JP6656172B2 - Emulsion stabilizer and emulsion stabilization method using the same - Google Patents
Emulsion stabilizer and emulsion stabilization method using the same Download PDFInfo
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Images
Classifications
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- A—HUMAN NECESSITIES
- A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
- A23C—DAIRY PRODUCTS, e.g. MILK, BUTTER OR CHEESE; MILK OR CHEESE SUBSTITUTES; MAKING THEREOF
- A23C9/00—Milk preparations; Milk powder or milk powder preparations
Description
本発明は、ＭＦＧ−Ｅ８タンパク質を有効成分として含有する乳化安定剤及びそれを用いた乳化安定化方法に関する。 The present invention relates to an emulsion stabilizer containing MFG-E8 protein as an active ingredient, and an emulsion stabilization method using the same.
牛乳などに代表される、水に油が乳化された脂肪球の状態で混在する水中油型乳化物では、水中に混在された脂肪球径を適切な大きさに調整することで、これらの脂肪球が水中で安定的に分散され、水中油型乳化物の乳化安定性が高まる。 Oil-in-water emulsions, such as milk, are mixed in the form of fat globules in which oil is emulsified in water.By adjusting the diameter of fat globules mixed in water to an appropriate size, these fats The spheres are stably dispersed in water, and the emulsion stability of the oil-in-water emulsion is increased.
一方、これらの脂肪球は、水中油型乳化物の保持時間が長くなるにつれ、水中で他の脂肪球との脂肪球同士での会合をすることにより、その脂肪球径が大きくなり、脂肪分の浮上（クリーム浮上）が発生する。そのため、脂肪球径を適切な大きさに調整することは、水中油型乳化物の品質を管理する上で、非常に重要な工程である。 On the other hand, as the retention time of the oil-in-water emulsion becomes longer, these fat globules are associated with other fat globules in water, thereby increasing the fat globule diameter and increasing the fat content. Floating (cream floating) occurs. Therefore, adjusting the diameter of fat globules to an appropriate size is a very important step in controlling the quality of the oil-in-water emulsion.
すなわち、水中油型乳化物に混在された脂肪球は、一般的に、水中油型乳化物の保持時間が長くなるに従い、脂肪球同士が会合し、脂肪球の集合体へと変化する。そのため、水中油型乳化物に混在された脂肪球径は、最初は小さくても、保持時間が長くなるにつれ、次第に大きくなる傾向にある。それゆえに、長期間にわたり、水中油型乳化物の乳化を安定化させることは難しい。 That is, the fat globules mixed in the oil-in-water emulsion generally associate with each other as the retention time of the oil-in-water emulsion becomes longer, and change into an aggregate of fat globules. Therefore, the fat globule diameter mixed in the oil-in-water emulsion tends to gradually increase as the holding time becomes longer, even though it is initially small. Therefore, it is difficult to stabilize the emulsification of the oil-in-water emulsion over a long period of time.
水中油型乳化物に混在された脂肪球径を適切な大きさに調整し、水中油型乳化物の乳化を安定化させる方法は、以下のようなものがある。例えば、水中油型乳化物に混在された脂肪球を均質機などで機械的にせん断することにより、その脂肪球径を小さくする（均質化する）方法、及び水中油型乳化物に乳化剤を添加し、水中油型乳化物の乳化を安定化させる方法、などが公知である。 Methods for adjusting the diameter of fat globules mixed in the oil-in-water emulsion to an appropriate size and stabilizing the emulsification of the oil-in-water emulsion include the following. For example, a method in which fat globules mixed in an oil-in-water emulsion are mechanically sheared with a homogenizer or the like to reduce the diameter of the fat globules (homogenization), and an emulsifier is added to the oil-in-water emulsion. Methods for stabilizing the emulsification of oil-in-water emulsions are known.
水中油型乳化物に添加する乳化剤は、例えば、水と油になじみやすいグリセリン脂肪酸エステルなどの化学合成品としての乳化剤、及び大豆由来の乳化剤、又は卵由来の乳化剤などの食物由来としての乳化剤が公知である。 The emulsifier to be added to the oil-in-water emulsion is, for example, an emulsifier as a chemically synthesized product such as glycerin fatty acid ester which is easily compatible with water and oil, and an emulsifier as a food source such as a soybean-based emulsifier or an egg-based emulsifier. It is known.
一方、化学的合成品としての乳化剤は、人工的なイメージを想起することから、近年の消費者の天然素材を評価する傾向に反する。また、食物由来としての乳化剤は、例えば、大豆由来の乳化剤を牛乳成分由来の乳飲料に添加するように、水中油型乳化物の由来が異なる場合には、食物アレルギーを懸念している消費者にとって好ましくない。 On the other hand, the emulsifier as a chemically synthesized product is contrary to the tendency of consumers to evaluate natural materials in recent years because it recalls an artificial image. Further, the emulsifier as a food source is, for example, when the origin of the oil-in-water emulsion is different, such as adding a soybean-derived emulsifier to a milk component-derived milk drink, consumers who are concerned about food allergies Not preferred for
そこで、水中油型乳化物を食品及び医薬品として経口摂取（投与）する場合には、水中油型乳化物と同じ由来の乳化剤を使用することが好ましい。例えば、水中油型乳化物が牛乳の場合には、牛乳由来の乳化剤として、カゼイン及び／又はカゼインナトリウムを使用することが公知である。また、牛乳の脂肪球の膜の画分を乳化剤として利用できることも公知である（特許文献１）。 Therefore, when the oil-in-water emulsion is orally ingested (administered) as a food or a pharmaceutical, it is preferable to use an emulsifier having the same origin as the oil-in-water emulsion. For example, when the oil-in-water emulsion is milk, it is known to use casein and / or sodium caseinate as an emulsifier derived from milk. It is also known that the fraction of the fat globule membrane of milk can be used as an emulsifier (Patent Document 1).
ところで、水中油型乳化物の一つである哺乳類の母乳中には、ＭＦＧ−Ｅ８（ｍｉｌｋ ｆａｔ ｇｌｏｂｕｌｅ−ＥＧＦ ｆａｃｔｏｒ ８）という分泌タンパク質を含有することが公知である。ＭＦＧ−Ｅ８は、母乳中に混在する脂肪球の膜中のリン脂質（フォスファチジルセリン）に結合する膜結合タンパク質であるが、乳脂肪球膜の構成成分として、本来必要とされる膜結合タンパク質ではないと言われている（非特許文献１）。一方、ＭＦＧ−Ｅ８の有する機能性は、殆ど知られていないため、その新たな機能性を見出すことを目的に、ＭＦＧ−Ｅ８遺伝子欠損マウスが作製されている（非特許文献２）。 By the way, it is known that mammalian breast milk, which is one of the oil-in-water emulsions, contains a secreted protein called MFG-E8 (milk fat globule-EGF factor 8). MFG-E8 is a membrane-bound protein that binds to phospholipids (phosphatidylserine) in fat globule membranes mixed in breast milk. It is said that it is not a protein (Non-Patent Document 1). On the other hand, since the functionality of MFG-E8 is almost unknown, MFG-E8 gene-deficient mice have been produced for the purpose of finding new functionality (Non-Patent Document 2).
そして、このＭＦＧ−Ｅ８質遺伝子欠損マウスを利用した研究を通じて、ＭＦＧ−Ｅ８には、生体内でのアポトーシス細胞の除去、及び、乳腺細胞内での乳脂肪球と上皮細胞間との相互作用の調節よる授乳期・離乳後の母乳中の脂肪球の調節などの機能を有することが知られている（非特許文献３）。 Through the study using the MFG-E8 quality gene-deficient mouse, MFG-E8 was found to be capable of removing apoptotic cells in vivo and of the interaction between milk fat globules and epithelial cells in mammary gland cells. It is known that it has functions such as regulation of fat globules in breast milk after lactation and weaning by regulation (Non-Patent Document 3).
本発明の課題は、比較的少量の含有（添加）であっても、水中油型乳化物に混在する脂肪球が水中で安定的に分散され、水中油型乳化物の乳化安定性が高まる乳化安定剤、及びこの乳化安定剤を用いた乳化安定化方法を提供することである。また、本発明の課題は、乳及び／又は乳製品を含有する乳性食品において、比較的少量の含有（添加）であっても、乳性食品に混在する脂肪球が水中で安定的に分散され、乳性食品の乳化安定性が高まる乳化安定剤、及びこの乳化安定剤を用いた乳化安定化方法を提供することである。 An object of the present invention is to provide an emulsion in which fat globules mixed in an oil-in-water emulsion are stably dispersed in water even if the content (addition) is relatively small, and the emulsion stability of the oil-in-water emulsion is enhanced. An object of the present invention is to provide a stabilizer and an emulsion stabilization method using the emulsion stabilizer. Further, an object of the present invention is to provide a milk food containing milk and / or dairy products, in which the fat globules mixed with the milk food are stably dispersed in water even if a relatively small amount is contained (added). It is an object of the present invention to provide an emulsion stabilizer, which enhances the emulsion stability of dairy foods, and a method for stabilizing the emulsion using the emulsion stabilizer.
本発明者らは、上記課題を解決することを目的に、鋭意検討したところ、タンパク質であるＭＦＧ−Ｅ８を有効成分として含有することで、水中油型乳化物の乳化安定性が高まることを見出した。具体的には、ＭＦＧ−Ｅ８遺伝子欠損マウスの母乳の脂肪球径は、ＭＦＧ−Ｅ８遺伝子が欠損されていない通常のマウスの母乳の脂肪球径と比較して、特に、母乳の保持時間が長くなるにつれ、母乳中で他の脂肪球との脂肪球同士での会合をすることにより、その脂肪球径が大きくなることを確認した。 Means for Solving the Problems The present inventors have conducted intensive studies with the aim of solving the above problems, and found that the inclusion of the protein MFG-E8 as an active ingredient enhances the emulsion stability of an oil-in-water emulsion. Was. Specifically, the fat globule diameter of the breast milk of the MFG-E8 gene-deficient mouse is longer than the fat globule diameter of the breast milk of a normal mouse in which the MFG-E8 gene is not deleted. As a result, it was confirmed that the fat globule diameter was increased by associating fat globules with other fat globules in breast milk.
また、ＭＦＧ−Ｅ８遺伝子欠損マウスの母乳にＭＦＧ−Ｅ８を添加したところ、母乳の保持時間が長くなっても、母乳中で他の脂肪球との脂肪球同士での会合を抑制でき、その脂肪球径が大きくならずに、長期間にわたり乳化安定性が高まることを確認した。さらに、搾乳後に均質化処理をしていない牛乳にＭＦＧ−Ｅ８を添加したところ、母乳の保持時間が長くなっても、母乳中で他の脂肪球との脂肪球同士での会合を抑制でき、その脂肪球径が大きくならずに、長期間にわたり乳化安定性が高まることを確認した。 In addition, when MFG-E8 was added to the milk of MFG-E8 gene-deficient mice, the association between fat globules and other fat globules with other fat globules in breast milk could be suppressed even if the retention time of the milk was prolonged, and the fat It was confirmed that the emulsion stability was enhanced over a long period without increasing the sphere diameter. Furthermore, when MFG-E8 was added to milk that had not been homogenized after milking, even if the retention time of breast milk was long, the association between fat globules and other fat globules in breast milk could be suppressed, It was confirmed that the emulsion stability was improved over a long period of time without increasing the fat globule diameter.
