JP5270122B2 - Acidic mucopolysaccharide-producing microorganism, method for producing acidic mucopolysaccharide, and whitening agent containing acidic mucopolysaccharide as an active ingredient - Google Patents
Acidic mucopolysaccharide-producing microorganism, method for producing acidic mucopolysaccharide, and whitening agent containing acidic mucopolysaccharide as an active ingredient Download PDFInfo
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- JP5270122B2 JP5270122B2 JP2007216324A JP2007216324A JP5270122B2 JP 5270122 B2 JP5270122 B2 JP 5270122B2 JP 2007216324 A JP2007216324 A JP 2007216324A JP 2007216324 A JP2007216324 A JP 2007216324A JP 5270122 B2 JP5270122 B2 JP 5270122B2
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- acidic mucopolysaccharide
- acidic
- mucopolysaccharide
- acid
- microorganism
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Landscapes
- Micro-Organisms Or Cultivation Processes Thereof (AREA)
- Cosmetics (AREA)
- Pharmaceuticals Containing Other Organic And Inorganic Compounds (AREA)
- Polysaccharides And Polysaccharide Derivatives (AREA)
- Coloring Foods And Improving Nutritive Qualities (AREA)
- Preparation Of Compounds By Using Micro-Organisms (AREA)
Abstract
Description
本発明は酸性ムコ多糖を産生する新規微生物およびそれを用いた酸性ムコ多糖の改良製造法、並びに該微生物の産生する酸性ムコ多糖を有効成分として配合した美白剤に関する。さらには、外用美白剤、外用美白剤を含む化粧料、経口美白剤、経口美白剤を含む飲食物(美白用健康食品)に関する。 The present invention relates to a novel microorganism producing acidic mucopolysaccharide, an improved method for producing acidic mucopolysaccharide using the same, and a whitening agent containing acidic mucopolysaccharide produced by the microorganism as an active ingredient. Furthermore, the present invention relates to an external whitening agent, a cosmetic containing an external whitening agent, an oral whitening agent, and a food and drink containing an oral whitening agent (a whitening health food).
皮膚の着色の原因となるメラニン色素は、一部不明な点もあるが、一般には、日焼け等により組織中にメラニンが産生することに起因しているものと考えられている。皮膚が日光に起因する紫外線の照射を受けると、皮膚内に存在しているチロシンが酵素チロシナーゼの作用により酵素的に酸化された後、数段階の反応を経て黒色のメラニンへ変化する過程がメラニン色素の生成過程である。そのメラニン色素の生成過程は、アミノ酸のチロシンがチロシナーゼによって酸化され、ドーパ(dihydroxyphenylalanine;DOPA)となり、さらにチロシナーゼの作用により酸化されてドーパキノン(dopaquinone)となり、最終的には、indole-5,6-quinoneが重合し、蛋白質と結合したものがメラニン色素である。したがって、チロシナーゼの活性を阻害することにより、ヒト皮膚のシミやそばかす等の発生を抑制することができる。 The melanin pigment that causes skin coloring is partially unknown, but is generally considered to be caused by the production of melanin in the tissue by sunburn or the like. When the skin is irradiated with ultraviolet rays caused by sunlight, tyrosine present in the skin is enzymatically oxidized by the action of the enzyme tyrosinase, and then changes to black melanin through several steps of reaction. This is the process of pigment production. In the melanin formation process, the amino acid tyrosine is oxidized by tyrosinase to become dopa (dihydroxyphenylalanine; DOPA), and further oxidized by the action of tyrosinase to become dopaquinone. Finally, indole-5,6- The quinone is polymerized and bound to the protein is the melanin pigment. Therefore, by inhibiting the activity of tyrosinase, it is possible to suppress the occurrence of spots and freckles in human skin.
そして従来から、上記メラニンの生成を抑制し、皮膚の美白状態を維持せしめる美白化粧料の組成物として、アルブチンなどのハイドロキノン誘導体、エラグ酸、アスコルビン酸及びその配糖体、コウジ酸及びその誘導体等が知られている。例えば、近年、優れた還元能を有するビタミンC(L−アスコルビン酸)誘導体を用いた化粧料が提案されているが、ビタミンC誘導体は安定性に難があるとともに、外用ではほとんど効果が認められていない。 Conventionally, as a whitening cosmetic composition that suppresses the production of melanin and maintains the whitening state of the skin, hydroquinone derivatives such as arbutin, ellagic acid, ascorbic acid and its glycosides, kojic acid and its derivatives, etc. It has been known. For example, in recent years, cosmetics using vitamin C (L-ascorbic acid) derivatives having excellent reducing ability have been proposed, but vitamin C derivatives have difficulty in stability and are almost effective for external use. Not.
一方、乳酸菌が産生するリン酸化多糖類がメラノーマ増殖抑制作用及びメラニン産生抑制作用を持ち、美白剤としての有用性が開示されている(特許文献1)。 On the other hand, phosphorylated polysaccharides produced by lactic acid bacteria have a melanoma growth inhibitory action and a melanin production inhibitory action, and the utility as a whitening agent is disclosed (Patent Document 1).
また、種々の海洋細菌が多糖類を産生することが知られてり、一部の菌種が酸性ムコ多糖類を産生することが報告されている (特許文献2、特許文献3、非特許文献1)。これらの酸性ムコ多糖類は、いずれも、グルクロン酸などのウロン酸残基やN−アセチルグルコサミン、N−アセチルガラクトサミンなどアミノ糖残基が一定の配列を繰り返して糖鎖を形成し、場合によってはグルコース、ガラクトースなどの中性糖残基も含み、又側鎖としてアミノ酸やピルビン酸基が結合した構造を有している点で中性多糖類とは異なる。
しかしこれら化粧料には、種々の問題点が残されており、実用性の面で問題があった。
例えば、美白成分としてのコウジ酸は発ガンの恐れがあること、アルブチンなどのハイドロキノン誘導体は発疹、かぶれなど安全性に問題があった。
また、動植物抽出物配合も知られてはいるが、その効果は満足出来るものではなかった。
In addition, various marine bacteria are known to produce polysaccharides, and some bacterial species have been reported to produce acidic mucopolysaccharides (Patent Literature 2, Patent Literature 3, Non-Patent Literature). 1). In these acidic mucopolysaccharides, uronic acid residues such as glucuronic acid and amino sugar residues such as N-acetylglucosamine and N-acetylgalactosamine repeat a certain sequence to form a sugar chain. It differs from neutral polysaccharides in that it also contains neutral sugar residues such as glucose and galactose and has a structure in which amino acids and pyruvate groups are bonded as side chains.
However, these cosmetics still have various problems and have problems in practical use.
For example, kojic acid as a whitening component may cause carcinogenesis, and hydroquinone derivatives such as arbutin have safety problems such as rash and rash.
Moreover, although the composition of animal and plant extracts is known, the effect is not satisfactory.
そこで本発明者は、優れた美白効果を有し、安定性及び皮膚に対して安全性の高い新規な成分を有効成分とする美白剤を提供するべく、海洋細菌が産生する式(1)の構造式で示される酸性ムコ多糖(以後の記述においては、単に「本酸性ムコ多糖」と表現することがある。)またはそれらの生理学的に許容される塩もしくは誘導体を有効成分として含む美白剤を開発した(特許文献4、非特許文献2)。
(上記の構造式において、GalNAcpはピラノース型N-アセチルガラクトサミン残基を、GlcUApはピラノース型グルクロン酸残基を、DはD型を、LはL型を、Pyrはピルビン酸を、nはゲルろ過クロマトグラフィーで測定した平均分子量がプルランを標準として100万〜150万であることを示す繰り返しの数をそれぞれ表す。)
Therefore, the present inventor has the formula of formula (1) produced by marine bacteria in order to provide a whitening agent having an excellent whitening effect and a novel ingredient having high stability and safety to the skin as an active ingredient. A whitening agent comprising an acidic mucopolysaccharide represented by the structural formula (in the following description, simply referred to as “the present acidic mucopolysaccharide”) or a physiologically acceptable salt or derivative thereof as an active ingredient. Developed (Patent Document 4, Non-Patent Document 2).
(In the above structural formula, GalNAcp is a pyranose-type N-acetylgalactosamine residue, GlcUAp is a pyranose-type glucuronic acid residue, D is a D-type, L is an L-type, Pyr is a pyruvate, and n is a gel. The average molecular weight measured by filtration chromatography represents the number of repetitions indicating that the average molecular weight is 1 to 1.5 million with pullulan as a standard.)
本酸性ムコ多糖に関してはこれまで、軟骨細胞の増殖促進効果、メラニン生成抑制効果、マクロファージ活性化効果等の生理活性が報告されている。 With respect to the acidic mucopolysaccharide, physiological activities such as chondrocyte proliferation promoting effect, melanin production inhibiting effect, macrophage activating effect and the like have been reported so far.
本酸性ムコ多糖は、シュードモナス属(Pseudomonas)の菌株によって産生される(特許文献2,非特許文献2、3)。しかし、これらの文献に記載された菌株は、市販規模では所望されるものではない。なぜなら、これは、ジャーファーメンターや大規模タンク培養等の振とう(または撹拌)培養においては選択的に同伴産生する硫酸多糖の除去が煩雑で高コストに繋がるからである。 This acidic mucopolysaccharide is produced by a strain of Pseudomonas (Patent Document 2, Non-Patent Documents 2 and 3). However, the strains described in these documents are not desired on a commercial scale. This is because, in shake (or agitation) culture such as jar fermenter or large-scale tank culture, the removal of the selectively produced sulfated polysaccharide is complicated and leads to high costs.
特に液体振とう培養(または撹拌培養)法を用いる本酸性ムコ多糖産生プロセスにおいて、選択的に同伴産生する硫酸多糖は、本酸性ムコ多糖の分離、精製に大きな諸問題を生ずる。従って微生物による本酸性ムコ多糖産生を実用的な規模のレベルで可能にするためには、硫酸多糖の同時産生を伴わない微生物が望まれる。 In particular, in the present acidic mucopolysaccharide production process using the liquid shaking culture (or stirring culture) method, the sulfated polysaccharide selectively produced along with it causes various problems in the separation and purification of the acidic mucopolysaccharide. Therefore, in order to enable production of the present acidic mucopolysaccharide by a microorganism at a practical scale level, a microorganism that does not involve simultaneous production of sulfated polysaccharide is desired.
本発明は上記事情に鑑みてなされたものであり、その目的は、色素沈着改善(美白)効果及び皮膚に対する安全性の両面を十分に満足させる化合物である本酸性ムコ多糖の産業的規模の生産にあたり、従来の微生物にない、硫酸多糖を同伴産生しない微生物、該微生物を用いて酸性ムコ多糖を効率的にかつ安価に生産する方法、並びに該微生物の産生する本酸性ムコ多糖を有効成分として配合した美白剤を提供することにある。さらには、外用美白剤、外用美白剤を含む化粧料、経口美白剤、経口美白剤を含む飲食物(美白用健康食品)を提供することにある。日焼け後の色素沈着・しみ・そばかす・肝斑等の淡色化などの美白効果は、化粧料のように日常的に使用されるものにより、あるいは飲食物のように日常的に摂取されるものにより、予防が構じられることが望まれており、本発明の目的は、副作用もなく、長期連用においても安全性が高く、飲食物や化粧料にも活用できる美白剤を提供することにある。 The present invention has been made in view of the above circumstances, and its purpose is to produce the acidic mucopolysaccharide on the industrial scale, which is a compound that sufficiently satisfies both the pigmentation improving (whitening) effect and the safety to the skin. In addition, microorganisms that do not produce sulfated polysaccharides that are not present in conventional microorganisms, a method for efficiently and inexpensively producing acidic mucopolysaccharides using the microorganisms, and the acidic mucopolysaccharides produced by the microorganisms as active ingredients It is to provide a whitening agent. Furthermore, it is providing the food / beverage (whitening health food) containing an external whitening agent, the cosmetics containing an external whitening agent, an oral whitening agent, and an oral whitening agent. Whitening effects such as pigmentation, blotches, freckles, and light spots after sunburn are caused by things that are used daily, such as cosmetics, or those that are taken daily, such as food and drink Therefore, it is desired that prevention be configured, and an object of the present invention is to provide a whitening agent that has no side effects, is highly safe even for long-term continuous use, and can be used for foods and beverages and cosmetics.
本発明者は、上記課題を解決すべく研究を重ねた結果、コベティア属(Cobetia)に属する微生物の中に硫酸多糖の同伴産生を伴わないで、「本酸性ムコ多糖」〔特許文献2の従来菌株で作った酸性ムコ多糖(登録商標:ウミノグリカン)と同一の物質(同一構造式、美白機能など同一の機能性)である。〕を産生する微生物を見出し、本発明を完成した。 As a result of repeated studies to solve the above-mentioned problems, the present inventors have found that the present acid mucopolysaccharide is not accompanied by the accompanying production of sulfated polysaccharide in microorganisms belonging to the genus Cobetia [conventional of Patent Document 2]. It is the same substance (same structural formula and the same functionality as whitening function) as acidic mucopolysaccharide (registered trademark: Uminoglycan) made from the strain. The present invention has been completed.
すなわち、驚くべきことに、本酸性ムコ多糖産生微生物株に対する本発明者のスクリーニング研究過程において、本発明者は、瀬戸内海の沿岸に生息するワカメから採取した海洋微生物より硫酸多糖の同伴産生を伴わずに本酸性ムコ多糖を産生する新規菌株を得ることに成功した。 That is, surprisingly, in the inventor's screening research process for this acidic mucopolysaccharide-producing microbial strain, the present inventor was accompanied by the accompanying production of sulfated polysaccharides from marine microorganisms collected from seaweeds that inhabit the coast of the Seto Inland Sea. And succeeded in obtaining a novel strain producing this acidic mucopolysaccharide.
本発明の新規菌株は本質的特性において特許文献2,非特許文献2、3に記載のシュードモナス属(Pseudomonas)の菌株と異なる。すなわち本発明の新規菌株は特許文献2および非特許文献2、3に記載の菌株の基本特性である硫酸多糖の同伴的産生がない。 The novel strain of the present invention differs from the strain of Pseudomonas described in Patent Document 2, Non-Patent Documents 2 and 3 in essential characteristics. That is, the novel strain of the present invention does not have the accompanying production of sulfated polysaccharide, which is a basic characteristic of the strains described in Patent Document 2 and Non-Patent Documents 2 and 3.
得られた本新規菌株の16S rDNA塩基配列(配列番号1)、さらには培養学的および生化学的特性を調べることにより、該新規菌株の同定を試み、コベティア属(Cobetia)に属する新規な微生物であることを明らかにした。次いで本新規菌株の培養液を分析し、本酸性ムコ多糖が産生されていること、および硫酸多糖の産生を欠くことを確認し、本酸性ムコ多糖の改良製造法を発明した。 By examining the 16S rDNA base sequence (SEQ ID NO: 1) of the obtained new strain, and further cultivating and biochemical characteristics, an attempt was made to identify the new strain, and a novel microorganism belonging to the genus Cobetia (Cobetia) It was revealed that. Subsequently, the culture solution of the novel strain was analyzed to confirm that the acidic mucopolysaccharide was produced and the production of sulfated polysaccharide was lacking, and an improved method for producing the acidic mucopolysaccharide was invented.
後述するとおり、上記新規菌株はコベティア属 (Cobetia) に属することが、16S rDNA塩基配列分析などの分類学的調査を通じて決定されたことより、該新規菌株をコベティア(Cobetia) sp. WAK-1Aと命名し、独立行政法人産業技術総合研究所 特許生物寄託センター (〒305−8566 日本国茨城県つくば市東1丁目1番地1中央第6)に寄託し、平成18年11月20日、寄託番号 FERM P-21101として受託された。 As will be described later, it was determined through taxonomic surveys such as 16S rDNA nucleotide sequence analysis that the new strain belongs to the genus Cobetia, so that the new strain was designated as Cobetia sp. WAK-1A. Named and deposited at the National Institute of Advanced Industrial Science and Technology Patent Biology Depositary Center (1st, 1st, 1st, 1st East, Tsukuba City, Ibaraki, Japan 305-8565), deposit number FERM on November 20, 2006 Commissioned as P-21101.
