JP2016175857A - Collagen production-promoting compositions which contain candida utilis-derived glucosylceramide - Google Patents
Collagen production-promoting compositions which contain candida utilis-derived glucosylceramide Download PDFInfo
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- JP2016175857A JP2016175857A JP2015056421A JP2015056421A JP2016175857A JP 2016175857 A JP2016175857 A JP 2016175857A JP 2015056421 A JP2015056421 A JP 2015056421A JP 2015056421 A JP2015056421 A JP 2015056421A JP 2016175857 A JP2016175857 A JP 2016175857A
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- glucosylceramide
- collagen
- collagen production
- yeast
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- 150000002305 glucosylceramides Chemical class 0.000 title claims abstract description 53
- 102000008186 Collagen Human genes 0.000 title claims abstract description 40
- 108010035532 Collagen Proteins 0.000 title claims abstract description 40
- 229920001436 collagen Polymers 0.000 title claims abstract description 40
- 239000000203 mixture Substances 0.000 title claims abstract description 34
- 241000235646 Cyberlindnera jadinii Species 0.000 title abstract description 5
- 230000037319 collagen production Effects 0.000 claims abstract description 33
- 108090000765 processed proteins & peptides Proteins 0.000 claims abstract description 31
- 230000001737 promoting effect Effects 0.000 claims abstract description 18
- 239000002537 cosmetic Substances 0.000 claims abstract description 10
- 235000013305 food Nutrition 0.000 claims abstract description 10
- 241000006364 Torula Species 0.000 claims description 16
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- 238000002360 preparation method Methods 0.000 claims description 5
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- 230000000694 effects Effects 0.000 abstract description 19
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- 238000000605 extraction Methods 0.000 description 15
- 239000000284 extract Substances 0.000 description 10
- 229940041514 candida albicans extract Drugs 0.000 description 8
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- 101000925662 Enterobacteria phage PRD1 Endolysin Proteins 0.000 description 1
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- CRJGESKKUOMBCT-VQTJNVASSA-N N-acetylsphinganine Chemical compound CCCCCCCCCCCCCCC[C@@H](O)[C@H](CO)NC(C)=O CRJGESKKUOMBCT-VQTJNVASSA-N 0.000 description 1
- 229910002651 NO3 Inorganic materials 0.000 description 1
- NHNBFGGVMKEFGY-UHFFFAOYSA-N Nitrate Chemical compound [O-][N+]([O-])=O NHNBFGGVMKEFGY-UHFFFAOYSA-N 0.000 description 1
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- LFVGISIMTYGQHF-UHFFFAOYSA-N ammonium dihydrogen phosphate Chemical compound [NH4+].OP(O)([O-])=O LFVGISIMTYGQHF-UHFFFAOYSA-N 0.000 description 1
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- 239000005018 casein Substances 0.000 description 1
- BECPQYXYKAMYBN-UHFFFAOYSA-N casein, tech. Chemical compound NCCCCC(C(O)=O)N=C(O)C(CC(O)=O)N=C(O)C(CCC(O)=N)N=C(O)C(CC(C)C)N=C(O)C(CCC(O)=O)N=C(O)C(CC(O)=O)N=C(O)C(CCC(O)=O)N=C(O)C(C(C)O)N=C(O)C(CCC(O)=N)N=C(O)C(CCC(O)=N)N=C(O)C(CCC(O)=N)N=C(O)C(CCC(O)=O)N=C(O)C(CCC(O)=O)N=C(O)C(COP(O)(O)=O)N=C(O)C(CCC(O)=N)N=C(O)C(N)CC1=CC=CC=C1 BECPQYXYKAMYBN-UHFFFAOYSA-N 0.000 description 1
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Abstract
Description
本発明は、トルラ酵母由来グルコシルセラミドと、コラーゲンペプチドを利用したコラーゲン産生促進効果に優れた組成物を提供するものである。 The present invention provides a composition excellent in the effect of promoting collagen production using Torula yeast-derived glucosylceramide and a collagen peptide.
真皮の主要なマトリックス成分であるコラーゲン線維の減少は、皮膚の厚みを減少させ、しわ・たるみの要因の一つとなっている。従って、しわやたるみの予防、改善のためにはコラーゲンの減少を防ぐことが重要であると考えられる。 The decrease in collagen fibers, the main matrix component of the dermis, reduces the thickness of the skin and is one of the causes of wrinkles and sagging. Therefore, it is considered important to prevent the decrease of collagen for the prevention and improvement of wrinkles and sagging.