本発明の乳化安定剤は、例えば、水中油型乳化物１００質量部に対して、ＭＦＧ−Ｅ８が０．００１〜１質量部という、比較的少量の含有（添加）であっても、水中油型乳化物に混在する脂肪球が水中で安定的に分散され、水中油型乳化物の乳化安定性が高まることを見出した。 The emulsion stabilizer of the present invention may be, for example, oil-in-water even if MFG-E8 is contained (added) in a relatively small amount of 0.001 to 1 part by mass with respect to 100 parts by mass of the oil-in-water emulsion. It has been found that fat globules mixed in a water-based emulsion are stably dispersed in water, and the emulsion stability of the oil-in-water emulsion is enhanced.
また、本発明の乳化安定剤を、水中油型乳化物のうち、例えば、乳及び／又は乳製品を含有する乳性食品に添加することで、乳性食品に混在する脂肪球が水中で安定的に分散され、乳性食品の乳化安定性が高まることを見出した。 In addition, by adding the emulsion stabilizer of the present invention to, for example, milk and / or dairy food containing dairy products among oil-in-water emulsions, fat globules mixed with dairy food are stable in water. And the emulsion stability of dairy foods is enhanced.
さらに、本発明の乳化安定剤が牛乳由来であり、水中油型乳化物が牛乳及び／又は牛乳由来の乳製品からなる乳性食品である場合には、乳化安定剤と水中油型乳化物が全て牛乳由来となり、「乳飲料」規格ではなく、「加工乳」規格又は「成分調整牛乳」規格などの付加価値の高い商品にすることができる。 Furthermore, when the emulsion stabilizer of the present invention is derived from milk, and the oil-in-water emulsion is a milk food comprising milk and / or milk-derived dairy products, the emulsion stabilizer and the oil-in-water emulsion are used. All products are derived from milk and can be made into high value-added products such as “processed milk” standard or “component-adjusted milk” standard instead of “milk drink” standard.
すなわち、本発明は以下の通りである。
（１）タンパク質であるＭＦＧ−Ｅ８（ｍｉｌｋ ｆａｔ ｇｌｏｂｕｌｅ−ＥＧＦ ｆａｃｔｏｒ ８）を有効成分として含有する水中油型乳化物用の乳化安定剤。
（２）ＭＦＧ−Ｅ８が乳由来である（１）に記載の水中油型乳化物用の乳化安定剤。
（３）ＭＦＧ−Ｅ８が脱脂乳由来である（１）に記載の水中油型乳化物用の乳化安定剤。
（４）ＭＦＧ−Ｅ８が脱脂乳の乳清由来である（１）に記載の水中油型乳化物用の乳化安定剤。
（５）水中油型乳化物にタンパク質であるＭＦＧ−Ｅ８を添加する工程を含む水中油型乳化物の乳化安定化方法。
（６）水中油型乳化物１００質量部に対し、０．００１〜１質量部のＭＦＧ−Ｅ８を添加する（５）に記載の水中油型乳化物の乳化安定化方法。
（７）水中油型乳化物が乳及び／又は乳製品を含有する乳性食品であり、ＭＦＧ−Ｅ８が乳由来である（５）又は（６）に記載の水中油型乳化物の乳化安定化方法。
（８）ＭＦＧ−Ｅ８が脱脂乳由来である（７）に記載の水中油型乳化物の乳化安定方法。
（９）ＭＦＧ−Ｅ８が脱脂乳の乳清由来である（７）に記載の水中油型乳化物の乳化安定方法。
（１０）ＭＦＧ−Ｅ８が牛乳由来であり、水中油型乳化物が牛乳及び／又は牛乳由来の乳製品からなる乳性食品である（７）〜（９）のいずれか１に記載の水中油型乳化物の乳化安定化方法。That is, the present invention is as follows.
(1) An emulsion stabilizer for an oil-in-water emulsion containing, as an active ingredient, MFG-E8 (milk fat globule-EGF factor 8) which is a protein.
(2) The emulsion stabilizer according to (1), wherein the MFG-E8 is derived from milk.
(3) The emulsion stabilizer according to (1), wherein MFG-E8 is derived from skim milk.
(4) The emulsion stabilizer according to (1), wherein MFG-E8 is derived from whey of skim milk.
(5) A method for stabilizing the emulsification of an oil-in-water emulsion, comprising a step of adding MFG-E8 as a protein to the oil-in-water emulsion.
(6) The method for stabilizing an oil-in-water emulsion according to (5), wherein 0.001 to 1 part by mass of MFG-E8 is added to 100 parts by mass of the oil-in-water emulsion.
(7) The emulsion stability of the oil-in-water emulsion according to (5) or (6), wherein the oil-in-water emulsion is a dairy food containing milk and / or dairy products, and MFG-E8 is derived from milk. Method.
(8) The method according to (7), wherein the MFG-E8 is derived from skim milk.
(9) The method according to (7), wherein the MFG-E8 is derived from whey of skim milk.
(10) The oil-in-water according to any one of (7) to (9), wherein MFG-E8 is derived from milk, and the oil-in-water emulsion is a dairy food comprising milk and / or milk-derived dairy products. Method for stabilizing emulsified emulsion.
本発明によれば、タンパク質であるＭＦＧ−Ｅ８の存在により、比較的少量の含有（添加）であっても、水中油型乳化物に混在する脂肪球が水中で安定的に分散され、水中油型乳化物の乳化安定性が高まることを見出した。本発明は、タンパク質であるＭＦＧ−Ｅ８（ｍｉｌｋ ｆａｔ ｇｌｏｂｕｌｅ−ＥＧＦ ｆａｃｔｏｒ ８）を有効成分として含有する水中油型乳化物用の乳化安定剤、及びこの乳化安定剤を添加する工程を含む水中油型乳化物の乳化安定化方法を提供することができる。 According to the present invention, due to the presence of MFG-E8 which is a protein, fat globules mixed in an oil-in-water emulsion can be stably dispersed in water even if the content is relatively small (addition), and oil-in-water It has been found that the emulsion stability of the emulsion of the type is enhanced. The present invention provides an emulsion stabilizer for an oil-in-water emulsion containing MFG-E8 (milk fat globule-EGF factor 8) which is a protein as an active ingredient, and an oil-in-water emulsion comprising a step of adding the emulsion stabilizer. An emulsion stabilization method for an emulsion can be provided.
以下、本発明について詳細に説明する。
＜乳化安定剤＞
上述のように、本発明の水中油型乳化物用の乳化安定剤は、タンパク質であるＭＦＧ−Ｅ８（ｍｉｌｋ ｆａｔ ｇｌｏｂｕｌｅ−ＥＧＦ ｆａｃｔｏｒ ８）を有効成分として含有することを特徴とする。Hereinafter, the present invention will be described in detail.
<Emulsion stabilizer>
As described above, the emulsion stabilizer for an oil-in-water emulsion of the present invention is characterized in that it contains MFG-E8 (milk fat globule-EGF factor 8) as an active ingredient.
本発明のＭＦＧ−Ｅ８は、母乳中に混在する脂肪球の膜中のリン脂質（フォスファチジルセリン）に結合する膜結合タンパク質であるが、乳脂肪球膜の構成成分として、本来必要とされる膜結合タンパク質ではないと言われていたものである。ＭＦＧ−Ｅ８は、例えば、ヒト由来、マウス由来、ラット由来、ブタ由来、及びウシ由来などでその存在が公知である。 The MFG-E8 of the present invention is a membrane-bound protein that binds to a phospholipid (phosphatidylserine) in a fat globule membrane mixed in breast milk, but is originally required as a component of the milk fat globule membrane. It is said that it is not a membrane-bound protein. The existence of MFG-E8 is known, for example, from human, mouse, rat, pig, and bovine.
ＭＦＧ−Ｅ８は、乳又は脱脂乳（例えば、脱脂粉乳を還元したもの）を、所定の条件で遠心分離し、カゼインを除去した上清画分（いわゆる乳清画分）に含有することが公知である（非特許文献１）。そして、この乳清画分を、公知のタンパク質の精製技術、例えば、各種クロマトグラフィー、塩析、透析、及び膜分離などにより精製することで、精製されたＭＦＧ−Ｅ８、又は部分的（例えば、フォスファチジルセリンなどの任意の段階まで）に精製されたＭＦＧ−Ｅ８を得ることができる。 MFG-E8 is known to contain milk or skim milk (for example, skim milk powder reduced) in a supernatant fraction (a so-called whey fraction) from which casein has been removed by centrifugation under predetermined conditions. (Non-Patent Document 1). Then, the whey fraction is purified by a known protein purification technique, for example, various chromatography, salting out, dialysis, membrane separation, etc., to obtain a purified MFG-E8 or partially (for example, MFG-E8 purified to any stage (such as phosphatidylserine) can be obtained.
すなわち、本発明のＭＦＧ−Ｅ８は、例えば、精製されたＭＦＧ−Ｅ８、部分的に精製されたＭＦＧ−Ｅ８、及び、乳又は脱脂乳（例えば、脱脂粉乳を還元したもの）を遠心分離し、カゼインを除去した上清画分（いわゆる乳清画分）を使用することができる。また、本発明のＭＦＧ−Ｅ８は、その形状に制限はなく、例えば、液状、ペースト状、粉末状、及び凍結状などである。また、本発明のＭＦＧ−Ｅ８は、その由来に制限はなく、例えば、乳由来（ヒト乳由来、マウス乳由来、ラット乳由来、ブタ乳由来、及びウシ乳由来など）、脱脂乳由来（ヒト乳由来、マウス乳由来、ラット乳由来、ブタ乳由来、及びウシ乳由来など）である。 That is, the MFG-E8 of the present invention is obtained by, for example, centrifuging purified MFG-E8, partially purified MFG-E8, and milk or skim milk (eg, reduced skim milk). The supernatant fraction from which casein has been removed (so-called whey fraction) can be used. The shape of the MFG-E8 of the present invention is not limited, and is, for example, a liquid, a paste, a powder, a frozen shape, or the like. In addition, the MFG-E8 of the present invention is not limited in its origin. For example, it is derived from milk (derived from human milk, mouse milk, rat milk, pig milk, and bovine milk), and derived from skim milk (human). Milk-derived, mouse-derived, rat-derived, pig-derived and bovine-derived).
本発明のＭＦＧ−Ｅ８の一態様である、乳又は脱脂乳（例えば、脱脂粉乳を還元したもの）を、所定の条件で遠心分離し、カゼインを除去した上清画分（いわゆる乳清画分）は、乳タンパク質原料として重要であるカゼインの製造工程において、いわゆる産業廃棄物とされる画分である。本発明のＭＦＧ−Ｅ８として、これらの乳清画分を使用することは、資源の有効利用、環境保護又はコスト削減などの観点から、好ましい。特に、乳清画分としては、本発明のＭＦＧ−Ｅ８を多く含有していることが、より好ましい。 Milk or skim milk (e.g., reduced skim milk), which is one embodiment of MFG-E8 of the present invention, is centrifuged under predetermined conditions to remove casein and a supernatant fraction (so-called whey fraction) ) Is a so-called industrial waste fraction in the production process of casein, which is important as a milk protein raw material. It is preferable to use these whey fractions as the MFG-E8 of the present invention from the viewpoint of effective use of resources, environmental protection or cost reduction. In particular, it is more preferable that the whey fraction contains a large amount of the MFG-E8 of the present invention.