すなわち、本発明は以下の(1)〜(4)に記載のコベティア属微生物を要旨とする。
(1)コベティア属微生物がコベティア(Cobetia) sp. WAK-1A(寄託番号FERM
P-21101)であって、該コベティア属微生物に適した培養条件での培養にて培養物を得、次いで培養物から下記式(1)の構造式で示される酸性ムコ多糖を硫酸多糖の選択的除去をすることなく分離回収可能であることを特徴とするコベティア属微生物。
(1)
(上記の構造式において、GalNAcpはピラノース型N-アセチルガラクトサミン残基を、GlcUApはピラノース型グルクロン酸残基を、DはD型を、LはL型を、Pyrはピルビン酸を、nは繰り返しの数をそれぞれ表す。)
(2)培養が、炭素源、窒素源および鉱物塩を含む培養培地を用いて固形培地、液体静置、振とう(または撹拌)条件で22℃から28℃の範囲で2日から7日間の培養を行うものである、上記(1)に記載のコベティア属微生物。
(3)以下の菌学的性質を有する上記(1)または(2)に記載のコベティア属微生物。
< 形態 >
細胞の形態:桿菌
運動性:有り
胞子形成:無し
<生育状態>
コロニーの形態:円形
コロニーの色調:クリーム色
コロニーの表面:スムーズ
生育温度:10℃、30℃、37℃および45℃で生育する。
生育pH:5.5〜9.5(至適生育pH:6.5〜7.5)
酸素要求性:好気性
<生理学的性質>
グラム染色性:陰性
カタラーゼ反応:陽性
オキシダーゼ反応:陰性
グルコースからの酸/ガス産生:陰性/陰性
O/Fテスト(酸化/発酵) :陰性/陰性
β−ガラクトシダーゼ活性:陽性
インドール産生:陰性
硝酸塩還元:陰性
でんぷん加水分解:陰性
ゼラチン加水分解:陰性
エスクリン加水分解:陰性
ウレアーゼ:陰性
アルギニンジヒドロラーゼ:陰性
資化性(D-グルコース、ラクトース、サッカロース、酢酸ナトリウム、クエン酸ナトリウム、グルコン酸ナトリウム、こはく酸ナトリウム):有り
(L-アラビノース、マルトース、D-マンノース、D-マンニトール、N-アセチル-D-グルコサミン、n-カプリン酸、アジピン酸、dl-リンゴ酸、酢酸フェニル、L-アラニン、L-ヒスチジン、L-セリン):無し
(4)16S rDNA塩基配列がコベティア マリナ(Cobetia marina)
DSM 4741のそれと99.7%の相同率を有し、かつ100%は一致しない配列番号1の塩基配列を有する上記(1)、(2)または(3)に記載のコベティア属微生物。
That is, the gist of the present invention is the Covetia microorganism described in the following (1) to ( 4 ).
(1) The microorganism of the genus Covetia is Cobetia sp. WAK-1A (deposit number FERM)
P-21101), a culture is obtained by culturing under a culture condition suitable for the microorganism of the genus Covetia , and then an acidic mucopolysaccharide represented by the structural formula of the following formula (1) is selected from the culture as a sulfated polysaccharide. Microbe of the genus Covetia, characterized in that it can be separated and recovered without selective removal.
(1)
(In the above structural formula, GalNAcp is a pyranose-type N-acetylgalactosamine residue, GlcUAp is a pyranose-type glucuronic acid residue, D is a D-type, L is an L-type, Pyr is pyruvic acid, and n is a repeat. Represents the number of each.)
(2) The culture is carried out for 2 days to 7 days in a range of 22 ° C. to 28 ° C. in a solid medium, liquid standing, and shaking (or stirring) conditions using a culture medium containing a carbon source, a nitrogen source and a mineral salt. The microorganism belonging to the genus Covetia according to (1), which is used for culture.
(3) The Covetia microorganism described in the above (1) or (2) having the following mycological properties.
<Form>
Cell morphology: Aspergillus motility: Yes Spore formation: None <Growth state>
Colony morphology: Circular colony color: Cream color Surface of colony: Smooth growth temperature: Grows at 10 ° C, 30 ° C, 37 ° C and 45 ° C.
Growth pH: 5.5 to 9.5 (optimum growth pH: 6.5 to 7.5)
Oxygen demand: Aerobic <Physiological properties>
Gram staining: Negative catalase reaction: Positive oxidase reaction: Acid / gas production from negative glucose: Negative / negative
O / F test (oxidation / fermentation): negative / negative β-galactosidase activity: positive indole production: negative nitrate reduction: negative starch hydrolysis: negative gelatin hydrolysis: negative esculin hydrolysis: negative urease: negative arginine dihydrolase: negative Assimilation (D-glucose, lactose, saccharose, sodium acetate, sodium citrate, sodium gluconate, sodium succinate): Yes (L-arabinose, maltose, D-mannose, D-mannitol, N-acetyl-D- Glucosamine, n-capric acid, adipic acid, dl-malic acid, phenyl acetate, L-alanine, L-histidine, L-serine): None (4) 16S rDNA nucleotide sequence is Cobetia marina ( Cobetia marina )
The microorganism of the genus Covetia according to the above (1), (2) or (3) having a base sequence of SEQ ID NO: 1 having a homology rate of 99.7% with that of DSM 4741 and 100% not matching.
また、本発明は以下の(5)または(6)に記載の酸性ムコ多糖の製造法を要旨とする。
(5)上記(1)ないし(4)のいずれかに記載のコベティア属微生物を培養して培地中で前記式(1)の構造式で示される酸性ムコ多糖を産生させ培養液を得る工程、および酸性ムコ多糖を培養液から硫酸多糖の選択的除去をすることなく単離する工程を含む酸性ムコ多糖の製造法。
(6)酸性ムコ多糖が美白剤用の酸性ムコ多糖である(5)に記載の酸性ムコ多糖の製造法。
In addition, the gist of the present invention is the method for producing acidic mucopolysaccharides described in the following ( 5 ) or ( 6 ).
( 5 ) A step of culturing the Covetia microorganism according to any one of (1) to ( 4 ) above to produce an acidic mucopolysaccharide represented by the structural formula of the formula (1) in a medium to obtain a culture solution, And a method for producing acidic mucopolysaccharide comprising isolating acidic mucopolysaccharide from a culture solution without selective removal of sulfate polysaccharide.
( 6 ) The method for producing acidic mucopolysaccharide according to ( 5 ), wherein the acidic mucopolysaccharide is an acidic mucopolysaccharide for whitening agents.
また、本発明は以下の(7)および(8)に記載の美白剤の製造方法を要旨とする。
(7)上記(1)ないし(4)のいずれかに記載のコベティア属微生物を培養して培地中で前記式(1)の構造式で示される酸性ムコ多糖を産生させ培養液を得る工程、および酸性ムコ多糖を培養液から硫酸多糖の選択的除去をすることなく単離する工程を含む製造法により酸性ムコ多糖を製造し、得られた酸性ムコ多糖をそのまま、またはそれらの生理学的に許容される塩もしくは脂肪酸によるアシル化物とし、該酸性ムコ多糖、またはそれらの生理学的に許容される塩もしくは脂肪酸によるアシル化物を有効成分として含む美白剤を製造することを特徴とする美白剤の製造方法。
(8)美白剤が皮膚外用剤である上記(7)記載の美白剤の製造方法。
Moreover, this invention makes a summary the manufacturing method of the whitening agent as described in the following (7) and (8).
(7) A step of culturing the Covetia microorganism according to any one of (1) to (4) above to produce an acidic mucopolysaccharide represented by the structural formula of the formula (1) in a medium to obtain a culture solution, And acidic mucopolysaccharides are produced by a production method comprising a step of isolating acidic mucopolysaccharides from a culture solution without selective removal of sulfate polysaccharides, and the obtained acidic mucopolysaccharides are intact or physiologically acceptable. and acylated by salt or fatty acid is, the production method of the acidic mucopolysaccharides, or whitening agent characterized by producing a whitening agent containing the acylated product as an active ingredient according to their physiologically acceptable salts or fatty acids .
(8) The method for producing a whitening agent according to the above (7), wherein the whitening agent is an external preparation for skin.
また、本発明は以下の(9)に記載の化粧料の製造方法を要旨とする。
(9)上記(1)ないし(4)のいずれかに記載のコベティア属微生物を培養して培地中で前記式(1)の構造式で示される酸性ムコ多糖を産生させ培養液を得る工程、および酸性ムコ多糖を培養液から硫酸多糖の選択的除去をすることなく単離する工程を含む製造法により酸性ムコ多糖を製造し、得られた酸性ムコ多糖をそのまま、またはそれらの生理学的に許容される塩もしくは脂肪酸によるアシル化物とし、該酸性ムコ多糖、またはそれらの生理学的に許容される塩もしくは脂肪酸によるアシル化物を有効成分として含む外用美白剤を製造し、得られた外用美白剤から、その外用美白剤を含む化粧料を製造することを特徴とする化粧料の製造方法。
Moreover, this invention makes the summary the manufacturing method of cosmetics as described in the following (9).
(9) A step of culturing the Covetia microorganism according to any one of (1) to (4) above to produce an acidic mucopolysaccharide represented by the structural formula of the formula (1) in a medium to obtain a culture solution, And acidic mucopolysaccharides are produced by a production method comprising a step of isolating acidic mucopolysaccharides from a culture solution without selective removal of sulfate polysaccharides, and the obtained acidic mucopolysaccharides are intact or physiologically acceptable. is an acylated by salts or fatty acids, to produce a topical lightening agent comprising acidic mucopolysaccharides, or acylated by their physiologically acceptable salts or fatty acids as an active ingredient, from the obtained external lightening agent, A cosmetic production method comprising producing a cosmetic comprising the external whitening agent.
また、本発明は以下の(10)に記載の飲食物の製造方法を要旨とする。
(10)上記(1)ないし(4)のいずれかに記載のコベティア属微生物を培養して培地中で前記式(1)の構造式で示される酸性ムコ多糖を産生させ培養液を得る工程、および酸性ムコ多糖を培養液から硫酸多糖の選択的除去をすることなく単離する工程を含む製造法により酸性ムコ多糖を製造し、得られた酸性ムコ多糖をそのまま、またはそれらの生理学的に許容される塩もしくは脂肪酸によるアシル化物とし、該酸性ムコ多糖、またはそれらの生理学的に許容される塩もしくは脂肪酸によるアシル化物を含む飲食物を製造することを特徴とする飲食物の製造方法。
Moreover, this invention makes a summary the manufacturing method of the food and drink as described in the following (10).
(10) A step of culturing the Covetia microorganism according to any one of (1) to (4) above to produce an acidic mucopolysaccharide represented by the structural formula of the formula (1) in a medium to obtain a culture solution, And acidic mucopolysaccharides are produced by a production method comprising a step of isolating acidic mucopolysaccharides from a culture solution without selective removal of sulfate polysaccharides, and the obtained acidic mucopolysaccharides are intact or physiologically acceptable. is an acylated by salts or fatty acids, method for producing the acidic mucopolysaccharides, or food, which comprises producing a food containing an acylated by their physiologically acceptable salts or fatty acids.
本発明によれば、硫酸多糖を同伴産生せずに本酸性ムコ多糖を培養中に産生することができる新規微生物を提供することができる。より具体的には、本発明によれば、独立行政法人産業技術総合研究所 特許生物寄託センター (〒305−8566 日本国茨城県つくば市東1丁目1番地1中央第6)に寄託し、平成18年11月20日 寄託番号 FERM P-21101として受託されている、単離されたコベティア(Cobetia) sp. WAK-1Aが提供される。
この微生物を用いることにより本酸性ムコ多糖をジャーファーメンターやタンク内で撹拌を伴って安全に大量生産することができ、本酸性ムコ多糖を容易に簡単な工程で大量、安価に分離精製できることから経済的な工業的生産規模を可能にする。
また、本発明による美白剤の製造方法によれば、メラニン生成抑制作用を有しており、日焼け後の色素沈着・しみ・そばかす・肝斑等の淡色化、美白に優れた効果を有すると共に、安全性にも優れた美白剤を製造することができる。
さらにまた、本発明によれば、メラニン生成抑制作用を有しており、日焼け後の色素沈着・しみ・そばかす・肝斑等の淡色化、美白に優れた効果を有すると共に、安全性にも優れた美白剤を提供することができる。
美白効果は、化粧料のように日常的に使用されるものにより、あるいは飲食物のように日常的に摂取されるものにより、予防が構じられることが望まれており、本発明によれば、副作用もなく、長期連用においても安全性が高く、飲食物や化粧料にも活用できる美白剤を提供することができる。
ADVANTAGE OF THE INVENTION According to this invention, the novel microorganisms which can produce this acidic mucopolysaccharide during culture | cultivation, without entraining and producing sulfated polysaccharide can be provided. More specifically, according to the present invention, it was deposited at the National Institute of Advanced Industrial Science and Technology, Patent Biological Deposit Center (1st, 1st, 1st East, 1-chome, Tsukuba City, Ibaraki 305-8586, Japan). November 20, Year An isolated Cobetia sp. WAK-1A, deposited under accession number FERM P-21101, is provided.
By using this microorganism, the acidic mucopolysaccharide can be safely and mass-produced with stirring in a jar fermenter or tank, and the acidic mucopolysaccharide can be easily and easily separated and purified in large quantities at a simple process. Enables economical industrial production scale.
In addition, according to the method for producing a whitening agent according to the present invention, it has a melanin production inhibitory action, and has an excellent effect on lightening whitening, such as pigmentation, blotches, freckles, and liver spots after sunburn, A whitening agent having excellent safety can be produced.
Furthermore, according to the present invention, it has an inhibitory action on melanin production, and has excellent effects on lightening and whitening of pigmentation, stains, freckles, and liver spots after sunburn, and also has excellent safety. Whitening agents can be provided.
The whitening effect is desired to be prevented by what is used daily such as cosmetics, or by what is taken daily such as food and drink. According to the present invention, Therefore, it is possible to provide a whitening agent that has no side effects, is highly safe even for long-term continuous use, and can be used for food and drink and cosmetics.
本発明の新規菌株であるコベティア(Cobetia) sp. WAK-1A(FERM P-21101)は、本酸性ムコ多糖を産生するシュードモナス属(Pseudomonas)菌株との区別において、特性として培養中に硫酸多糖を同伴的に産生蓄積しないで式(1)の構造式で示される本酸性ムコ多糖を蓄積することにある。この特性は、該新規菌株培養液から得られた多糖画分を以下の実施例に記載の分析によって確認された。 Cobetia sp. WAK-1A (FERM P-21101), which is a novel strain of the present invention, is characterized in that it is characterized by the presence of sulfated polysaccharides in the culture as distinctive from Pseudomonas strains that produce this acidic mucopolysaccharide. It is to accumulate the acidic mucopolysaccharide represented by the structural formula of the formula (1) without accumulating and accumulating it. This property was confirmed by analyzing the polysaccharide fraction obtained from the novel strain culture solution as described in the following Examples.
(上記の構造式において、GalNAcpはピラノース型N-アセチルガラクトサミン残基を、GlcUApはピラノース型グルクロン酸残基を、DはD型を、LはL型を、Pyrはピルビン酸を、nは繰り返しの数をそれぞれ表す。)
(In the above structural formula, GalNAcp is a pyranose-type N-acetylgalactosamine residue, GlcUAp is a pyranose-type glucuronic acid residue, D is a D-type, L is an L-type, Pyr is pyruvic acid, and n is a repeat. Represents the number of each.)
この単離された菌株(FERM P-21101)はグラム陰性桿菌で、運動能力があり、胞子が無く(non- sporing)、従属栄養性である。またMB2216寒天 (BectonDickinson, MD, USA)で充分に増殖し、粘稠性コロニーを生成した。単離された菌株はゼラチンを液化せず、でんぷんを加水分解せず、比較的少量の酸の生成を伴ってラクトースのような炭水化物を酸化したがガスは発生しなかった。この株はカタラーゼ陽性であったが、オキシダーゼおよびインドール陰性であった。 This isolated strain (FERM P-21101) is a Gram-negative gonococcus, has motor ability, is non-sporing and is heterotrophic. Moreover, it grew sufficiently on MB2216 agar (Becton Dickinson, MD, USA), and produced viscous colonies. The isolated strain did not liquefy gelatin, did not hydrolyze starch, oxidized carbohydrates such as lactose with the production of relatively small amounts of acid, but did not generate gas. This strain was catalase positive but oxidase and indole negative.
〈硫酸多糖の産生を同伴せずに本酸性ムコ多糖を産生する微生物〉
本発明の微生物は、コベティア属微生物に適した培養条件での培養にて培養液を得、次いで培養液から酸性ムコ多糖を硫酸多糖の選択的除去をすることなく分離回収可能であることを特徴とするコベティア属微生物である。より詳細には、本発明の微生物は、コベティア マリナ DSM 4741(Cobetia marina DSM 4741) (VALIDATION LIST No 88. Int.J.Syst.Evol.Microbiol., 52, 1915-1916, 2002., D.R.Arahalら:System. Appl. Microbiol., 25, 207-211, 2002)と99.7%の相同な16S rDNA塩基配列(配列番号1)を有し、かつ、100%の相同率は有しない微生物であり、硫酸多糖を同伴産生せずに本酸性ムコ多糖を産生する微生物である。
なお、Cobetia marina DSM4741株は、Cobetia marinaの標準株として位置付けられている。寄託番号と公の寄託先は下記のとおりである。
1.ATCC253741 (American Type Culture Collection, Manassas, VA, USA)
2.DSM4741 (DSMZ-Deutsche Sammlung von Mikroorganismen und Zellkulturen GmbH, Braunschweig, Germany)
3.NCIMB1877 (National Collection of Industrial and Marine Bacteria, Aberdeen, UK)
Cobetia 属も海洋細菌で、海洋資源の開発の進展の中で興味が持たれているものの一つであるが、本酸性ムコ多糖類を産生するものは見当らない。DSM4741株はArahalら(上記文献2)によってはじめて命名され、承認されているものである(上記文献1)。
<Microorganisms that produce this acid mucopolysaccharide without the production of sulfated polysaccharides>
The microorganism of the present invention is characterized in that a culture solution is obtained by culturing under a culture condition suitable for a microorganism of the genus Covetia, and then acidic mucopolysaccharide can be separated and recovered from the culture solution without selectively removing the sulfated polysaccharide. It is a microorganism of the genus Covetia. More specifically, the microorganisms of the present invention can be obtained from Cobetia marina DSM 4741 (VALIDATION LIST No 88. Int. J. Syst. Evol. Microbiol., 52, 1915-1916, 2002., DRArahal et al .: System. Appl. Microbiol., 25, 207-211, 2002) and 99.7% homologous 16S rDNA base sequence (SEQ ID NO: 1) and 100% homologous microorganism, sulfated polysaccharide It is a microorganism that produces this acidic mucopolysaccharide without accompanying production.
