212-1213, 1993 and Y. Ogawa et al., “Efficient Production of (Efficient Production of (γ) -polyglutamic acid by Natto in jar fermentation. -Polyglutamic Acid by Bacillus subtilis (natto) in Jar Fermentation) ", 61 (10), 1684-1687, 1997). Γ-polyglutamate (Na + , K + , NH 4 + , Ca ++ and Mg ++ isomers) are excellent in water absorption, and metal ions Ca ++ , Mg ++ , Zn ++ , Mn ++ , Se ++++ In addition, it has a good coordination ability to Cr +++ and forms a complex, and the polyanionic property of γ-polyglutamate has been studied for use in dissolving and stabilizing the metal ion in an aqueous system. 1 shows the molecular structures of γ-polyglutamic acid (γ-PGA) and γ-polyglutamate (Na + , K + , NH 4+ , Ca ++ , Mg ++ isomers). The 1 H-NMR, 13 C-NMR and FT-IR spectra are shown in Fig. 4. These spectra and analytical data are summarized in Table 1. Fig. 5 shows the pH titration curve.
γ−ポリグルタミン酸（γ−ＰＧＡ、Ｈ体）は、重合度が１０００から２００００までのグルタミン酸のバイオポリマーであり、グルタミック単位同士のγ−ペプチド結合のみから成る。γ−ポリグルタミン酸（γ−ＰＧＡ、Ｈ体）及びγ−ポリグルタメート（Ｎａ+、Ｋ+、ＮＨ4 +、Ｃａ++及びＭｇ++体）は末端アミンと多数のα−カルボン酸／カルボキシレート基とを含む。γ−ポリグルタミン酸（γ−ＰＧＡ、Ｈ体）及びγ−ポリグルタメート（Ｎａ+、Ｋ+、ＮＨ4 +、Ｃａ++及びＭｇ++体）はｐＨやイオン強度、その他のカチオン性種等の環境条件により、幾つかのコンホメーション状態、即ち、αヘリックス、ランダムコイル、βシート、ヘリックスコイル転移領域及び包み込まれた会合体（enveloped aggregation）として存在する。通常、円二色性（ＣＤ）を用いて、αヘリックス体の存在量を２２２ｎｍにおけるスペクトル強度の関数として測定する。ヘリックスコイル転移は均一な水溶液中で、約ｐＨ３〜５から生じ、γ−ポリグルタミン酸（γ−ＰＧＡ、Ｈ体）は非結合の形態となり、ｐＨがより高い５〜７にシフトすると結合した形態となる。ランダムコイルから包み込まれた会合体への転移は、γ−ＰＧＡの大規模のコンホメーション変化によって二価以上の金属イオンとの配位錯体が形成の場合に生じる。 γ-Polyglutamic acid (γ-PGA, H form) is a biopolymer of glutamic acid having a degree of polymerization of 1000 to 20000, and consists only of γ-peptide bonds between glutamic units. γ-polyglutamic acid (γ-PGA, H form) and γ-polyglutamate (Na + , K + , NH 4 + , Ca ++ and Mg ++ forms) are terminal amines and a number of α-carboxylic acids / carboxylates. Group. γ-polyglutamic acid (γ-PGA, H form) and γ-polyglutamate (Na + , K + , NH 4 + , Ca ++ and Mg ++ forms) are used for pH, ionic strength, other cationic species, etc. Depending on environmental conditions, there are several conformational states: alpha helix, random coil, beta sheet, helix coil transition region and enveloped aggregation. Usually, circular dichroism (CD) is used to measure the abundance of α-helix bodies as a function of spectral intensity at 222 nm. The helix coil transition occurs in a homogeneous aqueous solution from about pH 3-5, and γ-polyglutamic acid (γ-PGA, H form) is in an unbound form, and is bound when the pH is shifted to 5-7, Become. The transition from the random coil to the encapsulated aggregate occurs when a coordination complex with a divalent or higher metal ion is formed by a large-scale conformational change of γ-PGA.
本発明者らの研究により、γ−ポリグルタミン酸（γ−ＰＧＡ、Ｈ体）及びγ−ポリグルタメート（Ｎａ+、Ｋ+、ＮＨ4 +、Ｍｇ++及びＣａ++体）は、錯体形成によりカルシウム及びマグネシウムのいずれも溶解・安定化させるのに有効であり、食品や動物用飼料に栄養補助剤として用いる場合、カルシウム及びマグネシウムの生体利用性を高めるのに有効であることが分かった。 According to the studies by the present inventors, γ-polyglutamic acid (γ-PGA, H form) and γ-polyglutamate (Na + , K + , NH 4 + , Mg ++ and Ca ++ forms) It has been found that both calcium and magnesium are effective for dissolving and stabilizing, and when used as a nutritional supplement in foods and animal feeds, it is effective in enhancing the bioavailability of calcium and magnesium.
γ−ポリグルタミン酸（γ−ＰＧＡ、Ｈ体）はカルシウム塩やマグネシウム塩と反応し、安定した水溶性のカルシウムγ−ポリグルタメートやマグネシウムγ−ポリグルタメートをそれぞれ生成する。本発明者らは、インビトロの細胞培養研究により、配位錯体であるこれらカルシウムγ−ポリグルタメート及びマグネシウムγ−ポリグルタメートには高い吸収性、生体利用性があることを見出した。 γ-polyglutamic acid (γ-PGA, H form) reacts with calcium salt and magnesium salt to produce stable water-soluble calcium γ-polyglutamate and magnesium γ-polyglutamate, respectively. The present inventors have found through in vitro cell culture studies that these calcium γ-polyglutamate and magnesium γ-polyglutamate, which are coordination complexes, have high absorbability and bioavailability.
γ−ＰＧＡへの金属吸着には二つの可能な機構、即ち、（Ａ）金属イオンとカルボキシル部位との直接相互作用及び（Ｂ）ＣＯＯ-基により生じる静電ポテンシャル場による移動可能な重金属対イオンの保持が関与している。カルボキシレート基との相互作用の他に、アミド結合も弱い相互作用部位を提供することができる。γ−ＰＧＡの配座構造とイオン化に加え、水溶液中に存在する加水分解金属種の種類を知ることも重要である。種々の異なる化学種が生成するので、金属イオンに対する吸着能に差が生じ得る。 There are two possible mechanisms for metal adsorption on γ-PGA: (A) a direct interaction between a metal ion and a carboxyl moiety and (B) a movable heavy metal counterion due to an electrostatic potential field generated by a COO - group. Retention is involved. In addition to interaction with carboxylate groups, amide bonds can also provide weak interaction sites. In addition to the conformation structure and ionization of γ-PGA, it is also important to know the type of hydrolyzed metal species present in the aqueous solution. Since a variety of different chemical species are generated, there may be differences in the adsorption capacity for metal ions.
本発明者らは、鋭意研究を行った結果、γ−ポリグルタミン酸（γ−ＰＧＡ、Ｈ体）、γ−ポリグルタメートＮａ+体、γ−ポリグルタメートＫ+体、γ−ポリグルタメートＮＨ4 +体、γ−ポリグルタメートＣａ++体、γ−ポリグルタメートＭｇ++体又はこれらの混合物をカルシウム及びマグネシウムの錯形成吸着剤として添加し、可溶性及び生体利用性を向上させ腸におけるカルシウム吸収性を高めることを見出した。食料品によっては無機ミネラルの塩やミネラル粉末を含有させることによってミネラル分を増加させたのものもあるが、これらは他の共存物質と不溶な塩を生成する可能性がある。特定ミネラル種を過剰摂取すると他のミネラルの吸着を阻害する可能性があるため、体内のミネラル利用はあまり向上しない。例えば、カルシウムの大量摂取は鉄の吸収を阻害する。更に、食品がミネラル分を含み過ぎると食品の味という点において不利である。また、一般にミネラルは、小腸で吸収されるためには溶解した状態で存在しなければならないと考えられている。 As a result of intensive studies, the present inventors have found that γ-polyglutamic acid (γ-PGA, H form), γ-polyglutamate Na + form, γ-polyglutamate K + form, and γ-polyglutamate NH 4 + form. , Γ-polyglutamate Ca ++ isomer, γ-polyglutamate Mg ++ isomer or a mixture thereof is added as a calcium and magnesium complexing adsorbent to improve solubility and bioavailability and increase calcium absorption in the intestine I found out. Some foods have minerals increased by containing inorganic mineral salts or mineral powders, but these may produce salts that are insoluble with other coexisting substances. Since excessive intake of a specific mineral species may inhibit the adsorption of other minerals, the use of minerals in the body does not improve much. For example, large intakes of calcium inhibit iron absorption. Furthermore, if the food contains too much mineral, it is disadvantageous in terms of the taste of the food. In general, minerals are considered to have to exist in a dissolved state in order to be absorbed in the small intestine.