本発明のＭＦＧ−Ｅ８として使用する乳清画分は、乳又は脱脂乳（例えば、脱脂粉乳を還元したもの）を、遠心分離する条件は、ＭＦＧ−Ｅ８を得られる条件であれば、遠心力（回転数）、遠心分離に要する時間、遠心分離をする温度などに制限はない。 The whey fraction used as the MFG-E8 of the present invention is a milk or skim milk (for example, skim milk powder reduced), and the centrifugal separation is performed under the condition that MFG-E8 can be obtained. There are no restrictions on the (rotation speed), the time required for centrifugation, the temperature for centrifugation, and the like.
遠心力は、例えば、５０００〜１０００００×ｇ、６０００〜８００００×ｇ、７０００〜６００００×ｇ、８０００〜４００００×ｇ、９０００〜３００００×ｇ、１００００〜２００００×ｇ、１２０００〜１８０００×ｇ、及び１４０００〜１６０００×ｇである。遠心力が５０００×ｇ以上であれば、ＭＦＧ−Ｅ８の純度を高めることができ、好ましい。また、遠心力が１０００００×ｇ以下であれば、過剰な遠心力とはならず、効率よく遠心分離でき、好ましい。 The centrifugal force is, for example, 5000 to 100000 × g, 6000 to 80000 × g, 7000 to 60000 × g, 8000 to 40000 × g, 9000 to 30000 × g, 10000 to 20000 × g, 12000 to 18000 × g, and 14000. １６16000 × g. When the centrifugal force is 5000 × g or more, the purity of MFG-E8 can be increased, which is preferable. When the centrifugal force is 100000 × g or less, excessive centrifugal force does not occur and centrifugation can be performed efficiently, which is preferable.
遠心分離に要する時間は、例えば、２〜６０分間、４〜５０分間、６〜４０分間、８〜３０分間、１０〜２０分間、１２〜１８分間、及び１４〜１６分間などが挙げられる。遠心分離に要する時間が２分間以上であれば、ＭＦＧ−Ｅ８の純度を高めることができ、好ましい。また、遠心分離に要する時間が６０分以下であれば、過剰な遠心分離に要する時間とはならず、効率よく遠心分離でき、好ましい。 The time required for centrifugation includes, for example, 2 to 60 minutes, 4 to 50 minutes, 6 to 40 minutes, 8 to 30 minutes, 10 to 20 minutes, 12 to 18 minutes, and 14 to 16 minutes. If the time required for centrifugation is 2 minutes or more, the purity of MFG-E8 can be increased, which is preferable. In addition, if the time required for centrifugation is 60 minutes or less, the time required for excessive centrifugation is not reached, and efficient centrifugation can be performed, which is preferable.
遠心分離をする温度は、例えば、０〜２０℃、０〜１５℃、０〜１２℃、０〜１０℃、０〜８℃、０〜７℃、０〜６℃、０〜５℃、１〜５℃、２〜５℃、及び３〜５℃である。遠心分離をする温度が０℃以上であれば、乳又は脱脂乳（例えば、脱脂粉乳を還元したもの）が凍結しない状態で遠心分離でき、好ましい。また、遠心分離をする温度が２０℃以下であれば、特に予め加熱殺菌していない乳又は脱脂乳（例えば、脱脂粉乳を還元したもの）を衛生的に遠心分離でき、好ましい。 The temperature at which centrifugation is performed is, for example, 0-20 ° C, 0-15 ° C, 0-12 ° C, 0-10 ° C, 0-8 ° C, 0-7 ° C, 0-6 ° C, 0-5 ° C, -5 ° C, 2-5 ° C, and 3-5 ° C. When the temperature at which the centrifugation is performed is 0 ° C. or higher, the milk or skim milk (eg, skim milk powder reduced) can be centrifuged without being frozen, which is preferable. If the temperature at which the centrifugation is performed is 20 ° C. or lower, milk or skim milk that has not been heat-sterilized in advance (eg, reduced skim milk) can be centrifugally separated in a sanitary manner, which is preferable.
本発明のＭＦＧ−Ｅ８として使用する乳清画分は、これらの乳清画分中のＭＦＧ−Ｅ８の存在を、抗原抗体反応を利用し、定性的・定量的に確認することができる。抗原抗体反応は、例えば、抗ＭＦＧ−Ｅ８モノクローナル抗体と蛍光標識二次抗体による反応を利用して、乳清画分中のＭＦＧ−Ｅ８と反応させて、その蛍光強度により、乳清画分中のＭＦＧ−Ｅ８の存在や濃度を確認することができる。また、本発明のＭＦＧ−Ｅ８として使用する乳清画分は、実際の水中油型乳化物での乳化の安定性に応じて、水中油型乳化物への添加量を適宜設定することができる。 In the whey fraction used as MFG-E8 of the present invention, the presence of MFG-E8 in these whey fractions can be qualitatively and quantitatively confirmed using an antigen-antibody reaction. The antigen-antibody reaction is performed, for example, by reacting with MFG-E8 in the whey fraction by utilizing the reaction between the anti-MFG-E8 monoclonal antibody and the fluorescent-labeled secondary antibody, and determining the fluorescence intensity in the whey fraction. Of MFG-E8 can be confirmed. In addition, in the whey fraction used as MFG-E8 of the present invention, the amount to be added to the oil-in-water emulsion can be appropriately set according to the stability of the actual oil-in-water emulsion. .
本発明の乳化安定剤は、水中油型乳化物の乳化を安定化できれば、水中油型乳化物について特に制限はない。特に、哺乳類の乳由来の本発明の乳化安定剤は、化学合成品の乳化剤とは異なり、人工的なイメージが少なく、その食習慣から摂取による副作用の心配がないため、経口摂取できる水中油型乳化物への添加が好ましい。 The emulsion stabilizer of the present invention is not particularly limited as long as it can stabilize the emulsification of the oil-in-water emulsion. In particular, the emulsion stabilizer of the present invention derived from mammalian milk, unlike the emulsifier of a chemically synthesized product, has less artificial image and has no fear of side effects due to ingestion due to its eating habits. Addition to the emulsion is preferred.
ここでいう、経口摂取できる水中油型乳化物とは、例えば、水中油型乳化物である食品、水中油型乳化物である医薬品である。これらの水中油型乳化物の形状には、特に制限はなく、例えば、液状、ペースト状（糊状）、半固形状、及び固形状である。水中油型乳化物中に混在する脂肪球同士の会合は、流動性の高い系で起こりやすい。従って、本発明の乳化安定剤は、液状又は糊状などの流動性のある形状の水中油型乳化物に対し、乳化を安定化させる効果が期待できる。 The oil-in-water emulsion that can be taken orally here is, for example, a food that is an oil-in-water emulsion, and a pharmaceutical that is an oil-in-water emulsion. The shape of these oil-in-water emulsions is not particularly limited, and is, for example, liquid, paste-like (paste-like), semi-solid, or solid. The association of fat globules mixed in the oil-in-water emulsion tends to occur in a highly fluid system. Therefore, the emulsion stabilizer of the present invention can be expected to have an effect of stabilizing the emulsification of an oil-in-water emulsion having a fluid or pasty shape such as fluidity.
本発明における水中油型乳化物である食品には、例えば、乳（牛乳、山羊乳、水牛乳、羊乳、及び、めん羊乳などの獣乳、豆乳、及びココナツミルクなどの植物乳、コーヒーホワイトナーなどの人工乳）、乳製品、乳及び／又は乳製品を含有する乳性食品、及びマヨネーズなどが挙げられる。中でも、乳及び／又は乳製品を含有する乳性食品が好ましい。 Foods that are oil-in-water emulsions in the present invention include, for example, milk (animal milk such as milk, goat milk, buffalo milk, sheep milk, and sheep milk, soy milk, vegetable milk such as coconut milk, coffee). Artificial milk such as whitener), dairy products, milk and / or dairy food products containing dairy products, and mayonnaise. Among them, dairy foods containing milk and / or dairy products are preferred.
ここでいう、乳製品には、乳を加工したもの全般をいい、例えば濃縮乳、全脂粉乳、脱脂濃縮乳、脱脂粉乳、部分脱脂濃縮乳、部分脱脂粉乳、練乳、加糖練乳、クリーム、バター、発酵乳、チーズ、ホエイ、ホエイ粉、乳タンパク質濃縮物、及びホエイタンパク質濃縮物などがある。 As used herein, the dairy product refers to any processed milk, for example, concentrated milk, whole fat milk powder, skim concentrated milk, skim milk powder, partially skim concentrated milk, partially skim milk powder, condensed milk, sweetened condensed milk, cream, butter , Fermented milk, cheese, whey, whey powder, milk protein concentrate, and whey protein concentrate.
また、乳及び／又は乳製品を含有する乳性食品とは、例えば、牛乳、加工乳、成分調整乳、低脂肪乳、乳飲料、コーヒー入り乳飲料、フルーツ入り乳飲料、ミネラル強化乳飲料、ビタミン強化乳飲料、機能性素材強化乳飲料、ヨーグルト、乳酸菌飲料、乳清飲料、酸性乳飲料、チーズ、練乳、加糖練乳、生クリーム、コンパウンドクリーム、植物性脂肪クリーム、及びコーヒーホワイトナーなどがある。 The milky food containing milk and / or dairy products includes, for example, milk, processed milk, ingredient-modified milk, low-fat milk, milk drinks, coffee-containing milk drinks, fruit-containing milk drinks, mineral-enriched milk drinks, Vitamin-enriched milk drinks, functional ingredient-enriched milk drinks, yogurt, lactic acid bacteria drinks, whey drinks, acidic milk drinks, cheese, condensed milk, sweetened condensed milk, fresh cream, compound cream, vegetable fat cream, coffee whitener, etc. .
本発明における水中油型乳化物である医薬品は、公知の医薬品であれば特に制限されない。 The drug which is an oil-in-water emulsion in the present invention is not particularly limited as long as it is a known drug.
なお、本発明の乳化安定剤を水中油型乳化物である食品に使用する場合は、アレルギー源の混入を防止する目的から、同じ食品由来のＭＦＧ−Ｅ８、又は同じ動物由来のＭＦＧ−Ｅ８を使用することが好ましい。 When the emulsion stabilizer of the present invention is used in foods that are oil-in-water emulsions, MFG-E8 derived from the same food or MFG-E8 derived from the same animal is used for the purpose of preventing contamination of allergic sources. It is preferred to use.
水中油型乳化物である食品が、例えば、牛乳及び／又は牛乳由来の乳製品からなる乳性食品の場合には、本発明の乳化安定剤は、牛乳由来（ウシ由来）のＭＦＧ−Ｅ８であることが好ましい。これにより、乳化安定剤と水中油型乳化物が全て牛乳由来となり、「乳飲料」規格ではなく、「加工乳」規格又は「成分調整牛乳」規格などの付加価値の高い商品にすることができる。 In the case where the food that is an oil-in-water emulsion is, for example, a dairy food consisting of milk and / or milk-derived dairy products, the emulsion stabilizer of the present invention is a milk-derived (bovine-derived) MFG-E8. Preferably, there is. As a result, the emulsion stabilizer and the oil-in-water emulsion are all derived from milk, and can be made into high value-added products such as the "processed milk" standard or the "component adjusted milk" standard instead of the "milk drink" standard. .
