JP4978908B2 - Method for producing mannosyl erythritol lipid - Google Patents
Method for producing mannosyl erythritol lipid Download PDFInfo
- Publication number
- JP4978908B2 JP4978908B2 JP2006331073A JP2006331073A JP4978908B2 JP 4978908 B2 JP4978908 B2 JP 4978908B2 JP 2006331073 A JP2006331073 A JP 2006331073A JP 2006331073 A JP2006331073 A JP 2006331073A JP 4978908 B2 JP4978908 B2 JP 4978908B2
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- JP
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- Prior art keywords
- mel
- strain
- pseudozyma
- culture
- mannosyl erythritol
- Prior art date
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- -1 mannosyl erythritol lipid Chemical class 0.000 title claims description 55
- 238000004519 manufacturing process Methods 0.000 title claims description 50
- 244000005700 microbiome Species 0.000 claims description 46
- 241000893045 Pseudozyma Species 0.000 claims description 22
- 241000016347 Pseudozyma hubeiensis Species 0.000 claims description 21
- 241000759238 Sporisorium graminicola Species 0.000 claims description 17
- 125000002777 acetyl group Chemical group [H]C([H])([H])C(*)=O 0.000 claims description 15
- 239000000243 solution Substances 0.000 description 29
- 239000002609 medium Substances 0.000 description 28
- XEKOWRVHYACXOJ-UHFFFAOYSA-N Ethyl acetate Chemical compound CCOC(C)=O XEKOWRVHYACXOJ-UHFFFAOYSA-N 0.000 description 24
- 239000000203 mixture Substances 0.000 description 24
- 238000000034 method Methods 0.000 description 18
- UJEADPSEBDCWPS-SGJODSJKSA-N (2R,3R)-1-[(3S,4S,5S,6R)-3,4,5-trihydroxy-6-(hydroxymethyl)oxan-2-yl]butane-1,2,3,4-tetrol Chemical class C1([C@@H](O)[C@@H](O)[C@H](O)[C@H](O1)CO)C([C@H](O)[C@H](O)CO)O UJEADPSEBDCWPS-SGJODSJKSA-N 0.000 description 14
- CSNNHWWHGAXBCP-UHFFFAOYSA-L Magnesium sulfate Chemical compound [Mg+2].[O-][S+2]([O-])([O-])[O-] CSNNHWWHGAXBCP-UHFFFAOYSA-L 0.000 description 14
- 210000004027 cell Anatomy 0.000 description 14
- 239000003921 oil Substances 0.000 description 13
- CSCPPACGZOOCGX-UHFFFAOYSA-N Acetone Chemical compound CC(C)=O CSCPPACGZOOCGX-UHFFFAOYSA-N 0.000 description 12
- HEDRZPFGACZZDS-UHFFFAOYSA-N Chloroform Chemical compound ClC(Cl)Cl HEDRZPFGACZZDS-UHFFFAOYSA-N 0.000 description 12
- 150000004665 fatty acids Chemical class 0.000 description 12
- VWDWKYIASSYTQR-UHFFFAOYSA-N sodium nitrate Chemical compound [Na+].[O-][N+]([O-])=O VWDWKYIASSYTQR-UHFFFAOYSA-N 0.000 description 12
- 238000004809 thin layer chromatography Methods 0.000 description 12
- 125000004432 carbon atom Chemical group C* 0.000 description 11
- 239000003925 fat Substances 0.000 description 11
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- 125000004435 hydrogen atom Chemical group [H]* 0.000 description 9
- 239000007788 liquid Substances 0.000 description 9
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- 108700022487 rRNA Genes Proteins 0.000 description 9
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- 239000002994 raw material Substances 0.000 description 8
- 229910052943 magnesium sulfate Inorganic materials 0.000 description 7
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- IJGRMHOSHXDMSA-UHFFFAOYSA-N Atomic nitrogen Chemical compound N#N IJGRMHOSHXDMSA-UHFFFAOYSA-N 0.000 description 6
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- OKKJLVBELUTLKV-UHFFFAOYSA-N Methanol Chemical compound OC OKKJLVBELUTLKV-UHFFFAOYSA-N 0.000 description 6
- 241001661345 Moesziomyces antarcticus Species 0.000 description 6
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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 4
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- BASFCYQUMIYNBI-UHFFFAOYSA-N platinum Chemical compound [Pt] BASFCYQUMIYNBI-UHFFFAOYSA-N 0.000 description 4
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- 241000222120 Candida <Saccharomycetales> Species 0.000 description 3
- LFQSCWFLJHTTHZ-UHFFFAOYSA-N Ethanol Chemical compound CCO LFQSCWFLJHTTHZ-UHFFFAOYSA-N 0.000 description 3
- PEDCQBHIVMGVHV-UHFFFAOYSA-N Glycerine Chemical compound OCC(O)CO PEDCQBHIVMGVHV-UHFFFAOYSA-N 0.000 description 3
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- 229920001817 Agar Polymers 0.000 description 2
- QGZKDVFQNNGYKY-UHFFFAOYSA-N Ammonia Chemical compound N QGZKDVFQNNGYKY-UHFFFAOYSA-N 0.000 description 2
- NLXLAEXVIDQMFP-UHFFFAOYSA-N Ammonia chloride Chemical compound [NH4+].[Cl-] NLXLAEXVIDQMFP-UHFFFAOYSA-N 0.000 description 2
- 241000894006 Bacteria Species 0.000 description 2
- VTYYLEPIZMXCLO-UHFFFAOYSA-L Calcium carbonate Chemical compound [Ca+2].[O-]C([O-])=O VTYYLEPIZMXCLO-UHFFFAOYSA-L 0.000 description 2
- OKTJSMMVPCPJKN-UHFFFAOYSA-N Carbon Chemical compound [C] OKTJSMMVPCPJKN-UHFFFAOYSA-N 0.000 description 2
- 241000196324 Embryophyta Species 0.000 description 2
- VZCYOOQTPOCHFL-OWOJBTEDSA-N Fumaric acid Chemical compound OC(=O)\C=C\C(O)=O VZCYOOQTPOCHFL-OWOJBTEDSA-N 0.000 description 2
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- 239000001888 Peptone Substances 0.000 description 2
- 108010080698 Peptones Proteins 0.000 description 2
- VYPSYNLAJGMNEJ-UHFFFAOYSA-N Silicium dioxide Chemical compound O=[Si]=O VYPSYNLAJGMNEJ-UHFFFAOYSA-N 0.000 description 2
- FAPWRFPIFSIZLT-UHFFFAOYSA-M Sodium chloride Chemical compound [Na+].[Cl-] FAPWRFPIFSIZLT-UHFFFAOYSA-M 0.000 description 2
- CDXSJGDDABYYJV-UHFFFAOYSA-N acetic acid;ethanol Chemical compound CCO.CC(O)=O CDXSJGDDABYYJV-UHFFFAOYSA-N 0.000 description 2
- 239000008272 agar Substances 0.000 description 2
- 230000003266 anti-allergic effect Effects 0.000 description 2
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- 235000014113 dietary fatty acids Nutrition 0.000 description 2
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- UNXHWFMMPAWVPI-ZXZARUISSA-N erythritol Chemical compound OC[C@H](O)[C@H](O)CO UNXHWFMMPAWVPI-ZXZARUISSA-N 0.000 description 2
- 239000000469 ethanolic extract Substances 0.000 description 2
- 239000000194 fatty acid Substances 0.000 description 2
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- 230000002401 inhibitory effect Effects 0.000 description 2
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- 125000000311 mannosyl group Chemical group C1([C@@H](O)[C@@H](O)[C@H](O)[C@H](O1)CO)* 0.000 description 2
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- LWIHDJKSTIGBAC-UHFFFAOYSA-K potassium phosphate Substances [K+].[K+].[K+].[O-]P([O-])([O-])=O LWIHDJKSTIGBAC-UHFFFAOYSA-K 0.000 description 2
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Description
本発明は、バイオサーファクタントの一種であるマンノシルエリスリトールリピッドの中でも、特に量産が困難であったMEL-Cを効率よく生産できる微生物、およびその微生物を用いたMEL-Cを主成分とするマンノシルエリスリトールリピッドの製造方法に関する。 Among the mannosyl erythritol lipids which are a kind of biosurfactant, the present invention is a microorganism capable of efficiently producing MEL-C, which was particularly difficult to mass-produce, and a mannosyl erythritol lipid mainly composed of MEL-C using the microorganism. It relates to the manufacturing method.