The Cobetia marina DSM4741 strain is positioned as a standard strain of Cobetia marina. Deposit numbers and public deposit numbers are as follows.
1. ATCC253741 (American Type Culture Collection, Manassas, VA, USA)
2. DSM4741 (DSMZ-Deutsche Sammlung von Mikroorganismen und Zellkulturen GmbH, Braunschweig, Germany)
3. NCIMB1877 (National Collection of Industrial and Marine Bacteria, Aberdeen, UK)
The genus Cobetia is also a marine bacterium and is one of the interests in the development of marine resources, but there is no one that produces this acid mucopolysaccharide. The DSM4741 strain was first named and approved by Arahal et al. (Above reference 2) (above reference 1).
このような硫酸多糖の産生を同伴せずに本酸性ムコ多糖を産生する微生物の例として、本発明者が、瀬戸内海で採取した海藻サンプルから分離したWAK-1A菌株を挙げることができる。
〈本酸性ムコ多糖産生新規菌株の分離〉
本酸性ムコ多糖を産生する新規菌株を単離するために、ワカメより分離され、ペプトン、酵母エキス、蔗糖から成る海水培地で培養して本酸性ムコ多糖を産生することが確認されている保存中の菌株を用いて硫酸多糖類産生能を欠く菌株のスクリーニングを行った結果、WAK-1A菌株がその目的に適った性質を有することを見つけた。
As an example of a microorganism that produces the present acid mucopolysaccharide without accompanying the production of such sulfate polysaccharide, the WAK-1A strain isolated from the seaweed sample collected in the Seto Inland Sea by the present inventors can be mentioned.
<Isolation of a novel strain producing this acidic mucopolysaccharide>
In order to isolate a novel strain that produces this acid mucopolysaccharide, it has been confirmed that it is isolated from wakame and cultured in a seawater medium consisting of peptone, yeast extract and sucrose to produce this acid mucopolysaccharide. As a result of screening for strains lacking the ability to produce sulfate polysaccharides, it was found that the WAK-1A strain has properties suitable for its purpose.
BLASTを用いた細菌基準株の16S rDNA塩基配列データベースに対する相同性検索の結果から、WAK-1A菌株は、コベティア属(Cobetia)に属することが認められコベティア (Cobetia) sp. WAK-1Aと命名した。本菌株は、独立行政法人産業技術総合研究所 特許生物寄託センター (〒305−8566 日本国茨城県つくば市東1丁目1番地1中央第6 )に寄託し、平成18年11月20日 寄託番号 FERM P-21101として受託された。 Based on the results of homology searches against the 16S rDNA nucleotide sequence database of bacterial reference strains using BLAST, the WAK-1A strain was found to belong to the genus Cobetia and was named Cobetia sp. WAK-1A . This strain has been deposited with the National Institute of Advanced Industrial Science and Technology, Patent Biological Deposit Center (1st, 1st, 1st, 1st East, Tsukuba City, Ibaraki, Japan 305-8586). Deposit number: FERM on November 20, 2006 Commissioned as P-21101.
〈酸性ムコ多糖の調製〉
本酸性ムコ多糖の調製方法としては、各種の方法が知られているが、医薬として使用できる程度に精製されたものであれば、種々の方法で調製されたものを用いることができる。例えば、マツダらの方法(M.Matsudaら:Nippon Suisan Gakkaishi,58, 1735-1741, 1992)に準じて本新規菌株を炭素源として蔗糖、窒素源としてペプトン、酵母エキスを含有する海水および/または人工海水を寒天で固めた培地で培養して多糖類を生産し、採取、精製して得ることができる。より具体的には、例えば寒天平板培養では、炭素源として蔗糖、窒素源としてペプトン、酵母エキスを含有する多糖類産生用海水および/または人工海水培地を寒天で固めた培地において本菌株またはその変異株を培養し、寒天平板上に生じた粘質物中から本酸性ムコ多糖を分離、精製して得ることができる。
<Preparation of acidic mucopolysaccharide>
Various methods are known as methods for preparing this acidic mucopolysaccharide, but those prepared by various methods can be used as long as they are purified to the extent that they can be used as pharmaceuticals. For example, according to the method of Mazda et al. (M. Matsuda et al .: Nippon Suisan Gakkaishi, 58, 1735-1741, 1992), the novel strain is used as sucrose as a carbon source, peptone as a nitrogen source, seawater containing yeast extract and / or Polysaccharides can be produced by culturing artificial seawater in a medium hardened with agar and collected and purified. More specifically, for example, in agar plate culture, sucrose as a carbon source, peptone as a nitrogen source, a polysaccharide-producing seawater and / or artificial seawater medium containing yeast extract in a medium solidified with agar and the strain or its mutation The acidic mucopolysaccharide can be obtained by culturing the strain and separating and purifying the acidic mucopolysaccharide from the mucilage produced on the agar plate.
本酸性ムコ多糖を産生する微生物を培養する基本培地としては、多糖類を産生しうる微生物が生育できるものであって、少なくとも炭素源と、窒素源と、各種無機塩とおよび微量元素とを適量含有するものが用いられる。さらに好ましくは、上記基本培地として、コベティア属(Cobetia)に属する微生物が生育できるものが用いられる。炭素源としては、グルコース、サッカロース等の糖、あるいは糖蜜や廃糖蜜が挙げられる。炭素源として1種または2種以上を単独でまたは組み合わせて用いることができる。窒素源としては、硝酸塩、アンモニウム塩等の化合物やペプトン、酵母エキス、アミノ酸などの天然物が挙げられる。窒素源として1種または2種以上を単独でまたは組み合わせて用いることができる。無機塩としては、例えば、リン酸塩、マグネシウム塩、カリウム塩等が挙げられる。無機塩として1種または2種以上を単独でまたは組み合わせて用いることができる。固体培地の場合は寒天を用いる。 The basic medium for cultivating microorganisms that produce the acidic mucopolysaccharide can grow microorganisms that can produce polysaccharides, and contain at least a carbon source, a nitrogen source, various inorganic salts, and trace elements. What is contained is used. More preferably, a culture medium capable of growing microorganisms belonging to the genus Cobetia is used as the basic medium. Examples of the carbon source include sugars such as glucose and saccharose, molasses and waste molasses. One or two or more carbon sources can be used alone or in combination. Nitrogen sources include compounds such as nitrates and ammonium salts, and natural products such as peptone, yeast extract and amino acids. One or two or more nitrogen sources can be used alone or in combination. Examples of the inorganic salt include phosphate, magnesium salt, potassium salt and the like. One or two or more inorganic salts can be used alone or in combination. In the case of a solid medium, agar is used.
培養条件は使用する培地、培地のpH、培地への添加物、培養温度などは通常微生物の培養の際に用いられている条件をそのまま用いることができる。 The culture conditions used can be the same as those normally used for culturing microorganisms, such as the medium to be used, the pH of the medium, the additives to the medium, and the culture temperature.
本酸性ムコ多糖の産生に用いられる微生物としては、硫酸多糖を同伴産生することなく本酸性ムコ多糖を産生する能力のある微生物が用いられる。具体的にはコベティア属細菌が挙げられ、より具体的にはコベティア(Cobetia) sp. WAK-1A (FERM P-21101) またはその変異株が挙げられる。本菌株は本発明者が瀬戸内海においてワカメより分離した海洋性細菌であり、その分類学的特性は、以下の実施例に記載した。 As the microorganism used for production of the acidic mucopolysaccharide, a microorganism capable of producing the acidic mucopolysaccharide without accompanying the production of sulfated polysaccharide is used. Specific examples include bacteria belonging to the genus Covetia, and more specific examples include Cobetia sp. WAK-1A (FERM P-21101) or a mutant thereof. This strain is a marine bacterium isolated from seaweed in the Seto Inland Sea by the inventor, and its taxonomic characteristics are described in the following examples.
培養条件は、培養時のpHは微生物が生育し、かつ多糖類を生産する範囲(pH5.5〜9.5)であれば制限されないが、通常は6.5〜7.5の範囲のpHが好ましい。培養温度については微生物が生育し、かつ多糖類を産生する範囲であれば制限されないが、22℃から28℃の範囲が多糖類の産生には良好である。培養期間は培養のpHや温度により変化するが、通常2日から7日が適切である。 The culture conditions are not limited as long as the pH during the culture is a range in which microorganisms can grow and produce polysaccharides (pH 5.5 to 9.5), but usually a pH in the range of 6.5 to 7.5. Is preferred. The culture temperature is not limited as long as microorganisms grow and produce polysaccharides, but a range of 22 ° C. to 28 ° C. is good for producing polysaccharides. The culture period varies depending on the culture pH and temperature, but usually 2 to 7 days is appropriate.
上記した培地と微生物を用いて微生物を培養することにより、目的とする本酸性ムコ多糖が効率的に産生されることとなる。 By culturing a microorganism using the above-described medium and microorganism, the target acidic mucopolysaccharide can be efficiently produced.
上記本酸性ムコ多糖の化学構造は文献に詳しく記載されている(M.Matsudaら:Fisheries Science, 63, 983-988, 1997)。構成成分のモル比がN−アセチル−D−ガラクトサミン:D−グルクロン酸:N−アセチル−L−ガラクトサミン:ピルビン酸が2:1:1:1で、ゲル濾過クロマトグラフィーで測定した平均分子量がプルランを標準として100〜150万である。具体的には、Asahipak GFA-7M(昭和電工製)をカラムとする高速液体クロマトグラフィー(島津製)を使用し、0.1NNaClを移動相とし、分子量既知のプルラン(Shodex STANDARD KIT P-82、昭和電工製)を標準サンプルとして作成した分子量保持時間標準曲線を使用して測定することができる。構成成分の分析には、セルロースアセテート膜電気泳動、または高速液体クロマトグラフィーを用いることができる。この構成成分の分析には、多糖画分を2Mのトリフルオロ酢酸(TFA)、または4N−HClで100℃、12時間加水分解し、ロータリーエバポレイターでTFAまたはHClを除いたものを検体とし、中性糖、ウロン酸、有機酸、硫酸イオンおよびアミノ糖の分析を行う。特にウロン酸の分析は、多糖のカルボキシル基還元後に加水分解して中性糖を分析することにより行う。有機酸および硫酸基はイオンクロマト分析により行うが、有機酸は酵素法も用いることができる。また多糖画分を1HNMR分析することにより構成成分および糖鎖結合様式を解析することができる。 The chemical structure of the acid mucopolysaccharide is described in detail in the literature (M. Matsuda et al .: Fisheries Science, 63, 983-988, 1997). The molar ratio of the components is N-acetyl-D-galactosamine: D-glucuronic acid: N-acetyl-L-galactosamine: pyruvic acid is 2: 1: 1: 1, and the average molecular weight measured by gel filtration chromatography is pullulan. The standard is 1 to 1.5 million. Specifically, high performance liquid chromatography (manufactured by Shimadzu) using Asahipak GFA-7M (manufactured by Showa Denko) as a column, 0.1N NaCl as the mobile phase, pullulan having a known molecular weight (Shodex STANDARD KIT P-82, It can be measured using a molecular weight retention time standard curve prepared as a standard sample. Cellulose acetate membrane electrophoresis or high performance liquid chromatography can be used for the analysis of the constituent components. For analysis of this component, the polysaccharide fraction was hydrolyzed with 2M trifluoroacetic acid (TFA) or 4N-HCl at 100 ° C. for 12 hours, and TFA or HCl was removed by a rotary evaporator as a sample. Analysis of neutral sugar, uronic acid, organic acid, sulfate ion and amino sugar. In particular, the analysis of uronic acid is performed by analyzing the neutral sugar by hydrolysis after reduction of the carboxyl group of the polysaccharide. The organic acid and sulfuric acid group are analyzed by ion chromatography analysis, and the organic acid can also be an enzymatic method. In addition, the component and sugar chain binding mode can be analyzed by 1HNMR analysis of the polysaccharide fraction.
本酸性ムコ多糖の製造方法について、好ましい1実施形態としての本酸性ムコ多糖の製造方法は、(a)硫酸多糖の産生を伴わないで本酸性ムコ多糖を産生する新規菌株を分離する工程と、(b)本新規菌株を培養し本酸性ムコ多糖を産生する工程と、(c)産生された本酸性ムコ多糖を分離・回収する工程と、を有する。 About the production method of the present acidic mucopolysaccharide, the production method of the present acidic mucopolysaccharide as a preferred embodiment includes (a) a step of separating a novel strain that produces the present acidic mucopolysaccharide without the production of sulfated polysaccharide; (B) culturing the novel strain to produce the acidic mucopolysaccharide, and (c) separating and recovering the produced acidic mucopolysaccharide.
続いて、上記の実施形態を各工程毎に詳細に説明していく。
(a) 硫酸多糖の産生を伴わないで本酸性ムコ多糖を産生する新規菌株を分離する工程
本酸性ムコ多糖を産生する菌株として保存中の菌株についてこれをペプトン、酵母エキス、蔗糖からなり海水および/または人工海水で調製した培地で振とう培養して得られた培養物から多糖画分をエタノールによる沈殿により分離精製する。これを上記非特許文献1および2に記載の方法に準じてDEAE-celluloseカラムクロマト分析、化学分析、核磁気共鳴分析等により硫酸多糖産生の有無について試験する。
(b) 微生物を培養し多糖類を産生する工程
本酸性ムコ多糖を得るためには微生物としてWAK-1A菌株またはその変異株を用いる。微生物としてWAK-1A菌株またはその変異株を用いることで、本酸性ムコ多糖を効率的に生産することができる。液体培養は静置、振とう(攪拌)培養法、および固形培養には寒天平板法を用いることができる。
Subsequently, the above embodiment will be described in detail for each step.
(a) a step of isolating a novel strain that produces the acidic mucopolysaccharide without producing the sulfated polysaccharide, and a strain that is preserved as a strain that produces the acidic mucopolysaccharide is composed of peptone, yeast extract, sucrose, A polysaccharide fraction is separated and purified by precipitation with ethanol from a culture obtained by shaking culture in a medium prepared with artificial seawater. This is tested for the presence or absence of sulfated polysaccharide production by DEAE-cellulose column chromatography analysis, chemical analysis, nuclear magnetic resonance analysis, etc., according to the methods described in Non-Patent Documents 1 and 2 above.
(b) Step of culturing microorganisms to produce polysaccharides To obtain the present acidic mucopolysaccharide, the WAK-1A strain or a mutant thereof is used as the microorganism. By using the WAK-1A strain or a mutant thereof as the microorganism, the acidic mucopolysaccharide can be produced efficiently. Liquid culture can be performed by stationary, shaking (stirring) culture, and agar plate can be used for solid culture.
ここで、本酸性ムコ多糖は、下記式(1)の構造単位を有するものである。
(上記の構造式において、GalNAcpはピラノース型N-アセチルガラクトサミン残基を、GlcUApはピラノース型グルクロン酸残基を、DはD型を、LはL型を、Pyrはピルビン酸を、nは繰り返しの数をそれぞれ表す。)
そして、以下に説明する抽出・回収工程を経ることにより高純度の本酸性ムコ多糖を高収率で得ることが可能となる。
Here, this acidic mucopolysaccharide has a structural unit of the following formula (1).
(In the above structural formula, GalNAcp is a pyranose-type N-acetylgalactosamine residue, GlcUAp is a pyranose-type glucuronic acid residue, D is a D-type, L is an L-type, Pyr is pyruvic acid, and n is a repeat. Represents the number of each.)
And it becomes possible to obtain this acidic mucopolysaccharide of high purity by a high yield by passing through the extraction and collection | recovery process demonstrated below.