γ−ポリグルタミン酸（γ−ＰＧＡ、Ｈ体）及び各種γ−ポリグルタメート（Ｎａ+、Ｋ+、ＮＨ4 +、Ｃａ++、Ｍｇ++体）は小腸下部でミネラルに対し良好な溶解作用を有し、人体又は動物体内でカルシウム吸収を促進する。特に、本発明はγ−ポリグルタミン酸（γ−ＰＧＡ、Ｈ体）、γ−ポリグルタメートＮａ+体、γ−ポリグルタメートＮＨ4 +体、γ−ポリグルタメートＫ+体、γ−ポリグルタメートＣａ++体、γ−ポリグルタメートＭｇ++体又はこれらの混合物のダイエタリー製品において栄養補助剤として使用することに関する。 γ-polyglutamic acid (γ-PGA, H form) and various γ-polyglutamate (Na + , K + , NH 4 + , Ca ++ , Mg ++ form) have a good dissolving action on minerals in the lower part of the small intestine. And promotes calcium absorption in the human or animal body. In particular, the present invention relates to γ-polyglutamic acid (γ-PGA, H form), γ-polyglutamate Na + form, γ-polyglutamate NH 4 + form, γ-polyglutamate K + form, γ-polyglutamate Ca ++ The present invention relates to use as a dietary supplement in dietary products of the body, γ-polyglutamate Mg ++ body or mixtures thereof.
本発明の一実施形態において、ダイエタリー製品は栄養物質であり、前記ダイエタリー製品の全乾燥重量に対し０．００５重量％〜１００重量％の前記栄養補助剤を含む。他の実施形態において、ダイエタリー製品は食品又は飼料組成物であり、前記ダイエタリー製品の全乾燥重量に対し０．００５重量％〜５重量％の前記栄養補助剤を含む。 In one embodiment of the present invention, the dietary product is a nutritional substance and comprises 0.005% to 100% by weight of the nutritional supplement based on the total dry weight of the dietary product. In another embodiment, the dietary product is a food or feed composition and comprises 0.005% to 5% by weight of the nutritional supplement relative to the total dry weight of the dietary product.
本発明に係るダイエタリー製品は、５〜４０デキストロース当量のマルトデキストラン、乳タンパク質、大豆タンパク質単離物、グルコース、ラクトース、スクロース、フルクトース、小鎖オリゴ−フルクトース、グルカン又は他のオリゴ−ポリ多糖、コラーゲン、コラーゲンゼラチン加水分解物、α−デンプン、大豆タンパク質加水分解物、デンプン部分加水分解物、グリセロール、プロピレングリコール、エタノール、アラビアガム、グアーガム、カラギーナン（caragheneen）、セルロース及び他の変性セルロース、及びこれらの混合物から成る群から選択される成分を更に含むことができる。更に、前記ダイエタリー製品はソフトゲルカプセル又はハードゲルカプセル、錠剤又は液剤の形状とすることができる。 Dietary products according to the present invention include 5-40 dextrose equivalents of maltodextran, milk protein, soy protein isolate, glucose, lactose, sucrose, fructose, small chain oligo-fructose, glucan or other oligo-polypolysaccharide, collagen Collagen gelatin hydrolyzate, α-starch, soy protein hydrolyzate, starch partial hydrolysate, glycerol, propylene glycol, ethanol, gum arabic, guar gum, caragheneen, cellulose and other modified celluloses, and these It can further comprise a component selected from the group consisting of a mixture. Furthermore, the dietary product can be in the form of soft gel capsules or hard gel capsules, tablets or liquids.
本発明によれば、γ−ポリグルタミン酸（γ−ＰＧＡ、Ｈ体）、γ−ポリグルタメートＮａ+体、γ−ポリグルタメートＫ+体、γ−ポリグルタメートＮＨ4 +体、γ−ポリグルタメートＣａ++体及びγ−ポリグルタメートＭｇ++体の分子量は、それぞれ５０００〜２．５×１０6の範囲である。本発明者らは、インビトロの研究において１５０×１０3〜４５０×１０3ダルトンの範囲の分子量をそれぞれ有するγ−ポリグルタミン酸（γ−ＰＧＡ、Ｈ体）、γ−ポリグルタメートＮａ+体、γ−ポリグルタメートＫ+体、γ−ポリグルタメートＮＨ4 +体、γ−ポリグルタメートＣａ++体及びγ−ポリグルタメートＭｇ++体が骨芽細胞の増殖を促進することを見出し、インビボの研究において産卵鶏のパフォーマンスとブロイラー鶏の成長を向上させることを見出した。 According to the present invention, γ-polyglutamic acid (γ-PGA, H form), γ-polyglutamate Na + form, γ-polyglutamate K + form, γ-polyglutamate NH 4 + form, γ-polyglutamate Ca + form The molecular weights of the + form and the γ-polyglutamate Mg ++ form are in the range of 5000 to 2.5 × 10 6 , respectively. In the in vitro studies, the present inventors have found that γ-polyglutamic acid (γ-PGA, H form), γ-polyglutamate Na + form, γ- each having a molecular weight ranging from 150 × 10 3 to 450 × 10 3 daltons. It was found that polyglutamate K + body, γ-polyglutamate NH 4 + body, γ-polyglutamate Ca ++ body and γ-polyglutamate Mg ++ body promote the proliferation of osteoblasts, and lay eggs in in vivo studies. Found to improve chicken performance and broiler chicken growth.