コラーゲンを酵素分解などにより低分子量化し、吸収性を高めたコラーゲンペプチドが、コラーゲンの産生を促進する美容素材として知られ（例えば非特許文献１）、化粧品やサプリメントなどに多用されている。しかしながら、十分な効果を発現、実感させるためにはコラーゲンペプチドを大量摂取する必要がある。一方で、低分子量化したコラーゲンペプチドは特有の風味、臭気を有することから、使用量が制限される。従って、コラーゲンペプチドによるコラーゲン産生促進効果を増強させることが重要であると考えられる。 Collagen peptides having a low molecular weight by enzymatic degradation or the like and enhanced absorbability are known as cosmetic materials that promote collagen production (for example, Non-Patent Document 1), and are widely used in cosmetics and supplements. However, it is necessary to ingest a large amount of collagen peptide in order to realize and realize a sufficient effect. On the other hand, since the collagen peptide having a low molecular weight has a unique flavor and odor, the amount used is limited. Therefore, it is considered important to enhance the collagen production promoting effect by the collagen peptide.
グルコシルセラミドとは、スフィンゴイド塩基と脂肪酸がアミド結合したセラミド骨格に、１分子のグルコースが結合したスフィンゴ糖脂質の一種である。動植物や微生物に幅広く分布し、サプリメント等で摂取した場合、肌機能の改善効果（非特許文献２）や、大腸がんの予防効果（特許文献１）があることなどから近年、健康食品素材として注目を集めている。 Glucosylceramide is a type of glycosphingolipid in which one molecule of glucose is bound to a ceramide skeleton in which a sphingoid base and a fatty acid are amide-bonded. As a health food ingredient in recent years, it is widely distributed in animals, plants and microorganisms, and when taken as a supplement, it has an effect of improving skin function (Non-patent Document 2) and a preventive effect on colon cancer (Patent Document 1). It attracts attention.
本発明の課題は、コラーゲンペプチドのコラーゲン産生促進効果が相乗的に増強されて発揮でき、食品分野や化粧品、医薬品分野にて有用な組成物を提供することである。 An object of the present invention is to provide a composition useful in the field of food, cosmetics and pharmaceuticals, which can synergistically enhance the collagen production promoting effect of the collagen peptide.
本発明では、ヒト線維芽細胞におけるコラーゲン産生促進効果を確認したところ、コラーゲンペプチドとトルラ酵母由来のグルコシルセラミドを併用することで、コラーゲン産生効果が相乗的に増強されることを見出した。 In this invention, when the collagen production promotion effect in a human fibroblast was confirmed, it discovered that a collagen production effect was synergistically enhanced by using together a collagen peptide and glucosylceramide derived from Torula yeast.
また本発明では、コラーゲンペプチドとトルラ酵母由来のグルコシルセラミドとの併用による線維芽細胞におけるコラーゲン産生促進効果が、コメ、コーン、コンニャク由来のグルコシルセラミドとコラーゲンペプチドとの併用による効果に比較して優れていることを見出した。 In the present invention, the collagen production promotion effect in fibroblasts by the combined use of collagen peptide and Torula yeast-derived glucosylceramide is superior to the effect of the combined use of rice, corn, konjac glucosylceramide and collagen peptide. I found out.
本発明によると、コラーゲンペプチドに微量のトルラ酵母由来グルコシルセラミドを併用することで相乗的なコラーゲン産生促進効果が得られ、一般的なコラーゲン産生促進剤であるコラーゲンペプチドのコラーゲン産生促進効果を効率的に促進することが出来る。 According to the present invention, a synergistic collagen production promotion effect can be obtained by using a small amount of Torula yeast-derived glucosylceramide in combination with a collagen peptide, and the collagen production promotion effect of a collagen peptide, which is a general collagen production promoter, can be efficiently achieved. Can be promoted.
また本発明により、トルラ酵母由来グルコシルセラミドとコラーゲンペプチドの併用によるコラーゲン産生促進効果はコメ、コーン、こんにゃく由来のグルコシルセラミドとコラーゲンペプチドの併用による効果よりも優れていることを確認した。 In addition, according to the present invention, it was confirmed that the effect of promoting the production of collagen by the combined use of Torula yeast-derived glucosylceramide and collagen peptide is superior to the effect of the combined use of glucosylceramide derived from rice, corn, and konjac and collagen peptide.
さらに、トルラ酵母由来グルコシルセラミドは、トルラ酵母から酵母エキスを抽出して得られた酵母菌体から得ることが可能であり、酵母菌体を有効利用できるため、コスト、廃棄物削減の点でも、極めて有利で安価なコラーゲン産生促進組成物を提供することが可能である。 Furthermore, Torula yeast-derived glucosylceramide can be obtained from yeast cells obtained by extracting yeast extract from Torula yeast, and since yeast cells can be used effectively, in terms of cost and waste reduction, It is possible to provide a highly advantageous and inexpensive composition for promoting collagen production.
