JP2018123070A - Vessel endothelial function improving agent - Google Patents
Vessel endothelial function improving agent Download PDFInfo
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- JP2018123070A JP2018123070A JP2017014151A JP2017014151A JP2018123070A JP 2018123070 A JP2018123070 A JP 2018123070A JP 2017014151 A JP2017014151 A JP 2017014151A JP 2017014151 A JP2017014151 A JP 2017014151A JP 2018123070 A JP2018123070 A JP 2018123070A
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- endothelial function
- vascular endothelial
- phosphatidylcholine
- sphingomyelin
- phosphatidylserine
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Classifications
-
- Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
- Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
- Y02P—CLIMATE CHANGE MITIGATION TECHNOLOGIES IN THE PRODUCTION OR PROCESSING OF GOODS
- Y02P60/00—Technologies relating to agriculture, livestock or agroalimentary industries
- Y02P60/80—Food processing, e.g. use of renewable energies or variable speed drives in handling, conveying or stacking
- Y02P60/87—Re-use of by-products of food processing for fodder production
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- Coloring Foods And Improving Nutritive Qualities (AREA)
- Pharmaceuticals Containing Other Organic And Inorganic Compounds (AREA)
- Fodder In General (AREA)
Abstract
Description
本発明は、スフィンゴミエリン、ホスファチジルコリンおよびホスファチジルセリンから選ばれる１種類以上を有効成分とする血管内皮機能改善剤、及びスフィンゴミエリン、ホスファチジルコリンおよびホスファチジルセリンから選ばれる１種類以上を有効成分とすることを特徴とする血管内皮機能改善作用を有する食品および飼料に関する。 The present invention is characterized in that a vascular endothelial function improving agent comprising one or more selected from sphingomyelin, phosphatidylcholine and phosphatidylserine as an active ingredient, and one or more selected from sphingomyelin, phosphatidylcholine and phosphatidylserine as active ingredients. The present invention relates to foods and feeds that have an effect of improving vascular endothelial function.
食生活の乱れや慢性的な運動不足、過度のストレスなどにより、肥満や高血圧、高脂血症、糖尿病などの生活習慣病に対するリスクが増加している。これらの生活習慣病の症状が進行すると動脈硬化や心筋梗塞など、より重篤な病気を引き起こす可能性が高くなる。最近、上記したものを含む様々な疾患の要因の一つに血管の内皮機能の低下が関与していることが明らかとなってきた。よって、血管内皮細胞の機能を改善するような食品の開発は、多くの疾患の予防的側面からも大きな意義があると考えられる。
食品素材を原料とした血管内皮機能改善剤として、マメ目マメ科ゲンゲ属植物抽出物を有効成分とする血管内皮機能改善剤（特許文献１）、タマネギ、タマネギ処理物を有効成分とする血管内皮機能改善剤（特許文献２）、シトルリン、または、グルタチオンを有効成分とする血管内皮機能改善剤（特許文献３）などが開示されている。
一方、横山らは、リゾホスファチジルコリンとホスファチジルコリンが血管内皮細胞のＮＯ合成酵素を活性化し、ＮＯ産生量を増加させることで、血管を弛緩させることを報告しているが、血管内皮細胞の細胞死を抑制する手段として、リン脂質を用いることを開示した文献等はない。
Risks of lifestyle-related diseases such as obesity, hypertension, hyperlipidemia, and diabetes are increasing due to disordered diet, chronic lack of exercise, and excessive stress. As the symptoms of these lifestyle-related diseases progress, there is a high possibility of causing more serious diseases such as arteriosclerosis and myocardial infarction. Recently, it has become clear that one of the causes of various diseases including those described above is associated with a decrease in endothelial function of blood vessels. Therefore, the development of foods that improve the function of vascular endothelial cells is considered to have great significance from the preventive aspect of many diseases.
As a vascular endothelial function improving agent made from a food material, a vascular endothelial function improving agent (Patent Document 1) containing a leguminous leguminous genus plant extract as an active ingredient, an onion and a vascular endothelium containing an onion processed product as an active ingredient A function improving agent (Patent Document 2), citrulline, or a vascular endothelial function improving agent (Patent Document 3) containing glutathione as an active ingredient is disclosed.
On the other hand, Yokoyama et al. Reported that lysophosphatidylcholine and phosphatidylcholine activate the NO synthase of vascular endothelial cells and increase NO production, thereby relaxing the blood vessels. There is no literature that discloses the use of phospholipids as a means of suppression.
横山光宏、血管内皮細胞のシグナル伝達と脂質、動脈硬化、２２（６・７）：４５３−４５６、１９９４． Yokoyama Mitsuhiro, Vascular Endothelial Cell Signaling and Lipids, Arteriosclerosis, 22 (6.7): 453-456, 1994.
本発明は、食品に由来する成分を有効成分とする新たな血管内皮機能改善剤および新たな血管内皮機能改善作用を有する食品および飼料を提供することを課題とする。 An object of the present invention is to provide a new vascular endothelial function improving agent having a component derived from food as an active ingredient, and a food and feed having a new vascular endothelial function improving action.
本発明は、上記課題を解決するために、以下の解決手段を提供するものである。
（１）スフィンゴミエリン、ホスファチジルコリンおよびホスファチジルセリンからなる群から選ばれる１種類以上を有効成分とする血管内皮機能改善剤。
（２）スフィンゴミエリン、ホスファチジルコリンおよびホスファチジルセリンからなる群から選ばれる１種類以上を有効成分とする血管内皮機能改善用食品。
（３）スフィンゴミエリン、ホスファチジルコリンおよびホスファチジルセリンからなる群から選ばれる１種類以上を有効成分とする血管内皮機能改善用飼料。
（４）スフィンゴミエリン、ホスファチジルコリンおよびホスファチジルセリンからなる群から選ばれる１種類以上を有効成分とする血管内皮機能改善剤の製造方法であって、乳又は乳素材を原材料とし、孔径が０．１〜２μｍの分離膜または、分画分子量５〜５００ｋＤａの分離膜で処理する工程を含む（１）〜（３）に記載の製造方法。
（５）スフィンゴミエリン、ホスファチジルコリンおよびホスファチジルセリンからなる群から選ばれる１種類以上を有効成分とする血管内皮機能改善作用を有する（１）〜（３）の製造方法であって、スフィンゴミエリン、ホスファチジルコリンおよびホスファチジルセリンからなる群から選ばれる１種類以上を添加する工程を含む（１）〜（３）の製造方法。
The present invention provides the following means for solving the above-mentioned problems.