＜乳化安定化方法＞
上述のように、本発明の乳化安定化方法は、水中油型乳化物にタンパク質であるＭＦＧ−Ｅ８を添加する工程を含むことを特徴とする。ここで、水中油型乳化物へ添加する前の、本発明のＭＦＧ−Ｅ８は、例えば、精製されたＭＦＧ−Ｅ８、部分的に精製されたＭＦＧ−Ｅ８、及び、乳又は脱脂乳（例えば、脱脂粉乳を還元したもの）を遠心分離し、カゼインを除去した上清画分（いわゆる乳清画分）を使用することができる。<Emulsion stabilization method>
As described above, the emulsion stabilization method of the present invention is characterized by including a step of adding MFG-E8, which is a protein, to an oil-in-water emulsion. Here, before being added to the oil-in-water emulsion, the MFG-E8 of the present invention is, for example, purified MFG-E8, partially purified MFG-E8, and milk or skim milk (for example, The supernatant fraction (so-called whey fraction) from which casein has been removed by centrifuging the skim milk powder (reduced skim milk powder) can be used.
また、本発明のＭＦＧ−Ｅ８は、その形状に制限はなく、例えば、液状、ペースト状、粉末状、及び凍結状などである。また、本発明のＭＦＧ−Ｅ８は、その由来に制限はなく、例えば、乳由来（ヒト乳由来、マウス乳由来、ラット乳由来、ブタ乳由来、及びウシ乳由来など）、及び脱脂乳由来（ヒト乳由来、マウス乳由来、ラット乳由来、ブタ乳由来、及びウシ乳由来など）である。 The shape of the MFG-E8 of the present invention is not limited, and is, for example, a liquid, a paste, a powder, a frozen shape, or the like. The MFG-E8 of the present invention is not limited in its origin. For example, it is derived from milk (derived from human milk, derived from mouse milk, derived from rat milk, derived from pig milk, derived from bovine milk, etc.), and derived from skim milk ( Human milk, mouse milk, rat milk, pig milk, bovine milk, etc.).
本発明の乳化安定化方法において、水中油型乳化物へ添加する前の、本発明のＭＦＧ−Ｅ８の一態様である、乳又は脱脂乳（例えば、脱脂粉乳を還元したもの）を、所定の条件で遠心分離し、カゼインを除去した上清画分（いわゆる乳清画分）は、乳タンパク質原料として重要であるカゼインの製造工程において、いわゆる産業廃棄物とされる画分である。本発明のＭＦＧ−Ｅ８として、これらの乳清画分を使用することは、資源の有効利用、環境保護又はコスト削減などの観点から、好ましい。特に、乳清画分としては、本発明のＭＦＧ−Ｅ８を多く含有していることが、より好ましい。 In the emulsification stabilization method of the present invention, before adding to the oil-in-water emulsion, milk or skim milk (e.g., reduced skim milk), which is one embodiment of the MFG-E8 of the present invention, is added to a predetermined amount. The supernatant fraction from which casein has been removed by centrifugation under the conditions (so-called whey fraction) is a so-called industrial waste in the production process of casein which is important as a milk protein raw material. It is preferable to use these whey fractions as the MFG-E8 of the present invention from the viewpoint of effective use of resources, environmental protection or cost reduction. In particular, it is more preferable that the whey fraction contains a large amount of the MFG-E8 of the present invention.
本発明の乳化安定化方法において、水中油型乳化物へ添加する前の、本発明のＭＦＧ−Ｅ８として使用する乳清画分は、乳又は脱脂乳（例えば、脱脂粉乳を還元したもの）を、遠心分離して得られる。遠心分離の条件は、ＭＦＧ−Ｅ８を得られる条件であれば、遠心力（回転数）、遠心分離に要する時間、遠心分離をする温度などに制限はない。 In the emulsification stabilizing method of the present invention, the whey fraction used as the MFG-E8 of the present invention before adding to the oil-in-water emulsion may be milk or skim milk (e.g., reduced skim milk). , Obtained by centrifugation. The conditions for centrifugation are not limited, as long as MFG-E8 can be obtained, such as the centrifugal force (rotational speed), the time required for centrifugation, and the temperature for centrifugation.
遠心力は、例えば、５０００〜１０００００×ｇ、６０００〜８００００×ｇ、７０００〜６００００×ｇ、８０００〜４００００×ｇ、９０００〜３００００×ｇ、１００００〜２００００×ｇ、１２０００〜１８０００×ｇ、及び１４０００〜１６０００×ｇである。遠心力が５０００×ｇ以上であれば、ＭＦＧ−Ｅ８の純度を高めることができ、好ましい。また、遠心力が１０００００×ｇ以下であれば、過剰な遠心力とはならず、効率よく遠心分離でき、好ましい。 The centrifugal force is, for example, 5000 to 100000 × g, 6000 to 80000 × g, 7000 to 60000 × g, 8000 to 40000 × g, 9000 to 30000 × g, 10000 to 20000 × g, 12000 to 18000 × g, and 14000. １６16000 × g. When the centrifugal force is 5000 × g or more, the purity of MFG-E8 can be increased, which is preferable. When the centrifugal force is 100000 × g or less, excessive centrifugal force does not occur and centrifugation can be performed efficiently, which is preferable.
遠心分離に要する時間は、例えば、２〜６０分間、４〜５０分間、６〜４０分間、８〜３０分間、１０〜２０分間、１２〜１８分間、及び１４〜１６分間である。遠心分離に要する時間が２分間以上であれば、ＭＦＧ−Ｅ８の純度を高めることができ、好ましい。また、遠心分離に要する時間が６０分以下であれば、過剰な遠心分離に要する時間とはならず、効率よく遠心分離でき、好ましい。 The time required for centrifugation is, for example, 2 to 60 minutes, 4 to 50 minutes, 6 to 40 minutes, 8 to 30 minutes, 10 to 20 minutes, 12 to 18 minutes, and 14 to 16 minutes. If the time required for centrifugation is 2 minutes or more, the purity of MFG-E8 can be increased, which is preferable. In addition, if the time required for centrifugation is 60 minutes or less, the time required for excessive centrifugation is not reached, and efficient centrifugation can be performed, which is preferable.
遠心分離をする温度は、例えば、０〜２０℃、０〜１５℃、０〜１２℃、０〜１０℃、０〜８℃、０〜７℃、０〜６℃、０〜５℃、１〜５℃、２〜５℃、及び３〜５℃などが挙げられる。遠心分離をする温度が０℃以上であれば、乳又は脱脂乳（例えば、脱脂粉乳を還元したもの）が凍結しない状態で遠心分離でき、好ましい。また、遠心分離をする温度が２０℃以下であれば、特に予め加熱殺菌していない乳又は脱脂乳（例えば、脱脂粉乳を還元したもの）を衛生的に遠心分離でき、好ましい。 The temperature at which centrifugation is performed is, for example, 0-20 ° C, 0-15 ° C, 0-12 ° C, 0-10 ° C, 0-8 ° C, 0-7 ° C, 0-6 ° C, 0-5 ° C, To 5 ° C, 2 to 5 ° C, and 3 to 5 ° C. If the temperature at which the centrifugation is performed is 0 ° C. or higher, the milk or skim milk (eg, skim milk powder reduced) can be centrifuged without being frozen, which is preferable. If the temperature at which the centrifugation is performed is 20 ° C. or lower, milk or skim milk that has not been heat-sterilized in advance (eg, reduced skim milk) can be centrifugally separated in a sanitary manner, which is preferable.
本発明の乳化安定化方法において、水中油型乳化物へ添加する前の、本発明のＭＦＧ−Ｅ８として使用する乳清画分は、これらの乳清画分中のＭＦＧ−Ｅ８の存在を、抗原抗体反応を利用し、定性的・定量的に確認することができる。抗原抗体反応は、例えば、抗ＭＦＧ−Ｅ８モノクローナル抗体と蛍光標識二次抗体による反応を利用して、乳清画分中のＭＦＧ−Ｅ８と反応させて、その蛍光強度により、乳清画分中のＭＦＧ−Ｅ８の存在や濃度を確認することができる。また、本発明のＭＦＧ−Ｅ８として使用する乳清画分は、実際の水中油型乳化物での乳化の安定性に応じて、水中油型乳化物への添加量を適宜設定することができる。 In the emulsification stabilizing method of the present invention, the whey fraction used as the MFG-E8 of the present invention before adding to the oil-in-water emulsion, the presence of MFG-E8 in these whey fractions, It can be qualitatively and quantitatively confirmed using an antigen-antibody reaction. The antigen-antibody reaction is performed, for example, by reacting with MFG-E8 in the whey fraction by utilizing the reaction between the anti-MFG-E8 monoclonal antibody and the fluorescent-labeled secondary antibody, and determining the fluorescence intensity in the whey fraction. Of MFG-E8 can be confirmed. In addition, in the whey fraction used as MFG-E8 of the present invention, the amount to be added to the oil-in-water emulsion can be appropriately set according to the stability of the actual oil-in-water emulsion. .
本発明の乳化安定化方法において、本発明のＭＦＧ−Ｅ８を有効成分として含有する乳化安定剤を、水中油型乳化物に添加する時期は、水中油型乳化物の製造工程において、予め他の原料とともに調合する方法、製造工程の各段階において別途添加する方法、製造工程を経た後に別途添加する方法など、特に制限はない。 In the emulsion stabilization method of the present invention, the timing of adding the emulsion stabilizer containing the MFG-E8 of the present invention as an active ingredient to the oil-in-water emulsion is determined in advance in the production process of the oil-in-water emulsion. There is no particular limitation on the method of mixing with the raw materials, the method of separately adding at each stage of the production process, the method of separately adding after the production process, and the like.
本発明の乳化安定化方法において、本発明のＭＦＧ−Ｅ８を有効成分として含有する乳化安定剤を、水中油型乳化物に添加するにあたり、公知の水中油型乳化物の乳化の安定化方法を併用できる。ここでいう、公知の水中油型乳化物の乳化の安定化方法とは、例えば、水中油型乳化物に混在された脂肪球を均質機などで機械的にせん断することにより、その脂肪球径を小さくする（均質化する）方法、及び水中油型乳化物に乳化剤を添加し、水中油型乳化物の乳化を安定化させる方法などである。 In the emulsification stabilization method of the present invention, when adding an emulsion stabilizer containing MFG-E8 of the present invention as an active ingredient to an oil-in-water emulsion, a known method for stabilizing the emulsification of an oil-in-water emulsion is used. Can be used together. Here, the known method of stabilizing the emulsification of an oil-in-water emulsion is, for example, by mechanically shearing a fat globule mixed in the oil-in-water emulsion with a homogenizer or the like to obtain a fat globule diameter. And a method of adding an emulsifier to an oil-in-water emulsion to stabilize the emulsification of the oil-in-water emulsion.