(c) 産生された所定の多糖類を分離・回収する工程
上記製造方法で得られた培養物から本酸性ムコ多糖を抽出する方法としては、多糖の分離回収に用いられる従来公知の方法を用いることができる。例えば、培養物をそのまま、あるいは高温で殺菌した後で、遠心分離により菌体を除去し、これをそのまま、あるいは濃縮してから、2〜3倍量のエタノール、イソプロパノール、あるいはアセトン等を加え、沈殿を生じさせる。この沈殿物を再度、水あるいは1〜15重量%塩化ナトリウム溶液に溶解させた後で、アルコール等による沈殿を2〜3回繰り返し、水で透析を行い、噴霧乾燥や凍結乾燥機等を用いて乾燥させることにより、本酸性ムコ多糖を得る。これ以外にも電気透析法や限外濾過法も利用することができる。さらに精製するためには、イオン交換、ゲル濾過等の各種クロマトグラフィーや第4級アンモニウム塩による沈殿や塩析などを用いることができる。
(c) Step of separating and recovering the produced predetermined polysaccharide As a method of extracting the acidic mucopolysaccharide from the culture obtained by the above production method, a conventionally known method used for separating and recovering polysaccharides is used. be able to. For example, after sterilizing the culture as it is or at a high temperature, the cells are removed by centrifugation, and this is concentrated as it is or after adding 2-3 times amount of ethanol, isopropanol, acetone, or the like, This causes precipitation. This precipitate is dissolved again in water or 1 to 15% by weight sodium chloride solution, and then precipitation with alcohol or the like is repeated 2-3 times, dialyzed with water, and using a spray dryer or a freeze dryer. The acidic mucopolysaccharide is obtained by drying. In addition, electrodialysis and ultrafiltration can be used. For further purification, various chromatographies such as ion exchange and gel filtration, precipitation or salting out with a quaternary ammonium salt, and the like can be used.
培養液から菌体の除去に際しては、孔径が0.1〜0.45μmの中空糸膜モジュールを備えた膜分離装置を用いることができる。 When removing the cells from the culture solution, a membrane separation apparatus including a hollow fiber membrane module having a pore diameter of 0.1 to 0.45 μm can be used.
かくして本酸性ムコ多糖が産生および回収されることになる。
本発明により得られるコベティア(Cobetia) sp. WAK-1A(寄託番号FERM P-21101)の作る酸性ムコ多糖は、メラニン生成を強く抑制すると共に細胞毒性が極めて弱いことを実験的に裏付けることができた。したがって本発明に係る酸性ムコ多糖は美白剤の有効成分として有用である。前記美白剤は、例えば、医薬部外品を含む化粧品、医薬品、食品等種々の広い分野に応用される。
本発明に係る酸性ムコ多糖は、それらの生理学的に許容される塩、ナトリウム塩、カリウム塩等の塩の形もしくは誘導体、例えば脂肪酸によるアシル化物、好ましくはアセチル化物の形で配合してもよい。
本発明に係る酸性ムコ多糖のO-アシル誘導体は、例えば、酸性ムコ多糖を希酸水溶液中、均一系で無水酢酸、あるいは酸塩化物で処理することにより行うことができる。または、ホルムアミド等の有機溶媒に可溶な場合は、ピリジン及び無水酢酸で処理して酸性ムコ多糖のO-アセチル誘導体を得ることができる。その他例えば特開平3―143540号公報に記載の方法、特開平6―9707号公報に記載の方法により調製することもできる。O-アセチル化率は、アシル化の方法によって異なるが、本酸性ムコ多糖類の各繰り返し単位に1個以上、好ましくは3個以上、さらに好ましくは4個以上のO-アセチル基が導入されている。
The acidic mucopolysaccharide is thus produced and recovered.
Acidic mucopolysaccharides produced by Cobetia sp. WAK-1A (deposit number FERM P-21101) obtained by the present invention can experimentally support that melanin production is strongly suppressed and cytotoxicity is extremely weak. It was. Therefore, the acidic mucopolysaccharide according to the present invention is useful as an active ingredient of a whitening agent. The whitening agent is applied to various wide fields such as cosmetics including quasi-drugs, pharmaceuticals, and foods.
The acidic mucopolysaccharides according to the present invention may be formulated in the form of salts or derivatives thereof such as physiologically acceptable salts, sodium salts, potassium salts, etc., for example, acylated products with fatty acids, preferably acetylated products. .
The acidic mucopolysaccharide O-acyl derivative according to the present invention can be obtained, for example, by treating acidic mucopolysaccharide with acetic anhydride or acid chloride in a dilute aqueous acid solution in a homogeneous system. Alternatively, when soluble in an organic solvent such as formamide, an O-acetyl derivative of acidic mucopolysaccharide can be obtained by treatment with pyridine and acetic anhydride. In addition, for example, it can also be prepared by the method described in JP-A-3-143540 and the method described in JP-A-6-9707. Although the O-acetylation rate varies depending on the acylation method, 1 or more, preferably 3 or more, more preferably 4 or more O-acetyl groups are introduced into each repeating unit of the acidic mucopolysaccharide. Yes.
さらに、本発明に係る酸性ムコ多糖は組成物に有効成分として配合され組成物としての美白剤に調製される。 Furthermore, the acidic mucopolysaccharide according to the present invention is blended as an active ingredient in the composition and prepared as a whitening agent as the composition.
前記組成物としての美白剤において、前記酸性ムコ多糖は美白作用を発揮するに有効な量が含有されるが、一般にはこれら化合物を有効成分として全量中少なくとも0.001重量%以上、好ましくは0.01〜20.0重量%、好ましくは0.05〜10.0重量%含有するように添加すればよい。0.001重量%未満であると、本発明でいう効果が十分に発揮されず、20.0重量%を超えると製剤化が難しいので好ましくない。また、10.0重量%以上配合してもさほど大きな効果の向上はみられない。必ずしも有効成分を単離して使用する必要はなく、必要に応じて本発明の効果を損なわない範囲で、本発明の化合物を含む粗精製物を使用することができる。 In the whitening agent as the composition, the acidic mucopolysaccharide contains an effective amount for exerting a whitening action. Generally, these compounds are used as active ingredients in an amount of at least 0.001% by weight, preferably 0%. What is necessary is just to add so that it may contain 0.01 to 20.0 weight%, Preferably 0.05 to 10.0 weight%. If it is less than 0.001% by weight, the effect referred to in the present invention is not sufficiently exerted, and if it exceeds 20.0% by weight, it is difficult to make a preparation. Moreover, even if it mix | blends 10.0 weight% or more, the improvement of the big effect is not seen. It is not always necessary to isolate and use the active ingredient, and a crude product containing the compound of the present invention can be used as long as the effects of the present invention are not impaired.
前記組成物としての美白剤は、上記必須成分の酸性ムコ多糖の他に、本発明の効果を損なわない範囲で、化粧品、医薬部外品、医薬品等に通常用いられる他の成分を、必要に応じて適宜配合して製造することができる。
前記他の成分としては、例えば、油分、界面活性剤、粉末、色剤、水、アルコール類、増粘剤、酸化防止剤、紫外線吸収剤、保湿剤等が挙げられる。さらに、エデト酸二ナトリウム、エデト酸三ナトリウム、クエン酸ナトリウム、ポリリン酸ナトリウム、メタリン酸ナトリウム、グルコン酸等の金属封鎖剤、カフェイン、タンニン、ベラパミル、トラネキサム酸及びその誘導体、甘草抽出物、グラブリジン、カリンの果実の熱水抽出物、各種生薬、酢酸トコフェロール、グリチルリチン酸及びその誘導体またはその塩等の薬剤、ビタミンC、アスコルビン酸リン酸マグネシウム、アスコルビン酸グルコシド、3−O−エチルアスコルビン酸、アルブチン、コウジ酸等の美白剤 、グルコース、フルクトース、マンノース、ショ糖、トレハロース等の糖類、レチノイン酸、レチノール、レチノール酢酸、レチノールパルミチン酸等のビタミンA誘導体類なども適宜配合することができる。
In addition to the acidic mucopolysaccharide as an essential component, the whitening agent as the composition requires other components that are usually used in cosmetics, quasi-drugs, pharmaceuticals, etc., as long as the effects of the present invention are not impaired. Accordingly, it can be appropriately blended and manufactured.
Examples of the other components include oils, surfactants, powders, colorants, water, alcohols, thickeners, antioxidants, ultraviolet absorbers, and humectants. In addition, metal sequestering agents such as disodium edetate, trisodium edetate, sodium citrate, sodium polyphosphate, sodium metaphosphate, gluconic acid, caffeine, tannin, verapamil, tranexamic acid and its derivatives, licorice extract, grabrizine , Hot water extract of karin fruit, various herbal medicines, drugs such as tocopherol acetate, glycyrrhizic acid and its derivatives or salts thereof, vitamin C, magnesium ascorbate phosphate, glucoside ascorbate, 3-O-ethylascorbic acid, arbutin Whitening agents such as kojic acid, saccharides such as glucose, fructose, mannose, sucrose, trehalose, vitamin A derivatives such as retinoic acid, retinol, retinol acetic acid, retinol palmitic acid, and the like can be appropriately blended.
前記組成物としての美白剤を投与する場合の投与経路は特に限定されず、経口、非経口、局所等何れの方法でも投与することができる。投与量は、対象(哺乳動物、特にはヒト)、年齢、性別、個人差、症状などによって適宜調整されるので特に限定されないが、例えば、本発明の酸性ムコ多糖として0.1〜500mg/kg、好ましくは0.5〜200mg/kgの用量を経口または非経口投与にて、1日1回または数回に分けて投与することができる。 The administration route in the case of administering the whitening agent as the composition is not particularly limited, and it can be administered by any method such as oral, parenteral and topical. The dosage is not particularly limited because it is appropriately adjusted depending on the subject (mammal, particularly human), age, sex, individual differences, symptoms, etc. For example, 0.1 to 500 mg / kg as the acidic mucopolysaccharide of the present invention. Preferably, a dose of 0.5 to 200 mg / kg can be administered orally or parenterally once or divided into several times a day.
前記組成物としての美白剤は、好適には皮膚に外用される皮膚外用剤の形態で、皮膚に直接塗布または散布する投与方法により使用され、例えば顔用、ボディー用として皮膚に塗布すると優れた美白効果を発揮する。 The whitening agent as the composition is preferably used in the form of a topical skin preparation externally applied to the skin, and is applied by a method of direct application or spraying to the skin. For example, it is excellent when applied to the skin for the face and body. Demonstrate whitening effect.
前記組成物としての美白剤の好適な形態である皮膚外用剤の剤型としては、本発明の効果を発揮できるものであれば特に限定されず、例えば、溶液系、可溶化系、乳化系、粉末分散系、水−油二層系、水−油−粉末三層系、固形、軟膏、ゲル、エアゾール、ムース等の任意の剤型をとることができる。 The dosage form of the external preparation for skin that is a suitable form of the whitening agent as the composition is not particularly limited as long as it can exert the effects of the present invention, and includes, for example, a solution system, a solubilization system, an emulsification system, Arbitrary dosage forms such as a powder dispersion system, a water-oil two-layer system, a water-oil-powder three-layer system, a solid, an ointment, a gel, an aerosol, and a mousse can be used.
前記組成物としての美白剤の好適な形態である皮膚外用剤は、例えば、医薬部外品を含む化粧品、医薬品の分野で応用され、その製品形態としては、例えば、軟膏、乳液、クリーム、ローション、ジェル、パック等の任意の形態をとることができる。また、前記皮膚外用剤は、ローション、乳液、クリーム、エッセンス(美容液),化粧水等の基礎化粧品、ファンデーション,口紅等のメーキャップ化粧品、芳香化粧品、毛髪用化粧品、ボディ化粧品等の医薬部外品を含む化粧品の形態をとることができる。 The skin external preparation, which is a suitable form of the whitening agent as the composition, is applied, for example, in the field of cosmetics and pharmaceuticals including quasi-drugs, and the product forms include, for example, ointments, emulsions, creams, lotions. , Gels, packs, etc. can take any form. The topical skin preparations are quasi-drugs such as lotion, milky lotion, cream, essence (beauty serum), basic cosmetics such as lotion, makeup cosmetics such as foundation and lipstick, aromatic cosmetics, cosmetics for hair, body cosmetics, etc. Can take the form of cosmetics.
本発明に係る酸性ムコ多糖は、皮膚外用剤に配合することができ、特に、日焼け後の色素沈着・しみ・そばかす・肝斑等の予防および改善に有効で、皮膚の美白に優れた効果を有する美白用皮膚外用剤として有用である。 The acidic mucopolysaccharide according to the present invention can be incorporated into an external preparation for skin, and is particularly effective in preventing and improving pigmentation, stains, freckles, liver spots, etc. after sunburn, and has an excellent effect on skin whitening. It is useful as an external preparation for skin whitening.
前記皮膚外用剤において、前記酸性ムコ多糖は美白作用を発揮するに有効な量が含有されるが、一般にはこれら化合物を有効成分として全量中少なくとも0.001重量%以上、好ましくは0.01〜20.0重量%、好ましくは0.05〜10.0重量%含有するように添加すればよい。0.001重量%未満であると、本発明でいう効果が十分に発揮されず、20.0重量%を超えると製剤化が難しいので好ましくない。また、10.0重量%以上配合してもさほど大きな効果の向上はみられない。必ずしも有効成分を単離して使用する必要はなく、必要に応じて本発明の効果を損なわない範囲で、本発明の化合物を含む粗精製物を使用することができる。 In the above-mentioned external preparation for skin, the acidic mucopolysaccharide contains an effective amount for exerting a whitening effect. Generally, at least 0.001% by weight or more, preferably 0.01-% or more of the total amount of these compounds as active ingredients. What is necessary is just to add so that it may contain 20.0 weight%, Preferably 0.05-10.0 weight%. If it is less than 0.001% by weight, the effect referred to in the present invention is not sufficiently exerted, and if it exceeds 20.0% by weight, it is difficult to make a preparation. Moreover, even if it mix | blends 10.0 weight% or more, the improvement of the big effect is not seen. It is not always necessary to isolate and use the active ingredient, and a crude product containing the compound of the present invention can be used as long as the effects of the present invention are not impaired.
前記皮膚外用剤には、上記必須成分の酸性ムコ多糖の他に、本発明の効果を損なわない範囲内で、通常化粧品、医薬部外品、医薬品等の外用剤に用いられる他の成分、例えば他の美白剤 、保湿剤、酸化防止剤、油分、紫外線吸収剤、界面活性剤、増粘剤、アルコール類、粉末成分、色剤、水性成分、水、各種皮膚栄養剤等を必要に応じて適宜配合することができる。
さらに、エデト酸二ナトリウム、エデト酸三ナトリウム、クエン酸ナトリウム、ポリリン酸ナトリウム、メタリン酸ナトリウム、グルコン酸等の金属封鎖剤、カフェイン、タンニン、ベラパミル、トラネキサム酸及びその誘導体、甘草抽出物、グラブリジン、カリンの果実の熱水抽出物、各種生薬、酢酸トコフェロール、グリチルリチン酸及びその誘導体またはその塩等の薬剤、ビタミンC、アスコルビン酸リン酸マグネシウム、アスコルビン酸グルコシド、3−O−エチルアスコルビン酸、アルブチン、コウジ酸等の美白剤 、グルコース、フルクトース、マンノース、ショ糖、トレハロース等の糖類、レチノイン酸、レチノール、レチノール酢酸、レチノールパルミチン酸等のビタミンA誘導体類なども適宜配合することができる。
In addition to the above-mentioned essential component acidic mucopolysaccharide, the external preparation for skin includes other components that are usually used in external preparations such as cosmetics, quasi-drugs, and pharmaceuticals, as long as the effects of the present invention are not impaired. Other whitening agents, moisturizers, antioxidants, oils, UV absorbers, surfactants, thickeners, alcohols, powder components, colorants, aqueous components, water, various skin nutrients, etc. as required It can mix | blend suitably.
In addition, metal sequestering agents such as disodium edetate, trisodium edetate, sodium citrate, sodium polyphosphate, sodium metaphosphate, gluconic acid, caffeine, tannin, verapamil, tranexamic acid and its derivatives, licorice extract, grabrizine , Hot water extract of karin fruit, various herbal medicines, drugs such as tocopherol acetate, glycyrrhizic acid and its derivatives or salts thereof, vitamin C, magnesium ascorbate phosphate, glucoside ascorbate, 3-O-ethylascorbic acid, arbutin Whitening agents such as kojic acid, saccharides such as glucose, fructose, mannose, sucrose, trehalose, vitamin A derivatives such as retinoic acid, retinol, retinol acetic acid, retinol palmitic acid, and the like can be appropriately blended.
前記皮膚外用剤の剤型は特に限定されるものでなく、本発明の効果を発揮できるものであれば特に限定されず、例えば、溶液系、可溶化系、乳化系、粉末分散系、水−油二層系、水−油−粉末三層系、固形、軟膏、ゲル、エアゾール、ムース等の任意の剤型をとることができる。 The dosage form of the external preparation for skin is not particularly limited, and is not particularly limited as long as the effect of the present invention can be exhibited. For example, a solution system, a solubilization system, an emulsification system, a powder dispersion system, water- Any dosage form such as oil two-layer system, water-oil-powder three-layer system, solid, ointment, gel, aerosol, mousse and the like can be taken.