本発明で使用するコラーゲンペプチドとは、動物や魚介類、主に牛、豚、魚由来のコラーゲンを分解して低分子化したものであり、起原や分子量、組成は特に限定されるものではないが、食品衛生上、薬学的、又は香粧的に許容されるものが望ましい。 Collagen peptides used in the present invention are those obtained by decomposing and degrading collagen derived from animals and seafood, mainly cattle, pigs, and fish. Origin, molecular weight, and composition are not particularly limited. None, but food hygiene, pharmaceutically or cosmetically acceptable are desirable.
本発明で使用する酵母は、グルコシルセラミドを含有する酵母であればよい。特に好ましくは、一般名トルラ酵母と称されるＣａｎｄｉｄａ ｕｔｉｌｉｓである。酵母の培養形式はバッチ培養、あるいは連続培養のいずれかが用いられる。培地には炭素源として、ブドウ糖、酢酸、エタノール、グリセロール、糖蜜、亜硫酸パルプ廃液等が用いられ、窒素源としては、尿素、アンモニア、硫酸アンモニウム、塩化アンモニウム、硝酸塩などが使用される。リン酸、カリウム、マグネシウム源としては例えば過リン酸石灰、リン酸アンモニウム、塩化カリウム、水酸化カリウム、硫酸マグネシウム、塩化マグネシウム等が使用でき、その他亜鉛、銅、マンガン、鉄イオン等の無機塩を添加する。その他、ビタミン、アミノ酸、核酸関連物質等を添加したり、カゼイン、酵母エキス、肉エキス、ペプトン等の有機物を添加しても良い。培養温度は２１〜３７℃、好ましくは２５〜３４℃で、ｐＨは３．０〜８．０、特に３．５〜７．０が好ましい。培養条件によりアミノ酸や核酸の生産性が変動するため、目的とする酵母エキスの製品仕様に合わせて条件設定を行う。 The yeast used in the present invention may be a yeast containing glucosylceramide. Particularly preferred is Candida utilis, commonly called Torula yeast. As the culture format of yeast, either batch culture or continuous culture is used. In the medium, glucose, acetic acid, ethanol, glycerol, molasses, sulfite pulp waste liquor and the like are used as the carbon source, and urea, ammonia, ammonium sulfate, ammonium chloride, nitrate and the like are used as the nitrogen source. Examples of phosphoric acid, potassium, and magnesium sources include lime perphosphate, ammonium phosphate, potassium chloride, potassium hydroxide, magnesium sulfate, magnesium chloride, and other inorganic salts such as zinc, copper, manganese, and iron ions. Added. In addition, vitamins, amino acids, nucleic acid-related substances, etc. may be added, or organic substances such as casein, yeast extract, meat extract, and peptone may be added. The culture temperature is 21 to 37 ° C., preferably 25 to 34 ° C., and the pH is preferably 3.0 to 8.0, particularly 3.5 to 7.0. Since the productivity of amino acids and nucleic acids varies depending on the culture conditions, conditions are set according to the product specifications of the target yeast extract.
酵母菌体培養後に集菌し、得られた菌体から酵母エキスの抽出を行う。本願では、エキス抽出後の酵母を酵母残渣としている。酵母エキスの抽出法は、特に制限がないが、一般的に、自己消化法、熱水抽出法、酵素抽出法、酸、若しくはアルカリ抽出法、又はこれらの組み合わせにより行うことが可能である。 Bacteria are collected after culturing the yeast cells, and the yeast extract is extracted from the obtained cells. In the present application, the yeast after extraction of the extract is used as the yeast residue. The extraction method of the yeast extract is not particularly limited, but can generally be performed by an autolysis method, a hot water extraction method, an enzyme extraction method, an acid or alkali extraction method, or a combination thereof.
自己消化により酵母エキスを抽出する場合は、例えば集菌後洗浄して得られた菌体懸濁液を適温で適当な時間攪拌する。酵素抽出法であれば、細胞壁溶解酵素又はプロテアーゼ等で攪拌抽出する。酸抽出法であれば、硫酸等で酸性に調整後、抽出する。アルカリ抽出法であれば、アルカリに調整後、抽出する。又は、自己消化後に、酵素抽出をする等の組み合わせも可能である。 When extracting the yeast extract by self-digestion, for example, the cell suspension obtained by washing after collection is stirred at an appropriate temperature for an appropriate time. In the case of an enzyme extraction method, stirring and extraction is performed with a cell wall lytic enzyme or protease. In the case of an acid extraction method, extraction is performed after acidification with sulfuric acid or the like. If it is an alkali extraction method, it will extract after adjusting to an alkali. Alternatively, combinations such as enzyme extraction after autolysis are possible.
