JP2022155726A - Novel sophorolipid derivative - Google Patents
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- ZTOKUMPYMPKCFX-CZNUEWPDSA-N (E)-17-[(2R,3R,4S,5S,6R)-6-(acetyloxymethyl)-3-[(2S,3R,4S,5S,6R)-6-(acetyloxymethyl)-3,4,5-trihydroxyoxan-2-yl]oxy-4,5-dihydroxyoxan-2-yl]oxyoctadec-9-enoic acid Chemical class OC(=O)CCCCCCC/C=C/CCCCCCC(C)O[C@@H]1O[C@H](COC(C)=O)[C@@H](O)[C@H](O)[C@H]1O[C@H]1[C@H](O)[C@@H](O)[C@H](O)[C@@H](COC(C)=O)O1 ZTOKUMPYMPKCFX-CZNUEWPDSA-N 0.000 title claims abstract description 38
- 235000014113 dietary fatty acids Nutrition 0.000 claims abstract description 13
- 239000000194 fatty acid Substances 0.000 claims abstract description 13
- 229930195729 fatty acid Natural products 0.000 claims abstract description 13
- 125000004432 carbon atom Chemical group C* 0.000 claims abstract description 10
- -1 fatty acid ester Chemical class 0.000 claims abstract description 10
- 229910052739 hydrogen Inorganic materials 0.000 claims abstract description 8
- 239000001257 hydrogen Substances 0.000 claims abstract description 8
- 125000002777 acetyl group Chemical group [H]C([H])([H])C(*)=O 0.000 claims abstract description 5
- UFHFLCQGNIYNRP-UHFFFAOYSA-N Hydrogen Chemical compound [H][H] UFHFLCQGNIYNRP-UHFFFAOYSA-N 0.000 claims abstract description 4
- 150000002431 hydrogen Chemical group 0.000 claims abstract description 4
- 150000001875 compounds Chemical class 0.000 claims description 40
- 239000000203 mixture Substances 0.000 claims description 31
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- 229940079593 drug Drugs 0.000 claims description 7
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- 239000003905 agrochemical Substances 0.000 claims description 6
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- 235000013305 food Nutrition 0.000 claims description 6
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- 241001278026 Starmerella bombicola Species 0.000 abstract description 6
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- 238000004458 analytical method Methods 0.000 description 29
- CSCPPACGZOOCGX-UHFFFAOYSA-N Acetone Chemical compound CC(C)=O CSCPPACGZOOCGX-UHFFFAOYSA-N 0.000 description 21
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- PEDCQBHIVMGVHV-UHFFFAOYSA-N Glycerine Chemical compound OCC(O)CO PEDCQBHIVMGVHV-UHFFFAOYSA-N 0.000 description 17
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- 229930186217 Glycolipid Natural products 0.000 description 9
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- VYPSYNLAJGMNEJ-UHFFFAOYSA-N Silicium dioxide Chemical compound O=[Si]=O VYPSYNLAJGMNEJ-UHFFFAOYSA-N 0.000 description 7
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- HVCOBJNICQPDBP-UHFFFAOYSA-N 3-[3-[3,5-dihydroxy-6-methyl-4-(3,4,5-trihydroxy-6-methyloxan-2-yl)oxyoxan-2-yl]oxydecanoyloxy]decanoic acid;hydrate Chemical compound O.OC1C(OC(CC(=O)OC(CCCCCCC)CC(O)=O)CCCCCCC)OC(C)C(O)C1OC1C(O)C(O)C(O)C(C)O1 HVCOBJNICQPDBP-UHFFFAOYSA-N 0.000 description 5
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- HEMHJVSKTPXQMS-UHFFFAOYSA-M Sodium hydroxide Chemical compound [OH-].[Na+] HEMHJVSKTPXQMS-UHFFFAOYSA-M 0.000 description 3
- HIWPGCMGAMJNRG-ACCAVRKYSA-N Sophorose Natural products O([C@H]1[C@@H](O)[C@@H](O)[C@@H](CO)O[C@H]1O)[C@H]1[C@H](O)[C@@H](O)[C@@H](O)[C@@H](CO)O1 HIWPGCMGAMJNRG-ACCAVRKYSA-N 0.000 description 3
- YXFVVABEGXRONW-UHFFFAOYSA-N Toluene Chemical compound CC1=CC=CC=C1 YXFVVABEGXRONW-UHFFFAOYSA-N 0.000 description 3
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- BASFCYQUMIYNBI-UHFFFAOYSA-N platinum Chemical compound [Pt] BASFCYQUMIYNBI-UHFFFAOYSA-N 0.000 description 2
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- PZDOWFGHCNHPQD-VNNZMYODSA-N sophorose Chemical compound OC[C@@H](O)[C@@H](O)[C@H](O)[C@H](C=O)O[C@@H]1O[C@H](CO)[C@@H](O)[C@H](O)[C@H]1O PZDOWFGHCNHPQD-VNNZMYODSA-N 0.000 description 2
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- UFTFJSFQGQCHQW-UHFFFAOYSA-N triformin Chemical compound O=COCC(OC=O)COC=O UFTFJSFQGQCHQW-UHFFFAOYSA-N 0.000 description 2
- XLEYFDVVXLMULC-UHFFFAOYSA-N 2',4',6'-trihydroxyacetophenone Chemical compound CC(=O)C1=C(O)C=C(O)C=C1O XLEYFDVVXLMULC-UHFFFAOYSA-N 0.000 description 1
- 229920001817 Agar Polymers 0.000 description 1
- VHUUQVKOLVNVRT-UHFFFAOYSA-N Ammonium hydroxide Chemical compound [NH4+].[OH-] VHUUQVKOLVNVRT-UHFFFAOYSA-N 0.000 description 1
- 241000221198 Basidiomycota Species 0.000 description 1
- 101100377506 Drosophila melanogaster 14-3-3zeta gene Proteins 0.000 description 1
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- 235000019484 Rapeseed oil Nutrition 0.000 description 1
- QAOWNCQODCNURD-UHFFFAOYSA-L Sulfate Chemical compound [O-]S([O-])(=O)=O QAOWNCQODCNURD-UHFFFAOYSA-L 0.000 description 1