また、前記皮膚外用剤は、例えば、医薬部外品を含む化粧品、医薬品の分野で応用され、その製品形態としては、例えば、軟膏、乳液、クリーム、ローション、ジェル、パック等の任意の形態をとることができる。また、前記皮膚外用剤は、ローション、乳液、クリーム、エッセンス(美容液),化粧水等の基礎化粧品、ファンデーション,口紅等のメーキャップ化粧品、芳香化粧品、毛髪用化粧品、ボディ化粧品等の医薬部外品を含む化粧品の形態をとることができる。 Further, the external preparation for skin is applied in the fields of cosmetics and pharmaceuticals including quasi-drugs, for example, and the product forms include any forms such as ointments, emulsions, creams, lotions, gels, packs, etc. Can take. The topical skin preparations are quasi-drugs such as lotion, milky lotion, cream, essence (beauty serum), basic cosmetics such as lotion, makeup cosmetics such as foundation and lipstick, aromatic cosmetics, cosmetics for hair, body cosmetics, etc. Can take the form of cosmetics.
本発明は、本酸性ムコ多糖を有効成分として含有してなる日焼け後の色素沈着・しみ・そばかす・肝斑等の予防および改善と関連した美白剤を含有する飲食品または医薬品の形態の組成物、本酸性ムコ多糖を有効成分として含有し、日焼け後の色素沈着・しみ・そばかす・肝斑等の予防および改善するために用いられるものである旨の表示を付した飲食品に関するものである。したがって、本発明が対象とする医薬品または特定保健用食品は、配合した本酸性ムコ多糖が有効成分として含有されていることを特徴とする。 The present invention relates to a composition in the form of a food or drink or a medicine containing a whitening agent associated with prevention and improvement of pigmentation / stain / freckle / liver spots after sunburn comprising the acidic mucopolysaccharide as an active ingredient The present invention relates to foods and drinks that contain the present acidic mucopolysaccharide as an active ingredient and that are labeled for the prevention and improvement of pigmentation, stains, freckles, and liver spots after sunburn. Therefore, the medicinal product or food for specified health use according to the present invention is characterized in that the blended acidic mucopolysaccharide is contained as an active ingredient.
医薬品の形態のものである場合、1またはそれ以上の医薬上許容される担体とともに、本酸性ムコ多糖を含む日焼け後の色素沈着・しみ・そばかす・肝斑等の予防および改善させる組成物である。当該医薬組成物は、単独で、または1もしくはそれ以上の医薬上許容される担体と組み合わせて、本発明の薬理学的活性化合物の治療上有効量を含んでなる。
本酸性ムコ多糖を上述の製剤として使用する場合には、公知の方法によって実施することができる。具体的には、例えば、以下に記載するとおりに実施することができる。
When it is in the form of a pharmaceutical, it is a composition for preventing and improving post-tanning pigmentation / stain / freckle / liver plaque etc. containing this acidic mucopolysaccharide together with one or more pharmaceutically acceptable carriers . The pharmaceutical composition comprises a therapeutically effective amount of a pharmacologically active compound of the present invention alone or in combination with one or more pharmaceutically acceptable carriers.
When this acidic mucopolysaccharide is used as the above-mentioned preparation, it can be carried out by a known method. Specifically, for example, it can be carried out as described below.
本発明の製剤は、必要に応じて糖衣やコーティングを施した錠剤、丸剤、カプセル剤(ハードカプセル、ソフトカプセル、マイクロカプセルを含む)、散剤、顆粒剤、細粒剤、トローチ剤、液剤(シロップ剤、乳剤、懸濁剤を含む)などとして経口的に使用するのが好ましい。 The preparation of the present invention comprises tablets, pills, capsules (including hard capsules, soft capsules, microcapsules), powders, granules, fine granules, troches, liquids (syrups) with sugar coating or coating as necessary , Emulsions, suspensions, etc.).
本発明の製剤には、本発明の効果を阻害しない限り、生理学的に許容される担体などを配合することができる。生理学的に許容される担体などとしては、製剤材料として慣用の各種有機あるいは無機担体物質が用いられ、固形製剤における賦形剤、結合剤、崩壊剤、滑沢剤;液状製剤における溶剤、溶解補助剤、懸濁化剤、緩衝剤、増粘剤、乳化剤などがあげられる。また、必要に応じて、着色剤、甘味剤、抗酸化剤などの製剤添加剤も用いることができる。さらに本発明の製剤をコーティングしてもよい。 The preparation of the present invention can be mixed with a physiologically acceptable carrier or the like as long as the effects of the present invention are not inhibited. As physiologically acceptable carriers, various organic or inorganic carrier substances commonly used as pharmaceutical materials are used. Excipients, binders, disintegrants, lubricants in solid formulations; solvents in liquid formulations, dissolution aids Agents, suspending agents, buffering agents, thickeners, emulsifiers and the like. In addition, formulation additives such as coloring agents, sweetening agents, and antioxidants can be used as necessary. Furthermore, you may coat the formulation of this invention.
賦形剤としては、例えば、乳糖、白糖、D−マンニトール、D−ソルビトール、デンプン、α化デンプン、デキストリン、結晶セルロース(例えば、微結晶セルロースなど)、低置換度ヒドロキシプロピルセルロース、カルボキシメチルセルロースナトリウム、アラビアゴム、デキストリン、プルラン、軽質無水ケイ酸、合成ケイ酸アルミニウム、メタケイ酸アルミン酸マグネシウムなどがあげられる。結合剤としては、例えば、α化デンプン、ショ糖、ゼラチン、マクロゴール、アラビアゴム、メチルセルロース、カルボキシメチルセルロース、カルボキシメチルセルロースナトリウム、結晶セルロース、白糖、D−マンニトール、トレハロース、デキストリン、プルラン、ヒドロキシプロピルセルロース(HPC)、ヒドロキシプロピルメチルセルロース(HPMC)、ポリビニルピロリドン(PVP)などがあげられる。崩壊剤としては、例えば、乳糖、白糖、デンプン、カルボキシメチルセルロース、カルボキシメチルセルロースカルシウム、架橋ポリビニルピロリドン、カルメロースナトリウム、クロスカルメロースナトリウム、カルボキシメチルスターチナトリウム、軽質無水ケイ酸、低置換度ヒドロキシプロピルセルロース、陽イオン交換樹脂、部分α化でんぷん、トウモロコシデンプンなどがあげられる。滑沢剤としては、例えば、ステアリン酸、ステアリン酸マグネシウム、ステアリン酸カルシウム、タルク、ワックス類、コロイドシリカ、DL−ロイシン、ラウリル硫酸ナトリウム、ラウリル硫酸マグネシウム、マクロゴール、エアロジルなどがあげられる。 Examples of the excipient include lactose, sucrose, D-mannitol, D-sorbitol, starch, pregelatinized starch, dextrin, crystalline cellulose (for example, microcrystalline cellulose), low-substituted hydroxypropylcellulose, sodium carboxymethylcellulose, Examples include gum arabic, dextrin, pullulan, light anhydrous silicic acid, synthetic aluminum silicate and magnesium aluminate metasilicate. Examples of the binder include pregelatinized starch, sucrose, gelatin, macrogol, gum arabic, methylcellulose, carboxymethylcellulose, sodium carboxymethylcellulose, crystalline cellulose, sucrose, D-mannitol, trehalose, dextrin, pullulan, hydroxypropylcellulose ( HPC), hydroxypropylmethylcellulose (HPMC), polyvinylpyrrolidone (PVP), and the like. Examples of the disintegrant include lactose, sucrose, starch, carboxymethylcellulose, carboxymethylcellulose calcium, crosslinked polyvinylpyrrolidone, carmellose sodium, croscarmellose sodium, sodium carboxymethyl starch, light anhydrous silicic acid, low-substituted hydroxypropylcellulose, Examples include cation exchange resin, partially pregelatinized starch, and corn starch. Examples of the lubricant include stearic acid, magnesium stearate, calcium stearate, talc, waxes, colloidal silica, DL-leucine, sodium lauryl sulfate, magnesium lauryl sulfate, macrogol, and aerosil.
溶剤としては、例えば、注射用水、生理的食塩水、リンゲル液、アルコール、プロピレングリコール、ポリエチレングリコール、中鎖脂肪酸トリグリセリド(MCT)、植物油(例えば、サフラワー油、ゴマ油、トウモロコシ油、オリーブ油、綿実油、大豆レシチンなど)などがあげられる。溶解補助剤としては、例えば、ポリエチレングリコール、プロピレングリコール、D−マンニトール、トレハロース、安息香酸ベンジル、エタノール、トリスアミノメタン、コレステロール、トリエタノールアミン、炭酸ナトリウム、クエン酸ナトリウム、サリチル酸ナトリウム、酢酸ナトリウムなどがあげられる。懸濁化剤としては、例えば、ステアリルトリエタノールアミン、ラウリル硫酸ナトリウム、ラウリルアミノプロピオン酸、レシチン、塩化ベンザルコニウム、塩化ベンゼトニウム、モノステアリン酸グリセリンなどの界面活性剤;例えばポリビニルアルコール、ポリビニルピロリドン、カルボキシメチルセルロースナトリウム、メチルセルロース、ヒドロキシメチルセルロース、ヒドロキシエチルセルロース、ヒドロキシプロピルセルロースなどの親水性高分子;ポリソルベート類、ポリオキシエチレン硬化ヒマシ油などがあげられる。緩衝剤としては、例えば、リン酸塩、酢酸塩、炭酸塩、クエン酸塩などの緩衝液などがあげられる。増粘剤としては、例えば、天然ガム類、セルロース誘導体などがあげられる。乳化剤としては、例えば、脂肪酸エステル類(例えば、ショ糖脂肪酸エステル、グリセリン脂肪酸エステル、ソルビタン脂肪酸エステル、プロピレングリコール脂肪酸エステルなど)、ワックス(例えば、ミツロウ、菜種水素添加油、サフラワー水素添加油、パーム水素添加油、シトステロール、スチグマステロール、カンペステロール、ブラシカステロール、カカオ脂粉末、カルナウバロウ、ライスワックス、モクロウ、パラフィンなど)、レシチン(例えば、卵黄レシチン、大豆レシチンなど)などがあげられる。 Examples of the solvent include water for injection, physiological saline, Ringer's solution, alcohol, propylene glycol, polyethylene glycol, medium chain fatty acid triglyceride (MCT), vegetable oil (for example, safflower oil, sesame oil, corn oil, olive oil, cottonseed oil, soybean Lecithin, etc.). Examples of solubilizers include polyethylene glycol, propylene glycol, D-mannitol, trehalose, benzyl benzoate, ethanol, trisaminomethane, cholesterol, triethanolamine, sodium carbonate, sodium citrate, sodium salicylate, sodium acetate, and the like. can give. Examples of the suspending agent include surfactants such as stearyltriethanolamine, sodium lauryl sulfate, laurylaminopropionic acid, lecithin, benzalkonium chloride, benzethonium chloride, and glyceryl monostearate; for example, polyvinyl alcohol, polyvinylpyrrolidone, Hydrophilic polymers such as sodium carboxymethylcellulose, methylcellulose, hydroxymethylcellulose, hydroxyethylcellulose, hydroxypropylcellulose; polysorbates, polyoxyethylene hydrogenated castor oil, and the like. Examples of the buffer include buffer solutions of phosphate, acetate, carbonate, citrate, and the like. Examples of the thickener include natural gums and cellulose derivatives. Examples of emulsifiers include fatty acid esters (eg, sucrose fatty acid ester, glycerin fatty acid ester, sorbitan fatty acid ester, propylene glycol fatty acid ester), wax (eg, beeswax, rapeseed hydrogenated oil, safflower hydrogenated oil, palm Examples thereof include hydrogenated oil, sitosterol, stigmasterol, campesterol, brush casterol, cacao butter powder, carnauba wax, rice wax, molasses, paraffin, and the like, and lecithin (for example, egg yolk lecithin and soybean lecithin).
着色剤としては、例えば、水溶性食用タール色素(例、食用赤色2号および3号、食用黄色4号および5号、食用青色1号および2号などの食用色素、水不溶性レーキ色素(例、前記水溶性食用タール色素のアルミニウム塩など)、天然色素(例、β−カロチン、クロロフィル、ベンガラなど)などがあげられる。甘味剤としては、例えば、ショ糖、乳糖、サッカリンナトリウム、グリチルリチン酸二カリウム、アスパルテーム、ステビアなどがあげられる。抗酸化剤としては、例えば、亜硫酸塩、アスコルビン酸及びそれらのアルカリ金属塩、アルカリ土類金属塩などがあげられる。 Examples of the colorant include water-soluble edible tar dyes (eg, edible red Nos. 2 and 3, edible yellow Nos. 4 and 5, edible blue Nos. 1 and 2 and the like, water-insoluble lake dyes (eg, Examples of the water-soluble edible tar pigment include aluminum salts, natural pigments (eg, β-carotene, chlorophyll, bengara, etc.) Examples of the sweetener include sucrose, lactose, sodium saccharin, dipotassium glycyrrhizinate, Examples include aspartame, stevia, etc. Examples of the antioxidant include sulfite, ascorbic acid, and alkali metal salts and alkaline earth metal salts thereof.
錠剤、顆粒剤、細粒剤などに関しては、味のマスキング、光安定性の向上、外観の向上あるいは腸溶性などの目的のため、コーティング基材を用いて通常の方法でコーティングしてもよい。このコーティング基剤としては、糖衣基剤、水溶性フィルムコーティング基材、腸溶性フィルムコーティング基材などがあげられる。 Tablets, granules, fine granules and the like may be coated by a usual method using a coating substrate for the purpose of masking taste, improving light stability, improving appearance or enteric properties. Examples of the coating base include sugar coating base, water-soluble film coating substrate, enteric film coating substrate and the like.
糖衣基剤としては、例えば、白糖があげられ、さらにタルク、沈降炭酸カルシウム、ゼラチン、アラビアゴム、プルラン、カルナバロウなどから選ばれる1種または2種以上を併用してもよい。 Examples of the sugar coating base include sucrose, and one or more kinds selected from talc, precipitated calcium carbonate, gelatin, gum arabic, pullulan, carnauba wax and the like may be used in combination.
水溶性フィルムコーティング基剤としては、例えば、ヒドロキシプロピルセルロース(HPC)、ヒドロキシプロピルメチルセルロース(HPMC)、エチルセルロース、ヒドロキシエチルセルロース、メチルヒドロキシエチルセルロースなどのセルロース系高分子;ポリビニルアセタールジエチルアミノアセテート、アミノアルキルメタアクリレートコポリマーE(オイドラギットE(商品名)、ロームファルマ社)ポリビニルピロリドンなどの合成高分子;プルランなどの多糖類などがあげられる。腸溶性フィルムコーティング基剤としては、例えば、ヒドロキシプロピルメチルセルロースフタレート、ヒドロキシプロピルメチルセルロースアセテートサクシネート、カルボキシメチルエチルセルロース、酢酸フタル酸セルロースなどのセルロース系高分子;メタアクリル酸コポリマーL(オイドラギットL(商品名)ロームファルマ社)、メタアクリル酸コポリマーLD(オイドラギットL−30D55(商品名)ロームファルマ社)メタアクリル酸コポリマーS(オイドラギットS(商品名)ロームファルマ社)などのアクリル酸系高分子;セラックなどの天然物などがあげられる。これらのコーティング基剤は、単独で、または2種以上を適宜の割合で混合してコーティングしてもよく、また2種以上を順次コーティングしてもよい。 Examples of the water-soluble film coating base include cellulose polymers such as hydroxypropylcellulose (HPC), hydroxypropylmethylcellulose (HPMC), ethylcellulose, hydroxyethylcellulose, methylhydroxyethylcellulose; polyvinyl acetal diethylaminoacetate, aminoalkyl methacrylate copolymer Synthetic polymers such as E (Eudragit E (trade name), Rohm Pharma) polyvinylpyrrolidone; and polysaccharides such as pullulan. Examples of enteric film coating bases include cellulose polymers such as hydroxypropylmethylcellulose phthalate, hydroxypropylmethylcellulose acetate succinate, carboxymethylethylcellulose, and cellulose acetate phthalate; methacrylic acid copolymer L (Eudragit L (trade name)) Acrylic polymers such as Rohm Pharma), methacrylic acid copolymer LD (Eudragit L-30D55 (trade name) Rohm Pharma), methacrylic acid copolymer S (Eudragit S (trade name) Rohm Pharma), etc. Examples include natural products. These coating bases may be coated singly or in combination of two or more at an appropriate ratio, or two or more may be coated sequentially.