上記の酵母中のエキス抽出により、タンパク質や核酸が抽出除去されると共に、ステロール配糖体など一部の夾雑物が除去され、結果としてグルコシルセラミドが富化された酵母残渣が製造される。 Extraction of the above-mentioned yeast extract extracts and removes proteins and nucleic acids, and also removes some impurities such as sterol glycosides. As a result, a yeast residue enriched with glucosylceramide is produced.
エキス抽出後は遠心分離で酵母残渣を回収し、残渣を水に懸濁して遠心分離を行うことで洗浄し、このような洗浄を１回または複数回繰り返す。洗浄後の残渣を必要に応じて、凍結乾燥又は熱風乾燥することも可能である。得られた酵母残渣をトルラ酵母由来グルコシルセラミド原料とする。 After extraction of the extract, the yeast residue is recovered by centrifugation, washed by suspending the residue in water and centrifuging, and such washing is repeated one or more times. If necessary, the residue after washing can be freeze-dried or hot-air dried. Let the obtained yeast residue be a Torula yeast origin glucosylceramide raw material.
続いて上記原料を用いてグルコシルセラミドの精製を行う。精製の方法に制限はないが、食品として用いることができる精製法が望ましい。例えば、特開２００２−２８１９３６号公報に記載されている方法で精製することができる。アルコール抽出を行う場合は、トルラ酵母由来グルコシルセラミド原料の乾燥質量に対して約２倍量の９０％エタノールを使用し、攪拌によりグルコシルセラミドの抽出を行う。抽出後は抽出液を遠心分離で回収し、濃縮及び凍結乾燥または熱風乾燥させることでグルコシルセラミド組成物が得られる。なお、本願では、グルコシルセラミドの定性分析をＴＬＣで、定量分析をＨＰＬＣ−ＥＬＳＤで行った。 Subsequently, glucosylceramide is purified using the above raw materials. Although there is no restriction | limiting in the purification method, The purification method which can be used as a foodstuff is desirable. For example, it can be purified by the method described in JP-A-2002-281936. When alcohol extraction is performed, 90% ethanol of about twice the dry mass of the Torula yeast-derived glucosylceramide raw material is used, and glucosylceramide is extracted by stirring. After extraction, the extract is collected by centrifugation, concentrated and freeze-dried or hot-air dried to obtain a glucosylceramide composition. In the present application, qualitative analysis of glucosylceramide was performed by TLC, and quantitative analysis was performed by HPLC-ELSD.
必要に応じて、グルコシルセラミド組成物をさらに精製することにより、グルコシルセラミド含有量の高い組成物を製造することもできる。精製法は、既知の方法により精製可能であり、例えば、シリカゲルやイオン交換樹脂などのカラム精製、又はアルカリ処理や溶媒分画等を用いることができる。 If necessary, a composition having a high glucosylceramide content can also be produced by further purifying the glucosylceramide composition. The purification method can be purified by a known method. For example, column purification such as silica gel or ion exchange resin, alkali treatment, solvent fractionation, or the like can be used.
このようにして得られたトルラ酵母由来のグルコシルセラミドは、コラーゲンペプチドと組み合わせて用いることにより、コラーゲンペプチドのコラーゲン産生促進効果を相乗的に増強することが出来、これらを両方含有するものをコラーゲン産生促進組成物とする。 The glucosylceramide derived from Torula yeast thus obtained can be used in combination with a collagen peptide to synergistically enhance the collagen production promoting effect of the collagen peptide. Accelerating composition.
該組成物における、酵母由来グルコシルセラミドとコラーゲンペプチドの含有量の比は特に限定しないが、コラーゲンペプチドに対して微量の酵母由来グルコシルセラミドを配合しただけでも相乗的なコラーゲン産生促進効果を奏する。望ましい含有量の比は、コラーゲンペプチド１００質量部に対して、酵母由来のグルコシルセラミドが０．１〜１００質量部、より望ましくは０．５〜５０質量部、さらに望ましくは５〜３０質量部である。 The ratio of the content of the yeast-derived glucosylceramide and the collagen peptide in the composition is not particularly limited, but a synergistic collagen production promoting effect can be obtained by adding a trace amount of yeast-derived glucosylceramide to the collagen peptide. A desirable content ratio is 0.1 to 100 parts by mass, more preferably 0.5 to 50 parts by mass, and further preferably 5 to 30 parts by mass of glucosylceramide derived from yeast with respect to 100 parts by mass of collagen peptide. is there.