糖脂質は、脂質に１〜数十個の単糖が結合した物質であり、生体内において細胞間の情報伝達に関与し、神経系及び免疫系の機能維持にも重要な役割を果たしていることなどが明らかにされつつある。また、糖脂質は、糖の性質に由来する親水性と脂質の性質に由来する親油性の二つの性質を合わせ持つ両親媒性物質であり、このような性質を有する両親媒性物質は界面活性物質と呼ばれている。石油化学工業が隆盛となるまでは、レシチン、サポニン等の生体成分由来の界面活性剤（バイオサーファクタント）が利用されていた。近年、石油化学工業の発展により合成界面活性剤が開発され、その生産量が飛躍的に増加し、日常生活には無くてはならない物質となっている。現在、一般に用いられている合成界面活性剤の種類は、細かな構造の違いまで考慮すると、数千種類にものぼる。親水基・疎水基の種類が異なるものから、それぞれのドメインの組成が同じでも分子量や立体構造の違いによって親水性疎水性バランス（HLB）の異なる同族体など、あらゆる化合物が開発されており、これらを単一種類で用いるだけでなく、混合して用いることで、幅広い産業分野において要求される性能に対応している。しかしながら、このような合成界面活性剤の使用量の拡大に伴って環境汚染が広がり、社会問題が生じている。従って、安全性が高く、環境に対する負荷を低減できる生分解性の高い界面活性剤の開発が望まれている。 Glycolipids are substances in which 1 to several tens of monosaccharides are bound to lipids, are involved in information transmission between cells in vivo, and play an important role in maintaining the functions of the nervous system and immune system. Etc. are being revealed. Glycolipids are amphiphilic substances that have both hydrophilic properties derived from the properties of sugars and lipophilic properties derived from the properties of lipids. It is called a substance. Until the petrochemical industry prospered, surfactants (biosurfactants) derived from biological components such as lecithin and saponin were used. In recent years, synthetic surfactants have been developed due to the development of the petrochemical industry, and their production has increased dramatically, making them indispensable for daily life. At present, there are several thousand types of synthetic surfactants that are generally used in consideration of fine structural differences. A variety of compounds have been developed from different types of hydrophilic groups and hydrophobic groups, including homologues with different hydrophilic and hydrophobic balance (HLB) due to differences in molecular weight and steric structure even if the composition of each domain is the same. In addition to being used in a single kind, it can be used in combination to meet the performance required in a wide range of industrial fields. However, as the amount of such synthetic surfactants used increases, environmental pollution spreads and social problems arise. Therefore, development of a highly biodegradable surfactant that has high safety and can reduce the burden on the environment is desired.
微生物などが生産する界面活性物質であるバイオサーファクントは、生分解性が高く、低毒性で環境に優しく、新規な生理機能を持つといわれている。このことから、食品、化粧品、医薬品、化学工業、環境分野等にこれらのバイオサーファクタントを幅広く応用することは、環境調和型の社会を実現し、高機能な製品を提供する上で極めて有意義である。微生物が生産する界面活性物質は、糖脂質系、アシルペプタイド系、リン脂質系、脂肪酸系及び高分子系の界面活性物質の５つに分類されている。なかでも、糖脂質系の界面活性物質については最もよく研究されており、酵母が生産する糖脂質系の界面活性物質としてカンジダ属酵母によるソホロースリピッド（特許文献１参照）やマンノシルエリスリトールリピッド（下記非特許文献１から６参照）などが知られている。
Biosurfactants, which are surface-active substances produced by microorganisms, are said to have high biodegradability, low toxicity, environmental friendliness, and novel physiological functions. Therefore, the wide application of these biosurfactants to food, cosmetics, pharmaceuticals, chemical industry, environmental fields, etc. is extremely meaningful in realizing an environmentally conscious society and providing highly functional products. . Surfactants produced by microorganisms are classified into five types: surfactants of glycolipid type, acyl peptide type, phospholipid type, fatty acid type and polymer type. Among these, glycolipid-based surfactants have been most studied, and sophorose lipids produced by Candida yeast (see Patent Document 1) and mannosyl erythritol lipids (see below) are used as glycolipid-based surfactants produced by yeast. Non-Patent
マンノシルエリスリトールリピッド（mannosyl erythritol lipid:MEL）は、界面活性能以外にも抗微生物活性、白血病細胞、神経系細胞などの細胞分化誘導活性、糖タンパク質結合活性、抗炎症・抗アレルギー活性、アポトーシス誘導活性、癌細胞増殖抑制活性など多様な機能が知られている。MELの生産はこれまで酵母を中心に多くの報告があり、生産量向上を目的として菌株の選択、培養条件、培地組成が検討されている。酵母によるMEL生産に関する報告としては、たとえば、Candida sp. B-7株を用いて５質量％の大豆油から５日間で３５ｇ/Ｌ（生産速度：０．３ｇ/Ｌ/ｈ、原料収率：７０質量%）のMELを生産した例（非特許文献１及び２参照）、Candia antarctica T-34株を用いて８質量％の大豆油から８日間で３８ｇ/Ｌ（生産速度：０．２ｇ/Ｌ/ｈ、原料収率：４８質量％）のMELを生産した例（非特許文献３及び４参照）、Candia antarctica T-34株を用いて６日間隔で計３回の逐次流加により２４日後に２５質量％のピーナッツ油から１１０ｇ/Ｌ（生産速度：０．２ｇ/Ｌ/ｈ、原料収率：４４質量％）のMELを生産した例（非特許文献５参照）、Candida sp. SY-16株を用いて１０質量％の植物油脂から回分培養法により２００時間で５０ｇ/Ｌ（生産速度：０．２５ｇ/Ｌ/ｈ、原料収率：５０質量％）のMELを生産し、また、２０質量％の植物油から流加培養法により２００時間で１２０ｇ/Ｌ（生産速度：０．６ｇ/Ｌ/ｈ、原料収率：５０質量％）のMELを生産した例（非特許文献６参照）、Pseudozyma aphidis株を用いて８０質量％の植物油脂から流加培養法により２４時間で１３．９ｇ/Ｌ（生産速度：０．５７ｇ/Ｌ/ｈ、原料収率：９２質量％）のMELを生産した例（非特許文献７参照）などがある。また、醤油醸造工程において副産物として生産されるしょうゆ油を原料としてCandia antarctica T-34株を用いて７日間で８質量％のしょうゆ油から17g/L（生産速度：０．１ｇ/Ｌ/ｈ、原料収率：２１質量％）のMELの生産が可能であることも報告されている（特許文献２参照）。
Mannosyl erythritol lipid (MEL) has antimicrobial activity, cell differentiation-inducing activity such as leukemia cells and nervous system cells, glycoprotein binding activity, anti-inflammatory / anti-allergic activity, apoptosis-inducing activity in addition to its surface activity. Various functions such as cancer cell growth inhibitory activity are known. MEL production has so far been reported mainly in yeast, and selection of strains, culture conditions, and medium composition have been studied for the purpose of improving production. As a report on MEL production by yeast, for example, 35 g / L (production rate: 0.3 g / L / h, raw material yield: 5% by weight soybean oil from Candida sp. B-7 strain in 5 days) 70 mass%) of MEL produced (see Non-Patent
MELはマンノースにエリスリトールと２個の脂肪酸と２個のアセチル基が結合した糖脂質であり、アセチル基の結合位置と数によって、４種類のMEL（MEL-A、-B、-C、-D）が存在することが知られている。上記のように、MEL生産については多くの微生物菌株と培養技術が検討されているが、これまで量産可能なタイプは、MEL-A と MEL-B だけである。MEL-CとMEL-Dはより水溶性が高く、MEL-AやMEL-B とは異なる性質と用途を持つことが予想されているが、量産できないために、これまで全く実用化の見通しが立っていなかった。従って、MEL-C や MEL-D を量産できれば、マンノシルエリスリトールリピッドのバリエーションの拡充と実用化に大きく貢献できる。 MEL is a glycolipid in which erythritol, two fatty acids and two acetyl groups are bound to mannose. Depending on the binding position and number of acetyl groups, MEL (MEL-A, -B, -C, -D ) Is known to exist. As described above, many microorganism strains and culture techniques have been studied for MEL production, but MEL-A and MEL-B are the only types that can be mass-produced so far. MEL-C and MEL-D are more water-soluble and are expected to have different properties and applications from MEL-A and MEL-B. I didn't stand. Therefore, if MEL-C and MEL-D can be mass-produced, it will greatly contribute to the expansion and commercialization of mannosyl erythritol lipid variations.
本発明の課題は、マンノシルエリスリトールリピッドの中でも量産が困難であったMEL-Cを効率良く生産できる微生物を提供すること、および該微生物を用いてMEL-Cを主成分とするマンノシルエリスリトールリピッドを効率良く生産する方法を提供することを目的とする。 An object of the present invention is to provide a microorganism capable of efficiently producing MEL-C, which was difficult to mass-produce among mannosyl erythritol lipids, and to efficiently produce mannosyl erythritol lipids mainly composed of MEL-C using the microorganisms. The purpose is to provide a method of producing well.
本発明者は、上記課題を解決するため鋭意検討を重ねた結果、シュードザイマ属に属する微生物の中に、MEL-Cを主成分とするマンノシルエリスリトールリピッドを効率良く生産する能力を有する微生物を見出した。また、当該MEL-Cの液晶形成能を調べると、短時間で極めて容易にリオトロピック液晶、特にラメラ液晶の形成が可能であり、化粧品および医薬品素材として高い潜在能力を有することを見出した。本発明はかかる知見により完成されたものである。 As a result of intensive studies to solve the above problems, the present inventor found a microorganism having the ability to efficiently produce mannosylerythritol lipid mainly composed of MEL-C among microorganisms belonging to the genus Pseudozyma. . Further, by examining the liquid crystal forming ability of the MEL-C, it was found that lyotropic liquid crystal, particularly lamellar liquid crystal can be formed very easily in a short time, and has high potential as a cosmetic and pharmaceutical material. The present invention has been completed based on such findings.
すなわち、本発明は以下の発明を包含する。
（1) 配列番号１に示す塩基配列と95％以上相同な塩基配列を含むリボソームRNA遺伝子を有し、かつ、下記式(I)で表されるMEL-C高生産能を有するシュードザイマ（Pseudozyma）属に属する微生物。
That is, the present invention includes the following inventions.
(1) Pseudozyma, which has a ribosomal RNA gene containing a nucleotide sequence that is 95% or more homologous to the nucleotide sequence shown in SEQ ID NO: 1 and has a high MEL-C production ability represented by the following formula (I) A microorganism belonging to the genus.