(1) A vascular endothelial function improving agent comprising one or more selected from the group consisting of sphingomyelin, phosphatidylcholine and phosphatidylserine as active ingredients.
(2) A food for improving vascular endothelial function, comprising as an active ingredient at least one selected from the group consisting of sphingomyelin, phosphatidylcholine and phosphatidylserine.
(3) A vascular endothelial function improving feed comprising one or more selected from the group consisting of sphingomyelin, phosphatidylcholine and phosphatidylserine as active ingredients.
(4) A method for producing a vascular endothelial function improver comprising one or more selected from the group consisting of sphingomyelin, phosphatidylcholine and phosphatidylserine as active ingredients, wherein milk or a milk material is used as a raw material, and the pore size is 0.1 to The manufacturing method as described in (1)-(3) including the process processed with a 2 micrometers separation membrane or a separation membrane with a molecular weight cut-off of 5-500 kDa.
(5) The production method of (1) to (3) having an effect of improving vascular endothelial function, comprising one or more selected from the group consisting of sphingomyelin, phosphatidylcholine and phosphatidylserine, wherein the sphingomyelin, phosphatidylcholine and The manufacturing method of (1)-(3) including the process of adding 1 or more types chosen from the group which consists of phosphatidylserine.
本発明は、スフィンゴミエリン、ホスファチジルコリンおよびホスファチジルセリンから選ばれる１種類以上を有効成分とすることを特徴とする血管内皮機能改善剤およびスフィンゴミエリン、ホスファチジルコリン、ホスファチジルセリンから選ばれる１種類以上を有効成分とすることを特徴とする血管内皮機能改善作用を有する食品および飼料を提供するものである。 The present invention relates to a vascular endothelial function improving agent characterized by having one or more selected from sphingomyelin, phosphatidylcholine and phosphatidylserine as active ingredients, and one or more selected from sphingomyelin, phosphatidylcholine and phosphatidylserine as active ingredients The present invention provides foods and feeds that have an effect of improving vascular endothelial function.
（血管内皮機能改善作用）
本発明の血管内皮機能改善作用とは、血管内皮細胞の機能改善、又は血管内皮細胞の機能低下抑制をいう。
血管内皮機能改善作用は、血管内皮細胞の細胞死の抑制作用（生存率の向上）または血管内皮細胞自身の機能促進作用などにより評価することができる。例えば、以下の方法で評価することができる。
（１）血管内皮細胞の細胞死抑制活性の測定方法
血管内皮細胞の細胞死抑制活性を評価する方法の一様態を以下に示す。
細胞は、正常ヒト臍帯静脈内皮細胞（ＨＵＶＥＣ細胞）、培地はＨｕ−Ｍｅｄｉａ−ＥＧ２、または１％ウシ胎児血清（Ｆｅｔａｌ Ｂｏｖｉｎｅ Ｓｅｒｕｍ：ＦＢＳ）を含むロズウェルパーク記念研究所培地（Ｒｏｓｗｅｌｌ Ｐａｒｋ Ｍｅｍｏｒｉａｌ Ｉｎｓｔｉｔｕｔｅ Ｍｅｄｉｕｍ：ＲＰＭＩ）１６４０を用いる。細胞の培養は、３７℃、５％ＣＯ２インキュベータ内で行い、４〜６世代継代目の細胞を実験に供する。
初発細胞濃度を５×１０５／ｍｌに調整した細胞浮遊液（Ｈｕ−Ｍｅｄｉａ−ＥＧ２を使用）を６ウエルプレートに１ｍｌを播種後、６時間程度培養し、プレートに接着したことを確認する。その後、供試試料を含む１％ＦＢＳを含むＲＰＩＭ１６４０に交換して４０時間培養し、培養後の細胞を回収し、以下の染色を行う。なお、対照群には溶媒であるリン酸緩衝液のみを添加した培地を用いる。
染色には、ＡｎｎｅｘｉｎＶ−ＦＩＴＣ ｋｉｔ（ＢＤ Ｂｉｏｓｃｉｅｎｃｅｓ社製、ベルギー）Ｐｒｏｐｄｉｕｍ ｉｏｄｉｄｅ（ＳＩＧＭＡ ＡＬＤＲＩＣＨ社製、米国）を用いる。培養上清を回収し、冷リン酸緩衝液で細胞を２回洗浄し、１×ｂｉｎｄｉｎｇ ｂｕｆｆｅｒに再浮遊させる。１.５ｍｌのチューブに１００μｌの上記細胞浮遊液とＡｎｎｅｘｉｎＶ試薬を５μＬ、Ｐｒｏｐｄｉｕｍ ｉｏｄｉｄｅを２μＬ加え、穏やかに混和し、室温・暗所で１５分間反応させ、反応液中の生細胞数をフローサイトメーターで解析する。
（２）細胞増殖及び細胞運動能の亢進の指標である細胞外シグナル調節キナーゼ（Ｅｘｔｒａｃｅｌｌｕｌａｒ Ｓｉｎｇｎａｌ−ｒｅｇｕｌａｔｅｄ Ｋｉｎａｓｅ：ＥＲＫ）のリン酸化の評価
細胞増殖及び細胞運動能の亢進を評価する方法の一様態を以下に示す。
６ウエルプレートに１×１０６／ｍｌのＨＵＶＥＣ細胞を播種し、Ｈｕ−Ｍｅｄｉａ−ＥＧ２培地で一晩培養する。供試試料のリン酸緩衝液による懸濁液を添加して１５分間培養する。培養終了後、細胞を回収し、細胞をＰＢＳで洗浄後、ＲＩＰＡ ｌｙｓｉｓ ｂｕｆｆｅｒ（５０ｍＭ Ｔｒｉｓ−ＨＣｌ、ｐＨ７．５、１５０ｍＭ ＮａＣｌ、１％ＮＰ−４０、０．５％ＮａＤｏｃ、０．１％ＳＤＳ）を１００μｌ加え、細胞溶解液を回収する。本溶解液を氷上で１０分間静置後、１５，０００ｒｐｍで５分間遠心分離し、上清を得る。上清に６倍量のサンプルバッファー（０．３７５Ｍ Ｔｒｉｓ−ＨＣｌ ｐＨ６．７、１２％ＳＤＳ、３０％Ｓｕｃｒｏｓｅ、５％ ２−Ｍｅｒｃａｐｔｏｅｔｈａｎｏｌ、適量のＢｒｏｍｏｐｈｅｎｏｌ ｂｌｕｅ）を加え、９５℃、５分間加熱することでＳＤＳ化を行う。分離ゲル濃度が７．５％のポリアクリルアミドゲルを用いて、常法によりＳＤＳ−ＰＡＧＥを行い、ナイロン膜に転写し、ウエスタンブロットを実施する。
一次抗体に抗リン酸化Ｅｒｋ抗体、２次抗体に西洋ワサビオキシダーゼ（ＨｏｕｒｓｅＲａｄｉｓｈ Ｐｅｒｏｘｉｄａａｓｅ：ＨＲＰ）標識抗ラビットＩｇＧ抗体を用いて、ルミノイメージアナライザーによりバンドを検出する。
(Vascular endothelial function improving action)
The vascular endothelial function improving action of the present invention refers to improving the function of vascular endothelial cells or suppressing the decrease in the function of vascular endothelial cells.
