JP2010180219A - Lactoferrin composition - Google Patents
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- 239000000203 mixture Substances 0.000 title claims abstract description 124
- 235000021242 lactoferrin Nutrition 0.000 title claims abstract description 20
- CSSYQJWUGATIHM-IKGCZBKSSA-N l-phenylalanyl-l-lysyl-l-cysteinyl-l-arginyl-l-arginyl-l-tryptophyl-l-glutaminyl-l-tryptophyl-l-arginyl-l-methionyl-l-lysyl-l-lysyl-l-leucylglycyl-l-alanyl-l-prolyl-l-seryl-l-isoleucyl-l-threonyl-l-cysteinyl-l-valyl-l-arginyl-l-arginyl-l-alanyl-l-phenylal Chemical compound C([C@H](N)C(=O)N[C@@H](CCCCN)C(=O)N[C@@H](CS)C(=O)N[C@@H](CCCNC(N)=N)C(=O)N[C@@H](CCCNC(N)=N)C(=O)N[C@@H](CC=1C2=CC=CC=C2NC=1)C(=O)N[C@@H](CCC(N)=O)C(=O)N[C@@H](CC=1C2=CC=CC=C2NC=1)C(=O)N[C@@H](CCCNC(N)=N)C(=O)N[C@@H](CCSC)C(=O)N[C@@H](CCCCN)C(=O)N[C@@H](CCCCN)C(=O)N[C@@H](CC(C)C)C(=O)NCC(=O)N[C@@H](C)C(=O)N1CCC[C@H]1C(=O)N[C@@H](CO)C(=O)N[C@@H]([C@@H](C)CC)C(=O)N[C@@H]([C@@H](C)O)C(=O)N[C@@H](CS)C(=O)N[C@@H](C(C)C)C(=O)N[C@@H](CCCNC(N)=N)C(=O)N[C@@H](CCCNC(N)=N)C(=O)N[C@@H](C)C(=O)N[C@@H](CC=1C=CC=CC=1)C(O)=O)C1=CC=CC=C1 CSSYQJWUGATIHM-IKGCZBKSSA-N 0.000 title claims abstract description 17
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- UHVMMEOXYDMDKI-JKYCWFKZSA-L zinc;1-(5-cyanopyridin-2-yl)-3-[(1s,2s)-2-(6-fluoro-2-hydroxy-3-propanoylphenyl)cyclopropyl]urea;diacetate Chemical compound [Zn+2].CC([O-])=O.CC([O-])=O.CCC(=O)C1=CC=C(F)C([C@H]2[C@H](C2)NC(=O)NC=2N=CC(=CC=2)C#N)=C1O UHVMMEOXYDMDKI-JKYCWFKZSA-L 0.000 description 2
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- WQZGKKKJIJFFOK-QTVWNMPRSA-N D-mannopyranose Chemical compound OC[C@H]1OC(O)[C@@H](O)[C@@H](O)[C@@H]1O WQZGKKKJIJFFOK-QTVWNMPRSA-N 0.000 description 1
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Abstract
Description
本発明は、ラクトフェリン類と安定剤を含有させることで、高い耐熱性を有するラクトフェリン組成物に関する。 The present invention relates to a lactoferrin composition having high heat resistance by containing lactoferrins and a stabilizer.
ラクトフェリン（以下ＬＦという）は、鉄吸収促進作用、抗炎症作用、過酸化脂質生成抑制作用、免疫系の制御作用等様々な生理機能を有していることが報告されている。また、これらの生理機能を有効に利用するためにＬＦを含む様々な飲食品や飼料、医薬が開発されている。しかし、ＬＦは中性域において加熱に対して不安定であり、６２．５℃、３０分の加熱によりほぼ失活し、７０℃、１５分の加熱により完全に失活することが知られている（例えば、非特許文献１参照）。このことから、ＬＦを酸性領域において加熱処理する方法が一般的に行われている。この方法を用いれば天然のＬＦ は、加熱処理しても抗菌活性や鉄結合性等が殆ど変化しないことが報告されている（例えば、特許文献１参照）。また、イオン強度が低い状態でＬＦ を加熱した場合に、その生理活性を維持することも知られている。しかし、実際には、イオン強度が低い状態でＬＦ が利用されることは少ないため、イオン強度とｐＨを最適化して加熱安定性を付与する方法（例えば、特許文献２参照）や、ＬＦ に鉄を十分結合吸着させることで耐熱性を付与する方法（例えば、特許文献３参照）が開発された。 It has been reported that lactoferrin (hereinafter referred to as LF) has various physiological functions such as an iron absorption promoting action, an anti-inflammatory action, a lipid peroxide production inhibiting action, and an immune system controlling action. In order to effectively use these physiological functions, various foods, drinks, feeds and medicines containing LF have been developed. However, it is known that LF is unstable to heating in the neutral region, almost deactivated by heating at 62.5 ° C. for 30 minutes, and completely deactivated by heating at 70 ° C. for 15 minutes. (For example, refer nonpatent literature 1). For this reason, a method of heat-treating LF in an acidic region is generally performed. It has been reported that when this method is used, natural LF hardly changes in antibacterial activity, iron binding property, and the like even after heat treatment (see, for example, Patent Document 1). It is also known to maintain its physiological activity when LF is heated in a state of low ionic strength. In practice, however, LF is rarely used in a state where the ionic strength is low. Therefore, a method of optimizing the ionic strength and pH to impart heating stability (see, for example, Patent Document 2), or iron to LF A method of imparting heat resistance by sufficiently adsorbing and adsorbing (for example, see Patent Document 3) has been developed.
しかし、これらの方法でも、９０℃を超える殺菌、特にレトルト殺菌処理に対して、ラクトフェリン類（以下ＬＦ類という）の熱安定性は低く、下記に示す欠点を有していた。
（１）加熱耐性が十分でないため、９０℃以上の加熱殺菌ではＬＦ類が凝集・沈殿しやすい。
（２）特に、中性域（ｐＨ７程度）以上の環境下で加熱殺菌するとＬＦ類は失活しやすい。
従ってＬＦ類含有液を加熱処理する場合（特に９０℃以上）には、ＬＦ類が失活する可能性があり、レトルト殺菌処理等の超高温加熱処理を採用できないのが実状であり、ＬＦ類を失活させずに飲食品や飼料、医薬に配合する場合には制限があった。
However, even in these methods, the thermal stability of lactoferrins (hereinafter referred to as LFs) is low with respect to sterilization exceeding 90 ° C., particularly retort sterilization treatment, and has the following drawbacks.
(1) Since heat resistance is not sufficient, LFs are likely to aggregate and precipitate during heat sterilization at 90 ° C. or higher.
(2) In particular, when sterilized by heating in an environment of a neutral range (about pH 7) or higher, LFs are easily deactivated.
Therefore, when heat-treating the LF-containing liquid (particularly 90 ° C. or higher), the LFs may be deactivated, and it is actually impossible to employ ultra-high temperature heat treatment such as retort sterilization. There was a restriction when blended in foods, drinks, feeds and medicines without inactivating them.
本発明はＬＦ類の持つ上記のような欠点を解決するべく、さらにＬＦ類の熱安定化について鋭意検討を重ねた結果、ＬＦ類と大豆多糖類やキサンタンガム等の安定剤を含有させたラクトフェリン組成物（以下ＬＦ組成物という）にすると、ＬＦ類の耐熱性が格段に向上することを見出し、本発明を完成するに至った。すなわち、本発明は酸性から中性、アルカリ性の広いｐＨ範囲で、９０℃以上の高温加熱処理及び１２０℃以上のレトルト殺菌処理においても高い耐熱性を有するＬＦ組成物を提供することを課題とする。また、本発明は、ＬＦ類を失活させずに、飲食品や飼料、医薬に通常用いられる原材料等を混合してなるＬＦ組成物や、様々な飲食品や飼料、医薬へ配合するためのＬＦ組成物を提供することを課題とする。 In order to solve the above-mentioned drawbacks of LFs, the present invention has been further studied with regard to thermal stabilization of LFs. As a result, lactoferrin composition containing LFs and stabilizers such as soybean polysaccharide and xanthan gum When it was made into a product (hereinafter referred to as LF composition), it was found that the heat resistance of LFs was significantly improved, and the present invention was completed. That is, an object of the present invention is to provide an LF composition having high heat resistance even in a high temperature heat treatment of 90 ° C. or higher and a retort sterilization treatment of 120 ° C. or higher in a wide pH range from acidic to neutral and alkaline. . In addition, the present invention provides an LF composition obtained by mixing raw materials ordinarily used in foods and drinks, feeds, and medicines without deactivating LFs, and various foods and drinks, feeds, and medicines. It is an object to provide an LF composition.
本発明はＬＦ類と安定剤を含有する高い耐熱性を有するＬＦ組成物に関する。本発明においては、ＬＦ類を失活させることなく、ＬＦ組成物を９０℃以上で加熱することが可能である。 The present invention relates to an LF composition having high heat resistance containing LFs and a stabilizer. In the present invention, it is possible to heat the LF composition at 90 ° C. or higher without deactivating LFs.
本発明のＬＦ組成物は、酸性域から中性、アルカリ性域の幅広い範囲で加熱安定性が高く、飲食品、飼料及び医薬の製造に通常使用される高温加熱処理やレトルト殺菌処理が可能であり、粉末状であっても乾熱殺菌も可能である。従って、本発明のＬＦ組成物からなるＬＦ類を含有する液状、ゲル状、粉末状、顆粒状等様々な形態の飲食品や飼料、医薬を調製することができる。 The LF composition of the present invention has high heat stability in a wide range from an acidic range to a neutral and alkaline range, and can be subjected to high-temperature heat treatment and retort sterilization treatment that are usually used in the production of foods and drinks, feeds, and pharmaceuticals. Even in powder form, dry heat sterilization is possible. Accordingly, various forms of foods, drinks, feeds and medicines such as liquids, gels, powders and granules containing LFs comprising the LF composition of the present invention can be prepared.
本発明のＬＦ組成物には、ウシ、山羊、羊、ヒト等の哺乳類の初乳、移行乳、常乳、末期乳、または、これらの乳の処理物である脱脂乳、乳清等からイオン交換クロマトグラフィー等により分離されたままのラクトフェリン、ラクトフェリンを塩酸、クエン酸等により脱鉄したアポラクトフェリン、ラクトフェリンを鉄、銅、亜鉛、マンガン、セレン等の金属で飽和した金属飽和ラクトフェリン（特許第２８８４０４５号公報、特許第３２２３９５８号公報）等を用いる。また、これらのＬＦ類 の２つ以上を任意の割合で配合した混合物であってもよく、その他の物質との混合物であることを妨げない。 The LF composition of the present invention includes ions from mammals such as cows, goats, sheep, humans, colostrum, transitional milk, normal milk, terminal milk, or processed milk products of skim milk, whey, etc. Lactoferrin as isolated by exchange chromatography, apolactoferrin obtained by removing iron from lactoferrin with hydrochloric acid, citric acid, etc., and metal saturated lactoferrin saturated with lactoferrin with a metal such as iron, copper, zinc, manganese, selenium (Patent No. 2884045 No. 3, Patent No. 3223958) and the like are used. Moreover, the mixture which mix | blended two or more of these LFs in arbitrary ratios may be sufficient, and it does not prevent that it is a mixture with another substance.