(2) シュードザイマ（Pseudozyma）属に属する微生物が、シュードザイマ・ヒュベイエンシス（Pseudozyma hubeiensis）である、(1)に記載の微生物。
(3) シュードザイマ（Pseudozyma）属に属する微生物が、シュードザイマ・ヒュベイエンシス（Pseudozyma hubeiensis）KM-59株（FERM P-20987)である、(1)または(2)に記載の微生物。
(4) シュードザイマ（Pseudozyma）属に属する微生物を油脂類含有培地にて培養し、培養物中から下記式(I)で表されるMEL-Cを主成分とするマンノシルエリスリトールリピッドを採取することを特徴とする、マンノシルエリスリトールリピッドの製造方法。
(2) The microorganism according to (1), wherein the microorganism belonging to the genus Pseudozyma is Pseudozyma hubeiensis.
(3) The microorganism according to (1) or (2), wherein the microorganism belonging to the genus Pseudozyma is Pseudozyma hubeiensis KM-59 strain (FERM P-20987).
(4) culturing microorganisms belonging to the genus Pseudozyma in an oil-containing medium, and collecting mannosylerythritol lipids mainly composed of MEL-C represented by the following formula (I) from the culture. A method for producing a mannosyl erythritol lipid, which is characterized.
(5) シュードザイマ（Pseudozyma）属に属する微生物が、(1)〜(3)のいずれかに記載の微生物である、(4)に記載の製造方法。
(6) シュードザイマ（Pseudozyma）属に属する微生物が、シュードザイマ・グラミニコーラ（Pseudozyma graminicola）である、(4)に記載の製造方法。
(7) シュードザイマ（Pseudozyma）属に属する微生物が、シュードザイマ・グラミニコーラ（Pseudozyma graminicola）CBS 10092株である、(4)に記載の製造方法。
(8) (4)から(7)のいずれかに記載の方法により得られる、MEL-Cを主成分とするマンノシルエリスリトールリピッド。
(9) (8)に記載のマンノシルエリスリトールリピッドを含有するリオトピック液晶性組成物。
(5) The production method according to (4), wherein the microorganism belonging to the genus Pseudozyma is the microorganism according to any one of (1) to (3).
(6) The production method according to (4), wherein the microorganism belonging to the genus Pseudozyma is Pseudozyma graminicola.
(7) The production method according to (4), wherein the microorganism belonging to the genus Pseudozyma is Pseudozyma graminicola CBS 10092 strain.
(8) A mannosylerythritol lipid mainly comprising MEL-C, which is obtained by the method according to any one of (4) to (7).
(9) A Riotopic liquid crystalline composition containing the mannosyl erythritol lipid according to (8).
本発明によれば、MEL-Cを主成分とするマンノシルエリスリトールリピッドを効率良く生産する能力を有する微生物が提供される。従って、この微生物を植物油脂等の油脂類含有培地で培養することにより、これまで量産が困難であったMEL-Cを効率よく生産することができる。MEL-CはMEL-AやMEL-Bより水溶性が高く、より応用範囲が広いことが予想されており、マンノシルエリスリトールリピッドの新たな用途開拓ならびに構造・機能的バリエーションの拡充に貢献できる。本発明により得られたMEL-C は、水性媒体に容易に水和し、ラメラ液晶などの各種のリオトロピック液晶を形成できる。また、得られた液晶は糖骨格に起因する水素結合のネットワークにより、極めて安定性も良く、温度変化に対して強固であるから、化粧品や医薬品の安定化剤として有用である。 According to the present invention, there is provided a microorganism having an ability to efficiently produce mannosyl erythritol lipid containing MEL-C as a main component. Therefore, MEL-C, which has been difficult to mass-produce so far, can be efficiently produced by culturing this microorganism in a fat-containing medium such as vegetable oil. MEL-C has higher water solubility than MEL-A and MEL-B, and is expected to have a wider range of applications. It can contribute to the development of new uses for mannosyl erythritol lipid and the expansion of structural and functional variations. MEL-C obtained by the present invention is easily hydrated in an aqueous medium and can form various lyotropic liquid crystals such as lamellar liquid crystals. In addition, the obtained liquid crystal is extremely stable due to the hydrogen bond network resulting from the sugar skeleton and is strong against temperature changes, and thus is useful as a stabilizer for cosmetics and pharmaceuticals.
以下、本発明を詳細に説明する。
１．MEL-C高生産能を有する微生物
本発明の微生物は、配列番号１に示す塩基配列と95％以上相同な塩基配列を含むリボソームRNA遺伝子を有し、かつ、MEL-C高生産能を有するシュードザイマ（Pseudozyma）属に属する微生物である。
Hereinafter, the present invention will be described in detail.
1. Microorganism having high MEL-C production ability The microorganism of the present invention has a ribosomal RNA gene containing a nucleotide sequence of 95% or more homologous to the nucleotide sequence shown in SEQ ID NO: 1, and Pseudozyma having MEL-C high production ability It is a microorganism belonging to the genus (Pseudozyma).
このような微生物の例としては、本発明者らが、沖縄で採取した植物（葉）サンプルから分離したKM-59株を挙げることができる。本菌株は、ＹＭ寒天培地上にて２５℃２日間培養で、直径が２〜３ｍｍ程度のコロニーを形成する（形：円形、隆起状態：半レンズ状、周縁：波状、表面の形状：スムーズ、透明度：半透明、粘ちょう度：粘ちょう性）。また、子嚢胞子の形成は認められない。 As an example of such a microorganism, the KM-59 strain isolated from a plant (leaf) sample collected by the present inventors in Okinawa can be mentioned. This strain is cultured on a YM agar medium at 25 ° C. for 2 days to form colonies having a diameter of about 2 to 3 mm (shape: circular, raised state: half-lens shape, peripheral edge: wavy, surface shape: smooth, (Transparency: translucent, consistency: consistency). Also, no ascospore formation is observed.
KM-59株は、リボソームRNA遺伝子の塩基配列（rDNA配列）を決定し、DNAデータベース(DDBJ)にアクセスし、FASTAプログラム (http://www.ddbj.nig.ac.jp/search/fasta-j.html）を用いて相同性検索を行ったところ、シュードザイマ・ヒュベイエンシス（Pseudozyma hubeiensis）のリボソームRNA遺伝子のITS-1領域から5.8S 領域までの塩基配列と100％一致した。また、DNAデータベース上より入手したシュードザイマ（Pseudozyma）属および代表的な酵母種のリボソームRNA遺伝子のITS-1領域から5.8S 領域までの塩基配列を多重整列後、NJ法により分子系統解析を行った結果、本菌株の分子系統樹の位置はシュードザイマ・ヒュベイエンシス（Pseudozyma hubeiensis）と一致した（図１）。KM-59株は、マンノシルエリスリトールリピッド生産菌ではあるが、MEL-Cを量産できる点においてシュードザイマ属に属する既知のマンノシルエリスリトールリピッド生産菌とは異なる新菌株と判断されたため、本菌株をシュードザイマ・ヒュベイエンシス（Pseudozyma hubeiensis）KM-59株と命名した。本菌株は、平成18（2006）年8月11日付で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（IPOD）（茨城県つくば市東１−１−３）に受託番号FERM P-20987として寄託されている。 The KM-59 strain determines the base sequence (rDNA sequence) of the ribosomal RNA gene, accesses the DNA database (DDBJ), and accesses the FASTA program (http://www.ddbj.nig.ac.jp/search/fasta- The homology search using j.html) was 100% identical to the nucleotide sequence from the ITS-1 region to the 5.8S region of the ribosomal RNA gene of Pseudozyma hubeiensis. In addition, we performed molecular phylogenetic analysis by NJ method after multiple alignment of ITS-1 region to 5.8S region of ribosomal RNA genes of genus Pseudozyma and representative yeast species obtained from DNA database As a result, the position of the molecular phylogenetic tree of this strain coincided with Pseudozyma hubeiensis (FIG. 1). The KM-59 strain is a mannosyl erythritol lipid-producing bacterium, but was judged to be a new strain that differs from the known mannosyl erythritol lipid-producing bacterium belonging to the genus Pseudozyma in that MEL-C can be mass-produced. It was named Bayensis (Pseudozyma hubeiensis) KM-59 strain. This strain was deposited under the accession number FERM P-20987 at the National Institute of Advanced Industrial Science and Technology Patent Organism Depositary (IPOD) (1-1-3 East Tsukuba, Ibaraki) on August 11, 2006 Has been.
本発明の微生物には、上記のKM-59株のほか、その類縁微生物も含まれる。KM-59株の類縁微生物としては、例えば、配列番号１の塩基配列と95％以上、好ましくは97％以上、さらに好ましくは98％以上、最も好ましくは99％以上相同な塩基配列を含むリボソームRNA遺伝子を有し、かつ、MEL-C高生産能を有する微生物をいう。ここで、MEL-Cは、下記式(I)で表される構造を有するマンノシルエリスリトールリピッドである。 The microorganism of the present invention includes related microorganisms in addition to the above-mentioned KM-59 strain. Examples of related microorganisms of the KM-59 strain include ribosomal RNA containing a nucleotide sequence that is 95% or more, preferably 97% or more, more preferably 98% or more, most preferably 99% or more homologous to the nucleotide sequence of SEQ ID NO: 1. A microorganism having a gene and having a high productivity of MEL-C. Here, MEL-C is mannosyl erythritol lipid having a structure represented by the following formula (I).