本発明のコラーゲン産生促進組成物は、飲食品、化粧品、皮膚外用剤又は医薬品に適用することが出来る。 The composition for promoting collagen production of the present invention can be applied to foods and drinks, cosmetics, external preparations for skin, or pharmaceuticals.
本発明における飲食品とは、食料品、飲料品、嗜好品、サプリメント等、経口で摂取するものを指す。これらに、本発明のコラーゲン産生促進組成物を含有させることができるが、その形態は特に限定されるものではなく、パン類、麺類等主菜となりうるもの、チーズ、ハム、ウインナー、魚介加工品等副菜となりうるもの、果汁飲料、炭酸飲料、乳飲料等の飲料、クッキー、ケーキ、ゼリー、プリン、キャンディー、ヨーグルト等の嗜好品等とすることができる。また、サプリメントとしての形態も特に限定されるものではなく、錠剤、カプセル、ソフトカプセル、栄養ドリンク状の形態をとることもできる。 The food and drink in the present invention refers to foods, beverages, luxury goods, supplements and the like that are taken orally. These can contain the composition for promoting collagen production of the present invention, but the form is not particularly limited, and can be a main dish such as breads and noodles, cheese, ham, wiener, processed seafood It can be used as a side dish such as fruit juice beverages, carbonated beverages, milk beverages, etc., and favourites such as cookies, cakes, jelly, pudding, candy, yogurt and the like. Moreover, the form as a supplement is not specifically limited, It can also take the form of a tablet, a capsule, a soft capsule, and an energy drink form.
飲食品における上記組成物の配合量は特に限定されるものではなく、例えば該コラーゲン産生促進組成物が飲食品の０．００１〜５０質量％含まれていればよい。中でも０．０１〜１０質量％が好適であり、０．１〜５質量％はさらに好適である。 The compounding quantity of the said composition in food / beverage products is not specifically limited, For example, this collagen production promotion composition should just be contained 0.001-50 mass% of food / beverage products. Among these, 0.01 to 10% by mass is preferable, and 0.1 to 5% by mass is more preferable.
本発明の化粧品は、上記組成物を配合した化粧水、乳液、ファンデーション、口紅などを指す。 The cosmetics of the present invention refer to lotions, emulsions, foundations, lipsticks and the like containing the above composition.
化粧品における上記組成物の配合量は特に限定されるものではなく、例えば該コラーゲン産生促進組成物が化粧品の０．００１〜５０質量％含まれていればよい。中でも０．０１〜１０質量％が好適であり、０．１〜５質量％はさらに好適である。 The compounding quantity of the said composition in cosmetics is not specifically limited, For example, this collagen production promotion composition should just be contained 0.001-50 mass% of cosmetics. Among these, 0.01 to 10% by mass is preferable, and 0.1 to 5% by mass is more preferable.
また本発明の皮膚外用剤又は医薬品とは、上記組成物を配合したローション、クリーム、軟膏、スプレー、貼付剤（材）などの形状のものを指すが、その形態は特に限定されるものではなく、本発明の目的とする効果を発揮しうるものであればいかなる形態でもかまわない。 The topical skin preparation or pharmaceutical agent of the present invention refers to a shape such as a lotion, cream, ointment, spray, or patch (material) containing the above composition, but the form is not particularly limited. Any form may be used as long as it can exert the intended effect of the present invention.
皮膚外用剤又は医薬品における上記組成物の配合量の配合量は特に限定されるものではなく、例えば上記組成物が皮膚外用剤又は医薬品の０．００１〜５０質量％含まれていればよい。中でも０．０１〜１０質量％が好適であり、０．１〜５質量％はさらに好適である。 The blending amount of the composition in the external preparation for skin or medicine is not particularly limited. For example, the composition may be contained in an amount of 0.001 to 50% by mass of the external preparation for skin or medicine. Among these, 0.01 to 10% by mass is preferable, and 0.1 to 5% by mass is more preferable.
以下に、実施例を挙げて本発明を更に詳細に説明するが、本発明はこれら実施例に限定されるものではない。 Hereinafter, the present invention will be described in more detail with reference to examples. However, the present invention is not limited to these examples.