市販目的量のγ−ポリグルタミン酸（γ−ＰＧＡ、Ｈ体）及びその塩（即ち、γ−ポリグルタメートＮａ+体、γ−ポリグルタメートＫ+体、γ−ポリグルタメートＮＨ4 +体、γ−ポリグルタメートＭｇ++体、γ−ポリグルタメートＣａ++体）は、液内発酵プロセスにより、枯草菌（納豆菌）（Bacillus subtilis. Bacillus subtilis var. natto)（Ｈ．クボタ（Kubota）ら、「枯草菌Ｆ−２−０１によるポリ（γ−グルタミン酸）の生産（Production of Poly((-Glutamic Acid) by Bacillus subtitlis F-2-01）」、Ｂｉｏｓｃｉ．Ｂｉｏｔｅｃｈ．Ｂｉｏｃｈｅｍ．５７（７）、１２１２−１２１３、１９９３及びＹ．オガワ（Ogawa）ら、「ジャー内発酵（Jar Fermentation）における枯草菌（ナットウ）によるγ−ポリグルタミン酸の効果的な生産（Efficient Production of (-Polyglutamic Acid by Bacillus subtilis (natto) in Jar Fermentation）」、６１（１０）、１６８４−１６８７、１９９７参照）を用いて生産することができ、また、バシラス・リシェニフォルミス（Bacillus licheniformis）（ＪＰ０５−３１６９９９（１９９３年３月１２日公開）参照）を用いＬ−グルタミン酸及びグルコースを主な供給原料として使用して生産することができる。微生物培養培地には、炭素源、窒素源、各種無機ミネラル分及び他の栄養分が含まれている。通常、炭素源の一部としてＬ−グルタミン酸量を３〜１２％、グルコースを５〜１２％の濃度範囲で用い、クエン酸を０．２〜２％の濃度で用い、ペプトンと硫酸アンモニウム又は尿素とを窒素源として用い、酵母抽出物を栄養源として用い、Ｍｎ++、Ｍｇ++やＮａＣｌをミネラル源として用いる。培養は、適切な通気と撹拌の下、３０〜４０℃の温度で維持し、ｐＨを尿素溶液又は水酸化ナトリウム溶液を用いて６〜７．５に維持する。培養時間は通常４８〜８４時間である。γ−ポリグルタミン酸（γ−ＰＧＡ、Ｈ体）及びその塩（即ち、γ−ポリグルタメートＮａ+体、γ−ポリグルタメートＫ+体、γ−ポリグルタメートＮＨ4 +体、γ−ポリグルタメートＭｇ++体、γ−ポリグルタメートＣａ++体）は細胞外に蓄積される。 Commercial target amounts of γ-polyglutamic acid (γ-PGA, H form) and salts thereof (ie, γ-polyglutamate Na + form, γ-polyglutamate K + form, γ-polyglutamate NH 4 + form, γ-poly Glutamate Mg ++ and γ-polyglutamate Ca ++ forms Bacillus subtilis (Bacillus subtilis var. Natto) (H. Kubota et al. Production of Poly ((-Glutamic Acid) by Bacillus subtitlis F-2-01) by Fungus F-2-01, Biosci. Biotech. Biochem. 57 (7), 1212-1213 1993 and Y. Ogawa et al., “Efficient Production of (-Polyglutamic Acid by Bacillus su) by Bacillus subtilis (Natto). btilis (natto) in Jar Fermentation ”, 61 (10), 1684-1687, 1997), and Bacillus licheniformis (JP 05-316999 (1993)). (Published on March 12))) using L-glutamic acid and glucose as main feedstocks, including microbial culture medium, carbon source, nitrogen source, various inorganic minerals and other nutrients Usually, as part of the carbon source, the amount of L-glutamic acid is 3-12%, glucose is used in a concentration range of 5-12%, citric acid is used in a concentration of 0.2-2%, using the peptone and ammonium sulfate or urea as a nitrogen source, using yeast extract as a nutrient source, Mn ++, using Mg ++ and NaCl as mineral sources. cultivation is suitably The temperature is maintained at 30-40 ° C. under aeration and stirring, and the pH is maintained at 6-7.5 using urea solution or sodium hydroxide solution, and the culture time is usually 48-84 hours. Polyglutamic acid (γ-PGA, H form) and salts thereof (ie, γ-polyglutamate Na + form, γ-polyglutamate K + form, γ-polyglutamate NH 4 + form, γ-polyglutamate Mg ++ form, (γ-polyglutamate Ca ++ form) accumulates extracellularly.
γ−ポリグルタミン酸（γ−ＰＧＡ、Ｈ体）及びその塩（即ち、γ−ポリグルタメートＮａ+体、γ−ポリグルタメートＫ+体、γ−ポリグルタメートＮＨ4 +体、γ−ポリグルタメートＭｇ++体、γ−ポリグルタメートＣａ++体）は通常、発酵用ブロスから一連の手続により抽出するが、その手続には超遠心や加圧ろ過により細胞を分離し、３〜４倍のエタノールを添加してγ−ポリグルタミン酸（γ−ＰＧＡ、Ｈ体）やその塩（即ち、γ−ポリグルタメートＮａ+体、γ−ポリグルタメートＫ+体、γ−ポリグルタメートＮＨ4 +体、γ−ポリグルタメートＭｇ++体、γ−ポリグルタメートＣａ++体）を沈澱させる工程が含まれる。沈澱物を水に再度溶解し、新たにエタノールを使用しγ−ポリグルタミン酸（γ−ＰＧＡ、Ｈ体）やその塩（即ち、γ−ポリグルタメートＮａ+体、γ−ポリグルタメートＫ+体、γ−ポリグルタメートＮＨ4 +体、γ−ポリグルタメートＭｇ++体、γ−ポリグルタメートＣａ++体）を沈澱させる。この溶解−沈澱ステップを数回繰り返し、純粋なγ−ポリグルタミン酸（γ−ＰＧＡ、Ｈ体）やその塩（即ち、γ−ポリグルタメートＮａ+体、γ−ポリグルタメートＫ+体、γ−ポリグルタメートＮＨ4 +体、γ−ポリグルタメートＭｇ++体、γ−ポリグルタメートＣａ++体）を回収する。 γ-polyglutamic acid (γ-PGA, H form) and salts thereof (ie, γ-polyglutamate Na + form, γ-polyglutamate K + form, γ-polyglutamate NH 4 + form, γ-polyglutamate Mg ++ Body, γ-polyglutamate Ca ++ form) is usually extracted from the broth for fermentation by a series of procedures, in which cells are separated by ultracentrifugation or pressure filtration, and 3-4 times ethanol is added. Γ-polyglutamic acid (γ-PGA, H form) and salts thereof (ie, γ-polyglutamate Na + form, γ-polyglutamate K + form, γ-polyglutamate NH 4 + form, γ-polyglutamate Mg) ++ form, γ-polyglutamate Ca ++ form) is included. The precipitate is redissolved in water, and γ-polyglutamic acid (γ-PGA, H form) or a salt thereof (ie, γ-polyglutamate Na + form, γ-polyglutamate K + form, γ is newly used using ethanol. -Precipitating polyglutamate NH 4 + form, γ-polyglutamate Mg ++ form, γ-polyglutamate Ca ++ form). This dissolution-precipitation step is repeated several times to obtain pure γ-polyglutamic acid (γ-PGA, H form) or a salt thereof (ie, γ-polyglutamate Na + form, γ-polyglutamate K + form, γ-polyglutamate). NH 4 + isomer, γ-polyglutamate Mg ++ isomer, and γ-polyglutamate Ca ++ isomer) are recovered.
通常、γ−ポリグルタミン酸（γ−ＰＧＡ、Ｈ体）やその塩（即ち、γ−ポリグルタメートＮａ+体、γ−ポリグルタメートＫ+体、γ−ポリグルタメートＮＨ4 +体、γ−ポリグルタメートＭｇ++体、γ−ポリグルタメートＣａ++体）は、水やエタノール、メタノール等の適切な溶媒に溶解し、ｐＨは５．０〜７．５に調節する。定常的に撹拌しながら、この溶液に、適切に選択した多官能化学架橋剤、例えばポリグリセロールポリグリシジルエーテルやソルビトールポリグリシジルエーテル、ポリエチレングリコールジグリシジルエーテル、トリメチロールプロパントリアクリレートを添加する。添加量は、架橋剤の種類やヒドロゲルに要求される品質により異なるが、γ−ポリグルタミン酸（γ−ＰＧＡ、Ｈ体）又はその塩（即ち、γ−ポリグルタメートＮａ+体、γ−ポリグルタメートＫ+体、γ−ポリグルタメートＮＨ4 +体、γ−ポリグルタメートＭｇ++体、γ−ポリグルタメートＣａ++体）の重量の０．０１〜２０％の範囲である。通常、ゲル化反応は５０〜１２０℃の反応温度で１〜４時間で完了するが、使用する器具や条件により異なる。次に、生成したヒドロゲルを凍結乾燥し、乾燥した架橋γ−ポリグルタミン酸（γ−ＰＧＡ、Ｈ体）或いはその塩（即ち、γ−ポリグルタメートＮａ+体、γ−ポリグルタメートＫ+体、γ−ポリグルタメートＮＨ4 +体、γ−ポリグルタメートＭｇ++体、γ−ポリグルタメートＣａ++体）を生成する。これは、超吸水能を有し、非水溶性であり、水中で完全に膨張すると、無色透明で生分解可能なヒドロゲルを形成する。 Usually, γ-polyglutamic acid (γ-PGA, H form) and salts thereof (that is, γ-polyglutamate Na + form, γ-polyglutamate K + form, γ-polyglutamate NH 4 + form, γ-polyglutamate Mg) The ++ form and the γ-polyglutamate Ca ++ form are dissolved in an appropriate solvent such as water, ethanol, methanol, and the pH is adjusted to 5.0 to 7.5. With constant stirring, an appropriately selected polyfunctional chemical cross-linking agent such as polyglycerol polyglycidyl ether, sorbitol polyglycidyl ether, polyethylene glycol diglycidyl ether, trimethylolpropane triacrylate is added to this solution. The amount added varies depending on the type of crosslinking agent and the quality required for the hydrogel, but γ-polyglutamic acid (γ-PGA, H form) or a salt thereof (ie, γ-polyglutamate Na + form, γ-polyglutamate K) + Γ-polyglutamate NH 4 + isomer, γ-polyglutamate Mg ++ isomer, γ-polyglutamate Ca ++ isomer) in a range of 0.01 to 20%. Usually, the gelation reaction is completed in 1 to 4 hours at a reaction temperature of 50 to 120 ° C., but varies depending on the equipment and conditions used. Next, the produced hydrogel was freeze-dried, and the dried crosslinked γ-polyglutamic acid (γ-PGA, H form) or a salt thereof (ie, γ-polyglutamate Na + form, γ-polyglutamate K + form, γ- Polyglutamate NH 4 + form, γ-polyglutamate Mg ++ form, and γ-polyglutamate Ca ++ form). It is super water-absorbing, water-insoluble, and forms a colorless, transparent and biodegradable hydrogel when fully swollen in water.