また、「MEL-C高生産能」とは、MEL-Cを主成分とするマンノシルエリスリトールリピッドを効率良く生産する能力をいい、「主成分とする」とは、生産されるマンノシルエリスリトールリピッド全量に対してMEL-Cの占める割合がMEL-A、MEL-Bなどの他のマンノシルエリスリトールリピッドに比べて有意に高いことをいう。 “MEL-C high productivity” refers to the ability to efficiently produce mannosyl erythritol lipids based on MEL-C. “Main component” refers to the total amount of mannosyl erythritol lipids produced. On the other hand, the proportion of MEL-C is significantly higher than other mannosyl erythritol lipids such as MEL-A and MEL-B.
KM-59株の類縁微生物は、上記MEL-C高生産能と配列番号１に記載の塩基配列を指標として単離することができる。自然界から採取したサンプル（例えば、土壌、河川・湖沼の水、樹液、花、葉、果実など）を、油脂類含有培地にて２６〜３０℃にて３〜７日間培養し、培養液をGC/MS分析してMEL-C生産が認められたサンプルを選抜する（一次スクリーニング）。次に、選抜したサンプルの培養液をプレート上で培養し、コロニーを形成させて微生物を単離し、さらに単離した微生物を油脂類含有培地にて培養で培養し、GC/MS分析してMEL-C生産が認められた微生物を選択し（二次スクリーニング）、この微生物菌株のリボソームRNA遺伝子の塩基配列について、DDBJ のFASTAプログラム (http://www.ddbj.nig.ac.jp/search/fasta-j.html）の塩基配列データベースに対して相同性検索を行う。 A related microorganism of the KM-59 strain can be isolated using the above-mentioned MEL-C high productivity and the base sequence described in SEQ ID NO: 1 as indicators. Samples collected from nature (eg, soil, river / lake water, sap, flowers, leaves, fruits, etc.) are cultured in an oil and fat-containing medium at 26-30 ° C. for 3-7 days. Select samples with MEL-C production confirmed by MS / MS analysis (primary screening). Next, the culture solution of the selected sample is cultured on a plate, colonies are formed and microorganisms are isolated, and further, the isolated microorganisms are cultured in an oil-containing medium and subjected to GC / MS analysis and subjected to MEL. -Choose microorganisms that have been recognized for production (secondary screening), and the DDBJ FASTA program (http://www.ddbj.nig.ac.jp/search/ Perform a homology search against the base sequence database of fasta-j.html).
上記のスクリーニングに用いる培地としては、例えば、８０ｇ/Ｌの大豆油と酵母エキス１ｇ/Ｌ、硝酸ナトリウム１ｇ/Ｌ、リン酸２水素カリウム０．５ｇ/Ｌ、及び硫酸マグネシウム０．５ｇ/Ｌの組成の液体培地が挙げられる。 Examples of the culture medium used for the above screening include 80 g / L soybean oil and yeast extract 1 g / L, sodium nitrate 1 g / L, potassium dihydrogen phosphate 0.5 g / L, and magnesium sulfate 0.5 g / L. A liquid medium having a composition may be mentioned.
２．マンノシルエリスリトールリピッドの製造方法
本発明のマンノシルエリスリトールリピッドの製造方法には、シュードザイマ（Pseudozyma）属に属する微生物を用いることができる。シュードザイマ（Pseudozyma）属に属する微生物としては、MEL-C高生産能を有するものであれば特に制限はないが、例えば、配列番号１に示す塩基配列と80％以上、好ましくは90％以上、より好ましくは95％以上相同な塩基配列を含むリボソームRNA遺伝子を有するシュードザイマ（Pseudozyma）属に属する微生物；シュードザイマ・ヒュベイエンシス（Pseudozyma hubeiensis）；シュードザイマ・グラミニコーラ（Pseudozyma graminicola）などが挙げられる。特に、シュードザイマ・ヒュベイエンシス（Pseudozyma hubeiensis）KM-59株（FERM P-20987)、シュードザイマ・グラミニコーラ（Pseudozyma graminicola）CBS 10092株が好ましい。
2. Production method of mannosyl erythritol lipid A microorganism belonging to the genus Pseudozyma can be used in the production method of mannosyl erythritol lipid of the present invention. The microorganism belonging to the genus Pseudozyma is not particularly limited as long as it has high production ability for MEL-C. For example, the base sequence shown in SEQ ID NO: 1 is 80% or more, preferably 90% or more. Preferably, microorganisms belonging to the genus Pseudozyma having a ribosomal RNA gene having a base sequence homologous to 95% or more; Pseudozyma hubeiensis; Pseudozyma graminicola, and the like. In particular, Pseudozyma hubeiensis KM-59 strain (FERM P-20987) and
上記のような微生物を油脂類含有培地にて培養することにより培養物中に前記式(I)で表されるMEL-Cを主成分とするマンノシルエリスリトールリピッドを製造することができる。 By culturing the above microorganisms in a fat-and-oils-containing medium, mannosyl erythritol lipids containing MEL-C represented by the above formula (I) as the main component can be produced in the culture.
また、製造されるマンノシルエリスリトールリピッドは、下式（II)で表される。 In addition, the mannosyl erythritol lipid produced is represented by the following formula (II).
上記式（II）において、Ｒ１〜Ｒ５は、互いに同一であっても異なっていてもよく、水素原子、アセチル基、又は炭素原子数１〜１８、好ましくは３〜１２の飽和若しくは不飽和の脂肪酸残基を表す。 In the above formula (II), R 1 to R 5 may be the same or different from each other, and are a hydrogen atom, an acetyl group, or a saturated or unsaturated group having 1 to 18 carbon atoms, preferably 3 to 12 carbon atoms. Represents a fatty acid residue.
上記式（II）で表されるマンノシルエリスリトールリピッドには、主成分であるMEL-Cのほか、例えば、MEL-A（式（II)中、Ｒ１とＲ２が炭素原子数１〜１６の脂肪酸残基、Ｒ３とＲ4がアセチル基、Ｒ５が水素原子である化合物）、MEL-B（式（II）中、Ｒ１とＲ２が炭素原子数１〜１６の脂肪酸残基、Ｒ4がアセチル基、Ｒ３とＲ５が水素原子である化合物）、MEL-D（式（II）中、Ｒ１とＲ２が炭素原子数１〜１６の脂肪酸残基、Ｒ３とＲ4とＲ５が水素原子である化合物）、トリアシル型MEL（式（II）中、Ｒ１とＲ２が炭素原子数１〜１６の脂肪酸残基、Ｒ３とＲ4が水素原子またはアセチル基、Ｒ５が炭素原子数１〜１８の脂肪酸残基である化合物）、モノアシル型MEL（式（II）中、Ｒ１とＲ２のいずれか一方が炭素原子数１〜１６の脂肪酸残基で、いずれか一方が水素原子、Ｒ３とＲ4とＲ５が水素原子またはアセチル基である化合物）などが含まれる。 In addition to MEL-C as a main component, for example, MEL-A (in formula (II), R 1 and R 2 each have 1 to 16 carbon atoms are included in the mannosyl erythritol lipid represented by the above formula (II). A fatty acid residue, a compound in which R 3 and R 4 are acetyl groups, and R 5 is a hydrogen atom), MEL-B (in formula (II), R 1 and R 2 are fatty acid residues having 1 to 16 carbon atoms, R 4 is an acetyl group, R 3 and R 5 are hydrogen atoms), MEL-D (in formula (II), R 1 and R 2 are fatty acid residues having 1 to 16 carbon atoms, R 3 and R A compound in which 4 and R 5 are hydrogen atoms), triacyl type MEL (in formula (II), R 1 and R 2 are fatty acid residues having 1 to 16 carbon atoms, R 3 and R 4 are hydrogen atoms or acetyl groups) and R 5 is a fatty acid residue having 1 to 18 carbon atoms), in monoacyl type MEL (formula (II), one of R 1 and R 2 is a 1 to 16 carbon atoms In fatty acid residues, one is a hydrogen atom, and R 3 and R 4 and R 5 is a hydrogen atom or an acetyl group) and the like.
上記培養に用いる培地には、油脂類を添加する以外については特に制限はなく、酵母に対して一般に用いられる培地、たとえば、ＹＰＤ培地（イーストエキストラクト１０ｇ、ポリペプトン２０ｇ、及びグルコース２０ｇ）を使用することができる。培地中の油脂類の含有量としては、４〜１０質量％が好ましい。油脂類は培養開始時にその全量を添加しても、培養中に連続的または間欠的に添加してもよい。 There is no restriction | limiting in particular except adding fats and oils to the culture medium used for the said culture | cultivation, The culture medium generally used with respect to yeast, for example, YPD culture medium (yeast extract 10g, polypeptone 20g, and glucose 20g) is used. be able to. As content of fats and oils in a culture medium, 4-10 mass% is preferable. The total amount of fats and oils may be added at the start of the culture, or may be added continuously or intermittently during the culture.
油脂類としては、植物油脂又は動物油脂等のいずれでもよく、目的に応じて適宜選定することができる。植物油脂としては、大豆油、菜種油、コーン油、ピーナッツ油、綿実油、ベニバナ油、ごま油、オリーブ油、バーム油等が挙げられ、これらの中でも、大豆油が好ましい。動物油脂としては、牛脂、豚脂、魚脂等が挙げられる。これらは、１種を単独で又は２種以上を適宜混合して用いてもよい。 As fats and oils, any of vegetable oils and animal fats and the like may be used, and can be appropriately selected according to the purpose. Examples of vegetable oils include soybean oil, rapeseed oil, corn oil, peanut oil, cottonseed oil, safflower oil, sesame oil, olive oil, and balm oil. Among these, soybean oil is preferable. Examples of animal fats include beef tallow, pork tallow and fish tallow. You may use these individually by 1 type or in mixture of 2 or more types as appropriate.