The vascular endothelial function improving action can be evaluated by an action of suppressing vascular endothelial cell death (improving survival rate) or a function promoting action of the vascular endothelial cell itself. For example, it can be evaluated by the following method.
(1) Method for measuring cell death inhibitory activity of vascular endothelial cells
An embodiment of a method for evaluating the cell death inhibitory activity of vascular endothelial cells is shown below.
The cells are normal human umbilical vein endothelial cells (HUVEC cells), the medium is Hu-Media-EG2, or 1% fetal bovine serum (FBS) Roswell Park Memorial Institute Medium (Roswell Park Memorial Institute Medium: RPMI) 1640 is used. The cells are cultured in a 37 ° C., 5% CO 2 incubator, and the 4th to 6th generation passage cells are used for the experiment.
After seeding 1 ml of a cell suspension (using Hu-Media-EG2) adjusted to an initial cell concentration of 5 × 10 5 / ml in a 6-well plate, it is cultured for about 6 hours and confirmed to adhere to the plate. Thereafter, the sample is replaced with RPMI1640 containing 1% FBS containing the test sample and cultured for 40 hours, and the cultured cells are collected and stained as follows. In addition, the culture medium which added only the phosphate buffer which is a solvent is used for a control group.
For staining, Annexin V-FITC kit (BD Biosciences, Belgium) Propium iodide (SIGMA ALDRICH, USA) is used. The culture supernatant is collected, and the cells are washed twice with cold phosphate buffer and resuspended in a 1 × binding buffer. Add 100 μl of the above cell suspension and 5 μL of Annexin V reagent and 2 μL of Propium iodide to a 1.5 ml tube, mix gently, and allow to react for 15 minutes in the dark at room temperature. Analyze with.
(2) Evaluation of phosphorylation of extracellular signal-regulated kinase (ERK), which is an index of enhancement of cell proliferation and cell motility. One aspect of a method for evaluating cell proliferation and enhancement of cell motility. It is shown below.
1 × 10 6 / ml HUVEC cells are seeded in a 6-well plate and cultured overnight in Hu-Media-EG2 medium. A suspension of the test sample in phosphate buffer is added and incubated for 15 minutes. After completion of the culture, the cells were collected, and the cells were washed with PBS, followed by RIPA lysis buffer (50 mM Tris-HCl, pH 7.5, 150 mM NaCl, 1% NP-40, 0.5% NaDoc, 0.1% SDS). Add 100 μl and collect the cell lysate. The lysate is allowed to stand on ice for 10 minutes, and then centrifuged at 15,000 rpm for 5 minutes to obtain a supernatant. Add 6 times the amount of sample buffer (0.375M Tris-HCl pH 6.7, 12% SDS, 30% Sucrose, 5% 2-Mercaptoethanol, appropriate amount of Bromophenol blue) to the supernatant and heat at 95 ° C. for 5 minutes Perform SDS conversion. Using a polyacrylamide gel with a separation gel concentration of 7.5%, SDS-PAGE is performed by a conventional method, transferred to a nylon membrane, and Western blotting is performed.
A band is detected by a lumino image analyzer using an anti-phosphorylated Erk antibody as a primary antibody and a horseradish oxidase (HRP) -labeled anti-rabbit IgG antibody as a secondary antibody.
（有効成分）
本発明の血管内皮機能改善作用を有する有効成分は、スフィンゴミエリン、ホスファチジルコリンおよびホスファチジルセリンから選択される１種類以上である。これらは乳、大豆、卵黄などの食品素材等から調製したものを用いることができるが、化学的に合成したものでもよい。
また、本発明に用いるスフィンゴミエリン、ホスファチジルコリンおよびホスファチジルセリンは、分離・精製された高純度のものだけでなく、未精製の組成物であってもよい。すなわち、スフィンゴミエリン、ホスファチジルコリンおよびホスファチジルセリンからなる群から選択される１種類以上を含むものであれば、これらが分離されていないリン脂質の混合物である組成物を用いてもよい。
(Active ingredient)
The active ingredient having the vascular endothelial function improving action of the present invention is at least one selected from sphingomyelin, phosphatidylcholine and phosphatidylserine. These can be prepared from food materials such as milk, soybeans and egg yolk, but may be chemically synthesized.
The sphingomyelin, phosphatidylcholine, and phosphatidylserine used in the present invention may be not only separated and purified high-purity compositions but also unpurified compositions. That is, as long as it contains at least one selected from the group consisting of sphingomyelin, phosphatidylcholine, and phosphatidylserine, a composition that is a mixture of phospholipids in which these are not separated may be used.
（摂取量）
本発明で、血管内皮機能改善効果を発揮させるためには、成人の場合、スフィンゴミエリン、ホスファチジルコリンおよびホスファチジルセリンから選択される１種類以上を一日当たり１０〜２，５００ｍｇ、好ましくは、５０〜２，５００ｍｇを摂取すればよい。
また、スフィンゴミエリン、ホスファチジルコリンおよびホスファチジルセリンから選択される１種類以上を食品に配合する場合は、１食で上記の量を摂取してもよいし、数回に分けて、上記の摂取量になるように配合して摂取してもよい。
(Intake)
In the present invention, in order to exert the effect of improving vascular endothelial function, in the case of an adult, one or more selected from sphingomyelin, phosphatidylcholine and phosphatidylserine is 10 to 2,500 mg per day, preferably 50 to 2, It is sufficient to take 500 mg.