飲食品の形態の組成物、あるいは機能・効能などの表示を付した飲食品である場合、本発明の食品組成物あるいは飲食品は、機能性食品、健康食品、特定保健用食品 、病者用食品、サプリメントなどとして調製されてもよく、機能性食品として調製されるのが好ましい。このような本発明の食品組成物あるいは飲食品の形状としては、例えば、タブレット状、丸状、カプセル(ハードカプセル、ソフトカプセル、マイクロカプセルを含む)状、粉末状、顆粒状、細粒状、トローチ状、液状(シロップ状、乳状、懸濁状をふくむ)などがあげられ、タブレット状、カプセル状であるのが好ましい。 In the case of a composition in the form of a food or drink, or a food or drink with an indication of function or efficacy, the food composition or food or drink of the present invention is a functional food, health food, food for specified health use, or for the sick It may be prepared as a food, supplement or the like, and is preferably prepared as a functional food. Examples of the shape of the food composition or food or drink of the present invention include a tablet shape, a round shape, a capsule shape (including hard capsules, soft capsules, and microcapsules), a powder shape, a granular shape, a fine granular shape, a troche shape, Liquids (including syrups, milks, and suspensions) are included, and tablets and capsules are preferred.
本発明の食品組成物としては、特にタブレット状、カプセル状であるのが好ましく、とりわけタブレット状の機能性食品、カプセル状の機能性食品であるのが好ましい。 The food composition of the present invention is particularly preferably tablet-like or capsule-like, and particularly preferably a tablet-like functional food or a capsule-like functional food.
本発明の食品組成物は、例えば、公知の方法によって食品中に本酸性ムコ多糖を含有させることによって製造することができる。具体的には、例えば、タブレット状の食品組成物は、例えば、本酸性ムコ多糖、および、賦形剤(例えば、乳糖、白糖、マンニトールなど)、甘味剤、着香剤などの原料を添加、混合し、打錠機などで圧力をかけてタブレット状に成形することにより製造することができる。必要に応じて、その他の材料(例えば、ビタミンCなどのビタミン類、鉄などミネラル類、食物繊維など)を添加することもできる。カプセル状の食品組成物は、例えば、本酸性ムコ多糖を含有する液状、懸濁状、のり状、粉末状または顆粒状の食品組成物をカプセルに充填するか、またはカプセル基剤で被包成形して製造することができる。 The food composition of the present invention can be produced, for example, by incorporating the acidic mucopolysaccharide into the food by a known method. Specifically, for example, the tablet-shaped food composition includes, for example, the present acidic mucopolysaccharide and raw materials such as excipients (for example, lactose, sucrose, mannitol, etc.), sweeteners, flavoring agents, It can manufacture by mixing and shape | molding in a tablet shape by applying a pressure with a tableting machine etc. Other materials (for example, vitamins such as vitamin C, minerals such as iron, dietary fiber, etc.) can be added as necessary. Capsule-shaped food composition is, for example, filled with a liquid, suspension, paste, powder or granule-shaped food composition containing the acidic mucopolysaccharide, or encapsulated with a capsule base. Can be manufactured.
本発明の食品組成物には、本発明の効果を阻害しない限り、通常用いられる食品素材、食品添加物、各種の栄養素、ビタミン類、風味物質(例えば、チーズやチョコレートなど)などに加え、生理学的に許容される担体などを配合することができる。生理学的に許容される担体などとしては、慣用の各種有機あるいは無機担体物質が用いられ、賦形剤、結合剤、崩壊剤、滑沢剤、着色剤、甘味剤、防腐剤、抗酸化剤、増粘剤、乳化剤などがあげられる。また食品添加物としては、着色剤、甘味剤、防腐剤、抗酸化剤、着香剤などがあげられる。さらに、その他の材料、例えば、鉄などのミネラル類、ペクチン、カラギーナン、マンナンなどの食物繊維などを含有していてもよい。 As long as the effects of the present invention are not impaired, the food composition of the present invention can be used in addition to commonly used food materials, food additives, various nutrients, vitamins, flavor substances (for example, cheese and chocolate), and physiological May be blended with an acceptable carrier. As physiologically acceptable carriers, various conventional organic or inorganic carrier materials are used, and excipients, binders, disintegrants, lubricants, coloring agents, sweeteners, preservatives, antioxidants, Examples include thickeners and emulsifiers. Examples of food additives include colorants, sweeteners, preservatives, antioxidants, and flavoring agents. Furthermore, other materials, for example, minerals such as iron, dietary fibers such as pectin, carrageenan, and mannan may be contained.
賦形剤、結合剤、崩壊剤、滑沢剤、溶剤、溶解補助剤、懸濁化剤、緩衝剤、増粘剤、着色剤、甘味剤、防腐剤、抗酸化剤としては、それぞれ前記した本発明の製剤に用いられるものと同様のものがあげられる。 Excipients, binders, disintegrants, lubricants, solvents, solubilizers, suspending agents, buffers, thickeners, colorants, sweeteners, preservatives, and antioxidants are described above. The thing similar to what is used for the formulation of this invention is mention | raise | lifted.
ビタミン類としては、水溶性であっても脂溶性であってもよく、例えばパルミチン酸レチノール、トコフェロール、ビスベンチアミン、リボフラビン、塩酸ピリドキシン、シアノコバラミン、アスコルビン酸ナトリウム、コレカルシフェロール、ニコチン酸アミド、パントテン酸カルシウム、葉酸、ビオチン、重酒石酸コリンなどがあげられる。 Vitamins may be water-soluble or fat-soluble, such as retinol palmitate, tocopherol, bisbenchamine, riboflavin, pyridoxine hydrochloride, cyanocobalamin, sodium ascorbate, cholecalciferol, nicotinamide, pantothene. Examples include calcium acid, folic acid, biotin, and choline bitartrate.
タブレット状、顆粒状、細粒状の食品組成物などに関しては、味のマスキング、光安定性の向上、外観の向上あるいは腸溶性などの目的のため、コーティング基材を用いて自体公知の方法でコーティングしてもよい。そのコーティング基材としては、前記した本発明の製剤に用いられるものと同様のものがあげられ、同様にして実施することができる。 For tablet-like, granule-like, and fine-grained food compositions, etc., coating is carried out by a known method using a coating substrate for the purpose of masking taste, improving light stability, improving appearance, or enteric properties. May be. Examples of the coating substrate include those similar to those used in the preparation of the present invention described above, and can be carried out in the same manner.
本発明において、本酸性ムコ多糖は、安全であるので、ある個人が将来色素沈着・しみ・そばかす・肝斑等を発症し得る可能性に対する予防法として、あらゆる一般人に投与および/または給食してもよい。本発明の好ましい実施形態において、本酸性ムコ多糖は、色素沈着・しみ・そばかす・肝斑等のリスクが有ると疑われる個人に投与および/または給食してもよい。 In the present invention, the present acidic mucopolysaccharide is safe, so that it can be administered and / or fed to any general person as a preventive method for the possibility that a certain person may develop pigmentation, stains, freckles, liver spots, etc. in the future. Also good. In a preferred embodiment of the present invention, the acidic mucopolysaccharide may be administered and / or fed to an individual suspected of being at risk for pigmentation, stains, freckles, liver spots and the like.
本酸性ムコ多糖またはそれらの生理学的に許容される塩もしくはその誘導体をヒトが摂取する場合、個々人の年齢、体重および症状などによって用法用量が決定されるべきであるが、多くの場合有効な用量は本酸性ムコ多糖またはそれらの生理学的に許容される塩もしくはその誘導体を使用する場合、例えば、本発明の酸性ムコ多糖として0.1〜500mg/kg、好ましくは0.5〜200mg/kgの用量を経口または非経口投与にて、1日1回または数回に分けて投与することができる。あるいは1日1回または分割して食前、食後あるいは食事とともに摂取されるのが適当である。 When humans ingest the acid mucopolysaccharides or their physiologically acceptable salts or derivatives thereof, the dosage should be determined according to the age, weight and symptoms of the individual. When the present acidic mucopolysaccharide or a physiologically acceptable salt or derivative thereof is used, for example, the acidic mucopolysaccharide of the present invention is 0.1 to 500 mg / kg, preferably 0.5 to 200 mg / kg. The dose can be administered orally or parenterally once or divided into several times a day. Alternatively, it is appropriate to take once a day or in divided portions before meals, after meals or with meals.
本発明の好ましい実施形態において、本酸性ムコ多糖は、経口投与および/または給食されるが、皮下、吸入、静脈、くも膜下腔、直腸、または他のあらゆる好適な投与経路を含む、他の投与経路も利用されうる。本酸性ムコ多糖を含有する配合は、投与様式に依存して、標準的な手法及び化合物を用いて、変化し得る。 In preferred embodiments of the invention, the acidic mucopolysaccharide is administered orally and / or fed, but other administrations including subcutaneous, inhalation, intravenous, subarachnoid, rectal, or any other suitable route of administration. Routes can also be used. Formulations containing the present acidic mucopolysaccharides can be varied using standard techniques and compounds, depending on the mode of administration.
次に実施例を挙げ、本発明をさらに詳しく説明するが、本発明はこれら実施例になんら制約されるものではない。なお、特記しない限り、百分率(%)は重量基準であることはいうまでもない。 EXAMPLES Next, although an Example is given and this invention is demonstrated in more detail, this invention is not restrict | limited at all to these Examples. Needless to say, the percentage (%) is based on weight unless otherwise specified.
〈硫酸多糖の同伴産生を伴わない本酸性ムコ多糖産生菌の分離〉
ペプトン0.5%、 酵母エキス0.1%、蔗糖3%の組成を有し海水および/または人工海水で調製した培地を、温度121℃としたオートクレーブ中で15分間滅菌し、本酸性ムコ多糖産生菌として保存中の保存用斜面培養から、1白金耳を試験管中の上記滅菌培地(10ml)に接種し、25℃の温度で24時間振とう培養を行い、次いでこの前培養液を500ml容の三角フラスコ中に上記組成の培地200ml(121℃、15分間滅菌)に接種し、25℃の温度で3日間の振とう培養を行った。培養後培養液を濾過助剤(ラジオライト#500)を用いて濾過し、菌体を除いた上澄液にエタノールを加えて沈殿する画分を集め、水に溶解後5%第4級アンモニウム塩 (Cetavlon)溶液を加えて沈殿する画分を濾過により集めた。
これを4MNaClに溶解し再度エタノールを加えて沈殿する画分を集め、水に溶解後透析し凍結乾燥により多糖画分を得た。
この多糖画分は、構成成分のモル比がN−アセチル−D−ガラクトサミン:D−グルクロン酸:N−アセチル−L−ガラクトサミン:ピルビン酸が2:1:1:1で、ゲル濾過クロマトグラフィーで測定した平均分子量がプルランを標準として100〜150万であった。具体的には、Asahipak GFA-7M(昭和電工製)をカラムとする高速液体クロマトグラフィー(島津製)を使用し、0.1NNaClを移動相とし、分子量既知のプルラン(Shodex STANDARD KIT P-82、昭和電工製)を標準サンプルとして作成した分子量保持時間標準曲線を使用して測定した。
構成成分の分析には、セルロースアセテート膜電気泳動および高速液体クロマトグラフィーを用いた。この構成成分の分析には、該多糖画分を2Mのトリフルオロ酢酸(TFA)、または4N−HClで100℃、12時間加水分解し、ロータリーエバポレイターでTFAまたはHClを除いたものを検体とし、中性糖、ウロン酸、有機酸、硫酸イオンおよびアミノ糖の分析を行った。特にウロン酸の分析は、多糖のカルボキシル基還元後に加水分解して中性糖を分析することにより行った。有機酸の分析はイオンクロマト分析法で行い、この他に酵素法を用いた。硫酸基の分析は、加水分解後の試料をイオンクロマト分析法により実施した。
本多糖画分の1H核磁気共鳴分析(図1)によりアノメリックプロトンのシグナルδ=4.8 ppmの硫酸多糖のケミカルシフト(M.Matsudaら:Nippon Suisan Gakkaishi, 59, 535-538, 1993)が認められずまたイオンクロマト分析でも硫酸基の認められない菌株を探索した。
<Isolation of this acid mucopolysaccharide-producing bacterium without the accompanying production of sulfate polysaccharide>
A medium prepared with seawater and / or artificial seawater having a composition of 0.5% peptone, 0.1% yeast extract and 3% sucrose is sterilized for 15 minutes in an autoclave at a temperature of 121 ° C. From the slant culture for storage as a producing microorganism, inoculate one platinum loop into the above-mentioned sterilized medium (10 ml) in a test tube, perform shaking culture at a temperature of 25 ° C. for 24 hours, and then add 500 ml of this preculture solution. 200 ml (121 ° C., sterilized for 15 minutes) of the medium having the above composition was inoculated into an Erlenmeyer flask and cultured at 25 ° C. for 3 days with shaking. After incubation, the culture solution is filtered using a filter aid (Radiolite # 500), ethanol is added to the supernatant from which the cells have been removed, and the precipitated fraction is collected, dissolved in water, and then 5% quaternary ammonium. Fraction precipitated by addition of salt (Cetavlon) solution was collected by filtration.
This fraction was dissolved in 4M NaCl, and ethanol was added again to collect the precipitated fraction. The resulting fraction was dissolved in water, dialyzed, and freeze-dried to obtain a polysaccharide fraction.
This polysaccharide fraction has a molar ratio of constituents of N-acetyl-D-galactosamine: D-glucuronic acid: N-acetyl-L-galactosamine: pyruvic acid of 2: 1: 1: 1, and gel filtration chromatography. The measured average molecular weight was 1 to 1.5 million with pullulan as a standard. Specifically, high performance liquid chromatography (manufactured by Shimadzu) using Asahipak GFA-7M (manufactured by Showa Denko) as a column, 0.1N NaCl as the mobile phase, pullulan having a known molecular weight (Shodex STANDARD KIT P-82, It was measured using a molecular weight retention time standard curve created as a standard sample.
Cellulose acetate membrane electrophoresis and high performance liquid chromatography were used to analyze the constituent components. For analysis of this component, the polysaccharide fraction was hydrolyzed with 2M trifluoroacetic acid (TFA) or 4N-HCl at 100 ° C. for 12 hours, and the TFA or HCl was removed using a rotary evaporator as a sample. Neutral sugar, uronic acid, organic acid, sulfate ion and amino sugar were analyzed. In particular, uronic acid was analyzed by analyzing the neutral sugar by hydrolysis after reduction of the carboxyl group of the polysaccharide. Analysis of organic acids was performed by ion chromatography, and in addition to this, enzymatic methods were used. The sulfate group was analyzed by ion chromatography analysis of the sample after hydrolysis.
The 1 H nuclear magnetic resonance analysis of this polysaccharide fraction (Fig. 1) shows that the chemical shift of sulfate polysaccharide with an anomeric proton signal δ = 4.8 ppm (M. Matsuda et al .: Nippon Suisan Gakkaishi, 59, 535-538, 1993) We searched for strains that could not be recognized and that sulfate groups were not found by ion chromatography analysis.
本酸性ムコ多糖の1HNMRにおけるアノメリックプロトンのシグナル(500MHz,内部標準:TSP-d4、溶媒:D2O)、δ=4.50 (1H, J=7.6Hz), δ=4.58 (1H, J=8.3Hz), δ=4.57 (1H, J=8.1Hz), δ=5.07 (1H, J=2.9Hz)を図1に示した。 Anomeric proton signal (500 MHz, internal standard: TSP-d4, solvent: D2O), δ = 4.50 (1H, J = 7.6 Hz), δ = 4.58 (1H, J = 8.3 Hz) in 1 HNMR of this acidic mucopolysaccharide ), δ = 4.57 (1H, J = 8.1 Hz) and δ = 5.07 (1H, J = 2.9 Hz) are shown in FIG.
その結果、硫酸多糖の同伴産生を伴わずに本酸性ムコ多糖を産生する菌株を取得し、WAK-1A株と命名した。WAK-1A株は、独立行政法人産業技術総合研究所 特許生物寄託センター 〒305−8566 茨城県つくば市東1丁目1番地1中央第6 に寄託し、平成18年11月20日 寄託番号 FERM P-21101として受託されている。 As a result, a strain that produces this acidic mucopolysaccharide without accompanying production of sulfate polysaccharide was obtained and named WAK-1A strain. The WAK-1A strain was deposited at the National Institute of Advanced Industrial Science and Technology, Patent Biological Deposit Center, 1-6 Chuo, 1-chome Higashi 1-chome, Tsukuba City, Ibaraki Prefecture 305-8586. Deposit number FERM P- It has been entrusted as 21101.
