JP2006115751A - Method for producing protein micelle structure having nano size to which hydrophobic substance is adsorbed and retained - Google Patents
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本発明は、カゼイン等ミセル形成能を有するタンパク質と疎水性物質を水中で共存させることにより形成する粒径がナノサイズのミセル構造体の製造方法、及び透明で安定に可溶化された疎水性物質の過飽和溶液の製造方法に関するものである。 The present invention relates to a method for producing a micelle structure having a nano-size particle size formed by allowing a protein having a micelle-forming ability such as casein and a hydrophobic substance to coexist in water, and a hydrophobic substance that is transparent and stably solubilized. The present invention relates to a method for producing a supersaturated solution.
栄養、生理、薬理機能を持った化合物には、疎水性物質が多数存在する。そのような疎水性物質を食品や医薬品の成分として経口で摂取する際には、容易に摂取できる水溶液に可溶化されていることが望まれる。しかし、疎水性物質を水溶液にする場合、水に対する溶解度が低いという課題がある。疎水性物質の中には、水溶液の調製時に一旦溶解しても、その後、経時的に沈澱を生じ、疎水性物質含有水溶液としての保存安定性を保つことができず、品質低下の原因となっている。そのため、これらの疎水性物質の配合量が制限され、機能を満足に発揮できる製品を得ることが困難な場合がある。そこで、疎水性の高い化合物を水中に高濃度に且つ均一に可溶化するための技術の開発が望まれている。疎水性物質を高濃度で水溶液中に含有させるために、界面活性剤を用いた乳化技術、硬化剤を巧みに使用したマイクロカプセル技術などが開発されている。 Many hydrophobic substances exist in compounds having nutrition, physiology, and pharmacological functions. When such a hydrophobic substance is taken orally as a food or pharmaceutical ingredient, it is desired that it is solubilized in an aqueous solution that can be taken easily. However, when the hydrophobic substance is used as an aqueous solution, there is a problem that the solubility in water is low. Some hydrophobic substances, once dissolved during the preparation of the aqueous solution, subsequently precipitate over time, failing to maintain the storage stability of the hydrophobic substance-containing aqueous solution, leading to quality degradation. ing. Therefore, the blending amount of these hydrophobic substances is limited, and it may be difficult to obtain a product that can exhibit its functions satisfactorily. Accordingly, development of a technique for uniformly solubilizing a highly hydrophobic compound in water at a high concentration is desired. In order to contain a hydrophobic substance in an aqueous solution at a high concentration, an emulsification technique using a surfactant, a microcapsule technique using a curing agent, and the like have been developed.
疎水性物質を含むマイクロカプセルの製造方法としては、例えばコンプレックスコアセルベーション法（特許文献１）が代表的であるが、疎水性物質を可溶化するための溶剤や食用に適さないアルデヒド等の硬化剤が用いられることが多い。そこで、蛋白質と多糖類からなるコンプレックスコアセルベーション法において、蛋白架橋酵素であるトランスグルタミナーゼを被膜硬化剤として用いることを特徴とする可食性のマイクロカプセルの製造方法（特許文献２）や、蛋白と塩類の組合せにより塩析法で壁膜を形成し、トランスグルタミナーゼで壁膜を硬化するマイクロカプセルの製造方法（特許文献３）等、食用に適さない硬化剤を用いずにマイクロカプセルを製造する方法が開示されている。しかしながら、これらの文献に開示されているマイクロカプセルの大きさは平均粒径は数１０〜数１００μｍの大きさであり、後述の本発明の課題である「疎水性物質を過飽和濃度においても透明で安定に可溶化させる」ことはできない。 A typical method for producing a microcapsule containing a hydrophobic substance is, for example, a complex coacervation method (Patent Document 1). However, a solvent for solubilizing a hydrophobic substance or curing of an edible aldehyde or the like is used. Agents are often used. Therefore, in the complex coacervation method comprising protein and polysaccharide, transglutaminase, a protein cross-linking enzyme, is used as a film hardening agent (Patent Document 2), A method for producing a microcapsule without using an edible curing agent, such as a microcapsule production method (Patent Document 3) in which a wall membrane is formed by a salting-out method using a combination of salts and the wall membrane is cured with transglutaminase. Is disclosed. However, the size of the microcapsules disclosed in these documents has an average particle size of several tens to several hundreds of μm, which is the subject of the present invention described later, “hydrophobic substances are transparent even at supersaturated concentrations. It cannot be solubilized stably.