- HATRDXDCPOXQJX-UHFFFAOYSA-N Thapsigargin Natural products CCCCCCCC(=O)OC1C(OC(O)C(=C/C)C)C(=C2C3OC(=O)C(C)(O)C3(O)C(CC(C)(OC(=O)C)C12)OC(=O)CCC)C HATRDXDCPOXQJX-UHFFFAOYSA-N 0.000 description 1
- 241000192409 [Candida] floricola Species 0.000 description 1
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- RJGDLRCDCYRQOQ-UHFFFAOYSA-N anthrone Chemical compound C1=CC=C2C(=O)C3=CC=CC=C3CC2=C1 RJGDLRCDCYRQOQ-UHFFFAOYSA-N 0.000 description 1
- 230000000844 anti-bacterial effect Effects 0.000 description 1
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- IWXRPVHRDWXIEA-UHFFFAOYSA-N azanium formate hydrate Chemical compound [NH4+].O.[O-]C=O IWXRPVHRDWXIEA-UHFFFAOYSA-N 0.000 description 1
- VBUZIXJFGCVTJL-UHFFFAOYSA-N azanium;methanol;formate Chemical compound [NH4+].OC.[O-]C=O VBUZIXJFGCVTJL-UHFFFAOYSA-N 0.000 description 1
- 150000001720 carbohydrates Chemical class 0.000 description 1
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- 238000004113 cell culture Methods 0.000 description 1
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- 125000004079 stearyl group Chemical group [H]C([*])([H])C([H])([H])C([H])([H])C([H])([H])C([H])([H])C([H])([H])C([H])([H])C([H])([H])C([H])([H])C([H])([H])C([H])([H])C([H])([H])C([H])([H])C([H])([H])C([H])([H])C([H])([H])C([H])([H])C([H])([H])[H] 0.000 description 1
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- Cosmetics (AREA)
- Detergent Compositions (AREA)
- Fodder In General (AREA)
- General Preparation And Processing Of Foods (AREA)
- Non-Alcoholic Beverages (AREA)
- Saccharide Compounds (AREA)
- Medicinal Preparation (AREA)
Abstract
Description
本発明は、新規ソホロリピッド誘導体および該誘導体を含む組成物に関する。 The present invention relates to novel sophorolipid derivatives and compositions containing said derivatives.
親水基が糖で構成される糖型界面活性剤のうち、微生物由来の天然界面活性剤であり安全性の高い糖型バイオ界面活性剤は、優れた界面活性剤として知られている。これらのうちソホロリピッドは、糖脂質であり両親媒性構造を有するため強い界面活性作用を有し、生分解性と安全性が高いことから、バイオサーファクタントの主役として用途開発が進められている。 Among sugar-type surfactants in which the hydrophilic group is composed of sugar, sugar-type biosurfactants that are natural surfactants derived from microorganisms and have high safety are known as excellent surfactants. Among these, sophorolipids are glycolipids and have amphiphilic structures, so they have a strong surface-active action, and are highly biodegradable and safe. Therefore, their applications are being developed as main biosurfactants.
ソホロリピッドを生産する酵母としては、担子菌酵母であるスタルメレラ・ボンビコラ（Ｓｔａｒｍｅｒｅｌｌａ ｂｏｍｂｉｃｏｌａ）が代表的であり、そのバイオサーファクタントの生産力は培養液１Ｌ当たり４００ｇ以上にも達するため、商業ベースの生産に使用されている。
ソホロリピッドは、グルコースが２→１位でエーテル結合してできた二糖であるソホロースの1位に、ヒドロキシ脂肪酸がエーテル結合してできた下記式（３）で表されるラクトン型（ＬＳＬ）と、下記式（４）で表される酸型（ＡＳＬ）の分子構造を有する糖脂質である。微生物生産物中にはこれらが一種または複数種含まれる混合物として存在する。
Starmerella bombicola, which is a basidiomycete yeast, is a typical yeast that produces sophorolipids, and its biosurfactant productivity reaches 400 g or more per 1 L of culture solution, so it is used for commercial production. It is
Sophorolipid is a lactone type (LSL) represented by the following formula (3), which is formed by ether-bonding a hydroxy fatty acid to the 1-position of sophorose, a disaccharide formed by ether-bonding glucose at the 2 → 1-position. , is a glycolipid having an acid-type (ASL) molecular structure represented by the following formula (4). They are present as a mixture containing one or more of these in microbial products.
ソホロリピッドのラクトン型（非イオン型）と酸型（アニオン型）とでは、界面活性剤としての性質が大きく異なり、これらの組成の異なる混合物やこれらを作り分ける方法など、ソホロリピッド製品の構造や機能のバラエティを拡充する技術開発が行われている。
例えば、上記のスタルメレラ・ボンビコラ（Ｓｔａｒｍｅｒｅｌｌａ ｂｏｍｂｉｃｏｌａ）が生産するソホロリピッドは、一般的にラクトン型と酸型がおおよそ６～８：２～４の混合物として得られ、主成分であるラクトン型は酸型と比べて低濃度で優れた表面張力低下能を示す（非特許文献１）ほか、高い抗菌活性を示すことが報告されている（非特許文献２）。
The lactone type (non-ionic type) and acid type (anion type) of sophorolipid have greatly different properties as surfactants. Technological development is underway to expand the variety.
For example, the sophorolipid produced by the above-mentioned Starmerella bombicola is generally obtained as a mixture of the lactone type and the acid type at a ratio of approximately 6-8:2-4, and the lactone type, which is the main component, is mixed with the acid type. It has been reported that it exhibits excellent surface tension-lowering ability at a low concentration (Non-Patent Document 1) and high antibacterial activity (Non-Patent Document 2).
一方、加水分解によってラクトン環を開環して化学的に安定な高純度の酸型ソホロリピッドを得て、これを配合した洗浄剤が報告されている（特許文献１）。また、キャンディダ・フロリコラ（Ｃａｎｄｉｄａ ｆｌｏｒｉｃｏｌａ）を生産菌として培養することで、酸型ソホロリピッドのみを選択的に製造する方法が報告されている（特許文献２）。
さらに、複数存在する官能基が修飾されたソホロリピッド誘導体のほか、ソホロリピッド重合体などのラクトン型、酸型とは分類の異なる新規構造の誘導体も報告されている（特許文献３、非特許文献３）。
On the other hand, a chemically stable high-purity acid-type sophorolipid is obtained by hydrolysis to open the lactone ring, and a cleaning agent containing this is reported (Patent Document 1). Also, a method for selectively producing only acid-type sophorolipid by culturing Candida floricola as a producing strain has been reported (Patent Document 2).