In addition, when one or more selected from sphingomyelin, phosphatidylcholine and phosphatidylserine are blended in food, the above amount may be ingested in one meal, and the above ingestion amount is divided into several times. You may mix and ingest.
（製造方法）
乳由来のスフィンゴミエリン、ホスファチジルコリンおよびホスファチジルセリンからなる群から選択される１種類以上を含むリン脂質組成物（以下、リン脂質組成物）の製造方法一様態を以下に示す。
本発明のリン脂質組成物の原材料には、ウシ、ヤギ、ヒツジ、ウマ等の獣乳やヒト等の哺乳類の乳及びこれらに由来する乳素材や大豆、卵黄などの食品素材等を用いることができるが、このなかでウシ由来の乳や乳素材を用いることが好ましい。乳素材としては、バターゼーラムやバターミルクを用いることが好ましい。
バターゼーラムとは、脂肪分６０重量％以上の高脂肪クリーム又はバターを、遠心分離、加温、又はせん断処理することにより得られる脂肪分３０〜５１重量％の水層画分をいう。
バターミルクとは、バターを製造する際に生じた脂肪粒以外の部分をいい、乳脂肪分３０〜４０％に調製したクリームよりバターを製造する際に、チャーニングのような乳脂肪球同士の衝突による乳化破壊等の物理的分画操作より、バターと共に副産物として発生する淡黄色の液体である。なお、上記したバターは発酵バターであってもよい。
これらの原材料を用いて、乳由来リン脂質組成物は、以下のような方法等により調製することができる。
すなわち、乳または乳素材を精密ろ過（ＭＦ）膜または限外ろ過膜（ＵＦ）処理することにより、本発明のリン脂質組成物を得ることができる。
精密ろ過膜（ＭＦ）は、孔径０．１μｍ〜２．０μｍのものが好ましい。孔径が０．１μｍ未満になると、ホエイタンパク質等の夾雑物が濃縮液側に残存するようになり、固形当たりの脂質含量が減少することにより血管内皮機能改善剤としての効果が弱くなる。また、孔径が２．０μｍを越えると、脂肪球が膜を通過して透過液側に漏れるようになるため、血管内皮機能改善効果を有する脂質画分が濃縮画分から減少するために、血管内皮機能改善剤としての効果が弱くなる。混入するタンパク質の量やリン脂質の回収量を考慮すると、孔径０．１μｍ〜２．０μｍ程度の精密ろ過膜（ＭＦ）が最も好ましい。この孔径０．１μｍ〜２μｍの精密ろ過膜（ＭＦ）としては、たとえば、Ｍｅｍｂｒａｒｏｘ（ＳＣＴ、Ｓｏｃｉｅｔｉｅ Ｃｅｒａｍｉｃｓ Ｔｅｃｈｎｉｑｕｅｓｔ社製）を使用することができる。
限外ろ過膜（ＵＦ）は、分画分子量５〜５００ｋＤａのものが好ましい。５ｋＤａ未満になると、乳糖が濃縮され、脂質の割合が高くならないため、好ましくなく、５００ｋＤａはＵＦ膜の分画分子量の上限である。
なお、ＭＦ処理またはＵＦ処理を行う前に、上記原料に対して酸を加えてｐＨ４〜５程度に調整し、カゼインタンパク質を等電点沈殿させて除去しおくことで、膜処理における膜の汚れ付着を防止できるとともに、得られる濃縮液中に含まれる固形物当たりの脂質含量を高くすることが可能となり好ましい。
さらに、ｐＨを４〜５に調整した後に塩化カルシウムを加えること、カゼインタンパク質の沈殿がより促進されるのでより好ましい。塩化カルシウムの添加量は、全体の０．０１〜０．０５重量％が好ましい。また、ｐＨ調整の際に加える酸の種類は特に限定されないが、塩酸や硫酸等の無機酸等が好ましい。
(Production method)
A method for producing a phospholipid composition (hereinafter referred to as phospholipid composition) containing at least one selected from the group consisting of milk-derived sphingomyelin, phosphatidylcholine and phosphatidylserine is shown below.
As raw materials for the phospholipid composition of the present invention, animal milk such as cattle, goats, sheep and horses, milk from mammals such as humans, milk materials derived therefrom, food materials such as soybeans and egg yolks, and the like can be used. Among them, it is preferable to use cow-derived milk or milk material. As the milk material, it is preferable to use butterlam or buttermilk.
Bataselam refers to an aqueous layer fraction having a fat content of 30 to 51% by weight obtained by subjecting a high fat cream or butter having a fat content of 60% by weight or more to centrifugation, heating, or shearing.
Buttermilk refers to parts other than fat grains produced when producing butter. When producing butter from a cream prepared to have a milk fat content of 30 to 40%, It is a pale yellow liquid generated as a by-product with butter from physical fractionation operations such as emulsion breakage due to collision. The butter described above may be fermentation butter.
Using these raw materials, a milk-derived phospholipid composition can be prepared by the following method or the like.
That is, the phospholipid composition of the present invention can be obtained by treating milk or a milk material with a microfiltration (MF) membrane or an ultrafiltration membrane (UF).
The microfiltration membrane (MF) preferably has a pore diameter of 0.1 μm to 2.0 μm. When the pore size is less than 0.1 μm, contaminants such as whey protein remain on the concentrated liquid side, and the lipid content per solid is reduced, so that the effect as a vascular endothelial function improving agent is weakened. When the pore diameter exceeds 2.0 μm, fat globules pass through the membrane and leak to the permeate side, so that the lipid fraction having an effect of improving vascular endothelial function decreases from the concentrated fraction. The effect as a function improving agent is weakened. Considering the amount of protein to be mixed and the amount of phospholipid recovered, a microfiltration membrane (MF) having a pore size of about 0.1 μm to 2.0 μm is most preferable. As the microfiltration membrane (MF) having a pore diameter of 0.1 μm to 2 μm, for example, Membrarox (SCT, manufactured by Society Ceramics Technology) can be used.
The ultrafiltration membrane (UF) preferably has a molecular weight cut off of 5 to 500 kDa. If it is less than 5 kDa, lactose is concentrated and the proportion of lipid does not increase. Therefore, 500 kDa is the upper limit of the molecular weight cutoff of the UF membrane.
In addition, before performing MF processing or UF processing, the acid is added to the above-mentioned raw material to adjust the pH to about 4 to 5, and the casein protein is removed by isoelectric precipitation, thereby removing the contamination of the membrane in the membrane processing. Adhesion can be prevented and the lipid content per solid contained in the resulting concentrate can be increased, which is preferable.