ところでカゼインは乳タンパク質の主体をなすリンタンパク質であり、αs1-カゼイン、α s2-カゼイン、β-カゼイン、κ-カゼインと呼ばれる４種類のタンパク質成分からなる。これらのカゼイン成分は、それぞれ単独では特定の高次構造を持たないが、その両親媒性と高い界面活性から、水中でその濃度や環境に応じて分子集合体を形成することが知られている。最近の研究で、カゼイン分子が有する自己組織性とトランスグルタミナーゼによる架橋反応を組み合わせることで、カゼイン分子の集合体（カゼインミセル）を安定化できることが示されている（非特許文献１）が、この安定化カゼインミセル内に疎水性物質を吸着保持するという思想、疎水性物質を過飽和濃度で水に可溶化させるという思想は開示されていない。
本発明は、疎水性物質を過飽和濃度においても透明で安定に可溶化させる方法を提供することを目的とする。 It is an object of the present invention to provide a method for solubilizing and stably solubilizing hydrophobic substances even at supersaturated concentrations.
本発明者は前項記載の目的を達成すべく鋭意研究を行った結果、カゼイン等ミセル形成能を有するタンパク質と疎水性物質を水中又は水溶液中で共存させることにより、ミセル内部の疎水性領域に疎水性物質を吸着保持した粒径がナノサイズの構造体が得られること、カゼイン等ミセル形成能を有するタンパク質と疎水性物質を水中又は水溶液中に共存させた後、トランスグルタミナーゼのタンパク質分子内・分子間架橋形成能を利用して固定化することにより、より安定な、ミセル内部の疎水性領域に疎水性物質を吸着保持した構造体が得られること、および該構造体を水に溶解させると、該疎水性物質をその溶解度以上の濃度、すなわち過飽和濃度でも、透明で安定に可溶化できることを見出し、本発明を完成するに至った。 As a result of intensive studies to achieve the object described in the preceding paragraph, the present inventor made hydrophobicity in the hydrophobic region inside the micelle by coexisting a protein having a micelle-forming ability such as casein and a hydrophobic substance in water or an aqueous solution. A structure with a nano-sized particle size that adsorbs and retains an active substance, a protein having the ability to form micelles, such as casein, and a hydrophobic substance in water or in an aqueous solution, and then transglutaminase protein molecules and molecules By immobilizing using the ability to form intercrosslinks, it is possible to obtain a more stable structure that absorbs and holds a hydrophobic substance in the hydrophobic region inside the micelle, and when the structure is dissolved in water, The present inventors have found that the hydrophobic substance can be transparently and stably solubilized even at a concentration higher than its solubility, that is, a supersaturated concentration, and the present invention has been completed.
即ち、本発明は以下の通りである。
（１）ミセル形成能を有するタンパク質及び疎水性物質を水中又は水溶液中に共存させることによる、ミセル内部の疎水性領域に疎水性物質を吸着保持した構造体の製造方法。
（２）ミセル形成能を有するタンパク質及び疎水性物質を水中又は水溶液中に共存させた後、トランスグルタミナーゼを添加することによる、ミセル内部の疎水性領域に疎水性物質を吸着保持した構造体の製造方法。
（３）ミセル形成能を有するタンパク質がカゼインである（１）又は（２）記載の製造方法。
（４）疎水性物質を吸着保持した構造体の粒径分布が１μ未満である（１）乃至（３）のいずれかに記載の製造方法。
（５）（１）乃至（４）のいずれかに記載の製造方法で得られる疎水性物質を吸着保持した構造体を、該疎水性物質の濃度が該疎水性物質の過飽和濃度となるよう水又は水溶液に溶解させることを特徴とする透明な過飽和溶液の製造方法。
That is, the present invention is as follows.
(1) A method for producing a structure in which a hydrophobic substance is adsorbed and held in a hydrophobic region inside a micelle by coexisting a protein having a micelle-forming ability and a hydrophobic substance in water or an aqueous solution.
(2) Production of a structure in which a hydrophobic substance is adsorbed and held in a hydrophobic region inside a micelle by adding transglutaminase after coexisting a protein having a micelle-forming ability and a hydrophobic substance in water or an aqueous solution. Method.
(3) The production method according to (1) or (2), wherein the protein having micelle forming ability is casein.
(4) The production method according to any one of (1) to (3), wherein the particle size distribution of the structure holding the hydrophobic substance by adsorption is less than 1 μm.