（酵母の培養）
Ｃａｎｄｉｄａ ｕｔｉｌｉｓ ＣＳ７５２９株（ＦＥＲＭＰ−３３４０）を予めＹＰＤ培地（酵母エキス１％、ポリペプトン２％、グルコース２％）を含む三角フラスコで種母培養し、これを３０Ｌ容発酵槽に１８Ｌ培地に１〜２％植菌した。培地組成は、グルコース４％、燐酸一アンモニウム０．３％、硫酸アンモニウム０．１６１％、塩化カリウム０．１３７％、硫酸マグネシウム０．０８％、硫酸銅１．６ｐｐｍ、硫酸鉄１４ｐｐｍ、硫酸マンガン１６ｐｐｍ、硫酸亜鉛１４ｐｐｍを用いた。培養条件は、ｐＨ４．０、培養温度３０℃、通気量１ｖｖｍ、撹拌６００ｒｐｍで行い、アンモニアを添加しｐＨのコントロールを行った。１６時間菌体培養した後、培養液を回収し、遠心分離により集菌し、湿潤酵母菌体を得た。得られた湿潤菌体は、蒸留水に懸濁して遠心分離を繰り返すことで洗浄し、熱風乾燥させた。
(Yeast culture)
Candida utilis CS7529 strain (FERMP-3340) was seeded in an Erlenmeyer flask containing YPD medium (yeast extract 1%, polypeptone 2%, glucose 2%) in advance, and this was cultured in an 18 L medium in a 30 L fermentor. % Inoculated. Medium composition is 4% glucose, 0.3% monoammonium phosphate, 0.161% ammonium sulfate, 0.137% potassium chloride, 0.08% magnesium sulfate, 1.6 ppm copper sulfate, 14 ppm iron sulfate, 16 ppm manganese sulfate, 14 ppm of zinc sulfate was used. The culture conditions were pH 4.0, culture temperature 30 ° C., aeration rate 1 vvm, stirring 600 rpm, and ammonia was added to control the pH. After culturing the cells for 16 hours, the culture solution was collected and collected by centrifugation to obtain wet yeast cells. The obtained wet cells were washed by suspending in distilled water and repeating the centrifugal separation, and dried with hot air.
（酵母残渣の取得）
熱風乾燥させたトルラ酵母の乾燥菌体５ｋｇを蒸留水５０Ｌに懸濁し、２Ｎ ＮａＯＨでｐＨ１３．０に調整した後、７０℃で３０分間撹拌抽出した。エキス抽出後は遠心分離で酵母残渣を回収し、酵母残渣の蒸留水への懸濁と遠心分離を３回繰り返すことで洗浄した。洗浄後は酵母残渣を真空乾燥することで３．３ｋｇのエキス抽出酵母残渣が得られた。
(Acquisition of yeast residue)
5 kg of dried cells of Torula yeast dried with hot air were suspended in 50 L of distilled water, adjusted to pH 13.0 with 2N NaOH, and then stirred and extracted at 70 ° C. for 30 minutes. After extraction, the yeast residue was collected by centrifugation, and washed by suspending the yeast residue in distilled water and centrifuging three times. After washing, the yeast residue was vacuum dried to obtain 3.3 kg of extract-extracted yeast residue.
（グルコシルセラミドの精製）
得られた酵母残渣全量を２倍量の９０%エタノールに懸濁し、６０℃で１０時間撹拌してグルコシルセラミドを抽出した。遠心分離により抽出液を回収し、酵母残渣をエタノールで３回洗浄した洗浄液と合わせて濃縮した結果、抽出物３００ｇが得られた。これをグルコシルセラミド含有組成物とし、ＨＰＬＣ−ＥＬＳＤで分析した結果、グルコシルセラミドが３．０％含有されていた。またＴＬＣによる分析の結果、夾雑物のステロール配糖体は確認されなかった。コラーゲン産生促進効果の確認用のサンプルには、上記抽出物１５ｇをエタノールに溶解し、シリカカラムを用いて精製することでグルコシルセラミドを５０％含有する組成物０．６ｇが得られた。得られた酵母由来グルコシルセラミド組成物を用いてコラーゲン産生促進作用の評価を行った。
(Purification of glucosylceramide)
The total amount of the obtained yeast residue was suspended in twice the amount of 90% ethanol, and stirred at 60 ° C. for 10 hours to extract glucosylceramide. The extract was collected by centrifugation and concentrated together with a wash obtained by washing the yeast residue with ethanol three times. As a result, 300 g of extract was obtained. This was used as a glucosylceramide-containing composition and analyzed by HPLC-ELSD. As a result, 3.0% glucosylceramide was contained. Further, as a result of analysis by TLC, no sterol glycoside as a contaminant was confirmed. As a sample for confirming the effect of promoting collagen production, 0.6 g of a composition containing 50% glucosylceramide was obtained by dissolving 15 g of the extract in ethanol and purifying it using a silica column. Collagen production promoting action was evaluated using the obtained yeast-derived glucosylceramide composition.