分子量が５０００〜９０００００のγ−ポリグルタミン酸（γ−ＰＧＡ、Ｈ体）又はその塩（即ち、γ−ポリグルタメートＮａ+体、γ−ポリグルタメートＫ+体、γ−ポリグルタメートＮＨ4 +体、γ−ポリグルタメートＭｇ++体、γ−ポリグルタメートＣａ++体）は、選択された特定の反応条件（ｐＨ、温度、反応時間、γ−ポリグルタミン酸（γ−ＰＧＡ、Ｈ体）濃度）で制御された酸加水分解により生成することができる。ｐＨについては、ＨＣｌやＨ2ＳＯ4、他の有機酸等の適切な酸類（acidulants）でｐＨ２．５〜６．５の範囲にすることができる。加水分解の温度は、５０〜１２０℃の範囲にすることができる。反応時間は０．５〜５時間とすることができ、そして分子量１×１０6以上のγ−ポリグルタミン酸（γ−ＰＧＡ、Ｈ体）の濃度は、適宜必要ないかなる濃度とすることもできる。高純度で、低分子量から中程度の分子量のγ−ポリグルタミン酸（γ−ＰＧＡ、Ｈ体）又はその塩（即ち、γ−ポリグルタメートＮａ+体、γ−ポリグルタメートＫ+体、γ−ポリグルタメートＮＨ4 +体、γ−ポリグルタメートＭｇ++体、γ−ポリグルタメートＣａ++体）を選択的に生成するためには、反応終了後、透析や膜ろ過による更なる精製と乾燥を行う必要がある。酸加水分解速度はｐＨが低い程、温度が高い程、また、γ−ポリグルタミン酸（γ−ＰＧＡ、Ｈ体）の濃度が高い程速い。γ−ポリグルタメート塩（即ち、γ−ポリグルタメートＮａ+体、γ−ポリグルタメートＫ+体、γ−ポリグルタメートＮＨ4 +体、γ−ポリグルタメートＭｇ++体及びγ−ポリグルタメートＣａ++体）は、選択したγ−ポリグルタミン酸（γ−ＰＧＡ、Ｈ体）を、選択したＮａ+、Ｋ+、ＮＨ4 +、Ｃａ++又はＭｇ++の金属イオン塩基性水酸化物溶液又は酸化物と反応させることにより生産することができ、ｐＨは必要に応じて５．０〜７．２の所望の値に調節できる。 Γ-polyglutamic acid (γ-PGA, H form) having a molecular weight of 5000 to 900,000 or a salt thereof (that is, γ-polyglutamate Na + form, γ-polyglutamate K + form, γ-polyglutamate NH 4 + form, γ -Polyglutamate Mg ++ form and γ-polyglutamate Ca ++ form are controlled by selected specific reaction conditions (pH, temperature, reaction time, γ-polyglutamic acid (γ-PGA, H form) concentration) Can be produced by acid hydrolysis. The pH, HCl or H 2 SO 4, with a suitable acid such as other organic acids (acidulants) can be in the range of PH2.5～6.5. The temperature of hydrolysis can be in the range of 50-120 ° C. The reaction time can be 0.5 to 5 hours, and the concentration of γ-polyglutamic acid (γ-PGA, H form) having a molecular weight of 1 × 10 6 or more can be any required concentration. High purity, low to medium molecular weight γ-polyglutamic acid (γ-PGA, H form) or a salt thereof (ie, γ-polyglutamate Na + form, γ-polyglutamate K + form, γ-polyglutamate) NH 4 + form, γ-polyglutamate Mg ++ form, γ-polyglutamate Ca ++ form) must be further purified and dried by dialysis or membrane filtration after the reaction is completed. There is. The acid hydrolysis rate is faster as the pH is lower, the temperature is higher, and the concentration of γ-polyglutamic acid (γ-PGA, H form) is higher. γ-polyglutamate salt (ie, γ-polyglutamate Na + isomer, γ-polyglutamate K + isomer, γ-polyglutamate NH 4 + isomer, γ-polyglutamate Mg ++ isomer and γ-polyglutamate Ca ++ isomer) ) A selected γ-polyglutamic acid (γ-PGA, H form) with a selected metal ion basic hydroxide solution or oxide of Na + , K + , NH 4 + , Ca ++ or Mg ++ The pH can be adjusted to a desired value of 5.0 to 7.2 as necessary.
本発明を更に詳細に説明するため、以下に実施例を示し、本発明がどのように非常に改善されたカルシウム吸収を達成し、骨の成長と強度に健康上の利点をもたらし、カルシウム損失を減らし、骨粗鬆症の症状を軽減するのかを示す。しかしながら、本発明の範囲はこれら実施例によって何ら限定されるものではない。 To further illustrate the present invention, the following examples are provided to illustrate how the present invention achieves greatly improved calcium absorption, brings health benefits to bone growth and strength, and reduces calcium loss. Shows whether to reduce and reduce the symptoms of osteoporosis. However, the scope of the present invention is not limited by these examples.
＜実施例１＞ インビトロにおける細胞培養の研究−骨芽細胞の増殖促進
分子量９８０×１０3ダルトンのγ−ポリグルタミン酸（γ−ＰＧＡ、Ｈ体）ＨＭ、分子量８８０×１０3ダルトンのγ−ポリグルタメートＮａ+体ＨＭ及び分子量２５０×１０3ダルトンのγ−ポリグルタメートＮａ+体ＬＭを本研究に使用した。
γ−ポリグルタミン酸（γ−ＰＧＡ、Ｈ体）及び／又はγ−ポリグルタメートＮａ+体１０ｍｇを、１０％ウシ胎児血清（ＦＢＳ）含有Ｆ−１２ＤＭＥＭ培地（ダルベッコ変法イーグル培地）１ｍＬに加え段階希釈を適切に行うことにより、同一組成の栄養培地中におけるγ−ポリグルタミン酸（γ−ＰＧＡ、Ｈ体）及び／又はγ−ポリグルタメートＮａ+体の濃度が異なる（０．１％〜４．９×１０-5％）培地を調製し、各サンプル２００μＬを９６ウェル培養プレートに三連で添加した。１０％ウシ胎児血清（ＦＢＳ）のみを含むＦ−１２ＤＭＥＭ培地（ダルベッコ変法イーグル培地）をコントロールとして用いた。
<Example 1> Research of cell culture in vitro-Promotion of osteoblast proliferation γ-polyglutamic acid (γ-PGA, H form) HM having a molecular weight of 980 × 10 3 daltons, γ-polyglutamate having a molecular weight of 880 × 10 3 daltons Na + form HM and γ-polyglutamate Na + form LM having a molecular weight of 250 × 10 3 daltons were used in this study.