すなわち、例えば、本発明の乳化安定化方法において、本発明のＭＦＧ−Ｅ８を有効成分として含有する乳化安定剤を、水中油型乳化物に添加した後に、水中油型乳化物に混在された脂肪球を均質機などで機械的にせん断することにより、その脂肪球径を小さくする（均質化する）こともできる。これにより、本発明のＭＦＧ−Ｅ８を有効成分として含有する乳化安定剤の水中油型乳化物の添加が、水中油型乳化物を均質化する前の予備乳化に代わる方法として利用することができる。 That is, for example, in the emulsion stabilization method of the present invention, after adding the emulsion stabilizer containing MFG-E8 of the present invention as an active ingredient to the oil-in-water emulsion, the fat mixed in the oil-in-water emulsion can be used. The diameter of the fat globules can be reduced (homogenized) by mechanically shearing the globules with a homogenizer or the like. Thereby, the addition of the oil-in-water emulsion of the emulsion stabilizer containing MFG-E8 of the present invention as an active ingredient can be used as a method instead of the preliminary emulsification before homogenizing the oil-in-water emulsion. .
また、例えば、本発明の乳化安定化方法において、水中油型乳化物に混在された脂肪球を均質機などで機械的にせん断することにより、その脂肪球径を小さくした（均質化した）後に、本発明のＭＦＧ−Ｅ８を有効成分として含有する乳化安定剤を、水中油型乳化物に添加することもできる。これにより、本発明のＭＦＧ−Ｅ８を有効成分として含有する乳化安定剤の水中油型乳化物の添加が、水中油型乳化物の乳化の安定性を高め、本発明のＭＦＧ−Ｅ８を有効成分として含有する乳化安定剤の水中油型乳化物を添加しない場合と比較して、水中油型乳化物をより長期間保存できる。 Also, for example, in the emulsion stabilization method of the present invention, the fat globules mixed in the oil-in-water emulsion are mechanically sheared with a homogenizer or the like to reduce the fat globule diameter (after homogenization). An emulsion stabilizer containing MFG-E8 of the present invention as an active ingredient can also be added to an oil-in-water emulsion. Thus, the addition of the oil-in-water emulsion of the emulsion stabilizer containing MFG-E8 of the present invention as an active ingredient enhances the stability of the emulsification of the oil-in-water emulsion, and makes the MFG-E8 of the present invention an active ingredient. The oil-in-water type emulsion can be stored for a longer period of time as compared with the case where the oil-in-water type emulsion of the emulsion stabilizer contained as above is not added.
本発明の乳化安定化方法において、水中油型乳化物の乳化を安定化できれば、水中油型乳化物について特に制限はない。特に、哺乳類の乳由来の本発明の乳化安定剤は、化学合成品の乳化剤とは異なり、人工的なイメージが少なく、その食習慣から摂取による副作用の心配がないため、経口摂取できる水中油型乳化物への添加が好ましい。 In the emulsion stabilization method of the present invention, there is no particular limitation on the oil-in-water emulsion as long as the emulsification of the oil-in-water emulsion can be stabilized. In particular, the emulsion stabilizer of the present invention derived from mammalian milk, unlike the emulsifier of a chemically synthesized product, has less artificial image and has no fear of side effects due to ingestion due to its eating habits. Addition to the emulsion is preferred.
ここでいう、経口摂取できる水中油型乳化物とは、例えば、水中油型乳化物である食品、水中油型乳化物である医薬品である。これらの水中油型乳化物の形状には、特に制限はなく、例えば、液状、ペースト状（糊状）、半固形状、及び固形状である。水中油型乳化物中に混在する脂肪球同士の会合は、流動性の高い系で起こりやすい。従って、本発明の乳化安定剤は、液状又は糊状などの流動性のある形状の水中油型乳化物に対し、乳化が安定化する効果が期待できる。 The oil-in-water emulsion that can be taken orally here is, for example, a food that is an oil-in-water emulsion, and a pharmaceutical that is an oil-in-water emulsion. The shape of these oil-in-water emulsions is not particularly limited, and is, for example, liquid, paste-like (paste-like), semi-solid, or solid. The association of fat globules mixed in the oil-in-water emulsion tends to occur in a highly fluid system. Therefore, the emulsion stabilizer of the present invention can be expected to have an effect of stabilizing the emulsification with respect to an oil-in-water emulsion having a fluid or paste-like shape and having fluidity.
本発明の乳化安定化方法において、水中油型乳化物である食品には、例えば、乳（牛乳、山羊乳、水牛乳、羊乳、及び、めん羊乳などの獣乳、豆乳、ココナツミルクなどの植物乳、コーヒーホワイトナーなどの人工乳）、乳製品、乳及び／又は乳製品を含有する乳性食品、及びマヨネーズなどがあり、中でも、乳及び／又は乳製品を含有する乳性食品が好ましい。 In the emulsion stabilization method of the present invention, foods that are oil-in-water emulsions include, for example, milk (animal milk such as milk, goat milk, buffalo milk, sheep milk, and sheep milk, soy milk, coconut milk, and the like). Vegetable milk, artificial milk such as coffee whitener), dairy products, dairy foods containing milk and / or dairy products, and mayonnaise, among which dairy food products containing milk and / or dairy products are included. preferable.
ここでいう、乳製品には、乳を加工したもの全般をいい、例えば濃縮乳、全脂粉乳、脱脂濃縮乳、脱脂粉乳、部分脱脂濃縮乳、部分脱脂粉乳、練乳、加糖練乳、クリーム、バター、発酵乳、チーズ、ホエイ、ホエイ粉、乳タンパク質濃縮物、及びホエイタンパク質濃縮物などがある。また、乳及び／又は乳製品を含有する乳性食品とは、例えば、牛乳、加工乳、成分調整乳、低脂肪乳、乳飲料、コーヒー入り乳飲料、フルーツ入り乳飲料、ミネラル強化乳飲料、ビタミン強化乳飲料、機能性素材強化乳飲料、ヨーグルト、乳酸菌飲料、乳清飲料、酸性乳飲料、チーズ、練乳、加糖練乳、生クリーム、コンパウンドクリーム、植物性脂肪クリーム、及びコーヒーホワイトナーなどがある。 As used herein, the dairy product refers to any processed milk, for example, concentrated milk, whole fat milk powder, skim concentrated milk, skim milk powder, partially skim concentrated milk, partially skim milk powder, condensed milk, sweetened condensed milk, cream, butter , Fermented milk, cheese, whey, whey powder, milk protein concentrate, and whey protein concentrate. The milky food containing milk and / or dairy products includes, for example, milk, processed milk, ingredient-modified milk, low-fat milk, milk drinks, coffee-containing milk drinks, fruit-containing milk drinks, mineral-enriched milk drinks, Vitamin-enriched milk drinks, functional ingredient-enriched milk drinks, yogurt, lactic acid bacteria drinks, whey drinks, acidic milk drinks, cheese, condensed milk, sweetened condensed milk, fresh cream, compound cream, vegetable fat cream, coffee whitener, etc. .
本発明の乳化安定化方法において、水中油型乳化物である医薬品は、公知の医薬品であれば特に制限されない。 In the emulsification stabilization method of the present invention, the drug that is an oil-in-water emulsion is not particularly limited as long as it is a known drug.
本発明の乳化安定化方法において、本発明のＭＦＧ−Ｅ８を有効成分として含有する乳化安定剤を水中油型乳化物である食品に使用する場合は、アレルギー源の混入を防止する目的から、同じ食品由来のＭＦＧ−Ｅ８、又は同じ動物由来のＭＦＧ−Ｅ８を使用することが好ましい。 In the emulsification stabilizing method of the present invention, when the emulsion stabilizer containing MFG-E8 of the present invention as an active ingredient is used in an oil-in-water type emulsion, the same is used for the purpose of preventing contamination of allergens. It is preferred to use food-derived MFG-E8, or MFG-E8 from the same animal.
水中油型乳化物である食品が、例えば、牛乳及び／又は牛乳由来の乳製品からなる乳性食品の場合には、本発明のＭＦＧ−Ｅ８を有効成分として含有する乳化安定剤は、牛乳由来（ウシ由来）のＭＦＧ−Ｅ８であることが好ましい。これにより、乳化安定剤と水中油型乳化物が全て牛乳由来となり、「乳飲料」規格ではなく、「加工乳」規格又は「成分調整牛乳」規格などの付加価値の高い商品にすることができる。 When the food that is an oil-in-water emulsion is, for example, a dairy food consisting of milk and / or milk-derived dairy products, the emulsion stabilizer containing MFG-E8 of the present invention as an active ingredient is derived from milk. It is preferably MFG-E8 (of bovine origin). As a result, the emulsion stabilizer and the oil-in-water emulsion are all derived from milk, and can be made into high value-added products such as the "processed milk" standard or the "component-adjusted milk" standard instead of the "milk drink" standard. .
本発明の乳化安定化方法において、本発明のＭＦＧ−Ｅ８を有効成分として含有する乳化安定剤を、水中油型乳化物に添加するにあたり、そのＭＦＧ−Ｅ８の添加量は、水中油型乳化物１００質量部に対し、精製物換算で、例えば、０．００１〜１質量部、０．００５〜１質量部、０．０１〜０．１質量部、０．０１〜０．０５質量部、０．０１〜０．０３質量部、及び０．０２〜０．０３質量部である。 In the emulsification stabilizing method of the present invention, when adding the emulsion stabilizer containing MFG-E8 of the present invention as an active ingredient to an oil -in- water emulsion, the amount of the MFG-E8 to be added is controlled by the oil-in-water emulsion. For 100 parts by mass, for example, 0.001 to 1 part by mass, 0.005 to 1 part by mass, 0.01 to 0.1 part by mass, 0.01 to 0.05 part by mass, 0 0.01 to 0.03 parts by mass, and 0.02 to 0.03 parts by mass.
本発明の乳化安定化方法において、本発明のＭＦＧ−Ｅ８を有効成分として含有する乳化安定剤を、水中油型乳化物に添加するにあたり、そのＭＦＧ−Ｅ８の添加量が、水中油型乳化物１００質量部に対し、精製物換算で、０．００１質量部以上とすることにより、本発明の乳化安定化の効果が得られ、好ましい。また、本発明の乳化安定化方法において、本発明のＭＦＧ−Ｅ８を有効成分として含有する乳化安定剤を、水中油型乳化物に添加するにあたり、そのＭＦＧ−Ｅ８の添加量が、水中油型乳化物１００質量部に対し、精製物換算で、１質量部以下とすることにより、本発明の水中油系乳化物の性状（風味及び／又は物性）への影響を防ぐことができ、好ましい。 In the emulsification stabilization method of the present invention, when adding the emulsion stabilizer containing MFG-E8 of the present invention as an active ingredient to an oil -in- water emulsion, the amount of the MFG-E8 to be added may be an oil-in-water emulsion. When the amount is 0.001 part by mass or more in terms of a purified product with respect to 100 parts by mass, the effect of emulsification stabilization of the present invention can be obtained, which is preferable. In addition, in the emulsion stabilization method of the present invention, when the emulsion stabilizer containing MFG-E8 of the present invention as an active ingredient is added to an oil-in-water emulsion, the amount of the MFG-E8 added is an oil-in-water type. By adjusting the amount to 1 part by mass or less in terms of the purified product with respect to 100 parts by mass of the emulsion, it is possible to prevent the effect on the properties (flavor and / or physical properties) of the oil-in-water emulsion of the present invention, which is preferable.