〈新規菌株の分類学的位置〉
新たに単離した硫酸多糖の同伴産生を伴わない本酸性ムコ多糖産生菌株WAK-1A (FERM P-21101)を16S rDNA塩基配列の分析によって同定した。この新たに単離されたWAK-1A菌株の16S rDNAの塩基配列は、Cobetiaの16S rDNA塩基配列と99.7%の相同性を示しコベティア(Cobetia)に属すると決定された。しかし、この菌株は、コベティア属の基準株であるCobetia marina DSM4741とは、以下に記載されるいくつかの培養特性および生化学的特性によって明らかに区別される。
<Taxonomic position of new strains>
This acid mucopolysaccharide-producing strain WAK-1A (FERM P-21101) without accompanying production of newly isolated sulfate polysaccharide was identified by analysis of 16S rDNA nucleotide sequence. The 16S rDNA nucleotide sequence of this newly isolated WAK-1A strain was determined to belong to Cobetia, showing 99.7% homology with the Cobetia 16S rDNA nucleotide sequence. However, this strain is clearly distinguished from Covetia marina DSM4741, the reference strain of the genus Covetia, by several culture and biochemical characteristics described below.
WAK-1A菌株の16S rDNA-Full塩基配列による相同性検索を以下のようにして行った(表1:16S rDNA(16S rRNA遺伝子)塩基配列解析)。ゲノムの抽出操作はBIO RAD社のプロトコールに従った。抽出したゲノムDNAを鋳型とし、PCRにより16S ribosomal RNA遺伝子(16S rDNA)の全塩基配列1500〜1600bpの領域を増幅した。その後、増幅された16S rDNAをシーケンシングし16S rDNAの塩基配列(配列番号1)を決定した。 The homology search of the WAK-1A strain using the 16S rDNA-Full nucleotide sequence was performed as follows (Table 1: 16S rDNA (16S rRNA gene) nucleotide sequence analysis). The genome extraction procedure followed the BIO RAD protocol. Using the extracted genomic DNA as a template, a region of the entire base sequence 1500-1600 bp of the 16S ribosomal RNA gene (16S rDNA) was amplified by PCR. Thereafter, the amplified 16S rDNA was sequenced to determine the base sequence (SEQ ID NO: 1) of 16S rDNA.
BLASTを用いた細菌基準株データベースに対する相同性検索の結果、WAK-1A菌株の16S rDNA塩基配列(配列番号1)はCobetia由来の16S rDNAに対して高い相同性を示し、相同率99.7%でC.marina DSM4741株の16S rDNAに対し最も高い相同性を示した。 As a result of homology search against the bacterial reference strain database using BLAST, the 16S rDNA base sequence (SEQ ID NO: 1) of WAK-1A strain showed high homology to Cobetia-derived 16S rDNA, and the homology rate was 99.7%. It showed the highest homology to 16S rDNA of .marina DSM4741 strain.
GenBank/DDBJ/EMBLに対する相同性検索の結果においても、WAK-1A菌株の16S rDNAはC.marina由来の16S rDNAに対し高い相同性を示し、基準株ではC.marina DSM4741株の16S rDNAに対し相同率99.7%の相同性を示した。また、WAK-1A菌株の16S rDNAと細菌基準株データベースに対する相同性検索上位10株の16S rDNAを用いて行った簡易分子系統解析の結果、WAK-1A菌株はC.marina の16S rDNAと系統枝を形成し、近縁であることが示された。しかし、両者の16S rDNA間には4塩基の相違点があり、その内の3塩基は明らかに異なり、完全には一致しない。このことから、WAK-1A菌株がC.marinaに近縁であるものの、種としては異なる菌株であると考えられ、今回の16S rDNA塩基配列の結果からは、WAK-1A菌株をC.marinaに近縁なCobetia sp.とすることが妥当と判断した。図2は、WAK-1A菌株が16S rDNA塩基配列決定によって位置決めされた分子系統樹を示す。 As a result of homology search against GenBank / DDBJ / EMBL, the 16S rDNA of WAK-1A strain showed high homology to 16S rDNA derived from C. marina, and the reference strain compared to 16S rDNA of C. marina DSM4741 strain. The homology was 99.7%. In addition, as a result of simple molecular phylogenetic analysis using 16S rDNA of WAK-1A strain and 16S rDNA of the top 10 homology search against the bacterial reference strain database, WAK-1A strain was found to be C. marina 16S rDNA and phylogenetic branch. Was shown to be closely related. However, there is a difference of 4 bases between the 16S rDNAs, and 3 bases are clearly different and do not match completely. From this, although the WAK-1A strain is closely related to C.marina, it is considered that it is a different strain as a species.From the results of this 16S rDNA nucleotide sequence, the WAK-1A strain was changed to C.marina. It was judged that it was appropriate to use closely related Cobetia sp. FIG. 2 shows a molecular phylogenetic tree in which the WAK-1A strain was positioned by 16S rDNA sequencing.
WAK-1A菌株の培養学的および生化学的特性についてMB2216培地(Becton Dickinson, MD, USA)を用い、25℃、24時間培養により試験した。光学顕微鏡による観察およびBarrowら(Barrow,G.I.ら:Cowan and Steel’s Manual for the Identification of Medical Bacteria. 3 rd ed., Cambridge Press, 1993)の方法に基づき、カタラーゼ反応、オキシダーゼ反応、ブドウ糖からの酸/ガス産生、ブドウ糖の酸化/発酵(OF)について試験した。培養学的および生化学的特性についてはさらに試験用キットAP120NE(bioMerieux, Lyon, France)を用い当該キットのプロトコールに基づき実施した。表2(培養学的および生化学的試験)および表3(資化性試験)にその結果を示す。 The culture and biochemical characteristics of the WAK-1A strain were examined by culturing at 25 ° C. for 24 hours using MB2216 medium (Becton Dickinson, MD, USA). Based on observation by light microscope and Barrow et al. (Barrow, GI et al .: Cowan and Steel's Manual for the Identification of Medical Bacteria. 3rd ed., Cambridge Press, 1993), catalase reaction, oxidase reaction, acid from glucose / Tested for gas production, glucose oxidation / fermentation (OF). The culture and biochemical characteristics were further determined using a test kit AP120NE (bioMerieux, Lyon, France) according to the protocol of the kit. The results are shown in Table 2 (cultural and biochemical tests) and Table 3 (utilization test).
本菌株は、運動性を有するグラム陰性桿菌で、グルコースを酸化せず、カタラーゼ反応は陽性、オキシダーゼ反応は陰性を示した。またβ-ガラクトシダーゼ活性を示した。WAK-1A菌株は、10℃および45℃で生育し、でんぷんを加水分解せず、資化性試験ではD-グルコース、ラクトースおよびサッカロース等を資化し、L-アラビノース、マルトースおよびL-アラニン等を資化しなかった。これらの性状は16S rDNA塩基配列解析の結果において近縁性が示唆されたC.marinaに類似すると考えられる。しかし、45℃で生育する点、ラクトースおよびサッカロースを資化する点はC.marinaの基準株の性状と異なった。よって今回の試験結果から、本菌株をC.marinaに近縁なCobetia sp.と推定した。 This strain was a gram-negative gonococci having motility, did not oxidize glucose, showed a positive catalase reaction, and a negative oxidase reaction. It also showed β-galactosidase activity. The WAK-1A strain grows at 10 ° C and 45 ° C, does not hydrolyze starch, and assimilates D-glucose, lactose, saccharose, etc. in assimilation tests, and uses L-arabinose, maltose, L-alanine, etc. Not assimilated. These properties are considered to be similar to C.marina, which was suggested to be related in the results of 16S rDNA nucleotide sequence analysis. However, the point of growing at 45 ° C and the utilization of lactose and saccharose differed from those of C.marina reference strains. Therefore, from this test result, this strain was presumed to be Cobetia sp. Closely related to C. marina.
ペプトン0.5%、 酵母エキス0.1%、蔗糖3%の組成を有し海水および/または人工海水で調製した培地を、温度121℃としたオートクレーブ中で15分間滅菌し、WAK-1A菌株の斜面培養から、1白金耳を試験管中の上記滅菌培地(10ml)に接種し、25℃の温度で24時間振とう培養を行い、次いでこの前培養液を500ml容の三角フラスコ中に上記組成の培地200ml(121℃、15分間滅菌)に接種し、25℃の温度で3日間の静置培養を行った。培養後培養液を遠心分離または濾過助剤(ラジオライト#500)を用いて濾過し、菌体を除いた上澄液にエタノールを加えて沈殿する画分を集め、水に溶解後5%第4級アンモニウム塩 (Cetavlon)溶液を加えて沈殿する画分を濾過により集めた。これを4MNaClに溶解し再度エタノールを加えて沈殿する画分を集め、水に溶解後透析し凍結乾燥により多糖画分を得た。
なお、実用的な精製レベルとしては、培養濾過液から分子量5万カットの中空糸UF膜モジュール(Spectrum社製)を備えた膜濾過装置により得られる高分子画分を短時間のうちに濃縮、脱塩回収し、凍結乾燥により粉末化した。膜濾過装置は東洋紡エンジニアリング社製SYLS-SB04型を用いた。
このようにして得られた多糖画分については、セルロースアセテート膜電気泳動法を用いて均一性を確認すると共に、DEAE-セルロースイオン交換カラムクロマト分析、化学分析、核磁気共鳴分析により、公知の本酸性ムコ多糖であることを確認した。またこれらの標品について1HNMR分析およびイオンクロマト分析を行った結果、硫酸多糖類が含まれていないことが確認された。
A medium prepared with seawater and / or artificial seawater having a composition of 0.5% peptone, 0.1% yeast extract and 3% sucrose was sterilized in an autoclave at 121 ° C for 15 minutes, and strain WAK-1A From the slant culture, inoculate one platinum loop into the above sterilized medium (10 ml) in a test tube, shake culture at a temperature of 25 ° C. for 24 hours, and then place this preculture in the 500 ml Erlenmeyer flask. 200 ml of the medium having the composition (121 ° C., sterilized for 15 minutes) was inoculated, and static culture was performed at a temperature of 25 ° C. for 3 days. After culturing, the culture solution is centrifuged or filtered using a filter aid (Radiolite # 500), ethanol is added to the supernatant from which the cells have been removed, and the precipitated fraction is collected. A fraction that precipitated upon addition of a quaternary ammonium salt (Cetavlon) solution was collected by filtration. This fraction was dissolved in 4M NaCl, and ethanol was added again to collect the precipitated fraction. The resulting fraction was dissolved in water, dialyzed, and freeze-dried to obtain a polysaccharide fraction.
As a practical purification level, the polymer fraction obtained from the culture filtrate by a membrane filtration apparatus equipped with a hollow fiber UF membrane module (Spectrum) with a molecular weight of 50,000 cut is concentrated in a short time. Desalted and recovered, and powdered by lyophilization. The membrane filtration apparatus used was SYLS-SB04 manufactured by Toyobo Engineering.
The polysaccharide fraction thus obtained was confirmed for homogeneity by using cellulose acetate membrane electrophoresis, and by using DEAE-cellulose ion exchange column chromatography analysis, chemical analysis, and nuclear magnetic resonance analysis, The acid mucopolysaccharide was confirmed. Further, as a result of 1HNMR analysis and ion chromatography analysis of these samples, it was confirmed that the sulfated polysaccharide was not contained.
前培養までは実施例3と同様に処理し、次いでこの前培養液を500ml容の三角フラスコ中に上記組成を有する海水および/または人工海水から調製した滅菌培地200ml(121℃、15分間)に接種し、25℃の温度で3日間振とう培養を行った。次いで培養液を遠心分離または濾過助剤(ラジオライト#500)を用いて濾過し、上澄液にエタノールを加えて沈殿する画分を集め、水に溶解後5%第4級アンモニウム塩 (Cetavlon)溶液を加えて沈殿する画分を濾過により集めた。これを4MNaClに溶解し再度エタノールを加えて沈殿する画分を集め、水に溶解後透析し凍結乾燥により多糖画分を得た。なお、実用的な精製レベルとしては、培養濾液から分子量5万カットの中空糸UF膜モジュール(Spectrum社製)を備えた膜濾過装置により得られる高分子画分を短時間のうちに濃縮、脱塩回収し、凍結乾燥により粉末化した。膜濾過装置は東洋紡エンジニアリング社製SYLS-SB04型を用いた。このようにして得られた多糖画分については、セルロースアセテート膜電気泳動法を用いて均一性を試験すると共に、DEAE-セルロースイオン交換カラムクロマト分析、化学分析、核磁気共鳴分析により、公知の本酸性ムコ多糖であることを確認した。またこれらの標品について1HNMR分析およびイオンクロマト分析を行った結果、硫酸多糖類が含まれていないことが確認された。 Until the preculture, the same treatment as in Example 3 was performed, and then this preculture was put into 200 ml (121 ° C., 15 minutes) of a sterilized medium prepared from seawater having the above composition and / or artificial seawater in a 500 ml Erlenmeyer flask. Inoculated and cultured with shaking at 25 ° C. for 3 days. Next, the culture solution is centrifuged or filtered using a filter aid (Radiolite # 500), ethanol is added to the supernatant, and the precipitated fraction is collected, dissolved in water, and then dissolved in 5% quaternary ammonium salt (Cetavlon ) The fractions that precipitated upon addition of the solution were collected by filtration. This fraction was dissolved in 4M NaCl, and ethanol was added again to collect the precipitated fraction. The resulting fraction was dissolved in water, dialyzed, and freeze-dried to obtain a polysaccharide fraction. As a practical purification level, a polymer fraction obtained from a culture filtrate by a membrane filtration apparatus equipped with a hollow fiber UF membrane module (Spectrum) having a molecular weight of 50,000 cut is concentrated and removed in a short time. The salt was collected and pulverized by lyophilization. The membrane filtration apparatus used was SYLS-SB04 manufactured by Toyobo Engineering. The polysaccharide fraction thus obtained was tested for homogeneity using a cellulose acetate membrane electrophoresis method, and a known book was obtained by DEAE-cellulose ion exchange column chromatography analysis, chemical analysis, and nuclear magnetic resonance analysis. The acid mucopolysaccharide was confirmed. Further, as a result of 1HNMR analysis and ion chromatography analysis of these samples, it was confirmed that the sulfated polysaccharide was not contained.
前培養までは実施例3と同様に処理し、上記実施例3で述べた培地に寒天を1.5%添加した寒天平板培地250mlを平板(18×26cm)に広げて前培養液を塗沫し、25℃の温度で4日間培養を行った後、寒天平板の表面に生じた粘質物をかきとり、1%フエノール液に懸濁させ、濾過助剤(ラジオライト#500)で菌体を濾過により除いて得られた上澄液にエタノールを加えて沈殿する画分を集め、水に溶解後5%第4級アンモニウム塩 (Cetavlon)溶液を加えて沈殿する画分を濾過により集めた。これを4MNaClに溶解し再度エタノールを加えて沈殿する画分を集め、水に溶解後透析し凍結乾燥により多糖画分を得た。なお、実用的な精製レベルとしては、培養濾過液から分子量5万カットの中空糸UF膜モジュール(Spectrum社製)を備えた膜濾過装置により得られる高分子画分を短時間のうちに濃縮、脱塩回収し、凍結乾燥により粉末化した。
このようにして得られた多糖画分については、セルロースアセテート膜電気泳動法を用いて均一性を確認すると共に、化学分析、核磁気共鳴分析により公知の本酸性ムコ多糖であることを確認した。またこれらの標品について1HNMR分析およびイオンクロマト分析を行った結果、硫酸多糖類が含まれていないことが確認された。
Until the pre-culture, the same treatment as in Example 3 was performed, 250 ml of agar plate medium obtained by adding 1.5% of agar to the medium described in Example 3 above was spread on a flat plate (18 × 26 cm) and smeared with the pre-culture solution. After culturing at 25 ° C for 4 days, scrape the sticky material generated on the surface of the agar plate, suspend it in 1% phenol solution, and filter the cells with a filter aid (Radiolite # 500). Fraction precipitated by adding ethanol to the supernatant obtained by removing the solution by collecting the solution, and 5% quaternary ammonium salt (Cetavlon) solution was added after dissolution in water, and the fraction precipitated by filtration was collected by filtration. This fraction was dissolved in 4M NaCl, and ethanol was added again to collect the precipitated fraction. The resulting fraction was dissolved in water, dialyzed, and freeze-dried to obtain a polysaccharide fraction. As a practical purification level, the polymer fraction obtained from the culture filtrate by a membrane filtration apparatus equipped with a hollow fiber UF membrane module (Spectrum) with a molecular weight of 50,000 cut is concentrated in a short time. Desalted and recovered, and powdered by lyophilization.
About the polysaccharide fraction obtained in this way, while confirming uniformity using the cellulose acetate membrane electrophoresis method, it confirmed that it was this well-known acidic mucopolysaccharide by a chemical analysis and a nuclear magnetic resonance analysis. Further, as a result of 1HNMR analysis and ion chromatography analysis of these samples, it was confirmed that the sulfated polysaccharide was not contained.
なお、本実施例2〜5において用いられた上記の菌株は、独立行政法人産業技術総合研究所 特許生物寄託センター 〒305−8566 日本国茨城県つくば市東1丁目1番地1中央第6に寄託し、平成18年11月20日 寄託番号 FERM P-21101として受託されたものである。 The above strains used in Examples 2 to 5 were deposited in the 1st, 1st, 1st Street, 1-chome, Tsukuba City, Ibaraki Prefecture, Japan 305-8586, National Institute of Advanced Industrial Science and Technology. , November 20, 2006 Deposit No. FERM P-21101.