(5) A structure obtained by adsorbing and holding a hydrophobic substance obtained by the production method according to any one of (1) to (4) is washed with water so that the concentration of the hydrophobic substance becomes a supersaturated concentration of the hydrophobic substance. Or the manufacturing method of the transparent supersaturated solution characterized by making it melt | dissolve in aqueous solution.
本発明によると、疎水性物質をカゼインミセル内部に吸着保持した粒径がナノサイズの構造体が得られ、疎水性物質をその溶解度以上の濃度、すなわち過飽和濃度においても、透明で安定に可溶化することができる。 According to the present invention, a structure having a nano-sized particle size in which a hydrophobic substance is adsorbed and held inside casein micelles can be obtained, and the hydrophobic substance can be transparently and stably solubilized even at a concentration higher than its solubility, that is, at a supersaturated concentration. can do.
本発明に用いるミセル形成能を有するタンパク質はカゼイン等ミセル形成能を有するものであればよい。カゼインは、ミセル形成能があればいずれのカゼインを用いても良いが、好ましくはαs1-カゼイン、αs2-カゼイン、β-カゼインおよびκ-カゼインである。これらはそれぞれ単独で使用することができ、また２種以上を併用することもできる。 The protein having micelle forming ability used in the present invention may be any protein having ability to form micelles such as casein. Any casein may be used as long as it has the ability to form micelles, but αs1-casein, αs2-casein, β-casein and κ-casein are preferable. Each of these can be used alone or in combination of two or more.
本発明に用いられる疎水性物質は特に限定されるものではないが、例えば、疎水性の高いトリプトファン、チロシン、バリン、ロイシン、イソロイシンといったアミノ酸、食用色素、コーン油、大豆油、菜種油、落花生油、パーム油等の植物油、魚油、ラード、ヘッド等の動物油、レシチン、α−リノレン酸、エイコサペンタエン酸（ＥＰＡと略する）、ドコサヘキサエン酸（ＤＨＡと略する）といった脂肪酸等を挙げることができる。また、食用ワックス類を用いても何等差し支えない。これらはそれぞれ単独であるいは複数配合して用いられ、目的に応じ適宜選択することが出来る。これらの油脂に、フレーバー組成物、ビタミン等油溶性物質、調味料、香辛料、乳化剤等を含有させたり、あるいは着色剤を添加したりすることは何等差し支えない。要するに、これらを目的に応じて単独又は複数配合することができる。尚、ここで述べるフレーバー組成物とは例えば、ミートフレーバー、かつお節フレーバー等の畜肉魚介類に関連したものや、果実フレーバー、野菜フレーバー等が挙げられる。油溶性ビタミン類としては、ビタミンＡ、Ｄ、Ｅ、Ｆ、Ｋ等を挙げることができる。これらを、目的に応じて、単独叉は複数配合しても良い。 Although the hydrophobic substance used in the present invention is not particularly limited, for example, amino acids such as highly hydrophobic tryptophan, tyrosine, valine, leucine, and isoleucine, food coloring, corn oil, soybean oil, rapeseed oil, peanut oil, Examples include vegetable oils such as palm oil, animal oils such as fish oil, lard, and head, and fatty acids such as lecithin, α-linolenic acid, eicosapentaenoic acid (abbreviated as EPA), and docosahexaenoic acid (abbreviated as DHA). Moreover, there is no problem even if edible waxes are used. These may be used alone or in combination, and may be appropriately selected depending on the purpose. These fats and oils may contain a flavor composition, oil-soluble substances such as vitamins, seasonings, spices, emulsifiers, etc., or may be added with colorants. In short, these may be used alone or in combination according to the purpose. In addition, the flavor composition described here includes, for example, those relating to meat and seafood such as meat flavor and bonito flavor, fruit flavor, vegetable flavor and the like. Examples of oil-soluble vitamins include vitamins A, D, E, F, and K. These may be used alone or in combination according to the purpose.