本発明のＬＦ組成物を構成する安定剤に特に制限はないが、次の性質を有する成分を主成分とする安定剤がより望ましい。
（１）大豆多糖類等のように分子量が大きく、被膜性のある安定剤。
（２）キサンタンガム等のようにグルコース主鎖にマンノースとグルクロン酸が結合しているような、直鎖に対して側鎖の割合が大きい安定剤。
（３）ショ糖のヒドロキシル基に脂肪酸が反応してできるショ糖脂肪酸エステル等のように疎水基と親水基を持ち、多分子層吸着性を有する安定剤。
これらの性質を有する安定剤は、加熱時のＬＦ類の凝集・沈澱による失活を防ぎ、ＬＦ類の耐熱性を高める。
一方、本発明のＬＦ組成物に好ましくない安定剤としては次の性質を有するものがあげられる。
（１）ジェランガムのように直鎖状の構造をなす安定剤又はグアガムのように直鎖に対して側鎖の割合が大きくない安定剤。
これらの安定剤を使用すると加熱時のＬＦ類の耐熱性はそれほど向上しない。しかし、これらの安定剤を一部含む物はその限りでは無い。
Although there is no restriction | limiting in particular in the stabilizer which comprises LF composition of this invention, The stabilizer which has as a main component the component which has the following property is more desirable.
(1) A stabilizer having a large molecular weight and a coating property such as soybean polysaccharide.
(2) A stabilizer having a large proportion of side chains with respect to a straight chain, such as xanthan gum, in which mannose and glucuronic acid are bonded to the glucose main chain.
(3) A stabilizer having a hydrophobic group and a hydrophilic group, such as a sucrose fatty acid ester formed by reacting a fatty acid with a hydroxyl group of sucrose, and having a multimolecular layer adsorptivity.
Stabilizers having these properties prevent inactivation due to aggregation and precipitation of LFs during heating, and increase the heat resistance of LFs.
On the other hand, examples of the stabilizer which is not preferable for the LF composition of the present invention include those having the following properties.
(1) A stabilizer having a linear structure such as gellan gum or a stabilizer having a large ratio of side chains to the straight chain such as guar gum.
When these stabilizers are used, the heat resistance of LFs during heating is not so improved. However, the thing containing a part of these stabilizers is not limited.
したがって、本発明でＬＦ類と混合してＬＦ組成物を構成する安定剤としては、大豆多糖類、キサンタンガム、ローカストビーンガム、カラギナン、タマリンドガム、ショ糖脂肪酸エステル、グリセリン脂肪酸エステル、カゼインナトリウム、レシチン、ペクチン、カルボキシメチルセルロースが好ましく、これらの安定剤の少なくとも１種をＬＦ類と混合してＬＦ組成物を構成する。ここでいう安定剤とはＬＦ類の加熱安定性を高めるために使用される。また、これらの安定剤の中には乳化剤としての機能等、その他様々な機能を有している場合もあるが、全く問題なく使用できる。 Therefore, as a stabilizer that constitutes an LF composition by mixing with LFs in the present invention, soybean polysaccharide, xanthan gum, locust bean gum, carrageenan, tamarind gum, sucrose fatty acid ester, glycerin fatty acid ester, casein sodium, lecithin Pectin and carboxymethylcellulose are preferred, and at least one of these stabilizers is mixed with LFs to constitute the LF composition. The stabilizer here is used to increase the heat stability of LFs. Some of these stabilizers may have various other functions such as an emulsifier, but can be used without any problem.
また、本発明のＬＦ組成物中のＬＦ類と安定剤の含有量比に関しては、特に制限はないが、安定剤をＬＦ類に対して０．５〜１００（重量／重量）、好ましくは１〜４０（重量／重量）含有していることが好ましい。 The content ratio of the LFs and the stabilizer in the LF composition of the present invention is not particularly limited, but the stabilizer is 0.5 to 100 (weight / weight), preferably 1 with respect to the LFs. It is preferable to contain -40 (weight / weight).
本発明のＬＦ類と安定剤を混合してなるＬＦ組成物を調製する方法に特に制限はないが、例えば、溶液中で調製するには、ＬＦ類と安定剤を脱イオン水にそれぞれ懸濁あるいは溶解し、撹拌混合した後、飲食品や飼料、医薬の形態に調製して使用する。撹拌混合の条件としては、ＬＦ類と安定剤が十分に混合されればよく、必要に応じて４０〜８０℃程度に加熱しながら、ウルトラディスパーサー等を使用して撹拌混合することも可能である。また、このＬＦ組成物の溶液は、飲食品、飼料及び医薬に使用しやすいように、必要に応じて、ＵＦ膜等で濃縮したり、凍結乾燥等により乾燥して使用することができる。 There is no particular limitation on the method for preparing the LF composition comprising the LFs of the present invention and a stabilizer, but for example, in preparation in a solution, the LFs and the stabilizer are suspended in deionized water. Or after melt | dissolving and stirring and mixing, it prepares and uses for the form of food-drinks, feed, and a pharmaceutical. As conditions for stirring and mixing, it is sufficient that LFs and a stabilizer are sufficiently mixed, and stirring and mixing can be performed using an ultradisperser or the like while heating to about 40 to 80 ° C. as necessary. is there. Moreover, the solution of this LF composition can be used by concentrating with a UF film | membrane etc., or drying by freeze-drying etc. as needed so that it may be easy to use for food-drinks, feed, and a pharmaceutical.
本発明のＬＦ組成物は、酸性域から、中性、アルカリ性域の幅広い範囲で加熱安定性が高く、飲食品、飼料及び医薬の製造に通常使用される高温加熱処理やレトルト殺菌処理が可能であり、粉末状であっても乾熱殺菌も可能である。従って、本発明のＬＦ組成物からなる、ＬＦ類を含有する液状、ゲル状、粉末状、顆粒状等様々な形態の飲食品や飼料、医薬を調製することができる。 The LF composition of the present invention has high heat stability in a wide range from an acidic range to a neutral and alkaline range, and can be subjected to high-temperature heat treatment and retort sterilization treatment that are usually used in the production of food and drink, feed and medicine. Yes, dry heat sterilization is possible even in powder form. Therefore, various forms of foods and drinks, feeds, and medicines, including liquids, gels, powders, granules, etc., containing the LFs, comprising the LF composition of the present invention can be prepared.
本発明のＬＦ組成物においては、塩酸やリン酸等の無機酸およびクエン酸や酢酸等の有機酸、あるいは苛性ソーダや重曹等のアルカリ剤を使用し、ｐＨを調整する。また、ＬＦ組成物を取り巻く環境が当該ＬＦ組成物の加熱安定性を維持するｐＨであれば、特にｐＨを調整することなく高温加熱処理やレトルト殺菌処理が可能であるが、ＬＦ組成物からなる飲食品や飼料、医薬が求められる品質に応じて、加熱殺菌条件とｐＨを選択することができる。 In the LF composition of the present invention, the pH is adjusted using an inorganic acid such as hydrochloric acid or phosphoric acid and an organic acid such as citric acid or acetic acid, or an alkali agent such as caustic soda or sodium bicarbonate. In addition, if the environment surrounding the LF composition is a pH that maintains the heat stability of the LF composition, high-temperature heat treatment and retort sterilization can be performed without particularly adjusting the pH. The heat sterilization conditions and pH can be selected according to the quality required for food and drink, feed and medicine.
本発明のＬＦ組成物からなる飲食品や飼料、医薬とは、このＬＦ類と安定剤のみを含むＬＦ組成物からなる飲食品、飼料及び、医薬や、ＬＦ類と安定剤の他に糖類や脂質、フレーバー等、他の飲食品、飼料及び医薬に通常含まれる原材料等を含有するＬＦ組成物からなる飲食品、飼料及び医薬である。 The food and drink, feed and medicine comprising the LF composition of the present invention are the food and drink, feed and medicine comprising the LF composition containing only these LFs and stabilizers, saccharides in addition to LFs and stabilizers, and the like. It is food / beverage products, feed, and medicine which consist of LF composition containing the raw material etc. which are normally contained in other food / beverage products, feed, and a medicine, such as a lipid and flavor.
ＬＦ類の熱安定性は低く、９０℃を超える殺菌、特にレトルト殺菌処理をするとＬＦ類は凝集・沈殿し、また、中性域（ｐＨ７程度）以上の環境下で加熱殺菌するとＬＦ類は失活しやすいという従来の欠点を、本発明のＬＦ組成物を使用することにより解消することができるという予測出来ない程の格別の効果があった。
以下に、実施例及び試験例を示して本発明を詳細に説明するが、これらは単に本発明の実施態様を例示するのみであり、本発明はこれらによって何ら限定されるものではない。
The thermal stability of LFs is low. When sterilization exceeding 90 ° C, especially retort sterilization, LFs aggregate and precipitate, and when sterilized by heating in an environment of neutral range (about pH 7) or higher, LFs are lost. There was an unpredictable extraordinary effect that the conventional drawback of being easy to use can be eliminated by using the LF composition of the present invention.
EXAMPLES Hereinafter, the present invention will be described in detail with reference to examples and test examples. However, these are merely examples of the present invention, and the present invention is not limited to these examples.
［試験例１］
鉄飽和ラクトフェリンを２０ｍｇ％濃度で脱イオン水に溶解した（Ａ液）。安定剤として大豆多糖類０．４重量％を脱イオン水に溶解した（Ｂ液）。Ａ液とＢ液を混合し、ウルトラディスパーサー（ＵＬＴＲＡ−ＴＵＲＲＡＸ Ｔ−２５；ＩＫＡジャパン社製）にて、５０℃、８０００ｒｐｍで３分間撹拌混合してＬＦ組成物を調製した。次いで、このＬＦ組成物を乳酸または水酸化ナトリウム溶液を用いてｐＨ１〜１０の１０試料に調整した。これをアンプル管に２ｍｌずつ分注し、９０℃、１００℃、１１０℃、１２０℃、１３０℃にて４分間加熱した。比較として安定剤を含まない鉄飽和ラクトフェリンのみの溶液を上記と同様の方法で、ｐＨを調整して１１０℃にて４分間加熱した。
加熱処理後の各試料の凝集・沈殿状態を目視により判定し、その後、以下に示したポリアクリルアミドゲル電気泳動（ＳＤＳ−ＰＡＧＥ）により、ＬＦ類のバンドパターンの解析を行った。ＳＤＳ−ＰＡＧＥによる解析は、これにより加熱処理後のＬＦ類の分解を確認するためである。
ＳＤＳ−ＰＡＧＥ：各試料１５μｌをサンプルバッファー１５μｌ（０．５Ｍ Ｔｒｉｓ−ＨＣｌ（ｐＨ６．８） １．２５ｍｌ、グリセロール１．０ｍｌ、１０％ＳＤＳ ２．０ｍｌ、２−メルカプトエタノール０．５ｍｌ、０．１％ＢＰＢ ０．２５ｍｌ）にて希釈し、１００℃で５分間加熱した。その後各試料を１５μｌずつ１４％ポリアクリルアミドゲル（ＴＥＦＣＯ ＳＤＳ−ＰＡＧＥ ｍｉｎｉ）にて電気泳動した。分子量マーカーとしてはＫａｌｅｉｄｏｓｃｏｐｅ Ｐｒｅｓｔａｉｎｅｄ Ｓｔａｎｄａｒｄｓを用いた。
[Test Example 1]
Iron saturated lactoferrin was dissolved in deionized water at a concentration of 20 mg% (solution A). As a stabilizer, 0.4% by weight of soybean polysaccharide was dissolved in deionized water (Liquid B). A liquid and B liquid were mixed, and it stirred and mixed at 50 degreeC and 8000 rpm for 3 minutes with the ultradisperser (ULTRA-TURRAX T-25; IKA Japan company make), and prepared LF composition. Next, this LF composition was adjusted to 10 samples having a pH of 1 to 10 using lactic acid or sodium hydroxide solution. 2 ml of this was dispensed into an ampule tube and heated at 90 ° C., 100 ° C., 110 ° C., 120 ° C. and 130 ° C. for 4 minutes. For comparison, a solution containing only iron-saturated lactoferrin containing no stabilizer was adjusted to pH by the same method as described above and heated at 110 ° C. for 4 minutes.