以下、実施例による試験結果を示し、本発明をさらに詳細に説明する。なお、本発明はこれら実施例に限定されるものではない。 Hereinafter, the present invention will be described in more detail by showing test results according to examples. Note that the present invention is not limited to these examples.
〔試験１：乳試料の調製及び生化学分析〕
実験動物として、野生型Ｃ５７ＢＬ／６マウス（ＷＴ）（日本ＳＬＣ社より購入）、及び、ＭＦＧ−Ｅ８欠損Ｃ５７ＢＬ／６マウス（ＫＯ）（京都大学大学院医学研究科 長田重一教授より供与）を使用した。これらのマウスに対し、研究用の餌（日本ＳＬＣ社製）を与え、母マウスが出産した際に、マウスの同腹仔数が６〜８となるように調整した。[Test 1: Preparation of milk sample and biochemical analysis]
Wild-type C57BL / 6 mice (WT) (purchased from SLC Japan) and MFG-E8-deficient C57BL / 6 mice (KO) (provided by Professor Shigekazu Nagata, Graduate School of Medicine, Kyoto University) were used as experimental animals. did. These mice were fed with food for research (manufactured by SLC Japan) and adjusted so that the number of litters of mice became 6 to 8 when mother mice gave birth.
これらのマウスより、強制離乳の３時間後（Ｗ３ｈ）及び４８時間後（Ｗ４８ｈ）に、母乳を搾乳し、これを乳試料とした。これらの乳試料を、キャップ付きの試験管に分注し、５７μｇ／ｍｌのペニシリンＧ及び１００μｇ／ｍｌのストレプトマイシンの存在下となるよう、ペニシリンＧ及びストレプトマイシンを添加した後に、３７℃で２４時間又は４８時間、恒温となるよう保持した。 From these mice, breast milk was expressed at 3 hours (W3h) and 48 hours (W48h) after forced weaning, and this was used as a milk sample. These milk samples are dispensed into capped tubes and added for 24 hours at 37 ° C. after addition of penicillin G and streptomycin in the presence of 57 μg / ml penicillin G and 100 μg / ml streptomycin. It was kept at a constant temperature for 48 hours.
これらの乳試料の組成は、Ｔｒｉｇｌｙｃｅｒｉｄｅ Ｅ−ｔｅｓｔ ｋｉｔ（和光純薬工業社）による脂肪含量、及びＢＣＡ ａｓｓａｙ ｋｉｔ（Ｔｈｅｒｍｏ Ｆｉｓｈｅｒ Ｓｉｅｎｔｉｆｉｃ社）によるタンパク質含量を測定することで確認した。そして、これらの乳試料を、１５０００×ｇで１５分間、４℃で遠心分離をし、その上清画分を乳清画分とした。 The composition of these milk samples was confirmed by measuring the fat content by the Triglyceride E-test kit (Wako Pure Chemical Industries, Ltd.) and the protein content by the BCA assay kit (Thermo Fisher Scientific). Then, these milk samples were centrifuged at 15,000 × g for 15 minutes at 4 ° C., and the supernatant fraction was used as a whey fraction.
〔試験２：脂肪球の顕微鏡解析〕
乳試料における脂肪球の形態観察は、乳試料をリン酸緩衝生理食塩水（ＰＢＳ）で１０倍希釈したものを、３００×ｇで１０分間、１５℃で遠心分離をし、その上層の脂肪球（ＭＦＧ）画分を洗浄し、２％のパラホルムアルデヒドを用いて固定した後に、ＰＢＳに溶解した１％のＢＳＡでブロッキングしたものを観察試料とした。そして、これらの観察試料を位相差顕微鏡（ＩＸ７１，オリンパス社）で観察した。[Test 2: Microscopic analysis of fat globules]
To observe the morphology of fat globules in a milk sample, a milk sample diluted 10-fold with phosphate buffered saline (PBS) was centrifuged at 300 × g for 10 minutes at 15 ° C. (MFG) The fraction was washed, fixed with 2% paraformaldehyde, and then blocked with 1% BSA dissolved in PBS to obtain an observation sample. And these observation samples were observed with a phase contrast microscope (IX71, Olympus).
脂肪球におけるＭＦＧ−Ｅ８の存在は、脂肪球（ＭＦＧ）画分と、抗マウスＭＦＧ−Ｅ８抗体又は抗ウシＭＦＧ−Ｅ８モノクローナル抗体、並びにＡｌｅｘａ Ｆｌｕｏｒ ４８８標識二次抗体（Ｌｉｆｅ Ｔｅｃｈｎｏｌｏｇｙ社）とで抗原抗体反応させ、脂肪球を免疫蛍光染色したものを、位相差蛍光顕微鏡（ＩＸ７１，オリンパス社）で観察した。 The presence of MFG-E8 in fat globules was determined by the antigen in the fat globule (MFG) fraction, anti-mouse MFG-E8 antibody or anti-bovine MFG-E8 monoclonal antibody, and Alexa Fluor 488 labeled secondary antibody (Life Technology). An antibody reaction was performed, and fat globules immunofluorescently stained were observed with a phase-contrast fluorescence microscope (IX71, Olympus).
〔試験３：脂肪球径の分布〕
位相差蛍光顕微鏡（ＩＸ７１，オリンパス社）で観察した顕微鏡視野（２０４．８×２７２μｍ２）における脂肪球を観察し、脂肪球を脂肪球径ごとに以下の４グループ（小：１．０〜３．２μｍ、中：３．３〜６．５μｍ、大：６．６〜９．９μｍ、特大：１０μｍ以上）に分類し、各グループにおける脂肪球の個数を数えた。[Test 3: Fat globule diameter distribution]
The fat globules in the microscope visual field (204.8 × 272 μm 2 ) observed with a phase contrast fluorescence microscope (IX71, Olympus) were observed, and the fat globules were classified into the following four groups (small: 1.0 to 3) for each fat globule diameter. 0.2 μm, middle: 3.3 to 6.5 μm, large: 6.6 to 9.9 μm, extra large: 10 μm or more), and the number of fat globules in each group was counted.
〔試験４：ＭＦＧ−Ｅ８、及びＭＦＧ−Ｅ８を含有する乳清画分の添加による脂肪球の確認〕
ＭＦＧ−Ｅ８欠損Ｃ５７ＢＬ／６マウス（ＫＯ）の母乳（強制離乳の３時間後に母乳を搾乳した乳試料）を、キャップ付きの試験管に分注し、遊離のＭＦＧ−Ｅ８を含有する野生型Ｃ５７ＢＬ／６マウス（ＷＴ）の母乳の乳清画分（５０質量％）、又は牛乳から精製したＭＦＧ−Ｅ８（２５０μｇ／ｍｌ）を添加し、３７℃で２４時間又は４８時間、恒温となるよう保持した。その後、上述の試験２及び試験３に基づき、脂肪球の形態、及び脂肪球形の分布を確認した。[Test 4: Confirmation of fat globules by addition of MFG-E8 and whey fraction containing MFG-E8]
MFG-E8 deficient C57BL / 6 mouse (KO) breast milk (milk sample milked 3 hours after forced weaning) is dispensed into a capped test tube and wild-type C57BL containing free MFG-E8マ ウ ス mouse (WT) whey fraction (50% by mass) of mother's milk or MFG-E8 purified from cow's milk (250 μg / ml) is added and kept at 37 ° C. for 24 hours or 48 hours at constant temperature. did. Thereafter, based on
〔結果１：ＭＦＧ−Ｅ８欠損マウスの母乳と野生型マウスの母乳との間の脂肪球径の分布の相違〕
強制離乳の３時間後（Ｗ３ｈ）及び４８時間後（Ｗ４８ｈ）に母乳を搾乳した乳試料における脂肪球を、位相差顕微鏡で観察した。その結果を図１Ａに示す。[Result 1: Difference in distribution of fat globule diameter between breast milk of MFG-E8-deficient mouse and wild-type mouse]
Fat globules in a milk sample whose breast milk was milked 3 hours (W3h) and 48 hours (W48h) after forced weaning were observed with a phase contrast microscope. The result is shown in FIG. 1A.
図１Ａに示すように、ＭＦＧ−Ｅ８欠損Ｃ５７ＢＬ／６マウス（ＫＯ）では、強制離乳の３時間後（Ｗ３ｈ）に母乳を搾取した乳試料では、大（脂肪球径６．６〜９．９μｍ）又は特大（脂肪球径１０μｍ以上）と分類された脂肪球の個数の割合が少なく、強制離乳の４８時間後（Ｗ４８ｈ）に母乳を搾取した乳試料では、大又は特大と分類された脂肪球の個数の割合が増加した。一方、野生型Ｃ５７ＢＬ／６マウス（ＷＴ）では、強制離乳の３時間後（Ｗ３ｈ）に母乳を搾取した乳試料、強制離乳の４８時間後（Ｗ４８ｈ）に母乳を搾取した乳試料ともに、大又は特大と分類された脂肪球の個数の割合が少なかった。 As shown in FIG. 1A, in the MFG-E8 deficient C57BL / 6 mouse (KO), the milk sample whose breast milk was extracted 3 hours after forced weaning (W3h) had a large (fat globule diameter of 6.6 to 9.9 μm). ) Or extra-large (diameter of fat globules of 10 μm or more) is small, and in milk samples in which breast milk was extracted 48 hours after forced weaning (W48h), fat globules classified as large or extra-large The proportion of the number increased. On the other hand, in wild-type C57BL / 6 mice (WT), both the milk sample whose breast milk was squeezed 3 hours after forced weaning (W3h) and the milk sample whose breast milk was squeezed 48 hours after forced weaning (W48h) were both large or large. The percentage of fat globules classified as oversized was small.
脂肪球径の分布の結果を図１Ｂに示す。図１Ｂに示すように、ＭＦＧ−Ｅ８欠損Ｃ５７ＢＬ／６マウス（ＫＯ）の母乳から搾取した乳試料では、大（脂肪球径６．６〜９．９μｍ）又は特大（脂肪球径１０μｍ以上）と分類された脂肪球の個数の割合は、強制離乳の３時間後（Ｗ３ｈ）に母乳を搾取した乳試料、強制離乳の４８時間後（Ｗ４８ｈ）に母乳を搾取した乳試料ともに、野生型Ｃ５７ＢＬ／６マウス（ＷＴ）の母乳から同様に搾取した乳試料と比較して有意に高かった（Ｗ３ｈ：ｐ＜０．０５；Ｗ４８ｈ：ｐ＜０．０１）。 The results of the distribution of fat globule diameter are shown in FIG. 1B. As shown in FIG. 1B, the milk sample squeezed from the breast milk of M57-E8-deficient C57BL / 6 mouse (KO) was large (fat globule diameter of 6.6 to 9.9 μm) or oversized (fat globule diameter of 10 μm or more). The ratio of the number of classified fat globules was determined for both wild-type C57BL / milk samples that had milk extracted 3 hours after forced weaning (W3h) and milk samples that had been milked 48 hours after forced weaning (W48h). It was significantly higher than the milk sample similarly extracted from breast milk of 6 mice (WT) (W3h: p <0.05; W48h: p <0.01).