本発明に用いられるトランスグルタミナーゼ（以下、ＴＧと略記することがある）は、ＴＧ活性がある限りその起源を特に問わず、いずれの種類のＴＧを用いても良い。例えば、ストレプトベルチシリウム（Streptoverticillium）等に属する微生物由来のもの（特開平１−２７４７１号公報参照）、モルモット等の哺乳動物由来のもの（特公平１−５０３８２号公報参照）、水産動物由来のもの（関伸夫ら、Nippon Suisan Gakkaishi, 56(1), 125(1990)及び59(4), 711(1993)））、バイオテクノロジー技術を使用して遺伝子組換によって得られるもの（特開平１−３００８８９号公報参照）等を用いることができる。しかし、その中でもカルシウム非依存性であり、かつ大量に取得できる微生物由来トランスグルタミナーゼを用いる方が好ましい。 The transglutaminase used in the present invention (hereinafter sometimes abbreviated as TG) may be any type of TG, regardless of its origin, as long as it has TG activity. For example, those derived from microorganisms belonging to Streptoverticillium (see JP-A-1-27471), those derived from mammals such as guinea pigs (see Japanese Patent Publication No. 1-50382), marine animals (Nobuo Seki et al., Nippon Suisan Gakkaishi, 56 (1), 125 (1990) and 59 (4), 711 (1993))), obtained by genetic recombination using biotechnology (Japanese Patent Laid-Open No. 1) -300889) can be used. However, among these, it is preferable to use a microorganism-derived transglutaminase that is calcium-independent and can be obtained in large quantities.
ミセル内部の疎水性領域に疎水性物質を吸着保持した構造体の製造方法は、例えば、α-カゼインやβ-カゼイン等のミセル形成能を有するタンパク質を単独にまたは複数溶解した濃度０．１〜１０％の水溶液に疎水性アミノ酸等の疎水性物質を、水溶液の温度での溶解度から１００℃での溶解度に相当する量を添加する。疎水性物質が溶解するまで加温するか、所望の温度で加温をやめ、溶解できない疎水性物質を遠心分離等で除去後、２０〜８０℃程度まで冷却し、疎水性物質を吸着保持した構造体の溶液を得る。この溶液は、疎水性物質の濃度が該疎水性物質の過飽和濃度であり、このようにして該疎水性物質の透明な過飽和溶液が得られる。この過飽和溶液を噴霧乾燥、凍結乾燥等により乾燥しても良いし、膜濾過、ゲル濾過等により濃縮、分画しても良い。 A method for producing a structure in which a hydrophobic substance is adsorbed and held in a hydrophobic region inside a micelle is, for example, a concentration of 0.1 to 0.1, wherein a protein having the ability to form micelles such as α-casein and β-casein is dissolved alone or plurally. A hydrophobic substance such as a hydrophobic amino acid is added to a 10% aqueous solution in an amount corresponding to the solubility at 100 ° C. from the solubility at the temperature of the aqueous solution. Heat until the hydrophobic substance is dissolved, or stop heating at the desired temperature, remove the hydrophobic substance that cannot be dissolved by centrifugation, etc., cool to about 20-80 ° C., and hold the hydrophobic substance by adsorption A solution of the structure is obtained. In this solution, the concentration of the hydrophobic substance is the supersaturated concentration of the hydrophobic substance, and thus a transparent supersaturated solution of the hydrophobic substance is obtained. This supersaturated solution may be dried by spray drying, freeze drying, or the like, or may be concentrated and fractionated by membrane filtration, gel filtration, or the like.
さらに、ＴＧを用いる場合は、先ず、上に説明したと同じく、α-カゼインやβ-カゼイン等のミセル形成能を有するタンパク質を単独にまたは複数溶解した濃度０．１〜１０％の水溶液に疎水性アミノ酸等の疎水性物質を、水溶液の温度での溶解度から１００℃での溶解度に相当する量を添加する。疎水性物質が溶解するまで加温するか、所望の温度で加温をやめ、溶解できない疎水性物質を遠心分離等で除去後、２０〜８０℃程度まで冷却する。次に、ＴＧをタンパク質１ｇあたり０．００１〜５００単位（この単位については特開平１−２７４７１号公報参照）、好ましくは０．１〜１００単位添加する。これをＴＧによるタンパク質の分子内・分子間架橋反応が進行する時間、例えば２０〜４０℃で１分〜４８時間、好ましくは５分〜24時間置き、疎水性物質を包摂したカゼイン類を構成成分して形成されたミセル構造体の溶液を得る。この溶液は、疎水性物質の濃度が該疎水性物質の過飽和濃度であり、このようにして該疎水性物質の透明な過飽和溶液が得られる。この過飽和溶液を噴霧乾燥、凍結乾燥等により乾燥しても良いし、膜濾過、ゲル濾過等により濃縮、分画しても良い。ＴＧを用いることにより、ミセル構造体が安定化し、疎水性物質をより高濃度に溶解させることができる。 Further, when TG is used, first, as described above, a hydrophobic solution is added to an aqueous solution having a concentration of 0.1 to 10% in which a protein having a micelle-forming ability such as α-casein or β-casein is dissolved alone or plurally. An amount corresponding to the solubility at 100 ° C. from the solubility at a temperature of the aqueous solution is added to a hydrophobic substance such as a functional amino acid. Heat until the hydrophobic substance is dissolved or stop heating at a desired temperature, remove the hydrophobic substance that cannot be dissolved by centrifugation or the like, and then cool to about 20 to 80 ° C. Next, TG is added in an amount of 0.001 to 500 units per gram of protein (refer to JP-A-1-27471 for this unit), preferably 0.1 to 100 units. This is the time during which the intramolecular / intermolecular cross-linking reaction of the protein by TG proceeds, for example, at 20 to 40 ° C. for 1 minute to 48 hours, preferably 5 minutes to 24 hours, and the casein containing the hydrophobic substance is a constituent component. Thus, a solution of the micelle structure formed is obtained. In this solution, the concentration of the hydrophobic substance is the supersaturated concentration of the hydrophobic substance, and thus a transparent supersaturated solution of the hydrophobic substance is obtained. This supersaturated solution may be dried by spray drying, freeze drying, or the like, or may be concentrated and fractionated by membrane filtration, gel filtration, or the like. By using TG, the micelle structure is stabilized and the hydrophobic substance can be dissolved at a higher concentration.