The aggregation / precipitation state of each sample after the heat treatment was visually determined, and then the band pattern of LFs was analyzed by polyacrylamide gel electrophoresis (SDS-PAGE) shown below. The analysis by SDS-PAGE is for confirming decomposition | disassembly of LFs after heat processing by this.
SDS-PAGE: 15 μl of each sample was added to 15 μl of sample buffer (0.5 M Tris-HCl (pH 6.8) 1.25 ml, glycerol 1.0 ml, 10% SDS 2.0 ml, 2-mercaptoethanol 0.5 ml, 0.1 % BPB (0.25 ml) and heated at 100 ° C. for 5 minutes. Thereafter, 15 μl of each sample was electrophoresed on a 14% polyacrylamide gel (TEFCO SDS-PAGE mini). Kaleidoscope Prestained Standards were used as molecular weight markers.
この試験の結果を表１に示した。表１から分かるようにＬＦ組成物を含有する溶液は、ｐＨ ２〜９において目視による凝集・沈殿を生じず、また、ＳＤＳ−ＰＡＧＥによりＬＦ類のバンドを確認することができた。従って、このＬＦ組成物は、酸性側で安定なだけでなく、中性及びアルカリ性側でも加熱安定性が極めて高いことが分かった。さらに、加熱時間を延長して、ＬＦ類が確認できるかを検査したところ、ｐＨ ２〜９では、１２０℃で１０分間加熱しても凝集・沈殿せず、また、ＬＦ類のバンドが確認できた。この試験結果からみて、このＬＦ組成物は、レトルト殺菌処理することが十分に可能であることが明らかとなった。一方、鉄飽和ラクトフェリンのみを含む溶液は、ｐＨ ２〜９においてＬＦ類のバンドを全く確認することができなかった。 The results of this test are shown in Table 1. As can be seen from Table 1, the solution containing the LF composition did not cause visual aggregation / precipitation at pH 2 to 9, and the band of LFs could be confirmed by SDS-PAGE. Therefore, it was found that this LF composition is not only stable on the acidic side, but also extremely high in heat stability on the neutral and alkaline sides. Furthermore, when the heating time was extended to check whether LFs could be confirmed, at pH 2-9, even when heated at 120 ° C. for 10 minutes, no aggregation / precipitation was observed, and LF bands could be confirmed. It was. From this test result, it was revealed that this LF composition can be sufficiently subjected to retort sterilization. On the other hand, the solution containing only iron-saturated lactoferrin could not confirm any LF band at pH 2-9.
［試験例２］
ＬＦを１６ｍｇ％濃度で脱イオン水に溶解した（Ａ液）。安定剤としてキサンタンガムを０．０８％（重量％）を脱イオン水に溶解した（Ｂ液）。Ａ液とＢ液を混合し、ウルトラディスパーサー（ＵＬＴＲＡ−ＴＵＲＲＡＸ Ｔ−２５；ＩＫＡジャパン社製）にて、５０℃、９５００ｒｐｍで３分間撹拌混合してＬＦ組成物を調製した。次いで、このＬＦ組成物を乳酸または水酸化ナトリウム溶液を用いてｐＨ１〜１０の１０試料に調整した。これをアンプル管に２ｍｌずつ分注し、９０℃、１００℃、１１０℃、１２０℃、１３０℃にて４分間加熱した。加熱処理後の各試料の凝集・沈殿状態を目視により判定し、その後、以下に示したポリアクリルアミドゲル電気泳動（ＳＤＳ−ＰＡＧＥ）により、ＬＦ類のバンドパターンの解析を行った。ＳＤＳ−ＰＡＧＥによる解析は、これにより加熱処理後のＬＦ類の分解を確認するためである。
ＳＤＳ−ＰＡＧＥ：各試料１５μｌをサンプルバッファー１５μｌ（０．５Ｍ Ｔｒｉｓ−ＨＣｌ（ｐＨ６．８） １．２５ｍｌ、グリセロール１．０ｍｌ、１０％ＳＤＳ ２．０ｍｌ、２−メルカプトエタノール０．５ｍｌ、０．１％ＢＰＢ ０．２５ｍｌ）にて希釈し、１００℃で５分間加熱した。その後各試料を１５μｌずつ１４％ポリアクリルアミドゲル（ＴＥＦＣＯ ＳＤＳ−ＰＡＧＥ ｍｉｎｉ）にて電気泳動した。分子量マーカーとしてはＫａｌｅｉｄｏｓｃｏｐｅ Ｐｒｅｓｔａｉｎｅｄ Ｓｔａｎｄａｒｄｓを用いた。
[Test Example 2]
LF was dissolved in deionized water at a concentration of 16 mg% (solution A). As a stabilizer, xanthan gum (0.08% by weight) was dissolved in deionized water (solution B). A liquid and B liquid were mixed, and it stirred and mixed at 50 degreeC and 9500 rpm for 3 minutes with the ultradisperser (ULTRA-TURRAX T-25; IKA Japan company make), and prepared LF composition. Next, this LF composition was adjusted to 10 samples having a pH of 1 to 10 using lactic acid or sodium hydroxide solution. 2 ml of this was dispensed into an ampule tube and heated at 90 ° C., 100 ° C., 110 ° C., 120 ° C. and 130 ° C. for 4 minutes. The aggregation / precipitation state of each sample after the heat treatment was visually determined, and then the band pattern of LFs was analyzed by polyacrylamide gel electrophoresis (SDS-PAGE) shown below. The analysis by SDS-PAGE is for confirming decomposition | disassembly of LFs after heat processing by this.
SDS-PAGE: 15 μl of each sample was added to 15 μl of sample buffer (0.5 M Tris-HCl (pH 6.8) 1.25 ml, glycerol 1.0 ml, 10% SDS 2.0 ml, 2-mercaptoethanol 0.5 ml, 0.1 % BPB (0.25 ml) and heated at 100 ° C. for 5 minutes. Thereafter, 15 μl of each sample was electrophoresed on a 14% polyacrylamide gel (TEFCO SDS-PAGE mini). Kaleidoscope Prestained Standards were used as molecular weight markers.
この試験の結果を表２に示した。表２から分かるようにＬＦ組成物を含有する溶液は、ｐＨ ２〜９において目視による凝集・沈殿を生じず、また、ＳＤＳ−ＰＡＧＥによりＬＦ類のバンドを確認することができた。従って、このＬＦ組成物は、酸性側で安定なだけでなく、中性及びアルカリ性側でも加熱安定性が極めて高いことが分かった。さらに、加熱時間を延長して、ＬＦ類が確認できるかを検査したところ、ｐＨ ２〜９では、１３０℃で１０分間加熱しても凝集・沈殿せず、また、ＬＦ類のバンドが確認できた。この試験結果からみて、このＬＦ組成物は、レトルト殺菌処理することが十分に可能であることが明らかとなった。
The results of this test are shown in Table 2. As can be seen from Table 2, the solution containing the LF composition did not cause visual aggregation / precipitation at pH 2 to 9, and the band of LFs could be confirmed by SDS-PAGE. Therefore, it was found that this LF composition is not only stable on the acidic side, but also extremely high in heat stability on the neutral and alkaline sides. Furthermore, when the heating time was extended to check whether LFs could be confirmed, at pH 2-9, even when heated at 130 ° C. for 10 minutes, no aggregation or precipitation occurred, and LF bands could be confirmed. It was. From this test result, it was revealed that this LF composition can be sufficiently subjected to retort sterilization.
［試験例３］
塩酸で脱鉄したアポラクトフェリンを１０ｍｇ％濃度で脱イオン水に溶解した（Ａ液）。安定剤としてショ糖脂肪酸エステル０．２重量％を脱イオン水に溶解した（Ｂ液）。Ａ液とＢ液を混合し、ウルトラディスパーサー（ＵＬＴＲＡ−ＴＵＲＲＡＸ Ｔ−２５；ＩＫＡジャパン社製）にて、４０℃、８０００ｒｐｍで３分間撹拌混合してＬＦ組成物を調製した。次いで、このＬＦ組成物を乳酸または水酸化ナトリウム溶液を用いてｐＨ１〜１０の１０試料に調整した。これをアンプル管に２ｍｌずつ分注し、９０℃、１００℃、１１０℃、１２０℃、１３０℃にて４分間加熱した。
加熱処理後の各試料の凝集・沈殿状態を目視により判定し、その後、以下に示したポリアクリルアミドゲル電気泳動（ＳＤＳ−ＰＡＧＥ）により、ＬＦ類のバンドパターンの解析を行った。ＳＤＳ−ＰＡＧＥによる解析は、これにより加熱処理後のＬＦ類の分解を確認するためである。
ＳＤＳ−ＰＡＧＥ：各試料１５μｌをサンプルバッファー１５μｌ（０．５Ｍ Ｔｒｉｓ−ＨＣｌ（ｐＨ６．８） １．２５ｍｌ、グリセロール１．０ｍｌ、１０％ＳＤＳ ２．０ｍｌ、２−メルカプトエタノール０．５ｍｌ、０．１％ＢＰＢ ０．２５ｍｌ）にて希釈し、１００℃で５分間加熱した。その後各試料を１５μｌずつ１４％ポリアクリルアミドゲル（ＴＥＦＣＯ ＳＤＳ−ＰＡＧＥ ｍｉｎｉ）にて電気泳動した。分子量マーカーとしてはＫａｌｅｉｄｏｓｃｏｐｅ Ｐｒｅｓｔａｉｎｅｄ Ｓｔａｎｄａｒｄｓを用いた。
[Test Example 3]
Apolactoferrin deironated with hydrochloric acid was dissolved in deionized water at a concentration of 10 mg% (solution A). As a stabilizer, 0.2% by weight of sucrose fatty acid ester was dissolved in deionized water (solution B). A liquid and B liquid were mixed, and it stirred and mixed at 40 degreeC and 8000 rpm for 3 minutes with the ultradisperser (ULTRA-TURRAX T-25; made by IKA Japan), and prepared LF composition. Next, this LF composition was adjusted to 10 samples having a pH of 1 to 10 using lactic acid or sodium hydroxide solution. 2 ml of this was dispensed into an ampule tube and heated at 90 ° C., 100 ° C., 110 ° C., 120 ° C. and 130 ° C. for 4 minutes.
The aggregation / precipitation state of each sample after the heat treatment was visually determined, and then the band pattern of LFs was analyzed by polyacrylamide gel electrophoresis (SDS-PAGE) shown below. The analysis by SDS-PAGE is for confirming decomposition | disassembly of LFs after heat processing by this.
SDS-PAGE: 15 μl of each sample was added to 15 μl of sample buffer (0.5 M Tris-HCl (pH 6.8) 1.25 ml, glycerol 1.0 ml, 10% SDS 2.0 ml, 2-mercaptoethanol 0.5 ml, 0.1 % BPB (0.25 ml) and heated at 100 ° C. for 5 minutes. Thereafter, 15 μl of each sample was electrophoresed on a 14% polyacrylamide gel (TEFCO SDS-PAGE mini). Kaleidoscope Prestained Standards were used as molecular weight markers.