Furthermore, in addition to sophorolipid derivatives in which a plurality of existing functional groups are modified, derivatives with novel structures different from lactone-type and acid-type derivatives, such as sophorolipid polymers, have been reported (
式（３）
式（４）
Formula (4)
従来知られているソホロリピッドの構造は、おおよそ上記のラクトン型と酸型の二つのパターンしかなく、物性・機能の拡充のためには、構造のバラエティの拡張が求められている。
本発明は、飼料、肥料、飲食品、農薬、医薬品、医薬部外品、または化粧品などの広範囲の分野に適用することができる、新規なソホロリピッド誘導体を提供することを課題とする。
また、本発明は、新規ソホロリピッドを含む組成物、特に界面活性剤、洗浄剤、または乳化剤を提供することをその課題とする。
Conventionally known sophorolipids have only two patterns, the lactone type and the acid type, as described above, and there is a need to expand the variety of structures in order to expand their physical properties and functions.
An object of the present invention is to provide a novel sophorolipid derivative that can be applied to a wide range of fields such as feeds, fertilizers, food and drink, agricultural chemicals, pharmaceuticals, quasi-drugs, and cosmetics.
Another object of the present invention is to provide compositions containing novel sophorolipids, particularly surfactants, detergents or emulsifiers.
本発明者らはソホロリピッド生産菌として広く研究されているＳｔａｒｍｅｒｅｌｌａ ｂｏｍｂｉｃｏｌａの培養生産物中に、従来知られているソホロリピッドとは分子構造が異なる未知の成分が存在することを各種機器分析によって確認し、これらを単離・精製して構造解析を行い、新規構造のソホロリピッド誘導体であることを解明した。
物性解析によれば、これら新規ソホロリピッド誘導体は、従来のソホロリピッドとは異なる界面活性、自己組織化特性を示し、優れた洗浄成分として機能することを確認して、本発明の完成に至った。
The inventors of the present invention confirmed by various instrumental analyses, that the cultured products of Starmerella bombicola, which has been extensively studied as a sophorolipid-producing bacterium, contain an unknown component having a molecular structure different from that of conventionally known sophorolipids. After isolating and purifying these compounds, structural analysis was carried out, revealing that they are sophorolipid derivatives with novel structures.
According to physical property analysis, it was confirmed that these novel sophorolipid derivatives exhibit surface activity and self-organizing properties different from those of conventional sophorolipids and function as excellent cleansing ingredients, leading to the completion of the present invention.
本発明は、下記（１）、（２）に記載のソホロリピッド誘導体に関する。
（１）下記式（１）の一般式で示される、ソホロリピッド誘導体。
式（１）
（２）下記式（２）の化学式で表されるいずれかの化合物である、ソホロリピッド誘導体。
式（２）
(1) A sophorolipid derivative represented by the following general formula (1).
formula (1)
(2) A sophorolipid derivative which is any compound represented by the following chemical formula (2).
formula (2)
また、本発明は、下記（３）～（５）に記載の組成物に関する。
（３）上記（１）または（２）に記載のソホロリピッド誘導体を含む組成物。
（４）界面活性剤、洗浄剤、または乳化剤である、上記（３）に記載の組成物。
（５）飼料、肥料、飲食品、農薬、医薬品、医薬部外品、または化粧品、若しくはこれらの添加物である、上記（３）に記載の組成物。
The present invention also relates to compositions described in (3) to (5) below.
(3) A composition comprising the sophorolipid derivative according to (1) or (2) above.
(4) The composition according to (3) above, which is a surfactant, detergent, or emulsifier.
(5) The composition according to (3) above, which is feed, fertilizer, food and drink, agricultural chemicals, pharmaceuticals, quasi-drugs, cosmetics, or additives thereof.
本発明の新規ソホロリピッド誘導体は、従来のソホロリピッドとは異なる界面活性や自己集合特性を示し、優れた洗浄成分として機能するので、ソホロリピッド製品の構造・機能バラエティを拡充できる。従来のソホロリピッドと比較して、水溶性が高い非イオン型界面活性剤として機能する。
また、安全性の高い天然物由来のソホロリピッド誘導体であるから、各製品の安全性を高めることもでき、飼料、肥料、飲食品、農薬、医薬品、医薬部外品または化粧品などの広範囲の分野に適用できる。
The novel sophorolipid derivative of the present invention exhibits surface activity and self-assembly properties different from those of conventional sophorolipids, and functions as an excellent cleansing ingredient, so that the variety of structures and functions of sophorolipid products can be expanded. It functions as a nonionic surfactant with higher water solubility than conventional sophorolipids.
In addition, since it is a sophorolipid derivative derived from a highly safe natural product, it can improve the safety of each product, and can be used in a wide range of fields such as feeds, fertilizers, food and drink, agricultural chemicals, pharmaceuticals, quasi-drugs and cosmetics. Applicable.
本発明は、新規ソホロリピッド誘導体と、該誘導体を含む組成物に係るものである。
本発明のソホロリピッド誘導体（以下、「ＳＬ誘導体」ということがある。）は、下記の一般式（１）で示される。
式（１）
炭素数２～２２のいずれかの脂肪酸エステルは、－（Ｏ）Ｃ－炭化水素基で表され、この炭化水素基の炭素数は１～２１である。
また、ＳＬ基中のＲは、同一または異なってもよく、水素またはアセチル基を表し、ｎは１１～１９の整数を表す。
The present invention relates to novel sophorolipid derivatives and compositions containing said derivatives.
The sophorolipid derivative (hereinafter sometimes referred to as "SL derivative") of the present invention is represented by the following general formula (1).
formula (1)
Any fatty acid ester having 2 to 22 carbon atoms is represented by -(O)C-hydrocarbon group, and the hydrocarbon group has 1 to 21 carbon atoms.
In addition, R in the SL group may be the same or different, represents hydrogen or an acetyl group, and n represents an integer of 11-19.