Furthermore, it is more preferable to add calcium chloride after adjusting the pH to 4 to 5 and to accelerate the precipitation of casein protein. The amount of calcium chloride added is preferably 0.01 to 0.05% by weight of the whole. Moreover, the kind of acid added at the time of pH adjustment is not particularly limited, but inorganic acids such as hydrochloric acid and sulfuric acid are preferable.
リン脂質組成物の回収方法は特に限定されないが、フィルタープレス、デカンター等を用いることが好ましい。得られた濃縮液は、特に方法を限定しないが、凍結乾燥、噴霧乾燥等の操作により粉体あるいはペースト状の組成物にしておくことが保存の上で好ましい。
なお、このリン脂質組成物中のその他の成分は、特に限定されないが、上記したような方法によれば、全固形中、タンパク質を１５〜２０重量％、糖質を１０〜５０重量％、灰分を５〜１０重量％含有するものが得られる。また、組成物中の水分は５重量％以下である。また、リン脂質組成物はスフィンゴミエリン、ホスファチジルコリンおよびホスファチジルセリンを少なくとも１種類以上含み、その含有量が全固形物中１０重量％以上であることが望ましい。より望ましくは、１５％以上であり、２０％以上がよりいっそう望ましい。スフィンゴミエリン、ホスファチジルコリンまたはホスファチジルセリンのいずれか２種以上含む場合には、合計量が上記割合で含まれることが望ましい。
得られたリン脂質組成物は血管内皮機能改善剤や血管内皮機能改善作用を有する食品や飼料の有効成分として用いることができる。
また、上記したリン脂質組成物を各種クロマトグラフィー等の常法により、スフィンゴミエリン、ホスファチジルコリンおよびホスファチジルセリンの純度を高くしたものを調製することも可能である。
The method for recovering the phospholipid composition is not particularly limited, but it is preferable to use a filter press, a decanter or the like. The obtained concentrated solution is not particularly limited, but it is preferable in terms of storage to prepare a powder or paste-like composition by an operation such as freeze-drying or spray-drying.
The other components in the phospholipid composition are not particularly limited. However, according to the method as described above, 15-20% by weight of protein, 10-50% by weight of carbohydrates, and ash content in the total solid. Containing 5 to 10% by weight. Moreover, the water | moisture content in a composition is 5 weight% or less. Moreover, it is desirable that the phospholipid composition contains at least one kind of sphingomyelin, phosphatidylcholine and phosphatidylserine, and the content thereof is 10% by weight or more in the total solid. More desirably, it is 15% or more, and more desirably 20% or more. When any two or more of sphingomyelin, phosphatidylcholine, or phosphatidylserine are included, the total amount is desirably included in the above ratio.
The obtained phospholipid composition can be used as an active ingredient of food or feed having a vascular endothelial function improving agent or a vascular endothelial function improving action.
Moreover, it is also possible to prepare the above-described phospholipid composition with increased purity of sphingomyelin, phosphatidylcholine and phosphatidylserine by various methods such as chromatography.
血管内皮機能改善剤や血管内皮機能改善作用を有する食品や飼料の製造の際には、糖類、タンパク質、ビタミン類、ミネラル類やフレーバー等、他の飲食品や飼料に通常使用される原材料等、安定剤、賦型剤、結合剤、崩壊剤、滑沢剤、懸濁剤、コーティング剤、その他の任意の薬剤を混合した錠剤、カプセル剤、顆粒剤、散剤、粉末剤、シロップ剤等の製剤を用いることができる。
さらに、乳、乳飲料、発酵乳、チーズ、アイスクリームなどの乳製品、パン、スナック菓子、ケーキ、プリン、飲料、麺類、ソーセージ、各種粉乳や離乳食等の飲食品や飼料に配合することも可能である。
In the production of vascular endothelial function improving agent and food and feed having vascular endothelial function improving action, raw materials usually used in other food and drink, such as sugars, proteins, vitamins, minerals and flavors, etc. Stabilizers, excipients, binders, disintegrating agents, lubricants, suspensions, coating agents, and other pharmaceutical preparations such as tablets, capsules, granules, powders, powders, syrups, etc. Can be used.
In addition, it can be added to dairy products such as milk, milk drinks, fermented milk, cheese, ice cream, bread, snacks, cakes, puddings, beverages, noodles, sausages, various milk powders and baby foods, and feeds. is there.
以下に、実施例および試験例を示し、本発明についてより詳細に説明するが、本発明はこれらによって何ら限定されるものではない。 Hereinafter, the present invention will be described in more detail with reference to examples and test examples, but the present invention is not limited to these examples.
バターゼーラムを材料として、スフィンゴミエリン、ホスファチジルコリンおよびホスファチジルセリンの高濃度画分を得た。手順を下記に示す。
バターゼーラム粉（ＳＭ２、Ｃｏｒｍａｎ社製、ベルギー）の２５％溶液を調製し、５Ｍ塩酸を添加してｐＨ４．５に調整した。この溶液を５０℃で１時間静置し、カゼインタンパク質を沈殿させた。フィルタープレスを用いてこの沈殿を除去し、得られた水溶液を孔径１．０μｍのＭＦで処理して濃縮液画分を得た。この乳由来リン脂質含有組成物（実施例品１）は、全固形当たり脂質を５３％、リン脂質を３１％、タンパク質を２４％、糖質を１５％、灰分を８％含有していた。また、リン脂質中の８％がスフィンゴミエリン、８％がホスファチジルコリン、５％がホスファチジルセリンであった。
High concentration fractions of sphingomyelin, phosphatidylcholine and phosphatidylserine were obtained using butaselam. The procedure is shown below.
A 25% solution of butter-lamb flour (SM2, Corman, Belgium) was prepared and adjusted to pH 4.5 by adding 5M hydrochloric acid. This solution was allowed to stand at 50 ° C. for 1 hour to precipitate casein protein. This precipitate was removed using a filter press, and the resulting aqueous solution was treated with MF having a pore size of 1.0 μm to obtain a concentrated liquid fraction. This milk-derived phospholipid-containing composition (Example product 1) contained 53% lipid, 31% phospholipid, 24% protein, 15% carbohydrate, and 8% ash per total solid. Moreover, 8% in the phospholipid was sphingomyelin, 8% was phosphatidylcholine, and 5% was phosphatidylserine.