培地に添加する油脂類以外の他の成分としては、当該技術分野で通常用いられる炭素源、窒素源、無機塩類、及び必要な栄養源等が用いられる。炭素源としては、該微生物が資化し得るものであればよく、炭水化物（グルコース、マンノース、グリセロール、マンニトール等の単糖、ショ糖、麦芽糖、乳糖等のオリゴ糖、デンプン等）、有機酸（酢酸、プロピオン酸、マレイン酸、フマル酸、リンゴ酸等）、アルコール類（エタノール、プロパノール等）が用いられる。窒素源としては、塩化アンモニウム、硫酸アンモニウム、硝酸アンモニウム、リン酸アンモニウム等の無機酸若しくは有機酸のアンモニウム塩、又はペプトン、酵母エキス、麦芽エキス、肉エキス、コーンスチープリカー等の含窒素化合物が用いられる。無機塩類としては、リン酸第一カリウム、リン酸第二カリウム、リン酸マグネシウム、硫酸マグネシウム、塩化ナトリウム、硫酸第一鉄、硫酸マンガン、硫酸銅、炭酸カルシウム等が用いられる。 As other components other than fats and oils added to the medium, carbon sources, nitrogen sources, inorganic salts, necessary nutrient sources and the like that are usually used in the technical field are used. Any carbon source may be used as long as it can be assimilated by the microorganism, and includes carbohydrates (monosaccharides such as glucose, mannose, glycerol, and mannitol, oligosaccharides such as sucrose, maltose, and lactose, starch), organic acids (acetic acid) Propionic acid, maleic acid, fumaric acid, malic acid, etc.) and alcohols (ethanol, propanol, etc.) are used. As the nitrogen source, ammonium salts of inorganic acids or organic acids such as ammonium chloride, ammonium sulfate, ammonium nitrate, and ammonium phosphate, or nitrogen-containing compounds such as peptone, yeast extract, malt extract, meat extract, corn steep liquor and the like are used. As the inorganic salts, monopotassium phosphate, dipotassium phosphate, magnesium phosphate, magnesium sulfate, sodium chloride, ferrous sulfate, manganese sulfate, copper sulfate, calcium carbonate and the like are used.
培養温度は、２０〜３０℃、好ましくは２６〜３０℃である。培養日数としては、MEL-C生産量に応じて適宜設定すればよいが、３〜２０日間が好ましい。培養方法としては、振盪培養、通気撹拌培養等の公知の一般的な微生物の培養方法を適用することができる。良好な微生物の生育及びマンノシルマンニトールリピッドの生産のためには、培養液に酸素を供給することが好ましく、そのためには、ジャーファメンターを用いる場合では、空気を通気しながら撹拌するか、振とう培養を行えばよい。 The culture temperature is 20-30 ° C, preferably 26-30 ° C. The culture days may be appropriately set according to the production amount of MEL-C, but 3 to 20 days is preferable. As a culture method, known general microorganism culture methods such as shaking culture and aeration stirring culture can be applied. For good microbial growth and production of mannosyl mannitol lipids, it is preferable to supply oxygen to the culture medium. For this purpose, when using a jar fermenter, it is stirred or shaken with aeration of air. What is necessary is just to culture.
本発明において、前記シュードザイマ・ヒュベイエンシス（Pseudozyma hubeiensis）KM-59株またはシュードザイマ・グラミニコーラ（Pseudozyma graminicola）CBS 10092株を用いてMEL-Cを主成分とするマンノシルエリスリトールリピッドを生産する場合、増殖能の高い菌株を用いた方が、単位培養液及び単位時間当たりのマンノシルエリスリトールリピッド生産量が高くなるため、まず前培養によって菌体を活性化させ、これを本培養の培地に接種して培養を行うことが好ましい。例えば、種培養、本培養及びマンノシルエリスリトールリピッド生産培養の順にスケールアップしていくことが好ましい。これらの培養における、具体的な培地組成、培養条件を例示すると以下のとおりである。
In the present invention, when the Pseudozyma hubeiensis KM-59 strain or
グルコース５〜２０ｇ／Ｌ，酵母エキス０．１〜２ｇ／Ｌ，硝酸ナトリウム０．１〜５ｇ／Ｌ，リン酸２水素カリウム０．１〜２ｇ／Ｌ、及び硫酸マグネシウム０．１〜１ｇ／Ｌの組成の液体培地４ｍＬが入った試験管に、保存菌株を１白金耳接種し、２６〜３０℃で１〜３日振とう培養を行う（種培養）。次に、２０〜３００ｇ／Ｌ、好ましくは４０〜１００ｇ／Ｌの植物油脂等の油脂類を加えた上記と同じ組成の液体培地５０〜１００ｍＬの入った坂口フラスコに上記の種培養を行った培養液を接種して、２６〜３０℃で１〜７日間振とう培養を行う(本培養）。さらに、上記の本培養と同じ組成の液体培地１〜２Ｌが入ったジャーファメンターに上記の本培養を行った培養液を接種して、２６〜３０℃で１〜２Ｌ／分の通気速度と６００〜１０００ｒｐｍの撹拌速度で培養する(マンノシルエリスリトールリピッド生産培養)。このときの培養温度は２６〜３０℃、培養時間は、３〜２０日間である。この培養においては、培養途中から油脂類を培養容器中に流下させて、培地中の油脂類濃度を５０〜２００ｇ／Ｌに保持することが好ましい。 Glucose 5-20 g / L, yeast extract 0.1-2 g / L, sodium nitrate 0.1-5 g / L, potassium dihydrogen phosphate 0.1-2 g / L, and magnesium sulfate 0.1-1 g / L One platinum loop of the stock strain is inoculated into a test tube containing 4 mL of a liquid medium having the composition of (1), followed by shaking culture at 26 to 30 ° C. for 1 to 3 days (seed culture). Next, the culture which performed said seed culture to the Sakaguchi flask containing 50-100 mL of liquid culture medium of the same composition as the above which added fats and oils, such as vegetable fats and oils of 20-300 g / L, preferably 40-100 g / L The solution is inoculated and cultured with shaking at 26-30 ° C. for 1-7 days (main culture). Further, a jar fermenter containing 1 to 2 L of liquid medium having the same composition as that of the main culture is inoculated with the culture solution obtained by performing the main culture, and an aeration rate of 1 to 2 L / min at 26 to 30 ° C. Culturing is performed at a stirring speed of 600 to 1000 rpm (mannosylerythritol lipid production culture). The culture temperature at this time is 26 to 30 ° C., and the culture time is 3 to 20 days. In this culture, it is preferable that oils and fats flow down into the culture vessel from the middle of the culture to maintain the oils and fats concentration in the medium at 50 to 200 g / L.
微生物菌株の培地への使用量は、例えば、菌体を接種する場合、培地１Ｌあたり、１０〜２００ｍl、好ましくは５０〜１００ｍlの種培養液あるいは本培養液中に含まれる量であればよい。 The amount of the microorganism strain used in the medium may be, for example, 10 to 200 ml, preferably 50 to 100 ml of the seed culture solution or the main culture solution per liter of the medium when inoculating the cells.
本発明における培養は、培養液中の所望のMEL-C生成量が最高に達した時点で終了させることができるように、培養液中のMEL-Cをガスクロマトグラフィー、高速液体クロマトグラフィー、薄層クロマトグラフィー等の周知の方法により測定しながら行うことが好ましい。 The culture in the present invention can be terminated when the desired production amount of MEL-C in the culture solution reaches the maximum, so that the MEL-C in the culture solution is gas chromatographed, high performance liquid chromatography, It is preferable to perform the measurement while using a known method such as layer chromatography.
培養液中に蓄積されたマンノシルエリスリトールリピッドは、培養終了後、培養液を抽出することによって取得できる。抽出は、酢酸エチル等の有機溶媒を用いて行う。抽出は当該分野において通常行われる方法に従って行えばよく、例えば、培養液１Ｌあたり０．５〜１．５Ｌ程度の有機溶媒を用いて１〜数回抽出操作を行い、酢酸エチル層を合わせて溶媒を留去する。 Mannosyl erythritol lipid accumulated in the culture solution can be obtained by extracting the culture solution after completion of the culture. Extraction is performed using an organic solvent such as ethyl acetate. Extraction may be carried out according to a method usually performed in the art. For example, extraction is performed once or several times using about 0.5 to 1.5 L of an organic solvent per 1 L of a culture solution, and the ethyl acetate layer is combined with a solvent. Is distilled off.
酢酸エタノール抽出物に含まれるマンノシルエリスリトールリピッドから、MEL-Cを分画するには、シリカゲルカラムクロマトグラフィーによる分離精製を行う。酢酸エタノール抽出物をシリカゲルカラムに供し、クロロホルムとアセトンを溶出液として用い、当該抽出物中の成分を分離精製する。クロロホルムのみを流した後、クロロホルムとアセトンの混合液で当該抽出物中の成分を溶出する。この時、クロロホルムとアセトンの混合液中におけるアセトンの割合は、段階的に上げていく。例えば、９：１から始め、次に、８：２あるいは７：３としていく。各溶出液は収集し、TLCにて精製度を確認する。 To fractionate MEL-C from mannosyl erythritol lipid contained in an acetic acid ethanol extract, separation and purification by silica gel column chromatography is performed. The acetic acid ethanol extract is applied to a silica gel column, and components in the extract are separated and purified using chloroform and acetone as eluents. After flowing only chloroform, the components in the extract are eluted with a mixture of chloroform and acetone. At this time, the ratio of acetone in the mixed liquid of chloroform and acetone is increased stepwise. For example, start from 9: 1, then 8: 2 or 7: 3. Collect each eluate and check the purity by TLC.