本発明のＳＬ誘導体は、下記の化学式（２）で表される化合物のいずれか一つであってよい。
式（２）
formula (2)
本発明のＳＬ誘導体は、ＳＬ生産菌の培養物中から得られるが、ＳＬのグリセリドであるため化学的に合成することができる。
たとえば、ＳＬとグリセリンを非アルコール系の有機溶媒（クロロホルム、トルエン、アセトン等）中または無溶媒下で、リパーゼなどの固定化酵素を触媒としてエステル化（エステル交換または加水分解の逆反応）を行うことにより、または、グリセリンの代わりに植物油（トリグリセリド）を用いて、同様にエステル交換反応を行うことにより製造することができる。特に、反応性に優れたラクトン型（ＬＳＬ）を用いれば、グリセリン等と混合して加熱撹拌することで、本発明のＳＬ誘導体を製造することができる。
The SL derivative of the present invention can be obtained from cultures of SL-producing bacteria, and can be chemically synthesized since it is a glyceride of SL.
For example, SL and glycerin are esterified (reverse reaction of transesterification or hydrolysis) in a non-alcoholic organic solvent (chloroform, toluene, acetone, etc.) or in the absence of solvent using an immobilized enzyme such as lipase as a catalyst. or by using a vegetable oil (triglyceride) instead of glycerin and performing a transesterification reaction in the same manner. In particular, if a lactone type (LSL) with excellent reactivity is used, the SL derivative of the present invention can be produced by mixing with glycerin or the like and heating and stirring.
本発明の組成物は、本発明のＳＬ誘導体の有する界面活性に依る界面活性剤として、または乳化剤、分散剤として、飼料、肥料、飲食品、農薬、医薬品、医薬部外品または化粧品、及びこれらの添加物に適用することができる。
本発明の組成物に含まれるＳＬ誘導体の量は、特に限定されないが、０．０１～１００ｗｔ％、好ましくは０．１～５０ｗｔ％、より好ましくは１～３０ｗｔ％である。組成物におけるＳＬ誘導体の量が１ｗｔ％以下のように少ない場合、水溶性が小さくなり、一方、５０ｗｔ％以上のように多い場合、経済性が低下する。
［実施例］
The composition of the present invention can be used as a surfactant or as an emulsifier or dispersant depending on the surface activity of the SL derivative of the present invention, and can be used as feed, fertilizer, food and drink, agricultural chemicals, pharmaceuticals, quasi-drugs or cosmetics. can be applied to the additive of
The amount of SL derivative contained in the composition of the present invention is not particularly limited, but is 0.01 to 100 wt%, preferably 0.1 to 50 wt%, more preferably 1 to 30 wt%. If the amount of SL derivative in the composition is as low as 1 wt% or less, the water-solubility will be poor, while if it is as high as 50 wt% or more, economic efficiency will decrease.
[Example]
以下、実施例により本発明を更に具体的に説明するが、本発明は以下の実施例に制限されるものではない。実施例では、「％」は、「ｗｔ％」を意味する。 EXAMPLES The present invention will be described in more detail with reference to examples below, but the present invention is not limited to the following examples. In the examples, "%" means "wt%".
ソホロリピッドの生産・回収
スタルメレラ・ボンビコラ（Ｓｔａｒｍｅｒｅｌｌａ ｂｏｍｂｉｃｏｌａ）ＡＴＣＣ２２２１４株の培養
（１）種培養
保存培地（酵母エキス１０ｇ／Ｌ、ペプトン２０ｇ／Ｌ、グルコース２０ｇ／Ｌ、寒天２０ｇ／Ｌ）に保存しておいた上記のスタルメレラ・ボンビコラ（Ｓｔａｒｍｅｒｅｌｌａ ｂｏｍｂｉｃｏｌａ）ＡＴＣＣ２２２１４株を、酵母エキス１０ｇ／Ｌ、ペプトン２０ｇ／Ｌ、グルコース２０ｇ／Ｌの組成の液体培地４ｍＬが入った試験管に１白金耳接種し、２８℃で振とう培養を１日間行った。
（２）本培養
（１）で得られた菌体培養液を、１０ｇ／Ｌの酵母エキス、２０ｇ／Ｌのペプトン、１００ｇ／Ｌのグルコースおよび１００ｇ／Ｌのなたね油の組成の液体培地３０ｍＬが入った三角フラスコに接種し、振とう培養を２８℃にて７日間行った。
（３）ソホロリピッドの回収
（２）で得られた培養液を１時間静置することで下層にＳＬ相が生じる。このＳＬ相を回収し、水酸化ナトリウム水溶液で中和、水で洗浄することでＳＬ混合物を水溶液の状態で回収した。また、得られたＳＬ混合物水溶液について以降の実験を行うために、ヘキサンを添加して撹拌、遠心分離することでヘキサン相に残存油脂や脂肪酸を抽出・除去し、水相を凍結乾燥してＳＬ混合物固体を回収した。
（４）ラクトン型ソホロリピッド、酸型ソホロリピッド標品の精製
以降の実験で標品として利用するために既知の精製手法によって、ラクトン型ＳＬ（ＬＳＬ）および酸型ＳＬ（ＡＳＬ）を分離した。すなわち、上記ＳＬ混合物固体をアセトンに溶解してシリカゲル（ワコーゲルＣ－２００）をガラスカラム管に充填したシリカゲルカラムに供し、クロロホルムとアセトンの混合液を展開溶媒とするカラムクロマトグラフィー法によって精製した。クロロホルムとアセトンの割合は、８：２でＬＳＬを、続いて２：８で酸型ＳＬをそれぞれ分離回収し、それぞれの回収した画分が目的のＬＳＬ標品、ＡＳＬ標品であることを、後述する薄層クロマトグラフィー解析によって確認した。
Production and recovery of sophorolipid Culture of Starmerella bombicola ATCC22214 strain (1) Seed culture Preserved in a storage medium (yeast extract 10 g/L, peptone 20 g/L, glucose 20 g/L, agar 20 g/L). One platinum loop of the above Starmerella bombicola ATCC22214 strain was inoculated into a test tube containing 4 mL of a liquid medium containing 10 g/L of yeast extract, 20 g/L of peptone and 20 g/L of glucose, and incubated at 28°C. Shaking culture was performed for 1 day.