〔試験例１〕
実施例品１を使用して、乳由来リン脂質組成物の血管内皮細胞の細胞死に与える影響について試験した。
（１）試験方法
ＨＵＶＥＣ細胞（ＫＵＲＡＢＯ社製、東京）のを用いて、培地は、Ｈｕ−Ｍｅｄｉａ−ＥＧ２培地（ＫＵＲＡＢＯ社製）また、１％ＦＢＳを含むＲＰＭＩ１６４０（ＳＩＧＭＡ ＡＬＤＲＩＣＨ社製）を用いた。細胞の培養は、３７℃、５％ＣＯ２インキュベータ内で行い、４〜６世代継代目の細胞を実験に供した。
初発細胞濃度を５×１０５／ｍｌに調整した細胞浮遊液（Ｈｕ−Ｍｅｄｉａ−ＥＧ２を使用）を６ウエルプレート（Ｔｈｅｒｍｏ ｓｃｉｅｎｔｉｆｉｃ社製）の各ウエルに１ｍｌずつ播種した。６時間程度培養し、プレートに接着したことを確認した。その後、乳由来リン脂質組成物のリン酸緩衝液による懸濁液を含む１％ＦＢＳを含むＲＰＩＭ１６４０に交換して、４０時間培養後細胞を回収し、以下の染色を行った。なお、対照群には溶媒であるリン酸緩衝液のみを添加した培地を用いた。図１に示す乳由来リン脂質組成物濃度は、培養液中の濃度を示す。
染色には、ＡｎｎｅｘｉｎＶ−ＦＩＴＣ ｋｉｔ（ＢＤ Ｂｉｏｓｃｉｅｎｃｅｓ社製）とＰＩ（ＳＩＧＭＡ ＡＬＤＲＩＣＨ社）を用いて行った。培養上清を回収し、冷ＰＢＳで細胞を２回洗浄し、１ラｂｉｎｄｉｎｇ ｂｕｆｆｅｒに再浮遊させた。１．５ｍｌのチューブに１００μＬの上記細胞浮遊液とＡｎｎｅｘｉｎＶ試薬を５μＬ、ＰＩを２μＬ加え、穏やかに混和し、室温・暗所で１５分間反応させた。反応液をフローサイトメーター（ＦＡＣＳ Ｃａｌｉｂｅｒ、ＢＤ Ｂｉｏｓｃｉｅｎｃｅｓ社製）で解析を行った。染色された生細胞数の全細胞数に対する割合（％）を図１に示す。
（２）試験結果
培養液中への乳由来リン脂質組成物の添加により、無添加の場合に比べて血管内皮細胞の生細胞の割合が高く、添加濃度に依存して、細胞死の抑制が見られた（図１）。
したがって、本発明の乳由来リン脂質組成物が血管内皮細胞の細胞死を濃度依存的に抑制していることが明らかとなり、血管内皮機能改善剤として用いることが出来ることが分かった。
[Test Example 1]
Example product 1 was used to test the effect of milk-derived phospholipid composition on cell death of vascular endothelial cells.
(1) Test method Using HUVEC cells (manufactured by KURABO, Tokyo), the medium was Hu-Media-EG2 medium (manufactured by KURABO), or RPMI1640 (manufactured by SIGMA ALDRICH) containing 1% FBS. . The cells were cultured in a 37 ° C., 5% CO 2 incubator, and the 4th to 6th generation passage cells were subjected to the experiment.
1 ml of a cell suspension (using Hu-Media-EG2) adjusted to an initial cell concentration of 5 × 10 5 / ml was seeded in each well of a 6-well plate (manufactured by Thermo scientific). It was cultured for about 6 hours and confirmed to adhere to the plate. Then, the cells were recovered after 40 hours of culture by replacing with RPMI1640 containing 1% FBS containing a suspension of a phosphate-derived milk-derived phospholipid composition, and the following staining was performed. In addition, the culture medium which added only the phosphate buffer which is a solvent was used for the control group. The milk-derived phospholipid composition concentration shown in FIG. 1 indicates the concentration in the culture solution.
Staining was performed using AnnexinV-FITC kit (BD Biosciences) and PI (SIGMA ALDRICH). The culture supernatant was collected, the cells were washed twice with cold PBS, and resuspended in a 1 binding buffer. In a 1.5 ml tube, 100 μL of the cell suspension and 5 μL of Annexin V reagent and 2 μL of PI were added, mixed gently, and allowed to react at room temperature in the dark for 15 minutes. The reaction solution was analyzed with a flow cytometer (FACS Caliber, manufactured by BD Biosciences). The ratio (%) of the number of viable stained cells to the total number of cells is shown in FIG.
(2) Test results By adding the milk-derived phospholipid composition to the culture solution, the proportion of living vascular endothelial cells is high compared to the case of no addition, and depending on the added concentration, cell death can be suppressed. It was seen (Figure 1).
Therefore, it became clear that the milk-derived phospholipid composition of the present invention suppressed cell death of vascular endothelial cells in a concentration-dependent manner, and it was found that it can be used as a vascular endothelial function improving agent.