（ＨＰＬＣ−ＥＬＳＤ条件）
グルコシルセラミドの定量には高速液体クロマトグラフィー（ＨＰＬＣ）を用いた。カラムにはＧＬサイエンス社製Ｉｎｅｒｔｓｉｌ １００Ａを用い、グルコシルセラミドの検出は蒸発光散乱検出器（島津製ＥＬＳＤ−ＬＴＩＩ）で行った。溶出溶媒にはクロロホルム／メタノール：水＝９５：５（容量比）のグラジエントを用いた。カラム温度は３５℃、流速は１ｍＬ／ｍｉｎ、ドリフトチューブ温度は４０℃で窒素ガス圧力は３５０ｋＰａであった。グルコシルセラミド含有量は、グルコシルセラミド標準品（Ｇｌｕｃｏｃｅｌｅｂｒｏｓｉｄｅｓ、マトレヤ社製）及びグルコシルセラミド含有組成物をそれぞれ解析して得られるクロマトグラムにおける、グルコシルセラミドを示すピークの面積比より算出した。
(HPLC-ELSD conditions)
High performance liquid chromatography (HPLC) was used for quantification of glucosylceramide. The column was Inertsil 100A manufactured by GL Sciences, and glucosylceramide was detected with an evaporative light scattering detector (ELSD-LTII manufactured by Shimadzu). The elution solvent used was a gradient of chloroform / methanol: water = 95: 5 (volume ratio). The column temperature was 35 ° C., the flow rate was 1 mL / min, the drift tube temperature was 40 ° C., and the nitrogen gas pressure was 350 kPa. The glucosylceramide content was calculated from the area ratio of peaks indicating glucosylceramide in chromatograms obtained by analyzing a glucosylceramide standard product (Glucocerebrosides, manufactured by Matreya) and a glucosylceramide-containing composition, respectively.
（ＴＬＣ条件）
ＴＬＣはシリカゲルプレート（メルク社製Ｓｉｌｉｃａｇｅｌ ６０、層厚０．２５ｍｍ）を使用し、クロロホルム：メタノール：水＝６５：１６：２（容量比）で展開した。展開後はシリカゲルプレートを乾燥させ、アニスアルデヒド硫酸試薬を噴霧して加熱することで発色させた。
(TLC conditions)
TLC was developed using a silica gel plate (Silica Gel 60 manufactured by Merck & Co., Ltd., layer thickness: 0.25 mm) with chloroform: methanol: water = 65: 16: 2 (volume ratio). After the development, the silica gel plate was dried and developed by spraying with anisaldehyde sulfuric acid reagent and heating.
トルラ酵母、コメ、コーン、又はコンニャク由来グルコシルセラミドそれぞれ（単独区）のコラーゲン産生促進効果、及びそれらをコラーゲンペプチドと混合したもの（併用区）のコラーゲン産生促進効果を評価した。コメ由来グルコシルセラミドには、市販の米由来グルコシルセラミド３％含有組成物、コーン由来グルコシルセラミドには、市販のコーン由来グルコシルセラミド３％含有組成物、コンニャク由来グルコシルセラミドには、市販のコンニャク由来グルコシルセラミド３％含有組成物を使用した。 The collagen production promoting effect of Torula yeast, rice, corn, or konjac-derived glucosylceramide (single section) and the mixture of these with a collagen peptide (combined section) were evaluated. Rice-derived glucosylceramide contains 3% commercially available rice-derived glucosylceramide-containing composition, corn-derived glucosylceramide contains 3% commercially available corn-derived glucosylceramide-containing composition, and konjac-derived glucosylceramide contains commercially available konjac-derived glucosyl. A composition containing 3% ceramide was used.