上記培養において、微生物の形態は、特に限定されず、微生物の菌体、菌体処理物（例えば、菌体破砕物）などをいう。 In the above culture, the form of the microorganism is not particularly limited, and refers to a microorganism cell, a treated cell of the microorganism (for example, a disrupted cell).
微生物の菌体または菌体処理物は固定化して用いることもできる。固定化法としては、従来公知の担体結合法、架橋化法、包括法などの方法が挙げられる。担体結合法では、担体に菌体を固定化させるが、固定化は物理的吸着、イオン結合、共有結合のいずれであってもよい。担体としては多糖（アセチルセルロース、アガロース）、無機物質（多孔質ガラス、金属酸化物）、合成高分子（ポリアクリルアミド、ポリスチレン）等が用いられる。架橋化法では、グルタルアルデヒド等の二官能性試薬を用いて菌体同士を架橋、結合させることによって固定化する。また、包括法では、多糖（アルギン酸、カラギーナン）、ポリアクリルアミド、コラーゲン、ポリウレタン等の高分子ゲルの格子や半透膜カプセルに菌体を包み込むことによって固定化する。 The microbial cells or the processed microbial cells can be immobilized and used. Examples of the immobilization method include conventionally known methods such as a carrier binding method, a crosslinking method, and a comprehensive method. In the carrier binding method, the cells are immobilized on the carrier, and the immobilization may be any of physical adsorption, ionic bond, and covalent bond. As the carrier, polysaccharides (acetylcellulose, agarose), inorganic substances (porous glass, metal oxide), synthetic polymers (polyacrylamide, polystyrene) and the like are used. In the crosslinking method, the cells are immobilized by crosslinking and bonding with each other using a bifunctional reagent such as glutaraldehyde. In the inclusion method, the cells are immobilized by wrapping the cells in a lattice of a polymer gel such as polysaccharide (alginate, carrageenan), polyacrylamide, collagen, polyurethane, or a semipermeable membrane capsule.
３．MEL-Cを主成分とするマンノシルエリスリトールリピッドの用途
上記の方法により得られたMEL-Cを主成分とするマンノシルエリスリトールリピッド、及びMEL-Cは、温度範囲が室温から７０℃まで、濃度範囲が0.0001%から90%までの幅広い条件下でリオトロピック液晶を形成することが可能である。リオトロピック液晶は、高濃度の水溶性成分と油溶性成分の双方を内包安定化できるという性質を有している。従って、当該マンノシルエリスリトールリピッドは、医薬品、化粧品、食品等の種々の組成物に含有させることにより、その界面活性作用により主成分の分散性、製品の酸化安定性向上をはかることができる。化粧品の形態は特に限定はされないが、たとえばクリームやクレンジングジェル及び乳液等が挙げられる。また、医薬品の形態も特に限定はされないが、クリーム状の外用剤や分散安定化した注射剤などが挙げられる。
3. Use of mannosyl erythritol lipid mainly composed of MEL-C Mannosyl erythritol lipid mainly composed of MEL-C obtained by the above method and MEL-C have a temperature range from room temperature to 70 ° C and a concentration range. It is possible to form lyotropic liquid crystals under a wide range of conditions from 0.0001% to 90%. The lyotropic liquid crystal has the property that it can stabilize both the high-concentration water-soluble component and the oil-soluble component. Therefore, when the mannosyl erythritol lipid is contained in various compositions such as pharmaceuticals, cosmetics, and foods, the dispersibility of the main component and the oxidative stability of the product can be improved by the surface activity. The form of the cosmetic is not particularly limited, and examples thereof include creams, cleansing gels, and emulsions. Further, the form of the pharmaceutical is not particularly limited, and examples thereof include a creamy external preparation and a dispersion-stabilized injection.
また前記のとおり、マンノシルエリスリトールリピッドは界面活性能以外にも抗微生物活性、細胞分化誘導活性、糖タンパク質結合活性、抗炎症・抗アレルギー活性、アポトーシス誘導活性、癌細胞増殖抑制活性など種々の生理活性作用を有する。従って、MEL-Cを主成分とするマンノシルエリスリトールリピッドは、公知の種々の方法にて各種製剤形態に調製し、それ自体を有効成分とする医薬（例えば、抗癌剤など）として用いることができる。各種製剤は、製剤上通常用いられる賦形剤、増量剤、結合剤、湿潤剤、崩壊剤、潤滑剤、界面活性剤、分散剤、緩衝剤、保存剤、溶解補助剤、防腐剤、矯味矯臭剤、無痛化剤、安定化剤、等張化剤等などを適宜選択し、常法により製造することができる。 As described above, mannosyl erythritol lipid has a variety of physiological activities such as antimicrobial activity, cell differentiation-inducing activity, glycoprotein binding activity, anti-inflammatory / anti-allergic activity, apoptosis-inducing activity, and cancer cell growth-inhibiting activity in addition to its surface activity. Has an effect. Therefore, mannosyl erythritol lipid containing MEL-C as a main component can be prepared in various preparation forms by various known methods and used as a medicine (for example, an anticancer agent) containing itself as an active ingredient. Various preparations include excipients, extenders, binders, wetting agents, disintegrants, lubricants, surfactants, dispersants, buffering agents, preservatives, solubilizers, preservatives, flavoring agents that are commonly used in the formulation. Agents, soothing agents, stabilizers, tonicity agents and the like can be appropriately selected and produced by conventional methods.
以下、実施例により本発明をさらに具体的に説明する。但し、本発明はこれらに限定されるものではない。
（実施例１）KM-59株の単離と同定
沖縄で採取した植物（葉）サンプルから分離したKM-59株のリボソームRNA遺伝子の塩基配列（rDNA配列）による相同性検索を以下のようにして行った。ゲノムの抽出には、GenTLE (TaKaRa社製)を使用し、操作はTaKaRa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、リボゾームRNA遺伝子 (rDNA)に特異的なプライマーＡ（5’-gcatcgatgaagaacgcagc-3’:配列番号２）及びプライマーＢ（5’-tcctccgcttattgatatgc-3’:配列番号３）を用いて、PCRによりITS-1領域から5.8S 領域までの塩基配列を増幅した。その後、増幅された前記領域の塩基配列を決定した。決定した塩基配列を配列番号１に示す。得られたrDNA配列について、DNAデータベース(DDBJ)にアクセスし、FASTAプログラム (http://www.ddbj.nig.ac.jp/search/fasta-j.html）を用いて相同性検索を行ったところ、シュードザイマ・ヒュベイエンシス（Pseudozyma hubeiensis）の対応する前記領域のrDNA配列と100％一致した。また、DNAデータベース上より入手したシュードザイマ（Pseudozyma）属および代表的な酵母種のリボソームRNA遺伝子のITS-1領域から5.8S 領域までの塩基配列を多重整列後、NJ法により分子系統解析を行った結果、本菌株の分子系統樹の位置はシュードザイマ・ヒュベイエンシス（Pseudozyma hubeiensis）と一致した（図１）。従って、KM-59株は、は、シュードザイマ・ヒュベイエンシス（Pseudozyma hubeiensis）に分類された。KM-59株は、平成18（2006）年8月11日付で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（IPOD）（茨城県つくば市東１−１−３）に受託番号FERM P-20987として寄託されている。
Hereinafter, the present invention will be described more specifically with reference to examples. However, the present invention is not limited to these.
(Example 1) Isolation and identification of KM-59 strain The homology search based on the base sequence (rDNA sequence) of the ribosomal RNA gene of the KM-59 strain isolated from the plant (leaf) sample collected in Okinawa was as follows. I went. GenTLE (manufactured by TaKaRa) was used for genome extraction, and the operation followed the protocol of TaKaRa. Using the extracted genomic DNA as a template, primer A (5'-gcatcgatgaagaacgcagc-3 ': SEQ ID NO: 2) and primer B (5'-tcctccgcttattgatatgc-3': SEQ ID NO: 3) specific for the ribosomal RNA gene (rDNA) The base sequence from the ITS-1 region to the 5.8S region was amplified by PCR. Thereafter, the base sequence of the amplified region was determined. The determined base sequence is shown in SEQ ID NO: 1. The obtained rDNA sequence was accessed to the DNA database (DDBJ), and homology search was performed using the FASTA program (http://www.ddbj.nig.ac.jp/search/fasta-j.html) However, it was 100% identical with the corresponding rDNA sequence of Pseudozyma hubeiensis. In addition, we performed molecular phylogenetic analysis by NJ method after multiple alignment of ITS-1 region to 5.8S region of ribosomal RNA genes of genus Pseudozyma and representative yeast species obtained from DNA database As a result, the position of the molecular phylogenetic tree of this strain coincided with Pseudozyma hubeiensis (FIG. 1). Therefore, the KM-59 strain was classified as Pseudozyma hubeiensis. The KM-59 strain was registered with the Patent Organism Depositary (IPOD) (1-1-3 Higashi 1-1-3, Tsukuba City, Ibaraki Prefecture) on August 11, 2006. Has been deposited.