〔試験例２〕
試験例１で乳由来リン脂質組成物が血管内皮細胞の細胞死を抑制することが明らかとなったため、血管内皮細胞内のシグナルの変化を調べた。シグナルとしては、細胞増殖および細胞運動能の亢進の指標であるＥｒｋのリン酸化を測定した。乳由来リン脂質としては、スフィンゴミエリン（長良サイエンス社製、岐阜）、ホスファチジルコリン（長良サイエンス社製、岐阜）またはホスファチジルセリン（長良サイエンス社製、岐阜）を用いて添加濃度は３０μｇ/ｍｌとした。
（１）試験方法
６ウエルプレートに１×１０６／ｍｌのＨＵＶＥＣ細胞を播種し、Ｈｕ−Ｍｅｄｉａ−ＥＧ２培地で一晩培養した。各種の乳由来リン脂質のリン酸緩衝液による懸濁液を添加して１５分間培養した。培養終了後、細胞を回収し、細胞をＰＢＳで洗浄後、ＲＩＰＡ ｌｙｓｉｓ ｂｕｆｆｅｒ（５０ｍＭ Ｔｒｉｓ−ＨＣｌ、ｐＨ７．５、１５０ｍＭ ＮａＣｌ、１％ＮＰ−４０、０．５％ＮａＤｏｃ、０．１％ＳＤＳ）を１００μｌ加え、細胞溶解液を回収した。本溶解液を氷上で１０分間静置後、１５，０００ｒｐｍで５分間遠心分離し、上清を得た。コントロールとして培養液１ｍｌあたり１０μｌ のＮＮ−ジメチルスルホキシドを添加した。上清に６倍量のサンプルバッファー（０．３７５Ｍ Ｔｒｉｓ−ＨＣｌ ｐＨ６．７、１２％ ＳＤＳ、３０％ Ｓｕｃｒｏｓｅ、５％ ２−Ｍｅｒｃａｐｔｏｅｔｈａｎｏｌ、適量のＢｒｏｍｏｐｈｅｎｏｌ ｂｌｕｅ）を加え、９５℃、５分間加熱することでＳＤＳ化を行った。分離ゲル濃度が７．５％のポリアクリルアミドゲルを用いて、常法によりＳＤＳ−ＰＡＧＥを行い、ナイロン膜（Ａｍｅｒｓｈａｍ Ｂｉｏｓｃｉｅｎｃｅｓ、英国）に転写し、ウエスタンブロットを実施した。一次抗体に抗リン酸化Ｅｒｋ抗体（Ｃｅｌｌ Ｓｉｇｎａｌｉｎｇ Ｔｅｃｈｎｏｌｏｇｙ社製、米国）、２次抗体にＨＲＰ標識抗ラビットＩｇＧ抗体（Ｊａｃｋｓｏｎ ＩｍｍｕｎｏＲｅｓｅａｒｃｈ Ｌａｂｏｒａｔｏｒｉｅｓ社製、米国）を用いて、ＬＡＳ３０００ ｍｉｍｉ（ＦＵＪＩＦＩＬＭ社製、東京）によりバンドの検出を行った。
（２）試験結果
ＨＵＶＥＣ細胞を各種の乳由来リン脂質であるスフィンゴミエリン、ホスファチジルコリンまたはホスファチジルセリンで処理することで、Ｅｒｋのリン酸化がいずれにおいても亢進されていることが明らかとなった（図２）。
したがって、本発明の乳由来リン脂質であるスフィンゴミエリン、ホスファチジルコリンまたはホスファチジルセリンが血管内皮細胞のＥｒｋのリン酸化を促進して、血管内皮細胞の細胞死を抑制していることが明らかとなり、血管内皮機能改善剤として用いることが出来ることを確認した。
[Test Example 2]
Since it was revealed in Test Example 1 that the milk-derived phospholipid composition suppresses cell death of vascular endothelial cells, changes in the signal in vascular endothelial cells were examined. As a signal, phosphorylation of Erk, which is an index of enhancement of cell proliferation and cell motility, was measured. As the milk-derived phospholipid, sphingomyelin (manufactured by Nagara Science, Gifu), phosphatidylcholine (manufactured by Nagara Science, Gifu) or phosphatidylserine (manufactured by Nagara Science, Gifu) was used, and the addition concentration was 30 μg / ml.
(1) Test method 1 × 10 6 / ml HUVEC cells were seeded in a 6-well plate and cultured overnight in Hu-Media-EG2 medium. A suspension of various milk-derived phospholipids in a phosphate buffer was added and incubated for 15 minutes. After completion of the culture, the cells were collected, and the cells were washed with PBS, followed by RIPA lysis buffer (50 mM Tris-HCl, pH 7.5, 150 mM NaCl, 1% NP-40, 0.5% NaDoc, 0.1% SDS). Was added to collect cell lysate. The lysate was allowed to stand on ice for 10 minutes and then centrifuged at 15,000 rpm for 5 minutes to obtain a supernatant. As a control, 10 μl of NN-dimethyl sulfoxide was added per 1 ml of the culture solution. Add 6 times the amount of sample buffer (0.375M Tris-HCl pH 6.7, 12% SDS, 30% Sucrose, 5% 2-Mercaptoethanol, appropriate amount of Bromophenol blue) to the supernatant and heat at 95 ° C. for 5 minutes. SDS was performed. Using polyacrylamide gel with a separation gel concentration of 7.5%, SDS-PAGE was performed by a conventional method, transferred to a nylon membrane (Amersham Biosciences, UK), and Western blotting was performed. An anti-phosphorylated Erk antibody (manufactured by Cell Signaling Technology, USA) as the primary antibody and an HRP-labeled anti-rabbit IgG antibody (Jackson ImmunoResearch Laboratories, USA) as the secondary antibody, LAS3000 mimi (manufactured by FUJIFILM, Tokyo) The band was detected by.
(2) Test results It became clear that the phosphorylation of Erk was enhanced by treating HUVEC cells with various milk-derived phospholipids, sphingomyelin, phosphatidylcholine, or phosphatidylserine (FIG. 2). ).
Therefore, it has been clarified that sphingomyelin, phosphatidylcholine or phosphatidylserine, which are milk-derived phospholipids of the present invention, promotes phosphorylation of Erk in vascular endothelial cells and suppresses cell death of vascular endothelial cells. It was confirmed that it can be used as a function improver.
〔試験例３〕
乳由来リン脂質の精製品として、スフィンゴミエリン、ホスファチジルコリンおよびホスファチジルセリンを用いてそれぞれの血管内皮機能の改善効果を評価した。
（１）試験方法
試験例１と同様に長良サイエンス社製のスフィンゴミエリン、ホスファチジルコリンおよびホスファチジルセリンを培養液中の濃度が３０μｇ／ｍｌ及び３００μｇ／ｍｌとなるように培養液１ｍｌあたり１０μｌ添加し、試験例１と同様に血管内皮細胞の細胞死を測定した。コントロールとして培養液１ｍｌあたり１０μｌ のＮＮ−ジメチルスルホキシドを添加した。
（２）試験結果
スフィンゴミエリン、ホスファチジルコリンおよびホスファチジルセリンのいずれかを添加した場合は、コントロールに対して、ＨＵＶＥＣ細胞の生細胞の割合が高く、濃度依存的に細胞死を抑制した。（図３）。
したがって、スフィンゴミエリン、ホスファチジルコリンおよびホスファチジルセリンは、いずれも濃度依存的に血管内皮細胞の細胞死を抑制していることが明らかとなり、血管内皮機能改善剤として用いることが出来ることを確認した。
[Test Example 3]
As refined milk-derived phospholipids, sphingomyelin, phosphatidylcholine and phosphatidylserine were used to evaluate the effect of improving each vascular endothelial function.