以下に参考例及び実施例を挙げ、本発明をさらに詳しく説明する。本発明は、これらの実施例により何ら限定されるものではない。 Hereinafter, the present invention will be described in more detail with reference examples and examples. The present invention is not limited in any way by these examples.
５mg/mlの各種カゼイン溶液（50mM Tris塩酸緩衝液、ｐH7.5）を調製し、トランスグルタミナーゼ（1単位／ｇ）を0.5mg/ml添加し、３７℃で２４時間温置した。得られた反応溶液の光散乱（ＤＬＳ）をSysmex社製「Zetasizer Nano」にて測定し、粒度分布を求めた。下記第１表に示すように、５mg/ml濃度においてＴＧを添加しないα-カゼイン、β-カゼイン及びκ-カゼインでは、それぞれ、２９nm、３２nmおよび５５nmとなり、各種カゼインの混合物である「カゼイン」でも９１nmを示し、ナノサイズのミセルが形成された。これらのＴＧ処理によって、α-カゼインは２６nm、β-カゼインでは２６nm、κ-カゼインで４９nm、そしてカゼインでは７５nmとなり、未処理に比してコンパクトなナノサイズの構造体を形成した。 Various casein solutions (50 mM Tris hydrochloric acid buffer, pH 7.5) at 5 mg / ml were prepared, 0.5 mg / ml of transglutaminase (1 unit / g) was added, and the mixture was incubated at 37 ° C. for 24 hours. Light scattering (DLS) of the obtained reaction solution was measured with “Zetasizer Nano” manufactured by Sysmex to determine the particle size distribution. As shown in Table 1 below, α-casein, β-casein and κ-casein to which TG is not added at a concentration of 5 mg / ml are 29 nm, 32 nm and 55 nm, respectively, and “casein” which is a mixture of various caseins A nano-sized micelle was formed, showing 91 nm. By these TG treatments, α-casein was 26 nm, β-casein was 26 nm, κ-casein was 49 nm, and casein was 75 nm, forming a compact nano-sized structure as compared to untreated.