（ヒト線維芽細胞のコラーゲン産生量の評価）
正常ヒト線維芽細胞を２４ウェルプレートに播種（６×１０４ｃｅｌｌｓ／ｗｅｌｌ）し、２４時間前培養した（５％ ＣＯ２、３７℃）。培養液は、１％牛胎児血清を含むＤ−ＭＥＭ培地を使用した。その後、各原料由来グルコシルセラミド、コラーゲンペプチドをそれぞれ最終濃度が１０μｇ／ｍＬ、２００μｇ/ｍＬとなるように添加した新鮮な培地に交換し、７２時間培養した。対照は何も添加しない新鮮な培地に交換した。培養後、培養上清を１００μＬ採取し、０．５Ｍ酢酸に溶解した０．１質量％のシリウスレッド溶液３００μＬを上記の培養上清に加えて振とう後、１時間室温で染色した。これを１００００ｒｐｍで５分間遠心分離し、上清を捨てた後、コラーゲンとシリウスレッドの複合体形成による沈殿を回収した。回収した沈殿を１０ｍＭ ＨＣｌで２回洗浄した後、０．１Ｎ ＮａＯＨに溶解したものをサンプルとして、マイクロプレートリーダーを用いて５４０ｎｍの吸光度を測定した。対照の吸光度をコラーゲン産生率１００％とし、各サンプルを添加した培養上清の吸光度からそれぞれのコラーゲン産生率を求めた。
(Evaluation of collagen production by human fibroblasts)
Normal human fibroblasts were seeded in a 24-well plate (6 × 10 4 cells / well) and pre-cultured for 24 hours (5% CO 2 , 37 ° C.). As the culture solution, a D-MEM medium containing 1% fetal bovine serum was used. Thereafter, each raw material-derived glucosylceramide and collagen peptide were replaced with fresh media added to final concentrations of 10 μg / mL and 200 μg / mL, respectively, and cultured for 72 hours. The control was replaced with fresh medium without any addition. After the culture, 100 μL of the culture supernatant was collected, and 300 μL of a 0.1 mass% sirius red solution dissolved in 0.5 M acetic acid was added to the above culture supernatant and shaken, followed by staining at room temperature for 1 hour. This was centrifuged at 10,000 rpm for 5 minutes, the supernatant was discarded, and then the precipitate due to the complex formation of collagen and sirius red was collected. The collected precipitate was washed twice with 10 mM HCl, and the absorbance at 540 nm was measured using a sample dissolved in 0.1N NaOH using a microplate reader. The absorbance of the control was taken as 100% collagen production rate, and each collagen production rate was determined from the absorbance of the culture supernatant to which each sample was added.
各原料由来グルコシルセラミドとコラーゲンペプチドを併用した併用区のコラーゲン産生率の予測値は、各原料由来グルコシルセラミドの単独区、及びコラーゲンペプチドのみのコラーゲン産生率から以下の式で求めた。
予測値（％）＝｛単独区の産生率（％）＋コラーゲンペプチドの産生率（％）｝−１００（％）
The predicted value of the collagen production rate in the combination group in which each raw material-derived glucosylceramide and collagen peptide were used in combination was determined by the following formula from the single group of each raw material-derived glucosylceramide and the collagen production rate of only the collagen peptide.
Predicted value (%) = {Production rate of single section (%) + Production rate of collagen peptide (%)} − 100 (%)
各原料由来グルコシルセラミドとコラーゲンペプチドの併用による効果は、併用区の実測値と予測値との比較で評価した。（実測値）／（予測値）比が、１．１０以上の場合を相乗効果、０．９０超過１．１０未満の場合を相加効果、０．９０以下の場合を相殺効果と判定した。 The effect of the combined use of each raw material-derived glucosylceramide and collagen peptide was evaluated by comparing the measured value and the predicted value of the combination group. A case where the ratio of (actually measured value) / (predicted value) was 1.10 or higher was determined as a synergistic effect, a case where the ratio was more than 0.90 and less than 1.10 was an additive effect, and a case where the ratio was 0.90 or less was determined as an offset effect.
上記実施例の結果を表1に示す。表中の表示において◎は相乗効果、○は相加効果、×は相殺効果を示す。 The results of the above examples are shown in Table 1. In the table, ◎ indicates a synergistic effect, ○ indicates an additive effect, and X indicates an offset effect.
結果、トルラ酵母由来グルコシルセラミドとコラーゲンペプチドの併用区のみ、コラーゲン産生促進効果が相乗的に増強されることが確認された。すなわち、トルラ酵母由来のグルコシルセラミドは、コメ由来、コーン由来、コンニャク由来のグルコシルセラミドと比べて、コラーゲンペプチドのコラーゲン産生促進効果を顕著に向上させることが示された。 As a result, it was confirmed that the collagen production promoting effect was synergistically enhanced only in the combined use of Torula yeast-derived glucosylceramide and collagen peptide. That is, glucosylceramide derived from Torula yeast was shown to significantly improve the collagen production promoting effect of the collagen peptide as compared with glucosylceramide derived from rice, corn and konjac.
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| JP2002255847A (en) * | 2001-02-26 | 2002-09-11 | Miyagi Kagaku Kogyo Kk | Collagen production promoter, functional food and pharmaceutical preparation |
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