(1) Test method In the same manner as in Test Example 1, 10 μl of sphingomyelin, phosphatidylcholine and phosphatidylserine manufactured by Nagara Science Co., Ltd. were added per 1 ml of the culture solution so that the concentrations in the culture solution were 30 μg / ml and 300 μg / ml. In the same manner as in Example 1, cell death of vascular endothelial cells was measured. As a control, 10 μl of NN-dimethyl sulfoxide was added per 1 ml of the culture solution.
(2) Test results When any of sphingomyelin, phosphatidylcholine and phosphatidylserine was added, the proportion of living HUVEC cells was higher than that of the control, and cell death was suppressed in a concentration-dependent manner. (Figure 3).
Therefore, it was revealed that sphingomyelin, phosphatidylcholine, and phosphatidylserine all suppressed cell death of vascular endothelial cells in a concentration-dependent manner, and it was confirmed that they can be used as a vascular endothelial function improving agent.
（血管内皮機能改善用カプセル剤の調製）
表１に示す配合で原材料を混合した後、常法により造粒し、ソフトカプセルに充填して、本発明の血管内皮機能改善用カプセル剤を製造した。
(Preparation of capsules for improving vascular endothelial function)
After mixing the raw materials in the formulation shown in Table 1, the mixture was granulated by a conventional method and filled into a soft capsule to produce a capsule for improving vascular endothelial function of the present invention.
（血管内皮機能改善用錠剤の調製）
表２に示す配合で原材料を混合した後、常法により１ｇに成型、打錠して本発明の血管内皮機能改善用錠剤を製造した。
(Preparation of tablets for improving vascular endothelial function)
After mixing the raw materials with the formulation shown in Table 2, the tablets for vascular endothelial function improvement of the present invention were produced by molding and tableting into 1 g by a conventional method.
（血管内皮機能改善用液状栄養組成物）
実施例品１の乳由来リン脂質組成物５０ｇを４，９５０ｇの脱イオン水に溶解し、５０℃まで加熱後、ＴＫホモミクサー（ＴＫ ＲＯＢＯ ＭＩＣＳ；特殊機化工業社製）にて、６,０００ｒｐｍで３０分間撹拌混合して５０ｇ／５ｋｇのバターミルク溶液を得た。このバターミルク溶液５．０ｋｇに、カゼイン５．０ｋｇ、大豆タンパク質５．０ｋｇ、魚油１．０ｋｇ、シソ油３．０ｋｇ、デキストリン１７．０ｋｇ、ミネラル混合物６．０ｋｇ、ビタミン混合物１．９５ｋｇ、乳化剤２．０ｋｇ、安定剤４．０ｋｇ、香料０．０５ｋｇを配合し、２００ｍｌのレトルトパウチに分注し、レトルト殺菌機 (第１種圧力容器、ＴＹＰＥ: ＲＣＳ−４ＣＲＴＧＮ、日阪製作所製)で１２１℃、２０分間殺菌して、本発明の血管内皮機能改善用液状栄養組成物５０ｋｇを製造した。
(Liquid nutritional composition for improving vascular endothelial function)
50 g of the milk-derived phospholipid composition of Example Product 1 was dissolved in 4,950 g of deionized water, heated to 50 ° C., and then at 6,000 rpm with a TK homomixer (TK ROBO MICS; manufactured by Tokushu Kika Kogyo Co., Ltd.). And stirred for 30 minutes to obtain a 50 g / 5 kg buttermilk solution. 5.0 kg of this buttermilk solution, 5.0 kg of casein, 5.0 kg of soy protein, 1.0 kg of fish oil, 3.0 kg of perilla oil, 17.0 kg of dextrin, 6.0 kg of mineral mixture, 1.95 kg of vitamin mixture, emulsifier 2 1.0 kg, 4.0 kg stabilizer, 0.05 kg fragrance, dispensed into 200 ml retort pouch, 121 ° C. with retort sterilizer (type 1 pressure vessel, TYPE: RCS-4CRTGN, manufactured by Nisaka Seisakusho) Sterilized for 20 minutes to produce 50 kg of the liquid nutritional composition for improving vascular endothelial function of the present invention.
（血管内皮機能改善用飲料の製造）
脱脂粉乳３００ｇを４００ｇの脱イオン水に溶解した後、実施例品１の乳由来リン脂質組成物１０ｇを溶解し、５０℃まで加熱後、ウルトラディスパーサー（ＵＬＴＲＡ−ＴＵＲＲＡＸ Ｔ−２５；ＩＫＡジャパン社製）にて、９,５００ｒｐｍで３０分間撹拌混合した。マルチトール１００ｇ、酸味料２ｇ、還元水飴２０ｇ、香料２ｇ、脱イオン水１６６ｇを添加した後、１００ｍｌのガラス瓶に１００ｇずつ充填し、９５℃、１５秒間殺菌後、密栓し、本発明の血管内皮機能改善用飲料１０本（１００ｇ入り）を調製した。
(Manufacture of beverages for improving vascular endothelial function)
After dissolving 300 g of skim milk powder in 400 g of deionized water, 10 g of the milk-derived phospholipid composition of Example Product 1 was dissolved, heated to 50 ° C., and then Ultradisperser (ULTRA-TURRAX T-25; IKA Japan) The mixture was stirred and mixed at 9,500 rpm for 30 minutes. After adding 100 g maltitol, 2 g acidulant, 20 g reduced starch syrup, 2 g fragrance, and 166 g deionized water, each 100 g glass bottle is filled with 100 g, sterilized at 95 ° C. for 15 seconds, sealed, and vascular endothelial function of the present invention Ten drinks for improvement (with 100 g) were prepared.
Claims (5)
乳又は乳素材を原材料とし、孔径が０．１〜２μｍの分離膜または、分画分子量５〜５００ｋＤａの分離膜で処理する工程を含む請求項（１）〜（３）に記載の製造方法。 A method for producing a vascular endothelial function improving agent comprising as an active ingredient at least one selected from the group consisting of sphingomyelin, phosphatidylcholine and phosphatidylserine,
The production method according to any one of claims 1 to 3, further comprising a step of treating with milk or a milk material as a raw material and a separation membrane having a pore diameter of 0.1 to 2 µm or a separation membrane having a fractional molecular weight of 5 to 500 kDa.
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| JP2020068664A (en) * | 2018-10-29 | 2020-05-07 | 株式会社Adeka | Milk-derived phospholipid-containing composition |
| JP7350476B2 (en) | 2018-10-29 | 2023-09-26 | 株式会社Adeka | Milk-derived phospholipid-containing composition |
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