100mMリン酸緩衝液（pH6.2）にβ-カゼイン（シグマ社製）5mg/mｌを溶解し、37℃にて平衡化後、8-Anilinonaphthalene-1-sulfonate（ＡＮＳ）110μＭを加えた。ＡＮＳは、タンパク質の疎水領域と相互作用すると蛍光強度が著しく高まる疎水性プローブである。これに参考例１のＴＧを0.5mg/mL (カゼイン１ｇに対し０．１単位)添加し、37℃で24 時間温置し、ＡＮＳ包摂したミセル構造体を調製し、蛍光スペクトルの測定を行った（励起波長：350nm／測定波長：480nm）。同様の実験を100mMリン酸緩衝液（pH6.2）にβ-カゼイン（5mg/mL）を溶解し、37℃にて平衡化後、8-Anilinonaphthalene-1-sulfonate（ＡＮＳ）110μＭを加えたが、ＴＧ処理を行っていないサンプル（ＴＧ未処理カゼインミセル）についても同様の操作を行い、両者を比較した。図１に示したように、ＴＧ未処理カゼインミセル（図１ではβ−カゼインと表示）とＴＧ処理カゼインミセル（図１ではナノカプセルと表示）では、ほぼ同一の蛍光スペクトルを与え、両者疎水的空間は大きな変化は見られず、ＴＧ処理を行っていないカゼインミセル構造体でも、ＴＧ処理を行ったカゼインミセル構造体でも、同様に疎水性物質ＡＮＳが吸着していると判断される。
両者にトリプシン（50μg/ml）を加え、ＡＮＳ由来の蛍光強度変化を経時的に追跡したところ、図２に示したように、ＴＧ未処理カゼインミセルでは、疎水場の存在を示すＡＮＳ由来の蛍光がほぼ消失するのに対して、ＴＧ架橋化処理ミセルは蛍光強度が５０％以上保持された。また、ＴＧ処理を施したＡＮＳ包摂β-カゼインミセルについて、Sysmex社製「Zetasizer Nano」でトリプシン処理前後の粒径測定を行った。トリプシン処理前の粒径は36nmで、ＡＮＳ無添加のカゼインミセル（第１表、３２nm）と同等であった。トリプシン処理後の粒径は28nmとなり、ナノサイズの構造体が維持され、疎水性化合物ＡＮＳがそのナノサイズ構造体に保持されていることが明らかとなった。すなわち、カゼインミセルをＴＧ処理すると、その内部に疎水場を有する安定なカゼインミセル構造体が得られた。従って、ＴＧ処理ミセルは、ＴＧ処理によりトリプシン耐性を示すと共に、疎水性物質が内部疎水領域に保持することができていると判断された。
同様に、「カゼイン」（シグマ社製；αs1-カゼイン、α s2-カゼイン、β-カゼインおよびκ-カゼインの混合物）についても検討したところ、図３に示したように、いずれも、ＴＧ処理ミセルは、トリプシン処理してもＡＮＳを保持していることが確認された。
Β-Casein (manufactured by Sigma) 5 mg / ml was dissolved in 100 mM phosphate buffer (pH 6.2), equilibrated at 37 ° C., and then 8-Anilinonaphthalene-1-sulfonate (ANS) 110 μM was added. ANS is a hydrophobic probe that significantly increases fluorescence intensity when interacting with hydrophobic regions of proteins. To this was added 0.5 mg / mL of TG of Reference Example 1 (0.1 unit per 1 g of casein), incubated at 37 ° C. for 24 hours, and prepared an ANS-encapsulated micelle structure, and the fluorescence spectrum was measured. (Excitation wavelength: 350 nm / measurement wavelength: 480 nm). In the same experiment, β-casein (5 mg / mL) was dissolved in 100 mM phosphate buffer (pH 6.2), equilibrated at 37 ° C, and then 8-Anilinonaphthalene-1-sulfonate (ANS) 110 µM was added. The same operation was performed on samples not subjected to TG treatment (TG untreated casein micelles), and the two were compared. As shown in FIG. 1, TG-untreated casein micelles (shown as β-casein in FIG. 1) and TG-treated casein micelles (shown as nanocapsules in FIG. 1) give almost the same fluorescence spectrum, both hydrophobic. There is no significant change in the space, and it is determined that the hydrophobic substance ANS is adsorbed in the casein micelle structure not subjected to TG treatment or the casein micelle structure subjected to TG treatment.
Trypsin (50 μg / ml) was added to both, and changes in fluorescence intensity derived from ANS were traced over time. As shown in FIG. 2, in TG-untreated casein micelles, ANS-derived fluorescence indicating the presence of a hydrophobic field was observed. Almost disappeared, whereas the TG cross-linked micelles retained the fluorescence intensity of 50% or more. The ANS-containing β-casein micelle subjected to TG treatment was subjected to particle size measurement before and after trypsin treatment using “Zetasizer Nano” manufactured by Sysmex. The particle size before trypsinization was 36 nm, which was equivalent to casein micelles without ANS (Table 1, 32 nm). The particle size after trypsin treatment was 28 nm, indicating that the nano-sized structure was maintained and the hydrophobic compound ANS was retained in the nano-sized structure. That is, when casein micelles were treated with TG, a stable casein micelle structure having a hydrophobic field therein was obtained. Therefore, it was determined that the TG-treated micelles were resistant to trypsin by TG treatment and the hydrophobic substance could be retained in the internal hydrophobic region.
Similarly, “casein” (manufactured by Sigma; mixture of αs1-casein, αs2-casein, β-casein and κ-casein) was also examined. As shown in FIG. Was confirmed to retain ANS even after trypsin treatment.