Serial dilution by adding 10 mg of γ-polyglutamic acid (γ-PGA, H form) and / or γ-polyglutamate Na + form to 1 mL of F-12 DMEM medium (Dulbecco's modified Eagle medium) containing 10% fetal bovine serum (FBS) Are appropriately performed, the concentration of γ-polyglutamic acid (γ-PGA, H form) and / or γ-polyglutamate Na + form in the nutrient medium having the same composition is different (0.1% to 4.9 ×). 10 −5 %) medium was prepared and 200 μL of each sample was added in triplicate to a 96-well culture plate. F-12 DMEM medium (Dulbecco's modified Eagle medium) containing only 10% fetal bovine serum (FBS) was used as a control.
指数増殖期のヒト骨髄幹細胞から骨芽細胞のサンプルを、１２００ｒｐｍで６分間遠心することにより生育培地から分離し、１０ｍＬのリン酸緩衝溶液（ＰＢＳ）（０．０１Ｍホスフェート、ｐＨ７．４）で３回洗浄し、１０％ウシ胎児血清（ＦＢＳ）含有Ｆ−１２ＤＭＥＭ培地中で再度懸濁し、細胞密度を１×１０5個／ｍＬとした。活発に増殖している細胞の懸濁液２００μＬを各サンプルウェルに加えた。９６ウェル培養プレートのサンプルをボルテックスミキサーで十分に混合し、５％ＣＯ2雰囲気下ＲＨ７５％、３４℃で４８時間インキュベートした。ＭＴＴ［３−（４,５−ジメチルチアゾール−２−イル）−２,５−ジフェノールテトラゾリウムブロミド］溶液（１ｍｇ／ｍＬ）２０μLを各ウェルに加え、十分に混合し再度４時間インキュベートした。１０％ＳＤＳ／０．０１Ｎ ＨＣｌ緩衝液１００μＬを各サンプルウェルに加え、室温で一晩結晶を溶解させた。各サンプルウェルの光学濃度（ＯＤ570nm）を測定、記録し、骨芽細胞の増殖向上について計算した。 Samples of osteoblasts from exponentially growing human bone marrow stem cells are separated from the growth medium by centrifugation at 1200 rpm for 6 minutes and 3 with 10 mL of phosphate buffered saline (PBS) (0.01 M phosphate, pH 7.4). The cells were washed once and suspended again in F-12 DMEM medium containing 10% fetal bovine serum (FBS) to a cell density of 1 × 10 5 cells / mL. A 200 μL suspension of actively growing cells was added to each sample well. Samples from a 96-well culture plate were thoroughly mixed with a vortex mixer and incubated for 48 hours at 34 ° C., 75% RH in a 5% CO 2 atmosphere. 20 μL of MTT [3- (4,5-dimethylthiazol-2-yl) -2,5-diphenoltetrazolium bromide] solution (1 mg / mL) was added to each well, mixed well and incubated again for 4 hours. 100 μL of 10% SDS / 0.01N HCl buffer was added to each sample well to dissolve the crystals overnight at room temperature. The optical density (OD 570 nm ) of each sample well was measured and recorded and calculated for improved osteoblast proliferation.
骨芽細胞のミトコンドリアの脱水素酵素はＭＴＴ塩（無色のテトラゾリウム塩）と反応し、青色のホルマザン塩の結晶を生成することができる。次いで、ホルマザンの結晶はＳＤＳと反応し、可溶性の青色ホルマザンに変化した。青色ホルマザンの色の強度は骨芽細胞の増殖量を表す。骨芽細胞の増殖の向上は次に示すように計算できる。
増加(ＭＴＴ)％＝(（ＯＤ570）ａ−（ＯＤ570）ｃ)／(ＯＤ570)ｃ×１００％
(ＯＤ570)ａ：サンプルウェルの光学濃度
(ＯＤ570)ｃ：コントロールウェルの光学濃度
Osteoblast mitochondrial dehydrogenase can react with MTT salt (colorless tetrazolium salt) to produce blue formazan salt crystals. The formazan crystals then reacted with SDS and changed to a soluble blue formazan. The intensity of blue formazan color represents the amount of osteoblast proliferation. The improvement in osteoblast proliferation can be calculated as follows.
Increase (MTT)% = ((OD 570 ) a− (OD 570 ) c) / (OD 570 ) c × 100%
(OD 570 ) a: Optical density of sample well
(OD 570 ) c: Optical density of control well
＜実施例２＞
実施例１から得た増殖向上率の結果を表２に示す。
<Example 2>
The results of the growth improvement rate obtained from Example 1 are shown in Table 2.
結果から明らかなように、０．３９ｐｐｍという低濃度においても骨芽細胞の増殖は、γ−ポリグルタメートＮａ+体ＨＭの場合で１３０％以上、γ−ポリグルタメートＮａ+体ＬＭの場合で８０％以上、γ−ポリグルタミン酸（γ−ＰＧＡ、Ｈ体）ＨＭの場合で１２５％以上、向上した。より高分子量のγ−ポリグルタメートＮａ+体ＨＭの場合に、より良好な増殖向上が示された。 As is apparent from the results, even at a low concentration of 0.39 ppm, osteoblast proliferation is 130% or more in the case of γ-polyglutamate Na + form HM and 80% in the case of γ-polyglutamate Na + form LM. As described above, in the case of γ-polyglutamic acid (γ-PGA, H form) HM, the improvement was 125% or more. In the case of higher molecular weight γ-polyglutamate Na + form HM, better growth improvement was shown.
＜実施例３＞ インビトロにおける細胞培養の研究−骨の形成促進
分子量９８０×１０3ダルトンのγ−ポリグルタミン酸（γ−ＰＧＡ、Ｈ体）ＨＭ、分子量８８０×１０3ダルトンのγ−ポリグルタメートＮａ+体ＨＭ及び分子量２５０×１０3ダルトンのγ−ポリグルタメートＮａ+体ＬＭを本研究に使用した。
<Example 3> In vitro cell culture study-promotion of bone formation γ-polyglutamic acid (γ-PGA, H form) HM having a molecular weight of 980 × 10 3 daltons, γ-polyglutamate Na + having a molecular weight of 880 × 10 3 daltons Body HM and gamma-polyglutamate Na + form LM with a molecular weight of 250 × 10 3 daltons were used in this study.
γ−ポリグルタミン酸（γ−ＰＧＡ、Ｈ体）及び／又はγ−ポリグルタメートＮａ+体１０ｍｇを、１０％ウシ胎児血清（ＦＢＳ）含有Ｆ−１２ＤＭＥＭ培地（ダルベッコ変法イーグル培地）１ｍＬに加え段階希釈を適切に行うことにより、栄養培地中におけるγ−ポリグルタミン酸（γ−ＰＧＡ、Ｈ体）及び／又はγ−ポリグルタメートＮａ+体の濃度が異なる（０．１％〜３．１×１０-3％）培地を調製し、各サンプル１ｍＬを６ウェル培養プレートに三連で添加した。１０％ウシ胎児血清（ＦＢＳ）のみを含むＦ−１２ＤＭＥＭ培地（ダルベッコ変法イーグル培地）をコントロールとして用いた。 Serial dilution by adding 10 mg of γ-polyglutamic acid (γ-PGA, H form) and / or γ-polyglutamate Na + form to 1 mL of F-12 DMEM medium (Dulbecco's modified Eagle medium) containing 10% fetal bovine serum (FBS) Is appropriately performed, the concentration of γ-polyglutamic acid (γ-PGA, H form) and / or γ-polyglutamate Na + form in the nutrient medium is different (0.1% to 3.1 × 10 −3). %) Medium was prepared and 1 mL of each sample was added in triplicate to a 6-well culture plate. F-12 DMEM medium (Dulbecco's modified Eagle medium) containing only 10% fetal bovine serum (FBS) was used as a control.