この試験の結果を表３に示した。表３から分かるようにＬＦ組成物を含有する溶液は、ｐＨ ２〜９において目視による凝集・沈殿を生じず、また、ＳＤＳ−ＰＡＧＥによりＬＦ類のバンドを確認することができた。従って、このＬＦ組成物は、酸性側で安定なだけでなく、中性及びアルカリ性側でも加熱安定性が極めて高いことが分かった。さらに、加熱時間を延長して、ＬＦ類が確認できるかを検査したところ、ｐＨ ２〜９では、１２０℃で８分間加熱しても凝集・沈殿せず、また、ＬＦ類のバンドが確認できた。この試験結果からみて、このＬＦ組成物は、レトルト殺菌処理することが十分に可能であることが明らかとなった。
The results of this test are shown in Table 3. As can be seen from Table 3, the solution containing the LF composition did not cause visual aggregation / precipitation at pH 2 to 9, and the band of LFs could be confirmed by SDS-PAGE. Therefore, it was found that this LF composition is not only stable on the acidic side, but also extremely high in heat stability on the neutral and alkaline sides. Furthermore, when the heating time was extended to check whether LFs could be confirmed, at pH 2-9, even when heated at 120 ° C. for 8 minutes, no aggregation or precipitation occurred, and LF bands could be confirmed. It was. From this test result, it was revealed that this LF composition can be sufficiently subjected to retort sterilization.
［試験例４］
塩酸で脱鉄したアポラクトフェリンを１２ｍｇ％濃度で脱イオン水に溶解した（Ａ液）。安定剤としてローカストビーンガム０．１５重量％を脱イオン水に溶解した（Ｂ液）。Ａ液とＢ液を混合し、ウルトラディスパーサー（ＵＬＴＲＡ−ＴＵＲＲＡＸ Ｔ−２５；ＩＫＡジャパン社製）にて、４０℃、８０００ｒｐｍで４分間撹拌混合してＬＦ組成物を調製した。次いで、このＬＦ組成物を乳酸または水酸化ナトリウム溶液を用いてｐＨ １〜１０の１０試料に調整した。これをアンプル管に２ｍｌずつ分注し、９０℃、１００℃、１１０℃、１２０℃、１３０℃にて４分間加熱した。
加熱処理後の各試料の凝集・沈殿状態を目視により判定し、その後、以下に示したポリアクリルアミドゲル電気泳動（ＳＤＳ−ＰＡＧＥ）により、ＬＦ類のバンドパターンの解析を行った。ＳＤＳ−ＰＡＧＥによる解析は、これにより加熱処理後のＬＦ類の分解を確認するためである。
ＳＤＳ−ＰＡＧＥ：各試料１５μｌをサンプルバッファー１５μｌ（０．５Ｍ Ｔｒｉｓ−ＨＣｌ（ｐＨ６．８） １．２５ｍｌ、グリセロール１．０ｍｌ、１０％ＳＤＳ ２．０ｍｌ、２−メルカプトエタノール０．５ｍｌ、０．１％ＢＰＢ ０．２５ｍｌ）にて希釈し、１００℃で５分間加熱した。その後各試料を１５μｌずつ１４％ポリアクリルアミドゲル（ＴＥＦＣＯ ＳＤＳ−ＰＡＧＥ ｍｉｎｉ）にて電気泳動した。分子量マーカーとしてはＫａｌｅｉｄｏｓｃｏｐｅ Ｐｒｅｓｔａｉｎｅｄ Ｓｔａｎｄａｒｄｓを用いた。
[Test Example 4]
Apolactoferrin deironated with hydrochloric acid was dissolved in deionized water at a concentration of 12 mg% (solution A). As a stabilizer, locust bean gum 0.15% by weight was dissolved in deionized water (Liquid B). Liquid A and liquid B were mixed, and an ultradisperser (ULTRA-TURRAX T-25; manufactured by IKA Japan) was stirred and mixed at 40 ° C. and 8000 rpm for 4 minutes to prepare an LF composition. Next, this LF composition was adjusted to 10 samples having a pH of 1 to 10 using lactic acid or sodium hydroxide solution. 2 ml of this was dispensed into an ampule tube and heated at 90 ° C., 100 ° C., 110 ° C., 120 ° C. and 130 ° C. for 4 minutes.
The aggregation / precipitation state of each sample after the heat treatment was visually determined, and then the band pattern of LFs was analyzed by polyacrylamide gel electrophoresis (SDS-PAGE) shown below. The analysis by SDS-PAGE is for confirming decomposition | disassembly of LFs after heat processing by this.
SDS-PAGE: 15 μl of each sample was added to 15 μl of sample buffer (0.5 M Tris-HCl (pH 6.8) 1.25 ml, glycerol 1.0 ml, 10% SDS 2.0 ml, 2-mercaptoethanol 0.5 ml, 0.1 % BPB (0.25 ml) and heated at 100 ° C. for 5 minutes. Thereafter, 15 μl of each sample was electrophoresed on a 14% polyacrylamide gel (TEFCO SDS-PAGE mini). Kaleidoscope Prestained Standards were used as molecular weight markers.
この試験の結果、ＬＦ組成物を含有する溶液は、ｐＨ３〜８において目視による凝集・沈殿を生じず、また、ＳＤＳ−ＰＡＧＥによりＬＦ類のバンドを確認することができた。従って、このＬＦ組成物は、酸性側で安定なだけでなく、中性及びアルカリ性側でも加熱安定性が極めて高いことが分かった。さらに、加熱時間を延長して、ＬＦ類が確認できるかを検査したところ、ｐＨ３〜８では、１２０℃で７分間加熱しても凝集・沈殿せず、また、ＬＦ類のバンドが確認できた。この試験結果からみて、このＬＦ組成物は、レトルト殺菌処理することが十分に可能であることが明らかとなった。 As a result of this test, the solution containing the LF composition did not cause visual aggregation / precipitation at pH 3 to 8, and the band of LFs could be confirmed by SDS-PAGE. Therefore, it was found that this LF composition is not only stable on the acidic side, but also extremely high in heat stability on the neutral and alkaline sides. Furthermore, when the heating time was extended to check whether LFs could be confirmed, at pH 3-8, even when heated at 120 ° C. for 7 minutes, aggregation / precipitation did not occur, and a band of LFs could be confirmed. . From this test result, it was revealed that this LF composition can be sufficiently subjected to retort sterilization.
［試験例５］
鉄飽和ラクトフェリンを１８ｍｇ％濃度で脱イオン水に溶解した（Ａ液）。安定剤としてカラギナン０．１５重量％を脱イオン水に溶解した（Ｂ液）。Ａ液とＢ液を混合し、ウルトラディスパーサー（ＵＬＴＲＡ−ＴＵＲＲＡＸ Ｔ−２５；ＩＫＡジャパン社製）にて、４０℃、８０００ｒｐｍで３分間撹拌混合してＬＦ組成物を調製した。次いで、このＬＦ組成物を乳酸または水酸化ナトリウム溶液を用いてｐＨ１〜１０の１０試料に調整した。これをアンプル管に２ｍｌずつ分注し、９０℃、１００℃、１１０℃、１２０℃、１３０℃にて４分間加熱した。
加熱処理後の各試料の凝集・沈殿状態を目視により判定し、その後、以下に示したポリアクリルアミドゲル電気泳動（ＳＤＳ−ＰＡＧＥ）により、ＬＦ類のバンドパターンの解析を行った。ＳＤＳ−ＰＡＧＥによる解析は、これにより加熱処理後のＬＦ類の分解を確認するためである。
ＳＤＳ−ＰＡＧＥ：各試料１５μｌをサンプルバッファー１５μｌ（０．５Ｍ Ｔｒｉｓ−ＨＣｌ（ｐＨ６．８） １．２５ｍｌ、グリセロール１．０ｍｌ、１０％ＳＤＳ ２．０ｍｌ、２−メルカプトエタノール０．５ｍｌ、０．１％ＢＰＢ ０．２５ｍｌ）にて希釈し、１００℃で５分間加熱した。その後各試料を１５μｌずつ１４％ポリアクリルアミドゲル（ＴＥＦＣＯ ＳＤＳ−ＰＡＧＥ ｍｉｎｉ）にて電気泳動した。分子量マーカーとしてはＫａｌｅｉｄｏｓｃｏｐｅ Ｐｒｅｓｔａｉｎｅｄ Ｓｔａｎｄａｒｄｓを用いた。
[Test Example 5]
Iron saturated lactoferrin was dissolved in deionized water at a concentration of 18 mg% (solution A). As a stabilizer, 0.15% by weight of carrageenan was dissolved in deionized water (solution B). A liquid and B liquid were mixed, and it stirred and mixed at 40 degreeC and 8000 rpm for 3 minutes with the ultradisperser (ULTRA-TURRAX T-25; made by IKA Japan), and prepared LF composition. Next, this LF composition was adjusted to 10 samples having a pH of 1 to 10 using lactic acid or sodium hydroxide solution. 2 ml of this was dispensed into an ampule tube and heated at 90 ° C., 100 ° C., 110 ° C., 120 ° C. and 130 ° C. for 4 minutes.
The aggregation / precipitation state of each sample after the heat treatment was visually determined, and then the band pattern of LFs was analyzed by polyacrylamide gel electrophoresis (SDS-PAGE) shown below. The analysis by SDS-PAGE is for confirming decomposition | disassembly of LFs after heat processing by this.
SDS-PAGE: 15 μl of each sample was added to 15 μl of sample buffer (0.5 M Tris-HCl (pH 6.8) 1.25 ml, glycerol 1.0 ml, 10% SDS 2.0 ml, 2-mercaptoethanol 0.5 ml, 0.1 % BPB (0.25 ml) and heated at 100 ° C. for 5 minutes. Thereafter, 15 μl of each sample was electrophoresed on a 14% polyacrylamide gel (TEFCO SDS-PAGE mini). Kaleidoscope Prestained Standards were used as molecular weight markers.
この試験の結果、ＬＦ組成物を含有する溶液は、ｐＨ４〜８において目視による凝集・沈殿を生じず、また、ＳＤＳ−ＰＡＧＥによりＬＦ類のバンドを確認することができた。従って、このＬＦ組成物は、酸性側で安定なだけでなく、中性及びアルカリ性側でも加熱安定性が極めて高いことが分かった。さらに、加熱時間を延長して、ＬＦ類が確認できるかを検査したところ、ｐＨ４〜８では、１２０℃で６分間加熱しても凝集・沈殿せず、また、ＬＦ類のバンドが確認できた。この試験結果からみて、このＬＦ組成物は、レトルト殺菌処理することが十分に可能であることが明らかとなった。 As a result of this test, the solution containing the LF composition did not cause visual aggregation / precipitation at pH 4 to 8, and the band of LFs could be confirmed by SDS-PAGE. Therefore, it was found that this LF composition is not only stable on the acidic side, but also extremely high in heat stability on the neutral and alkaline sides. Further, when the heating time was extended to check whether LFs could be confirmed, at pH 4-8, even when heated at 120 ° C. for 6 minutes, no aggregation / precipitation was observed, and LF bands could be confirmed. . From this test result, it was revealed that this LF composition can be sufficiently subjected to retort sterilization.