(2) Main culture The cell culture solution obtained in (1) was added to 30 mL of a liquid medium having a composition of 10 g/L yeast extract, 20 g/L peptone, 100 g/L glucose and 100 g/L rapeseed oil. The strain was inoculated into an Erlenmeyer flask and cultured with shaking at 28°C for 7 days.
(3) Recovery of Sophorolipid The culture medium obtained in (2) is allowed to stand for 1 hour to form an SL phase in the lower layer. This SL phase was recovered, neutralized with an aqueous sodium hydroxide solution, and washed with water to recover an SL mixture in the form of an aqueous solution. In addition, in order to conduct subsequent experiments on the obtained SL mixture aqueous solution, hexane was added, stirred, and centrifuged to extract and remove residual fats and fatty acids in the hexane phase, and the aqueous phase was freeze-dried to obtain SL. A mixture solid was collected.
(4) Purification of lactone-type sophorolipid and acid-type sophorolipid preparations Lactone-type SL (LSL) and acid-type SL (ASL) were separated by a known purification method for use as preparations in subsequent experiments. That is, the above SL mixture solid was dissolved in acetone, applied to a silica gel column filled with silica gel (Wakogel C-200) in a glass column tube, and purified by column chromatography using a mixture of chloroform and acetone as a developing solvent. The ratio of chloroform and acetone was 8:2 to separate and recover LSL, followed by 2:8 to separate and recover acid-form SL. It was confirmed by the thin layer chromatography analysis described below.
ソホロリピッドの薄層クロマトグラフィー解析
実施例１で得られたＳＬ混合物固体について、メタノールに溶解して薄層クロマトグラフィー（ＴＬＣ）解析を行った。
まず、シリカゲルＴＬＣガラスプレート（Ｍｅｒｃｋ社製ＴＬＣシリカゲル６０Ｆ２５４ガラスプレート）を用いて、展開溶媒にクロロホルム／メタノール／アンモニア水＝８０／２０／２混合溶媒を用いた、従来の順相系でのＴＬＣ分析を行った。アンスロン硫酸指示薬で成分の検出を行うと、糖骨格を含む化合物は青緑色で検出される。その結果、非極性のＬＳＬが上部に、高極性のＡＳＬが下部に展開される結果となった（図１）。
次に、逆相修飾シリカゲルＴＬＣプレート（Ｍｅｒｃｋ社製ＴＬＣシリカゲル６０ＲＰ－１８Ｆ２５４Ｓガラスプレート）を用いて、展開溶媒にメタノール／水＝９５／５混合溶媒を用いた逆相系での分析を行ったところ、予想通り順相時とは逆に高極性のＡＳＬが上部に、非極性のＬＳＬが中部に展開されるとともに、さらに下部に従来法では検出されなかった構造未知の糖脂質と思われるスポットが検出された（図２）。
この結果から、実施例１で得られたＳＬ混合物には、従来知られているＬＳＬ、ＡＳＬとは異なる糖脂質が含まれていることが明らかとなった。
Analysis of Sophorolipid by Thin Layer Chromatography The SL mixture solid obtained in Example 1 was dissolved in methanol and analyzed by thin layer chromatography (TLC).
First, using a silica gel TLC glass plate (TLC silica gel 60F254 glass plate manufactured by Merck), using a mixed solvent of chloroform/methanol/ammonia water = 80/20/2 as a developing solvent, TLC analysis in a conventional normal phase system. did When components are detected with an anthrone sulfate indicator, compounds containing a sugar skeleton are detected in a blue-green color. The result was that the non-polar LSL was deployed on top and the highly polar ASL on the bottom (Fig. 1).
Next, using a reverse-phase modified silica gel TLC plate (TLC silica gel 60RP-18F254S glass plate manufactured by Merck), analysis was performed in a reverse-phase system using methanol/water = 95/5 mixed solvent as a developing solvent. As expected, highly polar ASL was developed in the upper part and non-polar LSL was developed in the middle part, contrary to normal phase. detected (Fig. 2).
These results revealed that the SL mixture obtained in Example 1 contained glycolipids different from conventionally known LSL and ASL.
ソホロリピッドの高速液体クロマトグラフィー解析
実施例１で得られたＳＬ混合物固体について、メタノールで溶解し高速液体クロマトグラフィー（ＨＰＬＣ）解析を行った。コロナ荷電化粒子検出器（ＣＡＤ）（Ｔｈｅｒｍｏ社製ＣｏｒｏｎａＴＭＶｅｏＴＭ）を搭載したＨＰＬＣ（Ｔｈｅｒｍｏ社製Ｔｈｅｒｍｏ Ｓｃｉｅｎｔｉｆｉｃ Ｕｌｔｉｍａｔｅ ３０００ ＨＰＬＣ）を用い、分析カラムは逆相系ＯＤＳカラム（ＧＬサイエンス社製ＩｎｅｒｔＳｕｓｔａｉｎＴＭ ＶＣ１８）を用いて、カラム温度は４０℃、溶離液は５ｍＭギ酸アンモニウムメタノール／５ｍＭギ酸アンモニウム水混合溶媒系で、グラジエントプログラムをメタノール７０％（ステップ０分→３分）、７０％→１００％（グラジエント３分→１８分）、１００％（ステップ１８分→２５分）、７０％（ステップ２５分→３５分）とし、流速０．３ｍＬ／ｍで成分分析を行った。得られたクロマトグラムを図３に示す。
分析の結果、保持時間３分～１０分にＡＳＬ、１２分～１６分ＬＳＬが検出された後、１８分前後、２０分前後、２２分前後にそれぞれ未知成分ピークが３つ（Ａ、Ｂ、Ｃ）検出された。これらの結果は、実施例２の逆相ＴＬＣ解析でＡＳＬ、ＬＳＬの後から未知糖脂質が検出される結果と一致するものであった。
High Performance Liquid Chromatography Analysis of Sophorolipid The SL mixture solid obtained in Example 1 was dissolved in methanol and subjected to high performance liquid chromatography (HPLC) analysis. HPLC (Thermo Scientific Ultimate 3000 HPLC) equipped with a corona charged particle detector (CAD) (Thermo Corona TM Veo TM ) was used, and the analysis column was a reversed-phase ODS column (GL Sciences InertSustain TM VC18), the column temperature is 40 ° C., the eluent is a mixed solvent system of 5 mM ammonium formate methanol / 5 mM ammonium formate water, and the gradient program is 70% methanol (
As a result of the analysis, after ASL was detected at a retention time of 3 to 10 minutes and LSL at 12 to 16 minutes, there were three unknown component peaks (A, B, C) detected. These results were consistent with the results of the reverse-phase TLC analysis in Example 2, in which unknown glycolipids were detected after ASL and LSL.