（実施例２）本発明微生物菌株によるマンノシルエリスリトールリピッド（MEL）の生産
(1) シュードザイマ・ヒュベイエンシス（Pseudozyma hubeiensis）KM-59株の培養
(a) 種培養
保存培地（麦芽エキス３ｇ/Ｌ、酵母エキス３ｇ/Ｌ、ペプトン５ｇ/Ｌ、グルコース１０ｇ/Ｌ、寒天３０ｇ/Ｌ）に保存しておいた上記のシュードザイマ・ヒュベイエンシス（Pseudozyma hubeiensis）KM-59株(以下、「KM-59株」という）を、グルコース２０ｇ/Ｌ、酵母エキス１ｇ/Ｌ、硝酸ナトリウム１ｇ/Ｌ、リン酸２水素カリウム０．５ｇ/Ｌ、及び硫酸マグネシウム０．５ｇ/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２６℃で振とう培養を１日間行った。
(b) 本培養
(a)で得られた菌体培養液を、８０ｇ/Ｌの大豆油と酵母エキス１ｇ/Ｌ、硝酸ナトリウム１ｇ/Ｌ、リン酸２水素カリウム０．５ｇ/Ｌ、及び硫酸マグネシウム０．５ｇ/Ｌの組成の液体培地２０ｍＬが入った坂口フラスコに接種し、振とう培養を２６℃にて７日間行った。
(Example 2) Production of mannosyl erythritol lipid (MEL) by the microbial strain of the present invention
(1) Cultivation of Pseudozyma hubeiensis KM-59 strain
(a) Seed culture Pseudozyma (Pseudozyma) above preserved in a preservation medium (malt extract 3 g / L, yeast extract 3 g / L, peptone 5 g / L, glucose 10 g / L, agar 30 g / L) hubeiensis) KM-59 strain (hereinafter referred to as “KM-59 strain”), glucose 20 g / L, yeast extract 1 g / L, sodium nitrate 1 g / L, potassium dihydrogen phosphate 0.5 g / L, and magnesium sulfate One platinum loop was inoculated into a test tube containing 4 mL of a liquid medium having a composition of 0.5 g / L and cultured at 26 ° C. for 1 day.
(b) Main culture
The bacterial cell culture solution obtained in (a) was prepared by adding 80 g / L soybean oil and yeast extract 1 g / L, sodium nitrate 1 g / L, potassium dihydrogen phosphate 0.5 g / L, and magnesium sulfate 0.5 g / L. A Sakaguchi flask containing 20 mL of a liquid medium having a composition of L was inoculated, and shaking culture was performed at 26 ° C. for 7 days.
(2) KM-59株のマンノシルエリスリトールリピッド（MEL）生産能の確認
上記(1)の培養終了後の培養液を採取し、これを用いてKM-59株のバイオサーファクタントの生産性を薄層クロマトグラフィー（TLC)で確認した（図２、レーンＡ）。マンノシルエリスリトールリピッドの標準として、Pseudozyma antarctica JCM 10317株を上記と同じ条件で培養し、原料油脂等の不純物を取り除いた精製標品を用いた（図２、レーンＳ）。なお、薄層クロマトグラフィーは、Silica gel 60F（Wako社製）を用い、展開溶媒はクロロホルム：メタノール：７N アンモニア＝６５:１５:２とした。検出は、展開後のプレートにアンスロン試薬を吹き付け、１１０℃で５分間加熱した。
(2) Confirmation of mannosyl erythritol lipid (MEL) production ability of KM-59 strain The culture solution after completion of the culture in (1) above was collected and used to reduce the biosurfactant productivity of the KM-59 strain. This was confirmed by chromatography (TLC) (FIG. 2, lane A). As a standard for mannosyl erythritol lipid,
なお、図２のマンノシルエリスリトールリピッド標準において、MEL-Aは前記式（II)中、Ｒ１とＲ２が炭素原子数１〜１６の脂肪酸残基、Ｒ３とＲ4がアセチル基、Ｒ５が水素原子である化合物、MEL-Bは同式中Ｒ１とＲ２が炭素原子数１〜１６の脂肪酸残基、Ｒ4がアセチル基、Ｒ３とＲ５が水素原子である化合物、MEL-CはＲ１とＲ２が炭素原子数１〜１６の脂肪酸残基、Ｒ３がアセチル基、Ｒ4とＲ５が水素原子である化合物である。 In the mannosyl erythritol lipid standard of FIG. 2, MEL-A is represented by the formula (II), wherein R 1 and R 2 are fatty acid residues having 1 to 16 carbon atoms, R 3 and R 4 are acetyl groups, R 5 MEL-B is a compound in which R 1 and R 2 are fatty acid residues having 1 to 16 carbon atoms, R 4 is an acetyl group, R 3 and R 5 are hydrogen atoms, MEL-B -C is a compound in which R 1 and R 2 are fatty acid residues having 1 to 16 carbon atoms, R 3 is an acetyl group, and R 4 and R 5 are hydrogen atoms.
図２に示すように、両株ともマンノシルエリスリトールリピッドを生産したが、マンノシルエリスリトールリピッドの標準（図２、レーンＳ）と対比して、KM-59株が主に生産しているバイオサーファクタントはマンノシルエリスリトールリピッドの中でもMEL-Cであることが確認できた（図２、レーンＡ）。 As shown in FIG. 2, both strains produced mannosyl erythritol lipids, but the biosurfactant mainly produced by the KM-59 strain was mannosyl in contrast to the mannosyl erythritol lipid standard (FIG. 2, lane S). Among the erythritol lipids, MEL-C was confirmed (FIG. 2, lane A).
（実施例３）本発明微生物菌株によるトリアシル型およびモノアシル型マンノシルエリスリトールリピッドの生産
実施例２と同様にして種培養を行ったKM-59株の培養液を、Yeast Nitrogen base ６．７ｇ/Ｌ、大豆油８０ｇ/Ｌの組成の液体培地（培地Ａ）、または８０ｇ/Ｌの大豆油と酵母エキス１ｇ/Ｌ、硝酸ナトリウム１ｇ/Ｌ、リン酸２水素カリウム０．５ｇ/Ｌ、及び硫酸マグネシウム０．５ｇ/Ｌ（培地Ｂ）を用い、実施例２と同様にして本培養を行った。
(Example 3) Production of triacyl-type and monoacyl-type mannosylerythritol lipids by the microbial strain of the present invention A culture solution of KM-59 strain that was seed-cultured in the same manner as in Example 2 was used in an amount of 6.7 g / L, Yeast Nitrogen base Liquid medium (medium A) with a composition of soybean oil 80 g / L, or 80 g / L soybean oil and yeast extract 1 g / L, sodium nitrate 1 g / L, potassium dihydrogen phosphate 0.5 g / L, and magnesium sulfate 0 Main culture was performed in the same manner as in Example 2 using 0.5 g / L (medium B).