１０％ウシ胎児血清（ＦＢＳ）含有Ｆ−１２ＤＭＥＭ（ダルベッコ変法イーグル培地）２ｍＬを各サンプルウェルに添加し十分に混合した。
指数増殖期のヒト骨髄幹細胞から骨芽細胞のサンプルを、１２００ｒｐｍで６分間遠心することにより成長培地から分離し、１０ｍＬのリン酸緩衝溶液（ＰＢＳ）（０．０１Ｍリン酸、ｐＨ７．４）で３回洗浄し、１０％ウシ胎児血清（ＦＢＳ）含有Ｆ−１２ＤＭＥＭ培地中で再度懸濁し、細胞密度を１×１０5個／ｍＬとした。活発に増殖している細胞の懸濁液１ｍＬを各サンプルウェルに加えた。６ウェル培養プレートのサンプルをボルテックスミキサーで十分に混合し、５％ＣＯ2雰囲気下ＲＨ７５％、３４℃で７日間インキュベートした。次いで、トリプシン溶液２００μＬを各ウェルに添加し、３４℃で約３０秒間反応させた。Ｆ−１２ＤＭＥＭ培地（ダルベッコ変法イーグル培地）１ｍＬを各サンプルウェルに添加し十分に混合した。懸濁した細胞を回収除去した。ｐＮＰＰ（１００μＬ）／ｐ−ニトロフェニルホスフェート溶液（ＳＫ−５９００、ベクター社）（２ｍＬ）を含有する０．０１Ｍ重炭酸／炭酸ナトリウム溶液（ｐＨ１０）１ｍＬを添加し、十分に混合して室温で３０分間反応させ、０．１Ｍ ＮａＯＨ溶液１ｍＬを添加し反応を停止させた。４１０ｎｍでの吸光度を測定し記録した。
2 mL of F-12DMEM (Dulbecco's modified Eagle medium) containing 10% fetal bovine serum (FBS) was added to each sample well and mixed well.
A sample of osteoblasts from exponentially growing human bone marrow stem cells is separated from the growth medium by centrifuging at 1200 rpm for 6 minutes, and 10 mL of phosphate buffered saline (PBS) (0.01 M phosphate, pH 7.4). The cells were washed three times and resuspended in F-12DMEM medium containing 10% fetal bovine serum (FBS) to a cell density of 1 × 10 5 cells / mL. A 1 mL suspension of actively growing cells was added to each sample well. Samples from 6-well culture plates were mixed well with a vortex mixer and incubated for 7 days at 34 ° C., 75% RH in 5% CO 2 atmosphere. Then, 200 μL of trypsin solution was added to each well and reacted at 34 ° C. for about 30 seconds. 1 mL of F-12 DMEM medium (Dulbecco's modified Eagle medium) was added to each sample well and mixed well. Suspended cells were collected and removed. Add 1 mL of 0.01 M bicarbonate / sodium carbonate solution (pH 10) containing pNPP (100 μL) / p-nitrophenyl phosphate solution (SK-5900, Vector) (2 mL), mix well, and mix at room temperature for 30 The reaction was allowed to proceed for 1 minute, and 1 mL of 0.1 M NaOH solution was added to stop the reaction. The absorbance at 410 nm was measured and recorded.
＜実施例４＞
実施例３から得た結果を次の表３に示した。
<Example 4>
The results obtained from Example 3 are shown in Table 3 below.
アルカリホスファターゼ活性は骨芽細胞が存在することを示すものである。アルカリホスファターゼ活性は細胞が石灰化する前に高くなり、細胞密度は骨前駆体の分化及び骨形成を規定する。ヒト骨髄幹細胞を１週間培養した場合、骨芽細胞の存在を示すアルカリホスファターゼ活性が出現する（ヴァインレープ（Weinreb）Ｍ．、スキナー（Schinar）Ｄ．Ｍ．及びロダン（Rodan）Ｇ．Ａ．１９９０、「ｉｎ ｓｉｔｕハイブリダイゼーションにより可視化したラットの骨発達におけるオステオデンティン及びオステオカルシンの発現、アルカリフォスファターゼの異なるパターン（Different Pattern of Alkaline Phosphatase、Osteodentin, and Osteocalcin Expression in Developing Rat Bone Visualized by in situ Hybridization）」、Ｊ．Ｂｏｎｅ Ｍｉｎｅｒ．Ｒｅｓ．５：８３８−８４２；ヤオ（Yao）Ｋ．Ｌ．、トデスカン（Todescan）Ｒ．Ｊｒ．及びソデック（Sodek）Ｊ．、１９９４、「成熟ラットの培養骨髄細胞による石灰化組織形成中のマトリックスタンパク質合成における一時的変化及びｍＲＮＡの発現（Temporal Changes in Matrix Protein Synthesis and mRNA Expression during Mineralized Tissue Formation by Adult Rat Bone Marrow Cells in Culture）」、Ｊ．Ｂｏｎｅ Ｍｉｎｅｒ．Ｒｅｓ．９：２３１−２４０；及びヘスベルトソン（Hesbertson）Ａ．及びオービン（Aubin）Ｊ．Ｅ．１９９５、「デキサメタゾンはラットの骨髄基質培養物のサブポピュレーション構成を変化させる（Dexamethasone, Alters the Subpopulation Make-Up of Rat Bone Marror Stromal Cultures)」Ｊ．Ｂｏｎｅ Ｍｉｎｅｒ．Ｒｅｓ．１０：２８５−２９４参照）。 Alkaline phosphatase activity indicates the presence of osteoblasts. Alkaline phosphatase activity is increased before the cells are mineralized, and cell density defines osteoprogenitor differentiation and bone formation. When human bone marrow stem cells are cultured for 1 week, alkaline phosphatase activity indicating the presence of osteoblasts appears (Weinreb M., Skinar DM and Rodan GA 1990). , “Different Pattern of Alkaline Phosphatase, Osteodentin, and Osteocalcin Expression in Developing Rat Bone Visualized by In Situ Hybridization” J. Bone Miner.Res.5: 838-842; Yao KL, Todescan R.Jr., and Sodek J., 1994, “Climate by cultured rat bone marrow cells. In the synthesis of matrix proteins during chemical tissue formation "Temporal Changes in Matrix Protein Synthesis and mRNA Expression during Mineralized Tissue Formation by Adult Rat Bone Marrow Cells in Culture", J. Bone Miner. Res. 9: 231-240; and Hesbertson A. and Aubin JE 1995, “Dexamethasone, Alters the Subpopulation Make-Up of Rat Bone Marror Stromal Cultures,” J. Bone. Miner. Res. 10: 285-294).
表３の結果はγ−ポリグルタメートＮａ+体ＨＭ及びγ−ポリグルタメートＮａ+体ヒドロゲルの双方が骨芽細胞の増殖促進において効果的であることを示しており、最適濃度はそれぞれγ−ポリグルタメートＮａ+体ＨＭの場合で５００ｐｐｍ（０．０５％）とγ−ポリグルタメートＮａ+体ヒドロゲルの場合で６．２ｐｐｍ（０．００６２％）である。低分子量のγ−ポリグルタメートＮａ+体ＬＭもまた骨芽細胞の増殖を促進するが、１０００ｐｐｍ（０．１％）という非常に高い濃度である。この結果は、γ−ポリグルタメートＮａ+体が、骨芽細胞とその前駆体による生体利用可能なカルシウムの吸収を効果的に促進することを示唆しており、骨の成長においてγ−ポリグルタメートＮａ+体の強い親水性と活性コイルコンホメーションが、骨成長因子（ＴＧＦ−β、アクチビン、骨形成因子（ＢＭＰ）、骨シアロタンパク質（ＢＳＰ）等）の機能を、この因子と効果的に錯体形成することにより促進し、骨芽細胞へ因子を輸送し、骨を形成及び成長させる。 The results in Table 3 show that both γ-polyglutamate Na + form HM and γ-polyglutamate Na + form hydrogel are effective in promoting osteoblast proliferation, and the optimum concentrations are respectively γ-polyglutamate. In the case of Na + form HM, it is 500 ppm (0.05%), and in the case of γ-polyglutamate Na + form hydrogel, it is 6.2 ppm (0.0062%). Low molecular weight γ-polyglutamate Na + form LM also promotes osteoblast proliferation, but at a very high concentration of 1000 ppm (0.1%). This result suggests that γ-polyglutamate Na + form effectively promotes the absorption of bioavailable calcium by osteoblasts and their precursors, and γ-polyglutamate Na in bone growth. + Strong hydrophilicity and active coil conformation of the body effectively complex the function of bone growth factors (TGF-β, activin, bone morphogenetic factor (BMP), bone sialoprotein (BSP), etc.) with this factor Promotes by forming, transports factors to osteoblasts, and forms and grows bone.