［試験例６］
ＬＦを２０ｍｇ％濃度で脱イオン水に溶解した（Ａ液）。安定剤としてタマリンドガム０．１５重量％を脱イオン水に溶解した（Ｂ液）。Ａ液とＢ液を混合し、ウルトラディスパーサー（ＵＬＴＲＡ−ＴＵＲＲＡＸ Ｔ−２５；ＩＫＡジャパン社製）にて、４０℃、８０００ｒｐｍで３分間撹拌混合してＬＦ組成物を調製した。次いで、このＬＦ組成物を乳酸または水酸化ナトリウム溶液を用いてｐＨ１〜１０の１０試料に調整した。これをアンプル管に２ｍｌずつ分注し、９０℃、１００℃、１１０℃、１２０℃、１３０℃にて４分間加熱した。
加熱処理後の各試料の凝集・沈殿状態を目視により判定し、その後、以下に示したポリアクリルアミドゲル電気泳動（ＳＤＳ−ＰＡＧＥ）により、ＬＦ類のバンドパターンの解析を行った。ＳＤＳ−ＰＡＧＥによる解析は、これにより加熱処理後のＬＦ類の分解を確認するためである。
ＳＤＳ−ＰＡＧＥ：各試料１５μｌをサンプルバッファー１５μｌ（０．５Ｍ Ｔｒｉｓ−ＨＣｌ（ｐＨ６．８） １．２５ｍｌ、グリセロール１．０ｍｌ、１０％ＳＤＳ ２．０ｍｌ、２−メルカプトエタノール０．５ｍｌ、０．１％ＢＰＢ ０．２５ｍｌ）にて希釈し、１００℃で５分間加熱した。その後各試料を１５μｌずつ１４％ポリアクリルアミドゲル（ＴＥＦＣＯ ＳＤＳ−ＰＡＧＥ ｍｉｎｉ）にて電気泳動した。分子量マーカーとしてはＫａｌｅｉｄｏｓｃｏｐｅ Ｐｒｅｓｔａｉｎｅｄ Ｓｔａｎｄａｒｄｓを用いた。
[Test Example 6]
LF was dissolved in deionized water at a concentration of 20 mg% (solution A). As stabilizer, 0.15% by weight of tamarind gum was dissolved in deionized water (solution B). A liquid and B liquid were mixed, and it stirred and mixed at 40 degreeC and 8000 rpm for 3 minutes with the ultradisperser (ULTRA-TURRAX T-25; made by IKA Japan), and prepared LF composition. Next, this LF composition was adjusted to 10 samples having a pH of 1 to 10 using lactic acid or sodium hydroxide solution. 2 ml of this was dispensed into an ampule tube and heated at 90 ° C., 100 ° C., 110 ° C., 120 ° C. and 130 ° C. for 4 minutes.
The aggregation / precipitation state of each sample after the heat treatment was visually determined, and then the band pattern of LFs was analyzed by polyacrylamide gel electrophoresis (SDS-PAGE) shown below. The analysis by SDS-PAGE is for confirming decomposition | disassembly of LFs after heat processing by this.
SDS-PAGE: 15 μl of each sample was added to 15 μl of sample buffer (0.5 M Tris-HCl (pH 6.8) 1.25 ml, glycerol 1.0 ml, 10% SDS 2.0 ml, 2-mercaptoethanol 0.5 ml, 0.1 % BPB (0.25 ml) and heated at 100 ° C. for 5 minutes. Thereafter, 15 μl of each sample was electrophoresed on a 14% polyacrylamide gel (TEFCO SDS-PAGE mini). Kaleidoscope Prestained Standards were used as molecular weight markers.
この試験の結果、ＬＦ組成物を含有する溶液は、ｐＨ４〜７において目視による凝集・沈殿を生じず、また、ＳＤＳ−ＰＡＧＥによりＬＦ類のバンドを確認することができた。従って、このＬＦ組成物は、酸性側で安定なだけでなく、中性側でも加熱安定性が極めて高いことが分かった。さらに、加熱時間を延長して、ＬＦ類が確認できるかを検査したところ、ｐＨ４〜７では、１２０℃で５分間加熱しても凝集・沈殿せず、また、ＬＦ類のバンドが確認できた。この試験結果からみて、このＬＦ組成物は、レトルト殺菌処理することが十分に可能であることが明らかとなった。 As a result of this test, the solution containing the LF composition did not cause visual aggregation / precipitation at pH 4 to 7, and the band of LFs could be confirmed by SDS-PAGE. Therefore, it was found that this LF composition is not only stable on the acidic side but also extremely high in heat stability on the neutral side. Further, when the heating time was extended to check whether LFs could be confirmed, at pH 4-7, even when heated at 120 ° C. for 5 minutes, no aggregation / precipitation was observed, and LF bands could be confirmed. . From this test result, it was revealed that this LF composition can be sufficiently subjected to retort sterilization.
［試験例７］
塩酸で脱鉄したアポラクトフェリンを１０ｍｇ％濃度で脱イオン水に溶解した（Ａ液）。安定剤としてグリセリン脂肪酸エステル０．１５重量％を脱イオン水に溶解した（Ｂ液）。Ａ液とＢ液を混合し、ウルトラディスパーサー（ＵＬＴＲＡ−ＴＵＲＲＡＸ Ｔ−２５；ＩＫＡジャパン社製）にて、４０℃、８０００ｒｐｍで３分間撹拌混合してＬＦ組成物を調製した。次いで、このＬＦ組成物を乳酸または水酸化ナトリウム溶液を用いて〜１０の１０試料に調整した。これをアンプル管に２ｍｌずつ分注し、９０℃、１００℃、１１０℃、１２０℃、１３０℃にて４分間加熱した。
加熱処理後の各試料の凝集・沈殿状態を目視により判定し、その後、以下に示したポリアクリルアミドゲル電気泳動（ＳＤＳ−ＰＡＧＥ）により、ＬＦ類のバンドパターンの解析を行った。ＳＤＳ−ＰＡＧＥによる解析は、これにより加熱処理後のＬＦ類の分解を確認するためである。
ＳＤＳ−ＰＡＧＥ：各試料１５μｌをサンプルバッファー１５μｌ（０．５Ｍ Ｔｒｉｓ−ＨＣｌ（ｐＨ６．８） １．２５ｍｌ、グリセロール１．０ｍｌ、１０％ＳＤＳ ２．０ｍｌ、２−メルカプトエタノール０．５ｍｌ、０．１％ＢＰＢ ０．２５ｍｌ）にて希釈し、１００℃で５分間加熱した。その後各試料を１５μｌずつ１４％ポリアクリルアミドゲル（ＴＥＦＣＯ ＳＤＳ−ＰＡＧＥ ｍｉｎｉ）にて電気泳動した。分子量マーカーとしてはＫａｌｅｉｄｏｓｃｏｐｅ Ｐｒｅｓｔａｉｎｅｄ Ｓｔａｎｄａｒｄｓを用いた。
[Test Example 7]
Apolactoferrin deironated with hydrochloric acid was dissolved in deionized water at a concentration of 10 mg% (solution A). As a stabilizer, 0.15% by weight of glycerin fatty acid ester was dissolved in deionized water (Liquid B). A liquid and B liquid were mixed, and it stirred and mixed at 40 degreeC and 8000 rpm for 3 minutes with the ultradisperser (ULTRA-TURRAX T-25; made by IKA Japan), and prepared LF composition. The LF composition was then adjusted to 10 samples of -10 using lactic acid or sodium hydroxide solution. 2 ml of this was dispensed into an ampule tube and heated at 90 ° C., 100 ° C., 110 ° C., 120 ° C. and 130 ° C. for 4 minutes.
The aggregation / precipitation state of each sample after the heat treatment was visually determined, and then the band pattern of LFs was analyzed by polyacrylamide gel electrophoresis (SDS-PAGE) shown below. The analysis by SDS-PAGE is for confirming decomposition | disassembly of LFs after heat processing by this.
SDS-PAGE: 15 μl of each sample was added to 15 μl of sample buffer (0.5 M Tris-HCl (pH 6.8) 1.25 ml, glycerol 1.0 ml, 10% SDS 2.0 ml, 2-mercaptoethanol 0.5 ml, 0.1 % BPB (0.25 ml) and heated at 100 ° C. for 5 minutes. Thereafter, 15 μl of each sample was electrophoresed on a 14% polyacrylamide gel (TEFCO SDS-PAGE mini). Kaleidoscope Prestained Standards were used as molecular weight markers.
この試験の結果、ＬＦ組成物を含有する溶液は、ｐＨ３〜７において目視による凝集・沈殿を生じず、また、ＳＤＳ−ＰＡＧＥによりＬＦ類のバンドを確認することができた。従って、このＬＦ組成物は、酸性側で安定なだけでなく、中性側でも加熱安定性が極めて高いことが分かった。さらに、加熱時間を延長して、ＬＦ類が確認できるかを検査したところ、ｐＨ３〜７では、１２０℃で６分間加熱しても凝集・沈殿せず、また、ＬＦ類のバンドが確認できた。この試験結果からみて、このＬＦ組成物は、レトルト殺菌処理することが十分に可能であることが明らかとなった。 As a result of this test, the solution containing the LF composition did not cause visual aggregation / precipitation at pH 3 to 7, and the band of LFs could be confirmed by SDS-PAGE. Therefore, it was found that this LF composition is not only stable on the acidic side but also extremely high in heat stability on the neutral side. Further, when the heating time was extended to check whether LFs could be confirmed, at pH 3-7, even when heated at 120 ° C. for 6 minutes, no aggregation / precipitation was observed, and LF bands could be confirmed. . From this test result, it was revealed that this LF composition can be sufficiently subjected to retort sterilization.
［試験例８］
鉄飽和ラクトフェリンを１０ｍｇ％濃度で脱イオン水に溶解した（Ａ液）。安定剤としてカゼインナトリウム ０．１５重量％を脱イオン水に溶解した（Ｂ液）。Ａ液とＢ液を混合し、ウルトラディスパーサー（ＵＬＴＲＡ−ＴＵＲＲＡＸ Ｔ−２５；ＩＫＡジャパン社製）にて、４０℃、９５００ｒｐｍで３分間撹拌混合してＬＦ組成物を調製した。次いで、このＬＦ組成物を乳酸または水酸化ナトリウム溶液を用いてｐＨ １〜１０の１０試料に調整した。これをアンプル管に２ｍｌずつ分注し、９０℃、１００℃、１１０℃、１２０℃、１３０℃にて４分間加熱した。
加熱処理後の各試料の凝集・沈殿状態を目視により判定し、その後、以下に示したポリアクリルアミドゲル電気泳動（ＳＤＳ−ＰＡＧＥ）により、ＬＦ類のバンドパターンの解析を行った。ＳＤＳ−ＰＡＧＥによる解析は、これにより加熱処理後のＬＦ類の分解を確認するためである。
ＳＤＳ−ＰＡＧＥ：各試料１５μｌをサンプルバッファー１５μｌ（０．５Ｍ Ｔｒｉｓ−ＨＣｌ（ｐＨ６．８） １．２５ｍｌ、グリセロール１．０ｍｌ、１０％ＳＤＳ ２．０ｍｌ、２−メルカプトエタノール０．５ｍｌ、０．１％ＢＰＢ ０．２５ｍｌ）にて希釈し、１００℃で５分間加熱した。その後各試料を１５μｌずつ１４％ポリアクリルアミドゲル（ＴＥＦＣＯ ＳＤＳ−ＰＡＧＥ ｍｉｎｉ）にて電気泳動した。分子量マーカーとしてはＫａｌｅｉｄｏｓｃｏｐｅ Ｐｒｅｓｔａｉｎｅｄ Ｓｔａｎｄａｒｄｓを用いた。
[Test Example 8]
Iron saturated lactoferrin was dissolved in deionized water at a concentration of 10 mg% (solution A). As a stabilizer, sodium caseinate (0.15% by weight) was dissolved in deionized water (solution B). A liquid and B liquid were mixed, and it stirred and mixed at 40 degreeC and 9500 rpm for 3 minutes with the ultradisperser (ULTRA-TURRAX T-25; made by IKA Japan), and prepared LF composition. Next, this LF composition was adjusted to 10 samples having a pH of 1 to 10 using lactic acid or sodium hydroxide solution. 2 ml of this was dispensed into an ampule tube and heated at 90 ° C., 100 ° C., 110 ° C., 120 ° C. and 130 ° C. for 4 minutes.
The aggregation / precipitation state of each sample after the heat treatment was visually determined, and then the band pattern of LFs was analyzed by polyacrylamide gel electrophoresis (SDS-PAGE) shown below. The analysis by SDS-PAGE is for confirming decomposition | disassembly of LFs after heat processing by this.