構造未知糖脂質の分離・精製
実施例３で新たに検出された３つのピーク成分を単離するために、シリカゲルカラムクロマトグラフィー法による成分分離を行った。まず実施例１（４）と同様のクロロホルム／アセトン混合溶媒を用いた順相カラム法により、クロロホルム／アセトン＝５０／５０溶媒でＬＳＬ画分をほぼ全量溶出させ、その後アセトン１００％やメタノール１００％溶媒でＡＳＬや目的の未知糖脂質が混在する残りの成分を回収した。次にオクタデシル基で修飾されたシリカゲル（ワコーゲル１００Ｃ１８）を充填したカラムを用いて、メタノール／水混合溶媒を展開溶媒とする逆相カラムクロマトグラフィー法により、ＡＳＬと未知糖脂質を分離する２段階のカラム精製法によって未知糖脂質を分離・回収した。分離した各成分は逆相ＴＬＣ解析および逆相ＨＰＬＣ解析によってほぼ単一スポット、単一ピークの成分であることを確認した。
以降は逆相カラムから溶出された順に化合物Ａ、Ｂ、Ｃとする。化合物Ａは透明粘稠な固体、ＢはＡよりも硬質な透明固体、Ｃは蝋状の白色固体であった。
Separation and Purification of Structure-Unknown Glycolipid In order to isolate the three peak components newly detected in Example 3, component separation was performed by silica gel column chromatography. First, by the normal phase column method using the same chloroform/acetone mixed solvent as in Example 1 (4), almost the entire amount of the LSL fraction was eluted with chloroform/acetone = 50/50 solvent, and then acetone 100% or methanol 100%. The remaining components mixed with ASL and the target unknown glycolipid were recovered with a solvent. Next, using a column packed with silica gel (Wakogel 100C18) modified with octadecyl groups, ASL and unknown glycolipids are separated by reversed-phase column chromatography using a methanol/water mixed solvent as a developing solvent. Unknown glycolipids were separated and recovered by a column purification method. It was confirmed by reversed-phase TLC analysis and reversed-phase HPLC analysis that each separated component had a single spot and a single peak.
Hereinafter, compounds A, B, and C are eluted from the reversed-phase column. Compound A was a transparent viscous solid, B was a transparent solid harder than A, and C was a waxy white solid.
構造未知糖脂質のマススペクトル解析
実施例４で分離した化合物Ａ、Ｂ、Ｃについて、液体クロマトグラフィー／マススペクトル（ＬＣ／ＭＳ）解析を行った。電子スプレーイオン化マススペクトロメトリー（ＥＳＩ－ＭＳ）（Ｔｈｅｒｍｏ社製Ｅｘａｃｔｉｖｅ Ｐｌｕｓ）を連結した実施例３に記載のＨＰＬＣを用い、分離条件は実施例３と同様の条件でＬＣ／ＭＳ解析を行った結果を、それぞれ図４～６に示す。
化合物Ａの主成分はｍ／ｚ＝１４６８、Ｂはｍ／ｚ＝２１５７、Ｃはｍ／ｚ＝１７３１（全て検出はネガティブモード）であり、その他各主成分から－４２の成分（１４２６、２１１５、１６８９）が混在していた。これは糖脂質型バイオ界面活性剤によくみられるパターンで、アセチル基が１個外れた化合物であるものと予想された。さらに、各成分から＋１１４の成分（１５８２、２２７１、１８４５）やそこから－４２の成分も混在しており、これらは全て主成分の官能基が一部修飾されたものと予想された。化合物Ａ～Ｃ中には、基本骨格は同じで脂肪酸組成の異なるものが混在していることが予想される。
Mass Spectral Analysis of Structure-Unknown Glycolipid Compounds A, B, and C separated in Example 4 were subjected to liquid chromatography/mass spectral (LC/MS) analysis. Electrospray ionization mass spectrometry (ESI-MS) (Exactive Plus manufactured by Thermo) was used, and the HPLC described in Example 3 was used, and the separation conditions were the same as in Example 3. Results of LC/MS analysis. are shown in FIGS. 4-6, respectively.
The main component of compound A is m / z = 1468, B is m / z = 2157, C is m / z = 1731 (all detection is negative mode), and -42 components (1426, 2115 , 1689) were mixed. This is a pattern often seen in glycolipid-type biosurfactants, and was expected to be a compound with one acetyl group removed. Furthermore, +114 components (1582, 2271, 1845) from each component and -42 components from them were mixed, and it was expected that all of these were partially modified functional groups of the main components. Compounds A to C are expected to contain a mixture of compounds having the same basic skeleton but different fatty acid compositions.
構造未知糖脂質の構造解析
実施例４で分離した化合物Ａ、Ｂ、Ｃについて、核磁気共鳴スペクトル（ＮＭＲ）解析により化学構造の同定を行った。Ｂｒｕｋｅｒ社製ＮＭＲ（ＡＶ－４００）を用いて、まず最も含有量の多い化合物Ｂについて１Ｈ－ＮＭＲ解析を行ったところ、構造既知のＡＳＬとほぼ同じパターンのスペクトルであることが確認された（図７）。
一方、糖骨格部分のスペクトルを拡大して比較したところ、５．３ｐｐｍ付近にＡＳＬとは異なる新しいピークが現れたとともに、４．１～４．４ｐｐｍ付近のピーク面積比が基準ピークとなるソホロース１位のピークと比較して明らか大きいことが確認された。これらのピークをさらに詳細に解析するために１Ｈ－１ＨＣＯＳＹ解析を行ったところ、元々４．１～４．４ｐｐｍ付近に現れるソホロース６位由来のピークとは別の新しいピークがこの位置で重なっており、これらが５．３ｐｐｍ付近の新しいピークと相関していることが分かった（図８）。
Structural Analysis of Structure-Unknown Glycolipid The chemical structures of compounds A, B, and C separated in Example 4 were identified by nuclear magnetic resonance spectroscopy (NMR) analysis. Using Bruker NMR (AV-400), 1 H-NMR analysis was first performed on compound B, which has the highest content, and it was confirmed that the spectrum had almost the same pattern as ASL with a known structure ( Figure 7).