＜実施例５＞ インビボにおける屋外給餌実験−ユアン−アン(Yuan-an)養鶏場での放牧養鶏（産卵鶏）
７３週齢の産卵鶏を本研究に用いた。全８区画（１区画８０００羽）を用いた。通常飼料（アホ（Aho）P.W. 2002, 「米国鶏肉産業案内（Introduction to the US chicken meat industry）」801〜818頁、「商用鶏肉・鶏卵の生産（Commercial Chicken Meat and Egg Production）」、第５版、ベル（Bell）, D. D.及びウェーバー・ジュニア（Weaver, junior）W.D.編、Kluwer Publishing；ノーウェル（Norwell） BS、ヘブンスタイン（Havenstein） G.B.ら、1994, 「１９５７年、１９９１年の「典型的」ブロイラー餌を給餌したときの、生育、生存率、飼料要求率に関する１９５７年対１９９１年の比較（Growth, livability, and feed conversion of 1957 vs 1991 broilers when fed "typical" 1957 and 1991 broiler diets）」、Poult. Sci. 73: 1785-1794）を４週間与え、次の４週間は１００ｐｐｍのγ−ポリグルタメートＮａ+体ＬＭ（分子量２００×１０3〜４００×１０3）を含有する通常飼料を与え、次いで２週間、通常飼料を与え、これら期間に亘って鶏卵サンプルを集めた。鶏卵サンプルについて、卵殻強度、卵殻厚、卵白及び卵黄のｐＨ、卵白高を決定し、ＨＵ値（鶏卵の品質の凡その鮮度指数又は健全性指数）を算出した
<Example 5> Outdoor feeding experiment in vivo-Grazing chicken (laying hen) in Yuan-an poultry farm
A 73-week-old laying hen was used in this study. A total of 8 compartments (1 compartment 8000 birds) were used. Normal feed (Aho PW 2002, “Introduction to the US chicken meat industry”, pages 801-818, “Commercial Chicken Meat and Egg Production”, 5th edition , Bell, DD and Weaver, junior WD, Kluwer Publishing; Norwell BS, Havenstein GB et al., 1994, “Atypical” broilers of 1957, 1991 1957 vs. 1991 broilers when fed "typical" 1957 and 1991 broiler diets), "Poult,""Growth, livability, and feed conversion of 1957 vs 1991 broilers when fed" typical "1957 and 1991 broiler diets" Sci. 73: 1785-1794) for 4 weeks, then for the next 4 weeks, a normal diet containing 100 ppm γ-polyglutamate Na + form LM (molecular weight 200 × 10 3 to 400 × 10 3 ) was given, and then 2 weeks, normal feed Given were collected egg samples over these periods. For egg samples, eggshell strength, eggshell thickness, pH of egg white and egg yolk, egg white height were determined, and HU value (approximate freshness index or health index of chicken egg quality) was calculated.
＜実施例６＞
実施例５で得た結果を表４、５及び６に示した。
<Example 6>
The results obtained in Example 5 are shown in Tables 4, 5 and 6.
注：１．７３週齢の産卵鶏を用いた。
２．４区画（８０００羽／区画）を用いた。
３．「*」は鶏卵１０個の平均値である。
４．ＨＵ値は次のように定義される：
ＨＵ＝ハウユニット・・・補正された卵アルブミンの高さの値
＝100＊log（H−Ｇ＊0.5（30＊Ｗ＊0.37−100）／100＋1.9
ここで、Ｈ−アルブミンの高さ（ｍｍ）
Ｗ−卵重（ｇ）
Ｇ−３２．２、重力定数
５．ＫＧＦ−卵殻強度の単位
Note: 1.73 weeks old laying hens were used.
2.4 sections (8000 birds / section) were used.
3. “ * ” Is the average value of 10 eggs.
4). The HU value is defined as follows:
HU = Howe unit: Corrected height of egg albumin
= 100 * log (HG * 0.5 (30 * W * 0.37-100) /100+1.9
Here, the height of H-albumin (mm)
W-Egg weight (g)
G-32.2, gravity constant 5. KGF-eggshell strength unit
表４、５及び６の結果から、卵黄、卵白の強度及び卵殻強度に顕著な改善が示された。これは、γ−ポリグルタメートＮａ+体を含有する飼料を与えた期間のＨＵ値の差の減少及びＫＧＦ値の差の減少に示されている。γ−ポリグルタメートＮａ+体を含有する飼料を与えた期間では、卵殻厚、卵白高、卵黄色の全てが改善された。 The results in Tables 4, 5 and 6 showed significant improvements in egg yolk, egg white strength and eggshell strength. This is shown in the decrease in the difference in HU values and the decrease in the difference in KGF values during the period of feeding the feed containing γ-polyglutamate Na + form. In the period when the feed containing the γ-polyglutamate Na + body was given, eggshell thickness, egg white height, and egg yolk were all improved.
＜実施例７＞ インビボにおける屋外給餌実験−イーチャン（Yee−Thiang）養豚場での放牧養豚
γ−ポリグルタミン酸（γ−ＰＧＡ、Ｈ体）、γ−ポリグルタメートＮａ+体及びγ−ポリグルタメートＣａ++体の栄養的・健康的側面について子豚（２群２セット）を用いた放牧養豚を３９日間行って調べた。標準飼料に対し１００ｐｐｍの濃度でγ−ポリグルタメートＮａ+体及びγ−ポリグルタメートＣａ++体を飼料補助剤として含む。表７に結果を示す。
<Example 7> Outdoor feeding experiment in vivo-grazing pigs in Yee-Thiang pig farm γ-polyglutamic acid (γ-PGA, H-form), γ-polyglutamate Na + form and γ-polyglutamate Ca + + The nutritional and health aspects of the body were investigated by conducting grazing pigs using piglets (2 groups, 2 sets) for 39 days. Γ-polyglutamate Na + form and γ-polyglutamate Ca ++ form are contained as feed supplements at a concentration of 100 ppm with respect to the standard feed. Table 7 shows the results.
＜実施例８＞
表７に実施例７の結果を示す。
<Example 8>
Table 7 shows the results of Example 7.
表７の結果より、γ−ポリグルタメートＣａ++体は体脂肪の減少及び子豚の飼料要求率（feed-to meat conversion）の減少に有効であるが、γ−ポリグルタメートＮａ+体ではこの飼料要求率が若干増加し、子豚の体脂肪減少においては効果はほとんど認められないことが明らかとなった。 From the results of Table 7, γ-polyglutamate Ca ++ body is effective in reducing body fat and feed-to meat conversion, but in γ-polyglutamate Na + body, The feed demand rate increased slightly, and it was clarified that there was almost no effect in reducing body fat in piglets.
＜実施例９＞ インビボにおける屋外給餌実験−トン−サン（Tong San）養鶏場での放牧養鶏（ブロイラー）
γ−ポリグルタメートＮａ+体の栄養的・健康的側面をブロイラーの雛（４群２セット）を用いた放牧養鶏を２５日間行い調べた。標準飼料に対し１００ｐｐｍの濃度で分子量２５０×１０3のγ−ポリグルタメートＮａ+体ＬＭを飼料補助剤として含む。表８に結果を示す。
<Example 9> In vivo outdoor feeding experiment-grazing chicken (broiler) at Tong San poultry farm
The nutritional and healthy aspects of the γ-polyglutamate Na + body were examined by carrying out grazing chickens using broiler chicks (4 groups, 2 sets) for 25 days. Γ-polyglutamate Na + form LM having a molecular weight of 250 × 10 3 and a concentration of 100 ppm relative to the standard feed is included as a feed supplement. Table 8 shows the results.
＜実施例１０＞
表８に実施例９の結果を示す。
<Example 10>
Table 8 shows the results of Example 9.