100mMリン酸緩衝液（pH6.2）にβ-カゼイン（シグマ社製）5mg/mｌとトリプトファン（Ｔｒｐ）90mMを60℃にて溶解した。これを40℃にて平衡化後、参考例１のＴＧを0.5 mg/mｌ(カゼイン１ｇに対し０．１単位) 添加し、24 時間温置した。これを25℃にて平衡化すると、カゼインを共存させていない対照溶液では溶解度を超えたＴｒｐが析出するのに比して、ＴＧ処理β-カゼインはＴｒｐの析出は認められず、溶解度を超えたＴｒｐはＴＧ処理ミセル構造体の内部に取りこまれ、透明な溶液を与えた。次に、これら溶液を遠心分離し、析出しているＴｒｐを完全に除去した後、α-キモトリプシン（50μg/mL）を加え、40℃にて24 時間温置し、得られる溶液中に含まれるＴｒｐをHPLC（カラムInertsil ODS-3, GLScience; グラジエント：水／アセトニトリル＝95/5→45/55 in 25min; 流速：0.5ml/min）により定量することで、包摂するＴｒｐ量を定量した。同様の実験をカゼイン非共存下ならびにＴＧ 未処理サンプルについても行った。
図４より、本実験条件下におけるＴｒｐの溶解度は70±3mM（control）と見積もられたのに対し、ＴＧ未処理カゼインミセル構造体（図４ではmicellと表示）では81±1mM、ＴＧ処理カゼインミセル構造体（図４ではcapsuleと表示）では89±7mM と、Ｔｒｐの溶解度が大きく向上し、Ｔｒｐの過飽和濃度においても透明な溶液が得られた。
Β-Casein (Sigma) 5 mg / ml and tryptophan (Trp) 90 mM were dissolved in 100 mM phosphate buffer (pH 6.2) at 60 ° C. After equilibrating this at 40 ° C., 0.5 mg / ml of TG of Reference Example 1 (0.1 unit per 1 g of casein) was added and incubated for 24 hours. When this was equilibrated at 25 ° C., Trp that exceeded the solubility was precipitated in the control solution not coexisting with casein, whereas TG-treated β-casein showed no Trp precipitation and exceeded the solubility. Trp was incorporated inside the TG treated micelle structure to give a clear solution. Next, these solutions are centrifuged to completely remove the deposited Trp, and then α-chymotrypsin (50 μg / mL) is added and incubated at 40 ° C. for 24 hours. By quantifying Trp by HPLC (column Inertsil ODS-3, GLScience; gradient: water / acetonitrile = 95/5 → 45/55 in 25 min; flow rate: 0.5 ml / min), the amount of Trp to be included was quantified. A similar experiment was performed in the absence of casein and on a sample not treated with TG.
From Fig. 4, the solubility of Trp under the present experimental conditions was estimated to be 70 ± 3 mM (control), whereas in the case of TG untreated casein micelle structure (indicated as micell in Fig. 4), 81 ± 1 mM, TG treatment In the casein micelle structure (labeled capsule in FIG. 4), the solubility of Trp was greatly improved to 89 ± 7 mM, and a transparent solution was obtained even at a supersaturated concentration of Trp.
実施例２と同様の操作で調製したＴＧ未処理Ｔｒｐ包摂カゼインミセル構造体とＴＧ処理Ｔｒｐ包摂カゼインミセル構造体各１ｍｌを透析チューブに加え、これを100ｍｌ の100mM リン酸緩衝液（pH6.2）に対して透析操作を施した。外水相に漏出したＴｒｐの濃度を吸光度変化により追跡した結果、Ｔｒｐの外水相への漏出速度が一次速度過程に従うと仮定しフィッティングを行い、見かけの半減期を算出すると、カゼインミセルでは13.4 分、ＴＧ処理Ｔｒｐ包摂カゼインミセル構造体では21.4 分となった。即ち、ＴＧ処理によりカゼインミセルのＴｒｐの保持力（＝Ｔｒｐに対する親和性）が増すことが判明した。 TG-untreated Trp-containing casein micelle structure and 1 ml of TG-treated Trp-containing casein micelle structure prepared in the same manner as in Example 2 were added to a dialysis tube, and this was added to 100 ml of 100 mM phosphate buffer (pH 6.2). A dialysis operation was performed on the test piece. As a result of tracing the concentration of Trp leaked into the outer aqueous phase by the change in absorbance, it was assumed that the rate of leakage of Trp into the outer aqueous phase followed the first-order rate process, and the apparent half-life was calculated to be 13.4 for casein micelles. Min, 21.4 min for the TG-treated Trp-containing casein micelle structure. That is, it was found that the Trp retention (= affinity with Trp) of casein micelles was increased by TG treatment.