(マウス培養細胞株によるメラニン生成抑制試験)
実施例4で得られた本酸性ムコ多糖を使用して,メラノーマ細胞に対する細胞増殖とメラニン生成抑制効果を調べ試料無添加の場合と比較して試料添加系での細胞増殖率,メラニン生成量を求めた。又陽性対照としてメラニン生成抑制作用を有することが知られている多糖類(Okazakiら:Fragrance Journal, 8, 61-64, 2004)を使用した。
(Inhibition test of melanin production using mouse cell lines)
Using the acidic mucopolysaccharide obtained in Example 4, cell growth and melanin production inhibitory effects on melanoma cells were examined, and the cell growth rate and melanin production amount in the sample addition system were compared with the case of no sample addition. Asked. As a positive control, a polysaccharide (Okazaki et al .: Fragrance Journal, 8, 61-64, 2004) known to have an inhibitory action on melanin production was used.
(細胞培養)
(1)前培養したB16F10細胞を12穴マルチウェルプレートの各ウェルに1.0x104細胞/ウェルの細胞数に播種した。播種48時間後、所定濃度の被験物質を含む0.5mMテオフィリン含有DMEM+10%FBS培地に交換した。被験物質の濃度は、25μg/ml、50μg/ml、100μg/mlに設定した。各試料濃度毎に3ウェルを割り当てアッセイに供した(N=3)。細胞増殖計数用のプレートとメラニン定量用のプレート2枚を1組として各試料のアッセイに使用した。
培地交換後、72時間培養を続けた。
(2)細胞増殖計数用プレートを用いて各ウェル内の細胞数(相対値)をWST-1法を用い450nmの吸光度を測定することにより、細胞の増殖度を測定した。この結果を図3に示す。
(Cell culture)
(1) Precultured B16F10 cells were seeded at a cell number of 1.0 × 10 4 cells / well in each well of a 12-well multiwell plate. 48 hours after sowing, the medium was replaced with 0.5 mM theophylline-containing DMEM + 10% FBS medium containing a test substance at a predetermined concentration. The concentration of the test substance was set to 25 μg / ml, 50 μg / ml, and 100 μg / ml. Three wells for each sample concentration were assigned for the assay (N = 3). A plate for counting cell proliferation and two plates for quantifying melanin were used as a set for assay of each sample.
After the medium change, the culture was continued for 72 hours.
(2) The number of cells in each well (relative value) was measured by measuring absorbance at 450 nm using the WST-1 method using a cell proliferation counting plate, thereby measuring the degree of cell proliferation. The result is shown in FIG.
(メラニン量の測定方法)
B16メラノーマ細胞を培養し、細胞中に産生したメラニンをアルカリで溶解し、吸光度を測定することによりメラニン生成量を求めた。
72時間培養後のメラニン定量用のプレートを用いて各ウェル内の細胞を10%トリクロロ酢酸溶液で処理した後にエタノール/エーテル(1:1(V/V))で処理した。
これに1N NaOH/10%DMSO溶液を加え、メラニンを可溶化した後吸光度475nmを測定した。合成メラニン(Sigma)を1N NaOH/10%DMSO溶液に溶解した溶液を所定の濃度に希釈し標準液として使用し、本標準液と測定溶液の吸光度を比較することにより測定溶液中のメラニン含有量を算出した。
(2)で求めたウェル内細胞数(相対値)と本ウェル内メラニン量からメラニン生成量(相対値)を計算した。
(Melanin content measurement method)
B16 melanoma cells were cultured, melanin produced in the cells was dissolved in alkali, and the amount of melanin produced was determined by measuring the absorbance.
Cells in each well were treated with a 10% trichloroacetic acid solution using a plate for melanin quantification after 72 hours of culture, and then treated with ethanol / ether (1: 1 (V / V)).
A 1N NaOH / 10% DMSO solution was added thereto to solubilize melanin, and then the absorbance was measured at 475 nm. A solution of synthetic melanin (Sigma) dissolved in 1N NaOH / 10% DMSO solution is diluted to a prescribed concentration and used as a standard solution. By comparing the absorbance of this standard solution and the measurement solution, the melanin content in the measurement solution Was calculated.
The amount of melanin production (relative value) was calculated from the number of cells in the well (relative value) obtained in (2) and the amount of melanin in this well.
図3から明らかなように,本酸性ムコ多糖の添加により濃度に依存してメラニン生成の抑制が認められ、この値は陽性対照(50μg/mlで23.21±2.34)に類似した。又本酸性ムコ多糖は,メラノーマ細胞の増殖抑制効果、すなわち細胞毒性は認められなかった。各濃度の本酸性ムコ多糖の添加で細胞毒性に伴う細胞の形態の変化も見られなかった。即ち、本酸性ムコ多糖は、細胞の生育に影響を与えない濃度範囲でメラニン生成抑制作用を示すことが認められた。 As is apparent from FIG. 3, the addition of the acidic mucopolysaccharide inhibited the melanin production depending on the concentration, and this value was similar to the positive control (23.21 ± 2.34 at 50 μg / ml). The acidic mucopolysaccharide was not observed to inhibit the proliferation of melanoma cells, ie, cytotoxicity. The addition of the acid mucopolysaccharide at each concentration did not change the cell morphology associated with cytotoxicity. That is, it was confirmed that the acidic mucopolysaccharide exhibits a melanin production inhibitory effect within a concentration range that does not affect the cell growth.
本発明に係る美白剤は種々の剤型で使用されるが一例として皮膚外用剤の配合例を実施例として説明する。表4に示す通りである。 The whitening agent according to the present invention is used in various dosage forms, but as an example, formulation examples of a skin external preparation will be described as examples. As shown in Table 4.
本発明に係る美白剤は種々の剤型で使用されるが一例として美容液の配合例を実施例として説明する。表5に示す通りである。 The whitening agent according to the present invention is used in various dosage forms, but an example of blending a cosmetic liquid will be described as an example. As shown in Table 5.
本発明の微生物、好ましくはコベティア(Cobetia) sp. WAK-1A(FERM P-21101)を用いることにより前記式(1)の構造式で示される酸性ムコ多糖をジャーファーメンターやタンク内で撹拌を伴って安全に大量生産することができ、ひいてはメラニン生成抑制作用を有しており、日焼け後の色素沈着・しみ・そばかす・肝斑等の淡色化、美白に優れた効果を有すると共に、安全性にも優れた美白剤および美白用皮膚外用剤を容易に簡単な工程で大量、安価に分離精製できることから経済的な工業的生産規模を可能にする。
本発明の美白剤は、そのまま、水等で希釈して、或は濃縮して、皮膚外用剤として投与できる。もしくはそのまま、水等で希釈して、濃縮して、或は粉末化又は顆粒化して、公知の医薬用担体と共に製剤化することにより、エアゾール剤、液剤、エキス剤、懸濁剤、乳剤、軟膏剤、パップ剤、リニメント剤、ローション剤の形態として提供できる。或は公知の化粧品、医薬部外品、医薬品に用いられる、水性成分、界面活性剤、油性成分、可溶化剤、保湿剤、粉末成分、アルコール類、pH調整剤、防腐剤、酸化防止剤、増粘剤、色素、顔料、香料などを必要に応じて適宜選択することにより調製される。皮膚外用剤として、ローション状、ゲル状、乳剤、軟膏等の剤形とすることができ、柔軟性化粧水、収斂性化粧水などの化粧水類、エモリエントクリーム、モイスチャアクリーム、マッサージクリーム等のクリーム類、エモリエント乳液、ナリシング乳液、クレンジング乳液等の乳液類、洗顔料、皮膚洗浄剤、ファンデーション、アイカラー、チークカラー、口紅等のメイクアップ化粧料、シャンプー、リンス、ヘアートリートメント剤、ヘアークリーム、整髪剤、ヘアートニック、養毛剤、育毛剤などの毛髪化粧料、バスオイル、バスソルト、フォームバスなどの入浴料等の種々の形態の化粧料として提供することができる。
また、本発明の美白剤は、そのまま、あるいは水等で希釈して、経口的に投与できる。もしくはこれを公知の医薬用担体と共に製剤化することにより調製される。例えば、抽出物をシロップ剤などの経口液状製剤として、または抽出物を、エキス、粉末などに加工して、薬学的に許容される担体と配合し、錠剤、カプセル剤、顆粒剤,散剤などの経口固形製剤として投与できる。薬学的に許容できる担体としては、製剤素材として慣用の各種有機あるいは無機担体物質が用いられ、固形製剤における賦形剤、滑沢剤、結合剤、崩壊剤、液状製剤における溶剤、賦形剤、懸濁化剤、結合剤などとして配合される。また、必要に応じて、防腐剤、抗酸化剤、着色料、甘味剤などの製剤添加物を用いることもできる。
By using the microorganism of the present invention, preferably Cobetia sp. WAK-1A (FERM P-21101), the acidic mucopolysaccharide represented by the structural formula of the above formula (1) is stirred in a jar fermenter or tank. It can be safely produced in large quantities, and thus has an inhibitory effect on melanin production, and has excellent effects on lightening and whitening of pigmentation, stains, freckles, and liver spots after sunburn, and safety. In addition, an excellent whitening agent and a skin whitening agent for whitening can be easily separated and purified in a large amount and at a low cost by a simple process, thereby enabling an economical industrial production scale.
The whitening agent of the present invention can be directly diluted with water or concentrated or administered as a skin external preparation. Alternatively, it can be diluted directly with water, concentrated, powdered or granulated, and formulated with a known pharmaceutical carrier to produce aerosols, solutions, extracts, suspensions, emulsions, ointments. It can be provided in the form of an agent, a poultice, a liniment, and a lotion. Or used in known cosmetics, quasi drugs, pharmaceuticals, aqueous ingredients, surfactants, oily ingredients, solubilizers, moisturizers, powder ingredients, alcohols, pH adjusters, preservatives, antioxidants, It is prepared by appropriately selecting a thickener, a dye, a pigment, a fragrance and the like as necessary. As an external preparation for skin, it can be in the form of lotion, gel, emulsion, ointment, etc., lotion such as flexible lotion, astringent lotion, emollient cream, moisture cream, massage cream etc. Makeup cosmetics such as creams, emollient emulsions, nourishing emulsions, cleansing emulsions, face wash, skin cleanser, foundation, eye color, teak color, lipstick, shampoo, rinse, hair treatment agent, hair cream, It can be provided as various forms of cosmetics such as hair cosmetics such as hair styling agents, hair nickings, hair nourishing agents, hair growth agents, etc., and bath oils such as bath oils, bath salts, and foam baths.
The whitening agent of the present invention can be administered orally as it is or after diluting with water or the like. Alternatively, it is prepared by formulating it with a known pharmaceutical carrier. For example, the extract as an oral liquid preparation such as a syrup, or the extract is processed into an extract, powder, etc., and blended with a pharmaceutically acceptable carrier, such as tablets, capsules, granules, powders, etc. It can be administered as an oral solid formulation. As the pharmaceutically acceptable carrier, various organic or inorganic carrier substances commonly used as pharmaceutical materials are used, and excipients, lubricants, binders, disintegrants in solid preparations, solvents, excipients in liquid preparations, Formulated as a suspending agent, binder, and the like. In addition, formulation additives such as preservatives, antioxidants, colorants, sweeteners, and the like can be used as necessary.
Claims (10)
(上記の構造式において、GalNAcpはピラノース型N-アセチルガラクトサミン残基を、GlcUApはピラノース型グルクロン酸残基を、DはD型を、LはL型を、Pyrはピルビン酸を、nは繰り返しの数をそれぞれ表す。) The microorganism of the genus Covetia is Cobetia sp. WAK-1A (deposit number FERM P-21101), and a culture is obtained by culturing under a culture condition suitable for the microorganism of the genus Covetia. 1. A Covetia microorganism characterized in that the acidic mucopolysaccharide represented by the structural formula (1) can be separated and recovered without selectively removing the sulfated polysaccharide.
(In the above structural formula, GalNAcp is a pyranose-type N-acetylgalactosamine residue, GlcUAp is a pyranose-type glucuronic acid residue, D is a D-type, L is an L-type, Pyr is pyruvic acid, and n is a repeat. Represents the number of each.)
< 形態 >
細胞の形態:桿菌
運動性:有り
胞子形成:無し
<生育状態>
コロニーの形態:円形
コロニーの色調:クリーム色
コロニーの表面:スムーズ
生育温度:10℃、30℃、37℃および45℃で生育する。
生育pH:5.5〜9.5(至適生育pH:6.5〜7.5)
酸素要求性:好気性
<生理学的性質>
グラム染色性:陰性
カタラーゼ反応:陽性
オキシダーゼ反応:陰性
グルコースからの酸/ガス産生:陰性/陰性
O/Fテスト(酸化/発酵) :陰性/陰性
β−ガラクトシダーゼ活性:陽性
インドール産生:陰性
硝酸塩還元:陰性
でんぷん加水分解:陰性
ゼラチン加水分解:陰性
エスクリン加水分解:陰性
ウレアーゼ:陰性
アルギニンジヒドロラーゼ:陰性
資化性(D-グルコース、ラクトース、サッカロース、酢酸ナトリウム、クエン酸ナトリウム、グルコン酸ナトリウム、こはく酸ナトリウム):有り
(L-アラビノース、マルトース、D-マンノース、D-マンニトール、N-アセチル-D-グルコサミン、n-カプリン酸、アジピン酸、dl-リンゴ酸、酢酸フェニル、L-アラニン、L-ヒスチジン、L-セリン):無し The microorganism of the genus Covetia according to claim 1 or 2, having the following mycological properties.
<Form>
Cell morphology: Aspergillus motility: Yes Spore formation: None <Growth state>
Colony morphology: Circular colony color: Cream color Surface of colony: Smooth growth temperature: Grows at 10 ° C, 30 ° C, 37 ° C and 45 ° C.
Growth pH: 5.5 to 9.5 (optimum growth pH: 6.5 to 7.5)
Oxygen demand: Aerobic <Physiological properties>
Gram staining: Negative catalase reaction: Positive oxidase reaction: Acid / gas production from negative glucose: Negative / negative
O / F test (oxidation / fermentation): negative / negative β-galactosidase activity: positive indole production: negative nitrate reduction: negative starch hydrolysis: negative gelatin hydrolysis: negative esculin hydrolysis: negative urease: negative arginine dihydrolase: negative Assimilation (D-glucose, lactose, saccharose, sodium acetate, sodium citrate, sodium gluconate, sodium succinate): Yes (L-arabinose, maltose, D-mannose, D-mannitol, N-acetyl-D- Glucosamine, n-capric acid, adipic acid, dl-malic acid, phenyl acetate, L-alanine, L-histidine, L-serine): None
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CL2011001286A1 (en) | 2011-05-31 | 2011-09-30 | Univ De Valparaiso | Bacterial strain of marine cobetia, non-pathogenic, useful in aquaculture that grows in sulfuric cyclic aromatic hydrocarbons as the only carbon source; method to obtain a biosurfactant extract; biosurfactant extract; use of biosurfactant to avoid infectious pathologies in aquaculture. |
FR2975906B1 (en) * | 2011-06-06 | 2014-03-14 | Courtage Et De Diffusion Codif Internat Soc D | COSMETICAL COMPOSITIONS OF EXOPOLYSACCHARIDES FROM MARINE BACTERIA |
FR2981847B1 (en) * | 2011-10-28 | 2013-11-15 | Courtage Et De Diffusion Codif Internat Soc D | COSMETIC OR PHARMACEUTICAL COMPOSITIONS OF AN EXOPOLYSACCHARIDE FROM A MARINE BACTERIUM |
JP5953078B2 (en) * | 2012-03-14 | 2016-07-13 | 株式会社ピカソ美化学研究所 | NOVEL MICROORGANISM AND ITS VARIANTS AND METHOD FOR PRODUCING POLYsaccharideS |
JP6251471B2 (en) * | 2012-05-31 | 2017-12-20 | 株式会社ピカソ美化学研究所 | Whitening agent |
JP2014001148A (en) * | 2012-06-15 | 2014-01-09 | Picaso Cosmetic Laboratory Ltd | Fibroblast proliferation promoter |
JP2014162748A (en) * | 2013-02-25 | 2014-09-08 | Kagawa Univ | Cutaneous light aging inhibitor |
AU2017240068B2 (en) | 2016-03-31 | 2022-12-15 | Gojo Industries, Inc. | Antimicrobial peptide stimulating cleansing composition |
JP2019510036A (en) | 2016-03-31 | 2019-04-11 | ゴジョ・インダストリーズ・インコーポレイテッド | A detergent composition comprising probiotic / prebiotic active ingredients |
EP3544575A1 (en) | 2016-11-23 | 2019-10-02 | GOJO Industries, Inc. | Sanitizer composition with probiotic/prebiotic active ingredient |
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