この結果、γ−ポリグルタメートＮａ+体はカルシウム吸収を促進し、ブロイラーの雌雄両方の全体重を増加させる。２５日間の給餌において、開始時体重ｋｇ当りの終了時体重の平均はγ−ポリグルタメートＮａ+体を含有させた通常飼料（ヘブンスタイン（Havenstein） G.B.ら、1994, 「１９５７年、１９９１年の「典型的」ブロイラー餌を給餌したときの、生育、生存率、飼料要求率に関する１９５７年対１９９１年の比較（Growth、livability、and feed conversion of 1957 vs 1991 broilers when fed "typical" 1957 and 1991 broiler diets）」、Poult. Sci. 73: 1795-1794）を与えた群では４．７１２ｋｇで、通常飼料（コントロール）を与えた群では４．６２９ｋｇである。脛骨の長さは、γ−ポリグルタメートＮａ+体を含有した飼料を適用した試験のブロイラー雄については９．３８ｃｍ、ブロイラー雌については９．１８ｃｍであり、通常飼料を適用したコントロールのブロイラー雄については９．２５ｃｍ、ブロイラー雌については９．１０ｃｍである。脛骨中のカルシウム含有量は通常飼料を適用したコントロールのブロイラー雄については１７．６６％、ブロイラー雌については１６．６９％である。 As a result, the γ-polyglutamate Na + form promotes calcium absorption and increases the overall weight of both male and female broilers. In 25 days of feeding, the average of the end body weight per kg body weight at the start was the normal feed containing γ-polyglutamate Na + body (Havenstein GB et al., 1994, “1957, 1991” 1957 vs. 1991 broilers when fed "typical" 1957 and 1991 broiler diets (Growth, livability, and feed conversion of 1957 vs 1991 broilers when fed "typical" 1957 and 1991 broiler diets ) ", Poult. Sci. 73: 1795-1794), the group was 4.712 kg, and the group fed the normal feed (control) was 4.629 kg. The length of the tibia is 9.38 cm for the broiler male in the test with the feed containing the γ-polyglutamate Na + body and 9.18 cm for the broiler female, and for the control broiler male with the normal feed applied. Is 9.25 cm and for broiler females is 9.10 cm. The calcium content in the tibia is 17.66% for control broiler males to which normal feed is applied and 16.69% for broiler females.
＜実施例１１＞ インビトロにおける細胞培養研究−３Ｔ３−Ｌ１細胞モデルによるＧＴＦ（ブドウ糖耐性因子）活性の促進
本細胞培養実験には、分子量８８０×１０3のγ−ポリグルタメートＮａ+体ＨＭ及び分子量２５０×１０3のγ−ポリグルタメートＮａ+体ＬＭを用いた。
脂肪前駆細胞である３Ｔ３−Ｌ１のサンプルを解凍し、これを３７℃、５％ＣＯ2のインキュベーターにおいて１０％ウシ胎児血清（ＦＢＳ）含有ＤＭＥＭ（ダルベッコＭＥＭ）培地中で一週間培養し、次いで分化培養を行った。分化培養培地ＤＭＥＭ（１０％ＦＢＳ、０．５ｍＭ ＩＢＭＸ、１μＭＤＸ及び１μg/ｍＬインスリン含有）における２日間の培養の後、脂肪前駆細胞を通常の１０％ＦＢＳを含有するＤＭＥＭ培地に移し、更に１０日間９０％を超える細胞が成熟脂肪細胞に変化するまで培養を継続した。ここで、細胞の形状は当初の星形から油滴状へと変化するため、培養プレートの下部に白色油状物質として視覚的に観察することができる。細胞分化完了後、次の実験を続ける。
<Example 11> Cell culture research in vitro-Promotion of GTF (glucose resistance factor) activity by 3T3-L1 cell model In this cell culture experiment, γ-polyglutamate Na + form HM having a molecular weight of 880 × 10 3 and a molecular weight of 250 were used. × 10 3 γ-polyglutamate Na + form LM was used.
A sample of 3T3-L1, which is a preadipocyte, was thawed and cultured for one week in DMEM (Dulbecco MEM) medium containing 10% fetal bovine serum (FBS) in an incubator at 37 ° C. and 5% CO 2 , and then differentiated. Culture was performed. After 2 days of culture in differentiation culture medium DMEM (containing 10% FBS, 0.5 mM IBMX, 1 μMDX and 1 μg / mL insulin), the preadipocytes are transferred to DMEM medium containing normal 10% FBS for another 10 days. The culture was continued until more than 90% of the cells changed to mature adipocytes. Here, since the shape of the cells changes from the original star shape to oil droplets, it can be visually observed as a white oily substance at the bottom of the culture plate. After cell differentiation is complete, continue with the next experiment.
この成熟脂肪細胞をまず、３７℃、５％ＣＯ2のインキュベーターにおいて２％ＦＢＳを含有した無糖のＤＭＥＭ培地中で１時間培養し、ＰＢＳ（０．０１Ｍリン酸緩衝溶液、ｐＨ７．４）で洗浄した。次いで、異なる濃度のγ−ポリグルタメートＮａ+体を含むテストサンプル２００μＬ及び１０ｎＭインスリンと２．５ｇ／Ｌグルコースとを含むＫＢＲ溶液２００μＬを添加し、３７℃、５％ＣＯ2で２時間インキュベートした。細胞を遠心分離し、清澄な上清を採取し、グルコース濃度をグルコースアナライザーを用いて分析した。反応前後のグルコース量の差は脂肪細胞による全グルコース摂取量で定義される。摂取されたグルコースの増加率は次に示すようにＧＴＦ活性として定義される。
ＧＴＦ活性の増加（％）＝（（サンプルによるグルコース摂取量−コントロールによるグルコース摂取量）／コントロールによるグルコース摂取量）×１００％
The mature adipocytes were first cultured in a sugar-free DMEM medium containing 2% FBS in an incubator at 37 ° C. and 5% CO 2 for 1 hour, and PBS (0.01 M phosphate buffer solution, pH 7.4) was used. Washed. Next, 200 μL of a test sample containing γ-polyglutamate Na + form at different concentrations and 200 μL of a KBR solution containing 10 nM insulin and 2.5 g / L glucose were added and incubated at 37 ° C., 5% CO 2 for 2 hours. The cells were centrifuged, the clear supernatant was collected, and the glucose concentration was analyzed using a glucose analyzer. The difference in glucose level before and after the reaction is defined by the total glucose intake by fat cells. The rate of increase in ingested glucose is defined as GTF activity as shown below.
Increase in GTF activity (%) = ((glucose intake by sample−glucose intake by control) / glucose intake by control) × 100%
＜実施例１２＞
図６に、γ−ポリグルタメートＮａ+体の分子量の変化の実施例１１のＧＴＦ活性へ及ぼす効果を示す。
この結果、高分子量、低分子量両方のγ−ポリグルタメートＮａ+体でＧＴＦ活性が増加することが示され、より高分子量のγ−ポリグルタメートＮａ+体ＨＭは約３００ｐｐｍで２１％という最も高い増加率を示し、低分子量のγ−ポリグルタメートＮａ+体ＬＭは、濃度が１５０〜６００ｐｐｍの範囲内において約３３．５％という最高のＧＴＦ活性を示した。
<Example 12>
FIG. 6 shows the effect of the change in molecular weight of the γ-polyglutamate Na + form on the GTF activity of Example 11.
As a result, it was shown that the GTF activity was increased in both high molecular weight and low molecular weight γ-polyglutamate Na + form, and the higher increase in high molecular weight γ-polyglutamate Na + form HM was 21% at about 300 ppm. The low molecular weight γ-polyglutamate Na + form LM showed the highest GTF activity of about 33.5% in the concentration range of 150-600 ppm.
この結果は、γ−ポリグルタメートＮａ+体が脂肪細胞のＧＴＦによるグルコースの消費を促進し、これは生体に対し、成長を維持し、健康上の効果を与えるのに非常に有効でありヒトの糖尿病の症状をコントロールするうえで、良好な生物学的機能を発揮し得ることを明らかに示唆している。 This result shows that the γ-polyglutamate Na + body promotes the consumption of glucose by GTF of adipocytes, which is very effective for maintaining growth and giving health effects to the living body. It clearly suggests that it can exert a good biological function in controlling the symptoms of diabetes.
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