実施例１と同様の操作で調製したＴＧ未処理ＡＮＳ包摂カゼインミセル構造体とＴＧ処理ＡＮＳ包摂カゼインミセル構造体をAmersham Pharmacia Boitec社製AKTA prime、カラムSuperdex 200 (HiLoad)にて、ゲル濾過を行い、各画分のカゼイン濃度と蛍光強度を測定した。5mg/mlの構造体２ｍｌより計約２００Ｌの画分を分取した。
図５に示したように、ＴＧ処理ＡＮＳ包摂カゼインミセル構造体の方が、見かけの分子量が大きく（ピークの画分は分子量約１００万）、分布も狭いことより、ＴＧ処理により、より安定な構造体が形成していることが確認された。ＴＧ処理ＡＮＳ包摂カゼインミセル構造体では、タンパク質と蛍光の検出が一致しており、かなり狭い範囲で溶出している。一方、ＴＧ未処理ＡＮＳ包摂カゼインミセル構造体では、後半の比較的広い範囲にタンパク質が出てきており、蛍光強度も低くなっている。このことは、ＴＧ処理ＡＮＳ包摂カゼインミセル構造体は大希釈条件下においても、疎水性の化合物を内包したまま保持できることを示しており、ＴＧ処理により、包摂コアがよりしっかり形成され、疎水性物質の溶解度が高まることを示している。
The TG-untreated ANS-incorporated casein micelle structure and TG-treated ANS-incorporated casein micelle structure prepared in the same manner as in Example 1 were subjected to gel filtration with AKTA prime, column Superdex 200 (HiLoad) manufactured by Amersham Pharmacia Boitec. The casein concentration and fluorescence intensity of each fraction were measured. A fraction of about 200 L in total was collected from 2 ml of the 5 mg / ml structure.
As shown in FIG. 5, the TG-treated ANS-incorporated casein micelle structure has a larger apparent molecular weight (the peak fraction has a molecular weight of about 1 million) and a narrow distribution. It was confirmed that a structure was formed. In the TG-treated ANS-encased casein micelle structure, the detection of protein and fluorescence are consistent, and they are eluted in a fairly narrow range. On the other hand, in the TG-untreated ANS-encased casein micelle structure, protein appears in a relatively wide range in the latter half, and the fluorescence intensity is low. This indicates that the TG-treated ANS-incorporated casein micelle structure can retain the hydrophobic compound even under large dilution conditions, and the inclusion core is more firmly formed by the TG treatment. It shows that the solubility of is increased.
本発明によると、疎水性物質をカゼインミセル内部に吸着保持した粒径がナノサイズの構造体が得られ、疎水性物質をその溶解度以上の濃度、すなわち過飽和濃度においても、透明で安定に可溶化することができる。近年、特定保健用食品や栄養食品の機能発揮の主体となる機能成分としては、疎水性物質が多く存在するが、本発明によれば、そのような脂溶性や難溶性の成分を可溶化することができるので、本発明は飲料、食品分野において極めて有用である。更に、疎水性物質を輸液や濃厚流動食としたり、医薬品を液状で供給することも本発明によって可能となるので、本発明は医薬品分野においても極めて有用である。 According to the present invention, a structure having a nano-sized particle size in which a hydrophobic substance is adsorbed and held inside casein micelles can be obtained, and the hydrophobic substance can be transparently and stably solubilized even at a concentration higher than its solubility, that is, at a supersaturated concentration. can do. In recent years, there are many hydrophobic substances as functional components that are the main components of the function of specific health foods and nutritional foods. According to the present invention, such fat-soluble and sparingly soluble components are solubilized. Therefore, the present invention is extremely useful in the beverage and food fields. Furthermore, since the present invention makes it possible to use a hydrophobic substance as an infusion solution or a concentrated liquid food, or to supply a pharmaceutical product in liquid form, the present invention is extremely useful in the pharmaceutical field.
Claims (5)
A structure obtained by adsorbing and holding a hydrophobic substance obtained by the production method according to any one of claims 1 to 4 is dissolved in water or an aqueous solution so that the concentration of the hydrophobic substance becomes a supersaturated concentration of the hydrophobic substance. A method for producing a transparent supersaturated solution.
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