〔結果２：ＭＦＧ−Ｅ８欠損マウスの母乳における脂肪球径の消長〕
ＭＦＧ−Ｅ８欠損Ｃ５７ＢＬ／６マウス（ＫＯ）の母乳から搾取した乳試料で、強制離乳の４８時間後（Ｗ４８ｈ）に母乳を搾取した乳試料の脂肪球の形態の再現を目的に、ＭＦＧ−Ｅ８欠損Ｃ５７ＢＬ／６マウス（ＫＯ）の母乳から搾取した乳試料をキャップ付きの試験管に分注し、５７μｇ／ｍｌのペニシリンＧ及び１００μｇ／ｍｌのストレプトマイシンの存在下となるよう、ペニシリンＧ及びストレプトマイシンを添加した後に、３７℃で２４時間又は４８時間、恒温となるよう保持した。対照として、野生型Ｃ５７ＢＬ／６マウス（ＷＴ）の母乳から搾取した乳試料も同様の条件で保持した。これらの保持した乳試料における脂肪球を、位相差顕微鏡で観察した。その結果を図２Ａに示す。図２Ａに示すように、２４時間（２４ｈ）又は４８時間（４８ｈ）保持に伴い、ＭＦＧ−Ｅ８欠損Ｃ５７ＢＬ／６マウス（ＫＯ）の乳試料中では、特大と分類された脂肪球の個数の割合が増加した。一方、２４時間（２４ｈ）又は４８時間（４８ｈ）保持に伴い、野生型Ｃ５７ＢＬ／６マウス（ＷＴ）の乳試料中では、特大と分類された脂肪球の個数の割合が増加しなかった。また、ＭＦＧ−Ｅ８欠損Ｃ５７ＢＬ／６マウス（ＫＯ）の母乳から搾取した乳試料中では、脂肪球同士の会合が位相差顕微鏡の形態観察で確認できた。なお、図２Ａの０ｈは、それぞれのマウスの母乳から搾取した（搾乳した）直後の状態を示す。[Result 2: Changes in fat globule diameter in breast milk of MFG-E8 deficient mouse]
For the purpose of reproducing the form of fat globules of a milk sample extracted from breast milk of M57-E8-deficient C57BL / 6 mouse (KO), the milk sample of which was extracted 48 hours after forced weaning (W48h). Milk samples squeezed from breast milk of defective C57BL / 6 mice (KO) were dispensed into capped tubes and penicillin G and streptomycin were added in the presence of 57 μg / ml penicillin G and 100 μg / ml streptomycin. After the addition, the temperature was kept constant at 37 ° C. for 24 hours or 48 hours. As a control, a milk sample squeezed from the breast milk of a wild-type C57BL / 6 mouse (WT) was maintained under the same conditions. Fat globules in these retained milk samples were observed with a phase contrast microscope. The result is shown in FIG. 2A. As shown in FIG. 2A, the ratio of the number of fat globules classified as extra-large in the milk sample of MFG-E8-deficient C57BL / 6 mice (KO) with the retention for 24 hours (24h) or 48 hours (48h). increased. On the other hand, in the milk sample of wild-type C57BL / 6 mice (WT), the proportion of the number of fat globules classified as extra-large did not increase with the retention for 24 hours (24 h) or 48 hours (48 h). In addition, in a milk sample extracted from breast milk of MFG-E8-deficient C57BL / 6 mouse (KO), association between fat globules was confirmed by morphological observation with a phase contrast microscope. Note that 0h in FIG. 2A indicates a state immediately after milking (milking) from the breast milk of each mouse.
ＭＦＧ−Ｅ８欠損Ｃ５７ＢＬ／６マウス（ＫＯ）の母乳から搾取した乳試料中に存在する、１５μｍ以上の脂肪球径の異常特大脂肪球とし、任意の１０視野中において、その個数を確認し、５頭のマウスの乳試料の平均値±標準偏差（ＳＤ）で示した。対照として、野生型Ｃ５７ＢＬ／６マウス（ＷＴ）の母乳から搾取した乳試料についても、同様の条件で、１５μｍ以上の脂肪球径の異常特大脂肪球の個数を確認し、５頭のマウスの乳試料の平均値±標準偏差（ＳＤ）で示した。これらの結果を図２Ｂに示す。 An abnormal extra-large fat globule having a fat globule diameter of 15 μm or more was present in a milk sample extracted from breast milk of MFG-E8-deficient C57BL / 6 mouse (KO), and its number was confirmed in any 10 visual fields. The results were shown as the mean ± standard deviation (SD) of the milk samples of the head mice. As a control, the number of abnormal extra-large fat globules having a fat globule diameter of 15 μm or more was confirmed under the same conditions for milk samples extracted from breast milk of wild-type C57BL / 6 mice (WT). The results are shown as the average value of the samples ± standard deviation (SD). These results are shown in FIG. 2B.
図２Ｂに示すように、１５μｍ以上の脂肪球径の異常特大脂肪球の数は、ＭＦＧ−Ｅ８欠損Ｃ５７ＢＬ／６マウス（ＫＯ）の母乳から搾取した乳試料では、３７℃で４８時間保持することでその個数は顕著に増加した。一方、野生型Ｃ５７ＢＬ／６マウス（ＷＴ）の母乳から搾取した乳試料では、３７℃で４８時間保持しても、１５μｍ以上の脂肪球径の異常特大脂肪球の数は変化しなかった。 As shown in FIG. 2B, the number of abnormal extra-large fat globules having a fat globule diameter of 15 μm or more should be maintained at 37 ° C. for 48 hours in a milk sample extracted from breast milk of MFG-E8-deficient C57BL / 6 mouse (KO). The number increased remarkably. On the other hand, in a milk sample squeezed from the breast milk of a wild-type C57BL / 6 mouse (WT), the number of abnormal extra-large fat globules having a fat globule diameter of 15 μm or more did not change even when the milk sample was kept at 37 ° C. for 48 hours.
〔結果３：ＭＦＧ−Ｅ８の添加による、ＭＦＧ−Ｅ８欠損マウスの母乳の脂肪球径の消長への影響〕
ＭＦＧ−Ｅ８欠損Ｃ５７ＢＬ／６マウス（ＫＯ）の母乳（強制離乳の３時間後に母乳を搾乳した乳試料）を、キャップ付きの試験管に分注し、遊離のＭＦＧ−Ｅ８を含有する野生型Ｃ５７ＢＬ／６マウス（ＷＴ）の母乳の乳清画分（５０質量％）、又は牛乳から精製したＭＦＧ−Ｅ８（２５０μｇ／ｍｌ）を添加し、３７℃で２４時間又は４８時間、恒温となるよう保持した。対照として、別のＭＦＧ−Ｅ８欠損Ｃ５７ＢＬ／６マウスの母乳の乳清画分（５０質量％）を添加し、３７℃で２４時間又は４８時間、恒温となるよう保持した。なお、全ての乳試料には、５７μｇ／ｍｌのペニシリンＧ及び１００μｇ／ｍｌのストレプトマイシンの存在下となるよう、ペニシリンＧ及びストレプトマイシンを添加した。これらの乳試料における脂肪球を、位相差顕微鏡で観察した。その結果を図３Ａに示す。[Result 3: Effect of addition of MFG-E8 on change in fat globule diameter in breast milk of MFG-E8 deficient mouse]
MFG-E8 deficient C57BL / 6 mouse (KO) breast milk (milk sample milked 3 hours after forced weaning) is dispensed into a capped test tube and wild-type C57BL containing free MFG-E8マ ウ ス mouse (WT) whey fraction (50% by mass) of mother's milk or MFG-E8 purified from cow's milk (250 μg / ml) is added and kept at 37 ° C. for 24 hours or 48 hours at constant temperature. did. As a control, a whey fraction (50% by mass) of mother's milk of another MFG-E8-deficient C57BL / 6 mouse was added, and kept at 37 ° C for 24 hours or 48 hours to be kept at a constant temperature. In addition, penicillin G and streptomycin were added to all milk samples so as to be in the presence of 57 μg / ml penicillin G and 100 μg / ml streptomycin. Fat globules in these milk samples were observed with a phase contrast microscope. The result is shown in FIG. 3A.
図３Ａに示すように、遊離のＭＦＧ−Ｅ８を含有する野生型Ｃ５７ＢＬ／６マウス（ＷＴ）の母乳の乳清画分（５０質量％）、又は牛乳から精製したＭＦＧ−Ｅ８（２５０μｇ／ｍｌ）を添加した乳試料では、３７℃で４８時間の保持中に、特大と分類された脂肪球の個数の割合が増加しなかった。一方、別のＭＦＧ−Ｅ８欠損Ｃ５７ＢＬ／６マウス（ＫＯ）の母乳の乳清画分（５０質量％）を添加した乳試料では、特大と分類された脂肪球の個数の割合が増加した。 As shown in FIG. 3A, whey fraction of breast milk of wild-type C57BL / 6 mouse (WT) containing free MFG-E8 (50% by mass), or MFG-E8 purified from cow's milk (250 μg / ml) Did not increase in the number of fat globules classified as oversized during the 48-hour hold at 37 ° C. On the other hand, in the milk sample to which the whey fraction (50% by mass) of the breast milk of another MFG-E8-deficient C57BL / 6 mouse (KO) was added, the ratio of the number of fat globules classified as oversized increased.
ＭＦＧ−Ｅ８欠損Ｃ５７ＢＬ／６マウス（ＫＯ）の母乳から搾取した乳試料中に存在する、１５μｍ以上の脂肪球径の異常特大脂肪球とし、任意の１０視野中において、その個数を確認し、５頭のマウスの乳試料の平均値±標準偏差（ＳＤ）で示した。野生型Ｃ５７ＢＬ／６マウス（ＷＴ）の母乳の乳清画分を添加したＭＦＧ−Ｅ８欠損Ｃ５７ＢＬ／６マウス（ＫＯ）の母乳（白三角）；牛乳から精製したＭＦＧ−Ｅ８を添加したＭＦＧ−Ｅ８欠損Ｃ５７ＢＬ／６マウス（ＫＯ）の母乳（白丸）；ＭＦＧ−Ｅ８欠損Ｃ５７ＢＬ／６マウス（ＫＯ）の母乳の乳清画分を添加したＭＦＧ−Ｅ８欠損Ｃ５７ＢＬ／６マウス（ＫＯ）マウスの母乳（黒三角）；リン酸緩衝生理食塩水を添加したＭＦＧ−Ｅ８欠損Ｃ５７ＢＬ／６マウス（ＫＯ）の母乳（黒丸）について、平均値±ＳＤで示した結果を図３Ｂに示す。 An abnormal extra-large fat globule having a fat globule diameter of 15 μm or more was present in a milk sample extracted from breast milk of MFG-E8-deficient C57BL / 6 mouse (KO), and its number was confirmed in any 10 visual fields. The results were shown as the mean ± standard deviation (SD) of the milk samples of the head mice. MFG-E8-deficient C57BL / 6 mouse (KO) breast milk (open triangles) supplemented with whey fraction of wild-type C57BL / 6 mouse (WT) breast milk; MFG-E8 supplemented with MFG-E8 purified from cow's milk Breast milk of deficient C57BL / 6 mouse (KO) (open circle); Breast milk of MFG-E8 deficient C57BL / 6 mouse (KO) mouse to which whey fraction of breast milk of MFG-E8 deficient C57BL / 6 mouse (KO) is added ( Black triangles); FIG. 3B shows the results shown as mean ± SD for breast milk (solid circles) of MFG-E8-deficient C57BL / 6 mice (KO) supplemented with phosphate buffered saline.