SDS-PAGE: 15 μl of each sample was added to 15 μl of sample buffer (0.5 M Tris-HCl (pH 6.8) 1.25 ml, glycerol 1.0 ml, 10% SDS 2.0 ml, 2-mercaptoethanol 0.5 ml, 0.1 % BPB (0.25 ml) and heated at 100 ° C. for 5 minutes. Thereafter, 15 μl of each sample was electrophoresed on a 14% polyacrylamide gel (TEFCO SDS-PAGE mini). Kaleidoscope Prestained Standards were used as molecular weight markers.
この試験の結果、ＬＦ組成物を含有する溶液は、ｐＨ５〜９において目視による凝集・沈殿を生じず、また、ＳＤＳ−ＰＡＧＥによりＬＦ類のバンドを確認することができた。従って、このＬＦ組成物は、酸性側で安定なだけでなく、中性及びアルカリ性側でも加熱安定性が極めて高いことが分かった。さらに、加熱時間を延長して、ＬＦ類が確認できるかを検査したところ、ｐＨ５〜９では、１２０℃で５分間加熱しても凝集・沈殿せず、また、ＬＦ類のバンドが確認できた。この試験結果からみて、このＬＦ組成物は、レトルト殺菌処理することが十分に可能であることが明らかとなった。 As a result of this test, the solution containing the LF composition did not cause visual aggregation / precipitation at pH 5 to 9, and the band of LFs could be confirmed by SDS-PAGE. Therefore, it was found that this LF composition is not only stable on the acidic side, but also extremely high in heat stability on the neutral and alkaline sides. Furthermore, when the heating time was extended to check whether LFs could be confirmed, at pH 5-9, even when heated at 120 ° C. for 5 minutes, no aggregation / precipitation was observed, and LF bands could be confirmed. . From this test result, it was revealed that this LF composition can be sufficiently subjected to retort sterilization.
［試験例９］
ＬＦを１６ｍｇ％濃度で脱イオン水に溶解した（Ａ液）。安定剤としてレシチン０．２５重量％を脱イオン水に溶解した（Ｂ液）。Ａ液とＢ液を混合し、ウルトラディスパーサー（ＵＬＴＲＡ−ＴＵＲＲＡＸ Ｔ−２５；ＩＫＡジャパン社製）にて、４０℃、８０００ｒｐｍで３分間撹拌混合してＬＦ組成物を調製した。次いで、このＬＦ組成物を乳酸または水酸化ナトリウム溶液を用いて〜１０の１０試料に調整した。これをアンプル管に２ｍｌずつ分注し、９０℃、１００℃、１１０℃、１２０℃、１３０℃にて４分間加熱した。
加熱処理後の各試料の凝集・沈殿状態を目視により判定し、その後、以下に示したポリアクリルアミドゲル電気泳動（ＳＤＳ−ＰＡＧＥ）により、ＬＦ類のバンドパターンの解析を行った。ＳＤＳ−ＰＡＧＥによる解析は、これにより加熱処理後のＬＦ類の分解を確認するためである。
ＳＤＳ−ＰＡＧＥ：各試料１５μｌをサンプルバッファー１５μｌ（０．５Ｍ Ｔｒｉｓ−ＨＣｌ（ｐＨ６．８） １．２５ｍｌ、グリセロール１．０ｍｌ、１０％ＳＤＳ ２．０ｍｌ、２−メルカプトエタノール０．５ｍｌ、０．１％ＢＰＢ ０．２５ｍｌ）にて希釈し、１００℃で５分間加熱した。その後各試料を１５μｌずつ１４％ポリアクリルアミドゲル（ＴＥＦＣＯ ＳＤＳ−ＰＡＧＥ ｍｉｎｉ）にて電気泳動した。分子量マーカーとしてはＫａｌｅｉｄｏｓｃｏｐｅ Ｐｒｅｓｔａｉｎｅｄ Ｓｔａｎｄａｒｄｓを用いた。
[Test Example 9]
LF was dissolved in deionized water at a concentration of 16 mg% (solution A). As a stabilizer, 0.25% by weight of lecithin was dissolved in deionized water (solution B). A liquid and B liquid were mixed, and it stirred and mixed at 40 degreeC and 8000 rpm for 3 minutes with the ultradisperser (ULTRA-TURRAX T-25; made by IKA Japan), and prepared LF composition. The LF composition was then adjusted to 10 samples of -10 using lactic acid or sodium hydroxide solution. 2 ml of this was dispensed into an ampule tube and heated at 90 ° C., 100 ° C., 110 ° C., 120 ° C. and 130 ° C. for 4 minutes.
The aggregation / precipitation state of each sample after the heat treatment was visually determined, and then the band pattern of LFs was analyzed by polyacrylamide gel electrophoresis (SDS-PAGE) shown below. The analysis by SDS-PAGE is for confirming decomposition | disassembly of LFs after heat processing by this.
SDS-PAGE: 15 μl of each sample was added to 15 μl of sample buffer (0.5 M Tris-HCl (pH 6.8) 1.25 ml, glycerol 1.0 ml, 10% SDS 2.0 ml, 2-mercaptoethanol 0.5 ml, 0.1 % BPB (0.25 ml) and heated at 100 ° C. for 5 minutes. Thereafter, 15 μl of each sample was electrophoresed on a 14% polyacrylamide gel (TEFCO SDS-PAGE mini). Kaleidoscope Prestained Standards were used as molecular weight markers.
この試験の結果、ＬＦ組成物を含有する溶液は、ｐＨ３〜８において目視による凝集・沈殿を生じず、また、ＳＤＳ−ＰＡＧＥによりＬＦ類のバンドを確認することができた。従って、このＬＦ組成物は、酸性側で安定なだけでなく、中性及びアルカリ性側でも加熱安定性が極めて高いことが分かった。さらに、加熱時間を延長して、ＬＦ類が確認できるかを検査したところ、ｐＨ３〜８では、１２０℃で６分間加熱しても凝集・沈殿せず、また、ＬＦ類のバンドが確認できた。この試験結果からみて、このＬＦ組成物は、レトルト殺菌処理することが十分に可能であることが明らかとなった。 As a result of this test, the solution containing the LF composition did not cause visual aggregation / precipitation at pH 3 to 8, and the band of LFs could be confirmed by SDS-PAGE. Therefore, it was found that this LF composition is not only stable on the acidic side, but also extremely high in heat stability on the neutral and alkaline sides. Further, when the heating time was extended to check whether LFs could be confirmed, at pH 3-8, no aggregation / precipitation occurred even when heated at 120 ° C. for 6 minutes, and a band of LFs could be confirmed. . From this test result, it was revealed that this LF composition can be sufficiently subjected to retort sterilization.
［試験例１０］
鉄飽和ラクトフェリンを１５ｍｇ％濃度で脱イオン水に溶解した（Ａ液）。安定剤としてペクチン０．６重量％を脱イオン水に溶解した（Ｂ液）。Ａ液とＢ液を混合し、ウルトラディスパーサー（ＵＬＴＲＡ−ＴＵＲＲＡＸ Ｔ−２５；ＩＫＡジャパン社製）にて、４０℃、８０００ｒｐｍで３分間撹拌混合してＬＦ組成物を調製した。次いで、このＬＦ組成物を乳酸または水酸化ナトリウム溶液を用いてｐＨ １〜１０の１０試料に調整した。これをアンプル管に２ｍｌずつ分注し、９０℃、１００℃、１１０℃、１２０℃、１３０℃にて４分間加熱した。
加熱処理後の各試料の凝集・沈殿状態を目視により判定し、その後、以下に示したポリアクリルアミドゲル電気泳動（ＳＤＳ−ＰＡＧＥ）により、ＬＦ類のバンドパターンの解析を行った。ＳＤＳ−ＰＡＧＥによる解析は、これにより加熱処理後のＬＦ類の分解を確認するためである。
ＳＤＳ−ＰＡＧＥ：各試料１５μｌをサンプルバッファー１５μｌ（０．５Ｍ Ｔｒｉｓ−ＨＣｌ（ｐＨ６．８） １．２５ｍｌ、グリセロール１．０ｍｌ、１０％ＳＤＳ ２．０ｍｌ、２−メルカプトエタノール０．５ｍｌ、０．１％ＢＰＢ ０．２５ｍｌ）にて希釈し、１００℃で５分間加熱した。その後各試料を１５μｌずつ１４％ポリアクリルアミドゲル（ＴＥＦＣＯ ＳＤＳ−ＰＡＧＥ ｍｉｎｉ）にて電気泳動した。分子量マーカーとしてはＫａｌｅｉｄｏｓｃｏｐｅ Ｐｒｅｓｔａｉｎｅｄ Ｓｔａｎｄａｒｄｓを用いた。
[Test Example 10]
Iron saturated lactoferrin was dissolved in deionized water at a concentration of 15 mg% (solution A). As a stabilizer, 0.6% by weight of pectin was dissolved in deionized water (solution B). A liquid and B liquid were mixed, and it stirred and mixed at 40 degreeC and 8000 rpm for 3 minutes with the ultradisperser (ULTRA-TURRAX T-25; made by IKA Japan), and prepared LF composition. Next, this LF composition was adjusted to 10 samples having a pH of 1 to 10 using lactic acid or sodium hydroxide solution. 2 ml of this was dispensed into an ampule tube and heated at 90 ° C., 100 ° C., 110 ° C., 120 ° C. and 130 ° C. for 4 minutes.
The aggregation / precipitation state of each sample after the heat treatment was visually determined, and then the band pattern of LFs was analyzed by polyacrylamide gel electrophoresis (SDS-PAGE) shown below. The analysis by SDS-PAGE is for confirming decomposition | disassembly of LFs after heat processing by this.
SDS-PAGE: 15 μl of each sample was added to 15 μl of sample buffer (0.5 M Tris-HCl (pH 6.8) 1.25 ml, glycerol 1.0 ml, 10% SDS 2.0 ml, 2-mercaptoethanol 0.5 ml, 0.1 % BPB (0.25 ml) and heated at 100 ° C. for 5 minutes. Thereafter, 15 μl of each sample was electrophoresed on a 14% polyacrylamide gel (TEFCO SDS-PAGE mini). Kaleidoscope Prestained Standards were used as molecular weight markers.
この試験の結果、ＬＦ組成物を含有する溶液は、ｐＨ３〜５において目視による凝集・沈殿を生じず、また、ＳＤＳ−ＰＡＧＥによりＬＦ類のバンドを確認することができた。従って、このＬＦ組成物は、酸性側で加熱安定性が極めて高いことが分かった。さらに、加熱時間を延長して、ＬＦ類が確認できるかを検査したところ、ｐＨ３〜５では、１２０℃で７分間加熱しても凝集・沈殿せず、また、ＬＦ類のバンドが確認できた。この試験結果からみて、このＬＦ組成物は、レトルト殺菌処理することが十分に可能であることが明らかとなった。 As a result of this test, the solution containing the LF composition did not cause visual aggregation / precipitation at pH 3 to 5, and the band of LFs could be confirmed by SDS-PAGE. Therefore, it was found that this LF composition has extremely high heat stability on the acidic side. Furthermore, when the heating time was extended to check whether LFs could be confirmed, at pH 3-5, even when heated at 120 ° C. for 7 minutes, no aggregation / precipitation was observed, and LF bands could be confirmed. . From this test result, it was revealed that this LF composition can be sufficiently subjected to retort sterilization.