On the other hand, when the spectra of the sugar skeleton portion were enlarged and compared, a new peak different from ASL appeared at around 5.3 ppm, and the peak area ratio around 4.1 to 4.4 ppm was the reference peak for
化合物ＢのＮＭＲデータを表１に示す。
以上の結果より、化合物ＢはＡＳＬの構造に加えて、これら２種類のピークを示す化学構造を持つＳＬ誘導体であることが分かった。これら５．３ｐｐｍ付近と４．１～４．４ｐｐｍ付近にピークが現れる典型的な化合物の例として、油脂（脂肪酸トリグリセリド）のグリセリン骨格が挙げられる。以上を踏まえて、化合物Ｂはグリセリンの３つの水酸基にＡＳＬがエステル結合した化学式（６）の化合物であると推定された。
実施例５で確認された化合物Ｂの主成分は、ｍ／ｚ＝２１５７であり、これは脂肪酸部位がＣ１８：１のＡＳＬが２個、Ｃ１８：２のＡＳＬが１個グリセリンにエステル結合したトリグリセリド構造の化合物の分子量と完全に一致した。以上を総合して、化合物Ｂは化学式（６）の化合物であると同定した。
式（６）
The main component of compound B confirmed in Example 5 has m/z = 2157, and is a triglyceride in which two ASLs with C18:1 fatty acid moieties and one C18:2 ASL is ester-bonded to glycerin. The molecular weights of the structural compounds were completely consistent. Based on the above, compound B was identified as the compound of chemical formula (6).
Formula (6)
同様の解析を化合物ＡとＣについて行った。化合物ＡとＣの１Ｈ－ＮＭＲ解析の結果を、図９と１０に示す。また、化合物ＡまたはＣのＮＭＲデータを、表２または表３に示す。
ＮＭＲ測定結果から推定される最も可能性の高い代表的な構造の化合物として、化合物ＡはグリセリンにＡＳＬが２個結合した化学式（５）の化合物、化合物ＣはグリセリンにＡＳＬが２個と長鎖脂肪酸が１個エステル結合した化学式（７）の化合物であると推定された。いずれも実施例５で確認された各化合物主成分の分子量と一致しており、この構造解析の結果を支持するものであった。
ただし、生産菌培養物中に含まれる誘導体はこれらに限られるものではない。
式（５）
However, the derivatives contained in the producing strain culture are not limited to these.
Formula (5)
新規ソホロリピッド誘導体Ａ～Ｃの水溶液
実施例４で分離した化合物Ａ、Ｂ、Ｃについて、それぞれ１０ｍｇをメスフラスコ中に秤取し、蒸留水を加えてメスアップすることで、１ｍｇ／ｍＬ（０．１ｗｔ％）水溶液を調製した（図１１）。
化合物Ａ、Ｂは完全に溶解して透明の水溶液が得られたが、Ｃは白濁した。さらＡ、Ｂについては同様の方法で、１０ｍｇ／ｍＬ（１ｗｔ％）水溶液を調製したところ、Ａは白濁し、Ｂは完全に溶解した。さらに、白濁した化合物Ａ、Ｃの水溶液を冷蔵庫中に静置したところ、どちらも完全に溶解して透明な水溶液となった。
この温度に応じた水溶液の状態変化は可逆的な現象であり、これは室温（２５℃以下）以下に曇点を有することを示すものであった。すなわち化合物ＡとＣが実施例６で構造決定された通り、非イオン性界面活性剤であることを支持するものであった。また以上の結果から、長鎖脂肪酸が結合している化合物Ｃと比べてそれが無い化合物Ａの方が高い水溶性を示し、さらに化合物Ｂはこれらと比べて高分子量でありながら極めて水溶性の高い非イオン性界面活性剤であることが確認された。
Aqueous Solution of Novel Sophorolipid Derivatives A to
Compounds A and B were completely dissolved to obtain a transparent aqueous solution, but C became cloudy. A 10 mg/mL (1 wt %) aqueous solution was prepared for A and B in the same manner, and A became cloudy and B dissolved completely. Furthermore, when the aqueous solutions of the cloudy compounds A and C were allowed to stand in a refrigerator, both dissolved completely to form transparent aqueous solutions.
This change in the state of the aqueous solution according to temperature is a reversible phenomenon, which indicates that it has a cloud point below room temperature (25° C. or lower). That is, it supported that compounds A and C, as determined in Example 6, are nonionic surfactants. In addition, from the above results, compared to compound C to which a long-chain fatty acid is attached, compound A without it exhibits higher water solubility, and compound B has a higher molecular weight than these, but is extremely water-soluble. It was confirmed to be a highly nonionic surfactant.
新規ＳＬ誘導体Ａ～Ｃの表面張力低下能
実施例４で分離した化合物Ａ、Ｂ、Ｃについて、各濃度の水溶液を調製し、接触角計（協和界面科学社製ＤＭｏ－５００）を使用してペンダントドロップ法（Ｙｏｕｎｇ Ｌａｐｌｈａｓ法）により水溶液の表面張力測定を行った。水溶液濃度－表面張力を対数プロットしたグラフを図１２に示す。
化合物Ｃは濃度によらず水溶液の表面張力値にほとんど変化が無かったが、Ａ、Ｂでは濃度の増加に伴って表面張力が大きく低下した。また、どちらも濃度の増加に伴いプロットの中で２段階の変曲点があることが確認され、表面張力値が一定になる後半の変曲点から臨界ミセル濃度（ＣＭＣ）を算出すると、化合物ＡはＣＭＣ＝１．９１ｇ／Ｌ（約１．３×１０－３Ｍ）、その時の表面張力値（γＣＭＣ）は４０．４ｍＮ／ｍ、化合物ＢはＣＭＣ＝２．９９g／Ｌ（約１．４×１０－３Ｍ）、γＣＭＣは４１．４ｍＮ／ｍであった。ＬＳＬ、ＡＳＬのこれらの値は文献よりそれぞれＬＳＬ：ＣＭＣ＝１．４×１０－５Ｍ、γＣＭＣ＝３２．３ｍＮ／ｍ、ＡＳＬ：ＣＭＣ＝１．２×１０－４Ｍ、γＣＭＣ＝３７．１ｍＮ／ｍであり、今回見出された新規ＳＬ誘導体はこれらと比較して非イオン性界面活性剤でありながら極めて水溶性が高く、穏やかな表面張力低下能を示すこと、すなわち水系での使用に非常に適した界面活性剤であることが示された。
Ability to Reduce Surface Tension of Novel SL Derivatives A to C For the compounds A, B, and C separated in Example 4, aqueous solutions of each concentration were prepared and measured using a contact angle meter (DMo-500 manufactured by Kyowa Interface Science Co., Ltd.). The surface tension of the aqueous solution was measured by the pendant drop method (Young Laplhas method). FIG. 12 shows a logarithmic plot of aqueous solution concentration versus surface tension.