図３Ｂに示すように、遊離のＭＦＧ−Ｅ８を含有する野生型Ｃ５７ＢＬ／６マウス（ＷＴ）の母乳の乳清画分（５０質量％）、及び牛乳から精製したＭＦＧ−Ｅ８（２５０μｇ／ｍｌ）を添加した乳試料では、３７℃で４８時間の保持によっても、１５μｍ以上の脂肪球径の異常特大脂肪球の数は増加しなかった。一方、別のＭＦＧ−Ｅ８欠損Ｃ５７ＢＬ／６マウス（ＫＯ）の母乳の乳清画分（５０質量％）、及びリン酸緩衝生理食塩水を添加した乳試料では、３７℃で４８時間の保持により、１５μｍ以上の脂肪球径の異常特大脂肪球の数は増加した。 As shown in FIG. 3B, whey fraction of wild-type C57BL / 6 mouse (WT) containing free MFG-E8 (50% by mass) and MFG-E8 purified from milk (250 μg / ml) The number of abnormal extra-large fat globules having a fat globule diameter of 15 μm or more did not increase even when the milk sample to which the was added was kept at 37 ° C. for 48 hours. On the other hand, the whey fraction (50% by mass) of the breast milk of another MFG-E8-deficient C57BL / 6 mouse (KO) and the milk sample to which phosphate buffered saline was added were kept at 37 ° C. for 48 hours. The number of abnormal extra-large fat globules having a fat globule diameter of 15 μm or more increased.
また、ＭＦＧ−Ｅ８欠損Ｃ５７ＢＬ／６マウス（ＫＯ）の母乳から搾取した乳試料に添加したＭＦＧ−Ｅ８が、ＭＦＧ−Ｅ８欠損Ｃ５７ＢＬ／６マウス（ＫＯ）の母乳に結合する様子を、試験２の抗原抗体反応による免疫染色で確認した。具体的には、３７℃で４８時間保持した上記の乳試料中の脂肪球を、抗ＭＦＧ−Ｅ８抗体を用いて免疫染色した。代表的な脂肪球の画像（位相差画像及び蛍光画像）、及びＭＦＧ−Ｅ８を添加しないで３７℃で４８時間保持した乳試料中（対照）の脂肪球の画像（位相差画像及び蛍光画像）を図３Ｃに示す。
図３Ｃに示すように、野生型Ｃ５７ＢＬ／６マウス（ＷＴ）の母乳の乳清画分由来のＭＦＧ−Ｅ８を添加した場合だけでなく、牛乳から精製したＭＦＧ−Ｅ８を添加した場合においても、ＭＦＧ−Ｅ８は、ＭＦＧ−Ｅ８欠損Ｃ５７ＢＬ／６マウス（ＫＯ）の母乳から搾取した乳試料の脂肪球に結合していることが確認された。 As shown in FIG. 3C, not only when MFG-E8 derived from the whey fraction of the breast milk of wild-type C57BL / 6 mice (WT) was added, but also when MFG-E8 purified from cow milk was added, It was confirmed that MFG-E8 was bound to fat globules of a milk sample extracted from breast milk of M57-E8-deficient C57BL / 6 mice (KO).
〔試験５：ＭＦＧ−Ｅ８、及びＭＦＧ−Ｅ８を含有する乳清画分の添加による、搾乳直後の牛乳の脂肪球の確認〕
搾乳直後の牛乳で、ＭＦＧ−Ｅ８が脂肪球同士の会合を抑制しているかを解明するために、搾乳直後の牛乳を、キャップ付きの試験管に分注し、遊離のＭＦＧ−Ｅ８を含有する野生型Ｃ５７ＢＬ／６マウス（ＷＴ）の母乳の乳清画分（５０質量％）、又は牛乳から精製したＭＦＧ−Ｅ８（２５０μｇ／ｍｌ（１０ｍＭトリス塩酸緩衝液、１５０ｍＭ塩化ナトリウム））を添加し、３７℃で１日間、２日間、３日間、４日間、又は７日間恒温で保持した。対照として、別のＭＦＧ−Ｅ８欠損Ｃ５７ＢＬ／６マウス（ＫＯ）の母乳の乳清画分（５０質量％）を添加し、３７℃で１日間、２日間、３日間、４日間、又は７日間恒温で保持した。また、それぞれにアジ化ナトリウムを終濃度０．１％となるように添加した。[Test 5: Confirmation of fat globules of milk immediately after milking by addition of MFG-E8 and a whey fraction containing MFG-E8]
In order to clarify whether MFG-E8 suppresses the association between fat globules in milk immediately after milking, milk immediately after milking is dispensed into a test tube with a cap and contains free MFG-E8. A whey fraction (50% by mass) of the milk of a wild-type C57BL / 6 mouse (WT) or MFG-E8 purified from cow's milk (250 μg / ml (10 mM Tris-HCl buffer, 150 mM sodium chloride)) was added. It was kept at 37 ° C. for 1 day, 2 days, 3 days, 4 days, or 7 days. As a control, a whey fraction (50% by mass) of breast milk of another MFG-E8-deficient C57BL / 6 mouse (KO) was added, and the mixture was added at 37 ° C. for 1, 2, 3, 4, or 7 days at 1 day. It was kept at a constant temperature. In addition, sodium azide was added to each so that the final concentration was 0.1%.
搾乳直後の牛乳の脂肪球画分の調製方法は、牛乳１ｍｌを遠心分離し（３００×ｇ、１０分間、２０℃）、乳清とカゼイン（３００×ｇ ｐｐｔ）を注射針により除去した後、５００μｌのＴＢＳ（１０ｍＭトリス塩酸緩衝液、１５０ｍＭ塩化ナトリウム）を添加し、再度遠心分離し（３００×ｇ、１０分間、２０℃）、脂肪球を洗浄した。ＴＢＳによる洗浄を３回繰り返し、これを脂肪球画分とした。 The method of preparing the fat globule fraction of milk immediately after milking is as follows: 1 ml of milk is centrifuged (300 × g, 10 minutes, 20 ° C.), whey and casein (300 × g ppt) are removed with a syringe needle, 500 μl of TBS (10 mM Tris-HCl buffer, 150 mM sodium chloride) was added and centrifuged again (300 × g, 10 minutes, 20 ° C.) to wash the fat globules. Washing with TBS was repeated three times to obtain a fat globule fraction.
〔結果４：搾乳直後の牛乳における脂肪球径の消長〕
搾乳直後の牛乳中の脂肪球を位相差顕微鏡で観察した結果を図４に示す。図４に示すように、搾乳直後の牛乳では、３７℃での保持に伴い、特大と分類された脂肪球の個数の割合が増加した。[Result 4: Change in fat globule diameter in milk immediately after milking]
FIG. 4 shows the result of observing fat globules in milk immediately after milking with a phase contrast microscope. As shown in FIG. 4, in milk immediately after milking, the ratio of the number of fat globules classified as oversized increased as the milk was kept at 37 ° C.
〔結果５：ＭＦＧ−Ｅ８、及びＭＦＧ−Ｅ８を含有する乳清画分の添加による、搾乳直後の牛乳の脂肪球の消長への影響〕
ＭＦＧ−Ｅ８、及びＭＦＧ−Ｅ８を含有する乳清画分の添加による、搾乳直後の牛乳中の脂肪球を位相差顕微鏡で観察した結果を図５、及び図６に示す。[Result 5: Effect of addition of MFG-E8 and a whey fraction containing MFG-E8 on the fate of fat globules in milk immediately after milking]
FIGS. 5 and 6 show the results of observing fat globules in milk immediately after milking by adding a MFG-E8 and a whey fraction containing MFG-E8 using a phase contrast microscope.
図５に示すように、牛乳から精製したＭＦＧ−Ｅ８（２５０μｇ／ｍｌ）を添加した搾乳直後の牛乳では、３７℃で７日間の保持中に、特大と分類された脂肪球の個数の割合が増加しなかった。一方、牛乳から精製したＭＦＧ−Ｅ８（２５０μｇ／ｍｌ）を添加しない搾乳直後の牛乳では、３７℃で７日間の保持中に、特大と分類された脂肪球の個数の割合が増加した。 As shown in FIG. 5, in milk immediately after milking to which MFG-E8 (250 μg / ml) purified from milk was added, the ratio of the number of fat globules classified as extra-large during the 7-day holding at 37 ° C. Did not increase. On the other hand, in milk immediately after milking to which MFG-E8 (250 μg / ml) purified from milk was not added, the ratio of the number of fat globules classified as extra-large increased during the 7-day holding at 37 ° C.
また、図６に示すように、遊離のＭＦＧ−Ｅ８を含有する野生型Ｃ５７ＢＬ／６マウス（ＷＴ）の母乳の乳清画分（５０質量％）を添加した搾乳直後の牛乳では、３７℃で７日間の保持中に、特大と分類された脂肪球の個数の割合が増加しなかった。一方、遊離のＭＦＧ−Ｅ８を含有しないＭＦＧ−Ｅ８欠損Ｃ５７ＢＬ／６マウス（ＫＯ）の母乳の乳清画分（５０質量％）を添加した搾乳直後の牛乳では、３７℃で７日間の保持中に、特大と分類された脂肪球の個数の割合が増加した。 As shown in FIG. 6, the milk immediately after milking to which the whey fraction (50% by mass) of the breast milk of the wild-type C57BL / 6 mouse (WT) containing free MFG-E8 was added, the temperature was 37 ° C. During the 7-day hold, the percentage of fat globules classified as oversized did not increase. On the other hand, the milk immediately after milking to which the whey fraction (50% by mass) of the breast milk of the MFG-E8-deficient C57BL / 6 mouse (KO) containing no free MFG-E8 was added was kept at 37 ° C. for 7 days. In addition, the percentage of fat globules classified as oversized increased.
本発明の水中油型乳化物の乳化安定剤は、比較的少量の含有（添加）であっても、水中油型乳化物に混在する脂肪球が水中で安定的に分散され、水中油型乳化物の乳化安定化に寄与することができる。特に、本発明の乳由来の乳化安定剤は、乳及び／又は乳製品を含有する乳性食品において、従来と比較して乳化安定剤の使用を少量に抑えることができる。 The emulsion stabilizer of the oil-in-water emulsion of the present invention can stably disperse fat globules mixed in the oil-in-water emulsion even in a relatively small amount of the oil-in-water emulsion. It can contribute to the stabilization of the emulsion of the product. In particular, the milk-derived emulsion stabilizer of the present invention can reduce the use of the emulsion stabilizer in milk and / or dairy food products containing dairy products, as compared with the conventional case.
本発明を特定の態様を用いて詳細に説明したが、本発明の意図と範囲を離れることなく様々な変更及び変形が可能であることは、当業者にとって明らかである。なお本出願は、２０１４年１２月２５日付で出願された日本特許出願（特願２０１４−２６２７８６）に基づいており、その全体が引用により援用される。 Although the present invention has been described in detail with particular embodiments, it will be apparent to those skilled in the art that various modifications and variations can be made without departing from the spirit and scope of the invention. This application is based on a Japanese patent application filed on Dec. 25, 2014 (Japanese Patent Application No. 2014-262786), the entire content of which is incorporated by reference.
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