培養終了後の培養液を採取し、これを用いてKM-59株のバイオサーファクタントの生産性を薄層クロマトグラフィー(TLC)で確認した[図３、レーンＡ（培地Ａによる培養液）、レーンＢ（培地Ｂによる培養液）]。マンノシルエリスリトールリピッドの標準として、Pseudozyma antarctica JCM 10317株を上記と同じ条件で培養し、原料油脂等の不純物を取り除いた精製標品（図３、レーンＳ）、トリアシル型およびモノアシル型マンノシルエリスリトールリピッドの精製標品（図３、レーンＳ１およびＳ２）を用いた。図３に示すように、培地Ａで培養した場合に、KM-59株はモノアシル型マンノシルエリスリトールリピッドを生産しており、培地Ｂで培養した場合、KM-59株はトリアシル型マンノシルエリスリトールリピッドを生産していることが確認できた。
The culture broth after completion of the culture was collected, and the biosurfactant productivity of the KM-59 strain was confirmed by thin layer chromatography (TLC) using the culture broth [FIG. 3, lane A (medium A culture broth), lane B (culture medium with medium B)]. As a standard for mannosyl erythritol lipid,
（実施例４）本発明微生物菌株により生産されるマンノシルエリスリトールリピッドの構造解析
(1) 精製標品の高速液体クロマトグラフィーによる分析
実施例２と同様にしてKM-59株の培養を行い、培養終了後の培養液を採取し、酢酸エチルで抽出し、酢酸エチル可溶分中のMELを高速液体クロマトグラフィー（HPLC）で検出した（図４、右のパネル）。また、比較例として既知のマンノシルエリスリトールリピッド生産酵母であるPseudozyma antarctica JCM 10317株を上記と同じ条件で培養し、同様にして高速液体クロマトグラフィー（HPLC)で検出した（図４、左のパネル）。高速液体クロマトグラフィー（HPLC)は、DP-8020（TOSOH社製）を用いた。カラムは、Inertsil SIL 100A 5μm, 4.6×250 mm（GL science 社製）を用い、クロロホルムとメタノールを 100:0 から 0:100 のグラジエントをかけて１ｍＬ/ｍｉｎの流速で流した。
(Example 4) Structural analysis of mannosylerythritol lipid produced by the microbial strain of the present invention
(1) Analysis of the purified sample by high-performance liquid chromatography The KM-59 strain is cultured in the same manner as in Example 2. The culture solution after completion of the culture is collected, extracted with ethyl acetate, and the ethyl acetate-soluble component is collected. MEL was detected by high performance liquid chromatography (HPLC) (FIG. 4, right panel). In addition,
図４に示すように、KM-59株のMEL生産パターンは、JCM 10317株のMEL生産パターンとは異なり、KM-59株のMEL生産パターンのメインピークは、JCM 10317株のMEL-Cと一致した。KM-59株は、高いMEL-C生産能を有し、一週間で約１３ｇ/ＬのMEL-Cを生産した。
As shown in Figure 4, the MEL production pattern of the KM-59 strain is different from the MEL production pattern of the
(2) 精製標品のNMRによる構造解析
上記の培養液の酢酸エチル可溶分から、主成分であるMEL-Cをシリカゲルカラムクロマトグラフィーによって精製分離した後、１H と 13C NMRによる構造解析を行った。結果を下記表１に示す。また、典型的なMELの構造解析結果の例としてPseudozyma antarctica JCM 10317株が生産するMELの分析結果も表１に示す。表１によれば、KM-59株が生産する物質は典型的なMEL-Cの構造を有することが確認できた。
(2) Structural analysis by NMR of the purified sample After the main component MEL-C was purified and separated by silica gel column chromatography from the ethyl acetate soluble content of the above culture broth, structural analysis by 1 H and 13 C NMR was performed. went. The results are shown in Table 1 below. Table 1 also shows the analysis results of MEL produced by the
（実施例５）マンノシルエリスリトールリピッドの大量生産（ジャー培養）
実施例２と同様にしてKM-59株の培養を行った後、得られた菌体培養液を、８０ｇ/Ｌの大豆油と酵母エキス１ｇ/Ｌ、硝酸ナトリウム１ｇ/Ｌ、リン酸２水素カリウム０．５ｇ/Ｌ、及び硫酸マグネシウム０．５ｇ/Ｌの組成の液体培地１．４Ｌが入ったジャーファメンターに接種して、２６℃で８００rpmの撹拌速度でさらに培養を７日間行った。この培養においては、培養途中から大豆油を培養容器中に流下させて、培地中の大豆油濃度を５０〜２００ｇ/Ｌに保持した。
(Example 5) Mass production of mannosyl erythritol lipid (jar culture)
After culturing the KM-59 strain in the same manner as in Example 2, the obtained cell culture broth was mixed with 80 g / L soybean oil and yeast extract 1 g / L, sodium nitrate 1 g / L, dihydrogen phosphate. A jar fermenter containing 1.4 L of a liquid medium having a composition of 0.5 g / L of potassium and 0.5 g / L of magnesium sulfate was inoculated, and further cultured at 26 ° C. and a stirring speed of 800 rpm for 7 days. In this culture, soybean oil was allowed to flow down into the culture vessel from the middle of the culture, and the soybean oil concentration in the medium was maintained at 50 to 200 g / L.
培養終了後、培養液を採取し、培養液中のMELを酢酸エチルで抽出し、その重量を測定した結果、MELの生産量は１４ｇ（１０ｇ/Ｌに相当する）であった。さらにMEL-C を精製分離した後、得られたMEL-Cの重量は２．８ｇであった。 After completion of the culture, the culture solution was collected, MEL in the culture solution was extracted with ethyl acetate, and the weight was measured. As a result, the production amount of MEL was 14 g (corresponding to 10 g / L). Further, after the MEL-C was purified and separated, the weight of the obtained MEL-C was 2.8 g.
（実施例６）マンノシルエリスリトールリピッドの液晶形成能
（1）MEL-C精製標品の液晶形成能
実施例４にて精製分離したMEL-Cのリオトロピック液晶形成能を進入法にて観察した。まず、スライドガラス上に上記MEL-Cを塗布してカバーガラスでシールし、カバーガラス中に水を進入させ、偏光顕微鏡（ニコン社製のECLIPSE E600）観察した（図５）。図５より明らかなように、非常に幅広い濃度領域において光学的異方相、すなわちリオトロピック液晶が観察された。
(Example 6) Liquid crystal forming ability of mannosyl erythritol lipid (1) Liquid crystal forming ability of purified MEL-C sample The lyotropic liquid crystal forming ability of MEL-C purified and separated in Example 4 was observed by an approach method. First, the above MEL-C was applied on a slide glass, sealed with a cover glass, water was allowed to enter the cover glass, and the polarizing microscope (Nikon Corporation ECLIPSE E600) was observed (FIG. 5). As apparent from FIG. 5, an optically anisotropic phase, that is, a lyotropic liquid crystal was observed in a very wide concentration range.
（2）MEL混合物の液晶形成能
未精製のMEL-A, -B, -C 混合物（MEL 混合物）のリオトロピック液晶形成能を上記と同様にして観察した（図６）。図６より明らかなように、MEL-Cを主成分とするMEL混合物は、MEL-Bを主成分とするMEL混合物に比べ、非常に幅広い濃度領域において光学的異方相、すなわちリオトロピック液晶が観察された。KM-59株が生産するMEL-A, -B, -C 混合物の主成分は図２に示されるようにMEL-Cであるが、その優れたリオトロピック液晶形成能は未精製の状態でも十分発揮できることがわかった。
(2) Liquid crystal forming ability of MEL mixture The lyotropic liquid crystal forming ability of an unpurified MEL-A, -B, -C mixture (MEL mixture) was observed in the same manner as described above (FIG. 6). As is clear from FIG. 6, the MEL mixture containing MEL-C as the main component shows an optically anisotropic phase, that is, a lyotropic liquid crystal in a much wider concentration range than the MEL mixture containing MEL-B as the main component. It was. The main component of the MEL-A, -B, -C mixture produced by the KM-59 strain is MEL-C as shown in Fig. 2, but its excellent lyotropic liquid crystal forming ability is fully demonstrated even in an unpurified state. I knew it was possible.
（実施例７）シュードザイマ・グラミニコーラ（Pseudozyma graminicola）によるマンノシルエリスリトールリピッド（MEL）の生産
(1) シュードザイマ・グラミニコーラ（Pseudozyma graminicola）CBS 10092株の培養
シュードザイマ・グラミニコーラ（Pseudozyma graminicola）CBS 10092株を、前記実施例２と同様にして種培養、本培養を行った。
(Example 7) Production of mannosyl erythritol lipid (MEL) by Pseudozyma graminicola
(1) Cultivation of
(2) CBS 10092株のマンノシルエリスリトールリピッド（MEL）生産能の確認
上記(1)の培養終了後の培養液を採取し、これを用いてCBS 10092株のバイオサーファクタントの生産性を、薄層クロマトグラフィー（TLC)で確認した（図７、レーンＡ）。マンノシルエリスリトールリピッドの標準として、Pseudozyma antarctica JCM 10317株を上記と同じ条件で培養し、原料油脂等の不純物を取り除いた精製標品を用いた（図７、レーンＳ）。なお、薄層クロマトグラフィー（TLC)は、実施例２の記載に従って実施した。
(2) Confirmation of mannosyl erythritol lipid (MEL) production ability of
図７に示すように、両株ともマンノシルエリスリトールリピッドを生産したが、マンノシルエリスリトールリピッドの標準（図７、レーンＳ）と対比して、CBS 10092株が主に生産しているバイオサーファクタントはマンノシルエリスリトールリピッドの中でもMEL-Cであることが確認できた（図７、レーンＡ）。
As shown in FIG. 7, both strains produced mannosyl erythritol lipids, but the biosurfactant mainly produced by the
（実施例８）シュードザイマ・グラミニコーラ（Pseudozyma graminicola）により生産されるマンノシルエリスリトールリピッドの構造解析
(1) 精製標品の高速液体クロマトグラフィーによる分析
実施例２と同様にしてシュードザイマ・グラミニコーラ（Pseudozyma graminicola）
CBS 10092株の培養を行い、培養終了後の培養液を採取し、酢酸エチルで抽出し、酢酸エチル可溶分中のMELを、高速液体クロマトグラフィー（HPLC）で検出した（図８、右のパネル）。また、比較例として既知のマンノシルエリスリトールリピッド生産酵母であるPseudozyma antarctica JCM 10317株を上記と同じ条件で培養し、同様にして高速液体クロマトグラフィー（HPLC)で検出した（図８、左のパネル）。なお、高速液体クロマトグラフィー（HPLC)は、実施例４の記載に従って実施した。
(Example 8) Structural analysis of mannosyl erythritol lipid produced by Pseudozyma graminicola
(1) Analysis of the purified sample by high performance liquid chromatography As in Example 2, Pseudozyma graminicola
図８に示すように、CBS 10092株のMEL生産パターンは、JCM 10317株のMEL生産パターンとは異なり、CBS 10092株のMEL生産パターンのメインピークは、JCM 10317株のMEL-Cと一致した。CBS 10092株は、高いMEL-C生産能を有し、一週間で約13g/LのMEL-Cを生産した。
As shown in FIG. 8, the MEL production pattern of the
(2) 精製標品のNMRによる構造解析
上記の培養液の酢酸エチル可溶分から、主成分であるMEL-Cをシリカゲルカラムクロマトグラフィーによって精製分離した後、１H と 13C NMRによる構造解析を行った。その分析結果は、前記表１と同じであり、シュードザイマ・グラミニコーラ（Pseudozyma graminicola）CBS 10092株もまた、生産する物質は典型的なMEL-Cの構造を有することが確認できた。
(2) Structural analysis by NMR of the purified sample After the main component MEL-C was purified and separated by silica gel column chromatography from the ethyl acetate soluble content of the above culture broth, structural analysis by 1 H and 13 C NMR was performed. went. The analysis results are the same as in Table 1 above, and it was confirmed that the
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