The surface tension of the aqueous solution of Compound C did not change significantly regardless of the concentration, but the surface tension of A and B decreased significantly as the concentration increased. In both cases, it was confirmed that there are two inflection points in the plot as the concentration increases. A has a CMC of 1.91 g/L (about 1.3×10 −3 M), and the surface tension value (γCMC) at that time is 40.4 mN/m. 4×10 −3 M) and γCMC was 41.4 mN/m. These values of LSL and ASL are LSL: CMC = 1.4 × 10 -5 M, γCMC = 32.3 mN/m, ASL: CMC = 1.2 × 10 -4 M, γCMC = 37.1 mN from the literature. /m, and the novel SL derivative discovered this time is a nonionic surfactant, but has extremely high water solubility compared to these, and exhibits a mild ability to reduce surface tension. It has been shown to be a very suitable surfactant.
ソホロリピッドのマトリックス支援レーザーイオン化飛行時間マススペクトル（ＭＡＬＤＩ－ＴＯＦ／ＭＳ）解析
実施例１で得られたＳＬ混合物固体について、日本分光社製ＪＭＳ－３０００ ＳｐｉｒａｌＴＯＦ－ＭＳを用い、マトリックスに２',４',６'-トリヒドロキシアセトフェノン（ＴＨＡＰ）を用いてＭＡＬＤＩ－ＴＯＦ／ＭＳ解析を行った（図１０）。
従来のＬＳＬ、ＡＳＬとは異なるｍ／ｚ＝８０３．４の構造未同定化合物のピークが検出された。これはＳＬがグリセリンにエステル結合した化合物Ａ～Ｃの構造を参考にして、分子量から算出すると、化学式（８）のようなグリセリンに脂肪酸部位がＣ１８：１のＡＳＬが１個エステル結合した化合物（Ｎａ付加体）であることが推定された。
式（８）
A peak of m/z=803.4 of an unidentified compound, which is different from conventional LSL and ASL, was detected. This is calculated from the molecular weight with reference to the structures of compounds A to C in which SL is ester-bonded to glycerin, and a compound ( Na adduct).
Formula (8)
本発明の新規ＳＬ誘導体は、水溶性、表面張力低下能、自己組織化特性に優れており、安全性の高い天然物由来のソホロリピッド誘導体であるから、飼料、肥料、飲食品、農薬、医薬品、医薬部外品および化粧品などの幅広い分野に適用できる。 The novel SL derivative of the present invention is a sophorolipid derivative derived from a natural product, which is excellent in water solubility, surface tension lowering ability, and self-organizing property, and is highly safe. It can be applied to a wide range of fields such as quasi-drugs and cosmetics.
また、本発明は、下記（３）、（４）に記載の界面活性剤、洗浄剤、分散剤、または乳化剤に関する。
（３）上記（１）または（２）に記載のソホロリピッド誘導体からなる界面活性剤、洗浄剤、分散剤、または乳化剤。
（４）飼料、肥料、飲食品、農薬、医薬品、医薬部外品、または化粧品用の、上記（３）に記載の界面活性剤、洗浄剤、分散剤、または乳化剤。
The present invention also relates to surfactants, detergents, dispersants, or emulsifiers described in (3) and ( 4 ) below.
(3) A surfactant, detergent, dispersant or emulsifier comprising the sophorolipid derivative according to (1) or (2) above.
(4) The surfactant, detergent, dispersant, or emulsifier according to (3) above for feeds, fertilizers, food and drink, agricultural chemicals, pharmaceuticals, quasi-drugs, or cosmetics.
Claims (5)
式（１）
（式中、Ｒ１～Ｒ３はそれぞれ独立して、水素、炭素数２～２２の脂肪酸エステル、または上記ＳＬ基であるが、Ｒ１～Ｒ３の少なくとも１つはＳＬ基である。ＳＬ基中のＲは、同一または異なって、水素またはアセチル基を表し、ｎは１１～１９の整数を表す。） A sophorolipid derivative represented by the following general formula (1).
formula (1)
(In the formula, R 1 to R 3 are each independently hydrogen, a fatty acid ester having 2 to 22 carbon atoms, or the above SL group, but at least one of R 1 to R 3 is SL group. SL R in the group is the same or different and represents hydrogen or an acetyl group, and n represents an integer of 11 to 19.)
式（２）
formula (2)
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| EP22777578.0A EP4317448A1 (en) | 2021-03-31 | 2022-03-18 | Novel sophorolipid derivative |
| BR112023003599A BR112023003599A2 (en) | 2021-03-31 | 2022-03-18 | NEW COMPOUND DERIVED FROM SOPHOROLIPID |
| PCT/JP2022/012536 WO2022210011A1 (en) | 2021-03-31 | 2022-03-18 | Novel sophorolipid derivative |
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| JP2014185106A (en) * | 2013-03-22 | 2014-10-02 | Saraya Kk | Sophorolipid powder in which degradation of lactone type sophorolipid is inhibited |
| JP2015507626A (en) * | 2011-12-28 | 2015-03-12 | エヴォニク インダストリーズ アーゲー | Aqueous composition for cleaning hair and skin comprising a biosurfactant |
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| JP2015507626A (en) * | 2011-12-28 | 2015-03-12 | エヴォニク インダストリーズ アーゲー | Aqueous composition for cleaning hair and skin comprising a biosurfactant |
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