［試験例１１］
ＬＦを１０ｍｇ％濃度で脱イオン水に溶解した（Ａ液）。安定剤としてカルボキシメチルセルロース０．１５重量％を脱イオン水に溶解した（Ｂ液）。Ａ液とＢ液を混合し、ウルトラディスパーサー（ＵＬＴＲＡ−ＴＵＲＲＡＸ Ｔ−２５；ＩＫＡジャパン社製）にて、４０℃、８０００ｒｐｍで３分間撹拌混合してＬＦ組成物を調製した。次いで、このＬＦ組成物を乳酸または水酸化ナトリウム溶液を用いてｐＨ １〜１０の１０試料に調整した。これをアンプル管に２ｍｌずつ分注し、９０℃、１００℃、１１０℃、１２０℃、１３０℃にて４分間加熱した。
加熱処理後の各試料の凝集・沈殿状態を目視により判定し、その後、以下に示したポリアクリルアミドゲル電気泳動（ＳＤＳ−ＰＡＧＥ）により、ＬＦ類のバンドパターンの解析を行った。ＳＤＳ−ＰＡＧＥによる解析は、これにより加熱処理後のＬＦ類の分解を確認するためである。
ＳＤＳ−ＰＡＧＥ：各試料１５μｌをサンプルバッファー１５μｌ（０．５Ｍ Ｔｒｉｓ−ＨＣｌ（ｐＨ６．８） １．２５ｍｌ、グリセロール１．０ｍｌ、１０％ＳＤＳ ２．０ｍｌ、２−メルカプトエタノール０．５ｍｌ、０．１％ＢＰＢ ０．２５ｍｌ）にて希釈し、１００℃で５分間加熱した。その後各試料を１５μｌずつ１４％ポリアクリルアミドゲル（ＴＥＦＣＯ ＳＤＳ−ＰＡＧＥ ｍｉｎｉ）にて電気泳動した。分子量マーカーとしてはＫａｌｅｉｄｏｓｃｏｐｅ Ｐｒｅｓｔａｉｎｅｄ Ｓｔａｎｄａｒｄｓを用いた。
[Test Example 11]
LF was dissolved in deionized water at a concentration of 10 mg% (solution A). As a stabilizer, 0.15% by weight of carboxymethylcellulose was dissolved in deionized water (Liquid B). A liquid and B liquid were mixed, and it stirred and mixed at 40 degreeC and 8000 rpm for 3 minutes with the ultradisperser (ULTRA-TURRAX T-25; made by IKA Japan), and prepared LF composition. Next, this LF composition was adjusted to 10 samples having a pH of 1 to 10 using lactic acid or sodium hydroxide solution. 2 ml of this was dispensed into an ampule tube and heated at 90 ° C., 100 ° C., 110 ° C., 120 ° C. and 130 ° C. for 4 minutes.
The aggregation / precipitation state of each sample after the heat treatment was visually determined, and then the band pattern of LFs was analyzed by polyacrylamide gel electrophoresis (SDS-PAGE) shown below. The analysis by SDS-PAGE is for confirming decomposition | disassembly of LFs after heat processing by this.
SDS-PAGE: 15 μl of each sample was added to 15 μl of sample buffer (0.5 M Tris-HCl (pH 6.8) 1.25 ml, glycerol 1.0 ml, 10% SDS 2.0 ml, 2-mercaptoethanol 0.5 ml, 0.1 % BPB (0.25 ml) and heated at 100 ° C. for 5 minutes. Thereafter, 15 μl of each sample was electrophoresed on a 14% polyacrylamide gel (TEFCO SDS-PAGE mini). Kaleidoscope Prestained Standards were used as molecular weight markers.
この試験の結果、ＬＦ組成物を含有する溶液は、ｐＨ３〜７において目視による凝集・沈殿を生じず、また、ＳＤＳ−ＰＡＧＥによりＬＦ類のバンドを確認することができた。従って、このＬＦ組成物は、酸性側で安定なだけでなく、中性側でも加熱安定性が極めて高いことが分かった。さらに、加熱時間を延長して、ＬＦ類が確認できるかを検査したところ、ｐＨ３〜７では、１２０℃で６分間加熱しても凝集・沈殿せず、また、ＬＦ類のバンドが確認できた。この試験結果からみて、このＬＦ組成物は、レトルト殺菌処理することが十分に可能であることが明らかとなった。 As a result of this test, the solution containing the LF composition did not cause visual aggregation / precipitation at pH 3 to 7, and the band of LFs could be confirmed by SDS-PAGE. Therefore, it was found that this LF composition is not only stable on the acidic side but also extremely high in heat stability on the neutral side. Further, when the heating time was extended to check whether LFs could be confirmed, at pH 3-7, even when heated at 120 ° C. for 6 minutes, no aggregation / precipitation was observed, and LF bands could be confirmed. . From this test result, it was revealed that this LF composition can be sufficiently subjected to retort sterilization.
［試験例１２］
試験例１で調製した試料を用いて、その抗原性を競合ＥＬＩＳＡ法により測定した。未加熱のＬＦ組成物の抗体との反応性に対する加熱処理したＬＦ組成物の抗体との反応性を百分率（％）で算出した。
[Test Example 12]
Using the sample prepared in Test Example 1, the antigenicity was measured by a competitive ELISA method. The reactivity of the heat-treated LF composition with respect to the reactivity of the unheated LF composition with the antibody was calculated as a percentage (%).
その結果を表４に示した。表４から、未加熱のＬＦ組成物の抗体との反応性に対する加熱処理したＬＦ組成物の抗体との反応性は、１３０℃までであれば５０％以上を維持していることが判明した。従って、本発明におけるＬＦ組成物はｐＨ２〜９の範囲であれば、１３０℃という超高温加熱処理に対して安定である。
The results are shown in Table 4. From Table 4, it was found that the reactivity of the heat-treated LF composition with respect to the reactivity of the unheated LF composition with the antibody maintained 50% or more up to 130 ° C. Therefore, the LF composition in the present invention is stable to an ultrahigh temperature heat treatment of 130 ° C. in the range of pH 2-9.
［試験例１３］
試験例２と同様の方法で調製したＬＦ組成物（ＬＦ８ｍｇ％、キサンタンガム０．０４重量％）を含有する溶液をｐＨ ２〜９に調整し、１５０ｍｌずつレトルトパウチに充填し、密封した。対照としてＬＦのみを含む溶液をｐＨ２〜９に調整したものを１５０ｍｌずつレトルトパウチに充填し、密封した。これらの溶液を レトルト殺菌機（第１種圧力容器、ＴＹＰＥ： ＲＣＳ−４ＣＲＴＧＮ、日阪製作所製）により１２０℃、４分間加熱した。加熱後のそれぞれの試料を２５℃で保存して、経時的に凝集・沈殿の有無を目視により観察し、また、ポリアクリルアミドゲル電気泳動（ＳＤＳ−ＰＡＧＥ）によりＬＦ類のバンドパターンの解析を行った。
その結果、対照としたＬＦのみを含む溶液をｐＨ２〜９に調整したものは、１日目で凝集・沈澱が確認できたが、ＬＦ組成物を含有する溶液をｐＨ ２〜９に調整したものは、保存後１ヶ月経っても凝集・沈澱は認められなかった。また、ＳＤＳ−ＰＡＧＥにてＬＦ類のバンドパターンを解析したところ、ＬＦ組成物を含む溶液をｐＨ ２〜９に調整したものは保存開始時から保存後１ヶ月においてもＬＦ類のバンドが確認でき、他の変化は認められなかった。この試験結果により、本発明によるＬＦ組成物はレトルト殺菌処理の場合に大変有効であることが判明した。
[Test Example 13]
A solution containing an LF composition (LF 8 mg%, xanthan gum 0.04 wt%) prepared in the same manner as in Test Example 2 was adjusted to pH 2-9, filled in a retort pouch 150 ml at a time, and sealed. As a control, a retort pouch was filled with 150 ml of a solution containing only LF adjusted to pH 2-9 and sealed. These solutions were heated at 120 ° C. for 4 minutes by a retort sterilizer (type 1 pressure vessel, TYPE: RCS-4CRTGN, manufactured by Nisaka Seisakusho). Each sample after heating is stored at 25 ° C., the presence or absence of aggregation / precipitation is visually observed over time, and the band pattern of LFs is analyzed by polyacrylamide gel electrophoresis (SDS-PAGE). It was.
As a result, the solution containing only LF as a control was adjusted to pH 2-9, and aggregation / precipitation was confirmed on the first day, but the solution containing the LF composition was adjusted to pH 2-9 No aggregation or precipitation was observed even after 1 month of storage. In addition, when the band pattern of LFs was analyzed by SDS-PAGE, the LF band was confirmed even after 1 month of storage from the start of storage when the solution containing the LF composition was adjusted to pH 2-9. No other changes were observed. From this test result, it was found that the LF composition according to the present invention is very effective in the case of retort sterilization treatment.
［試験例１４］
試験例２と同様の方法で調製したＬＦ組成物２００ｇ（ＬＦ８ｍｇ％、キサンタンガム０．０４重量％）に還元脱脂粉乳溶液（脱脂粉乳３重量％）８００ｇを混合し、ＬＦ組成物を含有する溶液（１）を調製した。対照としてＬＦ溶液（ＬＦ８ｍｇ％溶液）２００ｇに還元脱脂粉乳溶液（脱脂粉乳３重量％）８００ｇを混合した溶液（２）、及び還元脱脂粉乳溶液（脱脂粉乳３重量％）１０００ｇのみの溶液（３）を調製した。各溶液を１５０ｍｌずつレトルトパウチに充填し、密封した。これらの溶液をレトルト殺菌機 （第１種圧力容器、ＴＹＰＥ： ＲＣＳ−４ＣＲＴＧＮ、日阪製作所製）により１２０℃、２０分加熱した。その結果、溶液（１）、（３）においては凝集・沈殿は認められず、風味も良好であったが、（２）の溶液では凝集・沈殿が認められた。この試験結果により、本発明によるＬＦ組成物はレトルト殺菌処理の場合に大変有効であることが判明した。
[Test Example 14]
800 g of reduced skim milk powder solution (3% by weight of skim milk powder) was mixed with 200 g of LF composition (LF 8 mg%, xanthan gum 0.04% by weight) prepared in the same manner as in Test Example 2, and a solution containing the LF composition ( 1) was prepared. As a control, a solution (2) obtained by mixing 800 g of reduced skim milk powder solution (3% by weight of skim milk powder) with 200 g of LF solution (LF 8 mg% solution), and a solution of only 1000 g of reduced skim milk powder solution (3% by weight of skim milk powder) Was prepared. 150 ml of each solution was filled into a retort pouch and sealed. These solutions were heated at 120 ° C. for 20 minutes with a retort sterilizer (type 1 pressure vessel, TYPE: RCS-4CRTGN, manufactured by Nisaka Seisakusho). As a result, no aggregation / precipitation was observed in the solutions (1) and (3) and the flavor was good, but aggregation / precipitation was observed in the solution (2). From this test result, it was found that the LF composition according to the present invention is very effective in the case of retort sterilization treatment.
［比較例１］
安定剤として大豆多糖類、キサンタンガム、ジェランガム及びグアガムを選択し、試験例１と同様の方法でＬＦ組成物を調製した。なお、各安定剤は大豆多糖類０．４重量％、キサンタンガム０．０４重量％、ジェランガム０．０５重量％及びグアガム０．０５重量％にて試験を行った。これらのＬＦ組成物をｐＨ６．５に調整し、１２０℃で４分間加熱した。加熱された各試料の凝集・沈澱状態を目視により判定し、また、ポリアクリルアミドゲル電気泳動（ＳＤＳ−ＰＡＧＥ）によりＬＦ類のバンドパターンの解析を行った。
[Comparative Example 1]
Soy polysaccharides, xanthan gum, gellan gum and guar gum were selected as stabilizers, and an LF composition was prepared in the same manner as in Test Example 1. Each stabilizer was tested at 0.4% by weight of soybean polysaccharide, 0.04% by weight of xanthan gum, 0.05% by weight of gellan gum and 0.05% by weight of guar gum. These LF compositions were adjusted to pH 6.5 and heated at 120 ° C. for 4 minutes. The aggregation / precipitation state of each heated sample was visually determined, and the band pattern of LFs was analyzed by polyacrylamide gel electrophoresis (SDS-PAGE).
試験結果を表５に示した。加熱前のＬＦ組成物はいずれも凝集・沈殿せず、透明であり、ＬＦ類のバンドも確認できた。加熱後は大豆多糖類及びキサンタンガム含有ＬＦ組成物は透明であり、また、ＬＦ類のバンドも確認できたが、ジェランガム及びグアガム含有ＬＦ組成物は、いずれも半透明か、あるいは凝集・沈殿し、ＳＤＳ−ＰＡＧＥにてＬＦ類のバンドも確認できなかった。
The test results are shown in Table 5. All LF compositions before heating did not aggregate and precipitate, were transparent, and LF bands could be confirmed. After heating, soybean polysaccharides and xanthan gum-containing LF compositions were transparent, and LF bands were also confirmed, but gellan gum and guar gum-containing LF compositions were both translucent or agglomerated and precipitated, An LF band could not be confirmed by SDS-PAGE.
鉄飽和ラクトフェリンを２０ｍｇ％濃度で脱イオン水に溶解した（Ａ液、３００ｇ）。安定剤として大豆多糖類０．８重量％を脱イオン水に溶解した（Ｂ液、３００ｇ）。Ａ液とＢ液を混合し、ウルトラディスパーサー（ＵＬＴＲＡ−ＴＵＲＲＡＸ Ｔ−２５；ＩＫＡジャパン社製）にて、５０℃、９５００ｒｐｍで３分間撹拌混合して本発明のＬＦ組成物６００ｇを調製した。 Iron saturated lactoferrin was dissolved in deionized water at a concentration of 20 mg% (solution A, 300 g). As a stabilizer, 0.8% by weight of soybean polysaccharide was dissolved in deionized water (solution B, 300 g). A liquid and B liquid were mixed, and it stirred and mixed at 50 degreeC and 9500 rpm for 3 minutes with the ultradisperser (ULTRA-TURRAX T-25; made by IKA Japan), and prepared 600 g of LF compositions of this invention.
ＬＦを１６ｍｇ％濃度で脱イオン水に溶解した（Ａ液、１０ｋｇ）。安定剤としてキサンタンガムを０．１６重量％で脱イオン水に溶解した（Ｂ液、１０ｋｇ）。Ａ液とＢ液を混合し、ＴＫホモミクサー（ＭＡＲＫ ＩＩ １６０型、特殊機化工業製）にて、３６００ｒｐｍで３０分間撹拌混合し、さらに分画分子量１０ｋＤａのＵＦ膜にて濃縮して、本発明のＬＦ組成物１０ｋｇを調製した。 LF was dissolved in deionized water at a concentration of 16 mg% (A solution, 10 kg). As a stabilizer, xanthan gum was dissolved in deionized water at 0.16% by weight (solution B, 10 kg). Liquid A and liquid B are mixed, mixed and stirred for 30 minutes at 3600 rpm with a TK homomixer (MARK II 160 type, manufactured by Tokushu Kika Kogyo Co., Ltd.), and further concentrated on a UF membrane with a molecular weight cut off of 10 kDa. 10 kg of an LF composition was prepared.
塩酸で脱鉄したアポラクトフェリンを１０ｍｇ％濃度で脱イオン水に溶解した（Ａ液、１０００ｋｇ）。安定剤としてショ糖脂肪酸エステル０．４重量％を脱イオン水に溶解した（Ｂ液、１０００ｋｇ）。Ａ液とＢ液を混合し、ＴＫホモミクサー（ＭＡＲＫ ＩＩ ２５００型、特殊機化工業製）にて、４０℃、３６００ｒｐｍで４０分間撹拌混合し、さらに凍結乾燥して本発明のＬＦ組成物３．９ｋｇを調製した。 Apolactoferrin deironated with hydrochloric acid was dissolved in deionized water at a concentration of 10 mg% (solution A, 1000 kg). As a stabilizer, 0.4% by weight of sucrose fatty acid ester was dissolved in deionized water (solution B, 1000 kg). Liquid A and liquid B are mixed, mixed and stirred for 40 minutes at 40 ° C. and 3600 rpm in a TK homomixer (MARK II 2500 type, manufactured by Tokushu Kika Kogyo Co., Ltd.), and freeze-dried. 9 kg was prepared.
鉄飽和ラクトフェリンを２０ｍｇ％濃度で脱イオン水に溶解した（Ａ液、５００ｇ）。安定剤として大豆多糖類０．４重量％を脱イオン水に溶解した（Ｂ液、５００ｇ）。Ａ液とＢ液を混合し、ウルトラディスパーサー（ＵＬＴＲＡ−ＴＵＲＲＡＸ Ｔ−２５；ＩＫＡジャパン社製）にて、４０℃、９５００ｒｐｍで３分間撹拌混合した。その後、上記の溶液に、ソルビトール８０ｇ、酸味料４ｇ、香料４ｇ、ペクチン１０ｇ、乳清タンパク質濃縮物１０ｇ、乳酸カルシウム２ｇ、水８９０ｇを添加して、撹拌混合し、本発明のＬＦ組成物を調製した。２００ｍｌのチアパックに充填し、８５℃、２０分間殺菌後、密栓し、本発明のＬＦ組成物からなるゲル状食品１０袋を調製した。調製したゲル状食品は、すべて沈殿等は認められず、風味に異常は感じられなかった。 Iron saturated lactoferrin was dissolved in deionized water at a concentration of 20 mg% (solution A, 500 g). As a stabilizer, 0.4% by weight of soybean polysaccharide was dissolved in deionized water (solution B, 500 g). A liquid and B liquid were mixed, and it stirred and mixed at 40 degreeC and 9500 rpm for 3 minutes with the ultradisperser (ULTRA-TURRAX T-25; made by IKA Japan). Thereafter, 80 g of sorbitol, 4 g of acidulant, 4 g of fragrance, 10 g of pectin, 10 g of whey protein concentrate, 2 g of calcium lactate and 890 g of water are added to the above solution and mixed by stirring to prepare the LF composition of the present invention. did. 200 ml of cheer pack was filled, sterilized at 85 ° C. for 20 minutes, sealed, and 10 gel foods made of the LF composition of the present invention were prepared. In the prepared gel foods, no precipitation or the like was observed, and no abnormal flavor was felt.
鉄飽和ラクトフェリンを１００ｍｇ％濃度で脱イオン水に溶解した（Ａ液、２００ｇ）。安定剤として大豆多糖類４重量％を脱イオン水に溶解した（Ｂ液、２００ｇ）。マルチトール１００ｇ、還元水飴２０ｇ、酸味料２ｇ、香料２ｇ、前記Ａ液２００ｇ、前記Ｂ液２００ｇ、及び水４７６ｇを混合し、本発明のＬＦ組成物を調製した。このＬＦ組成物を５０ｍｌのガラス瓶に充填し、９０℃、１５分間殺菌後、密栓し、本発明ＬＦ組成物を含有する飲料２０本を調製した。調製した飲料は、すべて沈殿は認められず、風味に異常は感じられなかった。 Iron saturated lactoferrin was dissolved in deionized water at a concentration of 100 mg% (solution A, 200 g). As a stabilizer, 4% by weight of soybean polysaccharide was dissolved in deionized water (solution B, 200 g). Maltitol 100g, reduced starch syrup 20g, acidulant 2g, fragrance 2g, said A liquid 200g, said B liquid 200g, and water 476g were mixed, and LF composition of this invention was prepared. This LF composition was filled in a 50 ml glass bottle, sterilized at 90 ° C. for 15 minutes, sealed, and 20 beverages containing the LF composition of the present invention were prepared. In all the prepared beverages, no precipitation was observed, and no abnormal flavor was felt.
実施例２で調製したＬＦ組成物０．２ｋｇ（ラクトフェリン８ｍｇ％、キサンタンガム０．０８重量％）に大豆粕１２ｋｇ、脱脂粉乳１４ｋｇ、大豆油４ｋｇ、コーン油２ｋｇ、パーム油２８ｋｇ、トウモロコシ澱粉１５ｋｇ、小麦粉９ｋｇ、ふすま２ｋｇ、ビタミン混合物９ｋｇ、セルロース２．８ｋｇ、ミネラル混合物２ｋｇを配合し、１２０℃、４分間殺菌して、イヌ飼育用飼料１００ｋｇを調製した。 0.2 kg of LF composition prepared in Example 2 (lactoferrin 8 mg%, xanthan gum 0.08 wt%), soybean meal 12 kg, skim milk powder 14 kg, soybean oil 4 kg, corn oil 2 kg, palm oil 28 kg, corn starch 15 kg, wheat flour 9 kg, 2 kg of bran, 9 kg of vitamin mixture, 2.8 kg of cellulose, and 2 kg of mineral mixture were blended and sterilized at 120 ° C. for 4 minutes to prepare 100 kg of dog breeding feed.
実施例３で調製したＬＦ組成物３ｋｇ（アポラクトフェリン５ｍｇ％、ショ糖脂肪酸エステル０．２重量％）に、カゼイン５ｋｇ、大豆タンパク質５ｋｇ、魚油１ｋｇ、シソ油３ｋｇ、デキストリン１９ｋｇ、ミネラル混合物６ｋｇ、ビタミン混合物１．９５ｋｇ、乳化剤２ｋｇ、安定剤４ｋｇ、香料０．０５ｋｇを配合し、２００ｍｌのレトルトパウチに充填し、レトルト殺菌機 （第１種圧力容器、ＴＹＰＥ： ＲＣＳ−４ＣＲＴＧＮ、日阪製作所製）で１２１℃、２０分間殺菌して、経腸栄養剤５０ｋｇを調製した。 3 kg of LF composition prepared in Example 3 (apolactoferrin 5 mg%, sucrose fatty acid ester 0.2 wt%), casein 5 kg, soy protein 5 kg, fish oil 1 kg, perilla oil 3 kg, dextrin 19 kg, mineral mixture 6 kg, vitamins 1.95 kg of mixture, 2 kg of emulsifier, 4 kg of stabilizer, 0.05 kg of fragrance are blended and filled into a 200 ml retort pouch, and the retort sterilizer (type 1 pressure vessel, TYPE: RCS-4CRTGN, manufactured by Nisaka Seisakusho) Sterilized at 121 ° C. for 20 minutes to prepare 50 kg of enteral nutrient.
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Cited By (4)
| Publication number | Priority date | Publication date | Assignee | Title |
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| EP2984935A4 (en) * | 2013-03-28 | 2016-11-30 | Megmilk Snow Brand Co Ltd | Protein composition and production process therefor |
| JP2019115362A (en) * | 2019-04-24 | 2019-07-18 | 森永乳業株式会社 | Inhibitors of gastric acidic protease activity, methods for producing lactoferrin compositions and lactoferrin compositions |
| WO2019176246A1 (en) | 2018-03-12 | 2019-09-19 | 森永乳業株式会社 | Method for producing lactoferrin-containing aqueous solution |
| JP2019154409A (en) * | 2018-03-16 | 2019-09-19 | 森永乳業株式会社 | Lactoferrin containing acidic beverage and production method thereof |
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| JP2019115362A (en) * | 2019-04-24 | 2019-07-18 | 森永乳業株式会社 | Inhibitors of gastric acidic protease activity, methods for producing lactoferrin compositions and lactoferrin compositions |
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