JP4803435B2 - Novel mannosyl erythritol lipid and method for producing the same - Google Patents
Novel mannosyl erythritol lipid and method for producing the same Download PDFInfo
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- JP4803435B2 JP4803435B2 JP2006095882A JP2006095882A JP4803435B2 JP 4803435 B2 JP4803435 B2 JP 4803435B2 JP 2006095882 A JP2006095882 A JP 2006095882A JP 2006095882 A JP2006095882 A JP 2006095882A JP 4803435 B2 JP4803435 B2 JP 4803435B2
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- -1 mannosyl erythritol lipid Chemical class 0.000 title claims description 34
- 238000004519 manufacturing process Methods 0.000 title claims description 20
- 125000002252 acyl group Chemical group 0.000 claims description 15
- 150000002632 lipids Chemical class 0.000 claims description 15
- 244000005700 microbiome Species 0.000 claims description 14
- 239000003921 oil Substances 0.000 claims description 14
- 239000003925 fat Substances 0.000 claims description 11
- 125000001424 substituent group Chemical group 0.000 claims description 10
- 239000001257 hydrogen Substances 0.000 claims description 9
- 229910052739 hydrogen Inorganic materials 0.000 claims description 9
- UFHFLCQGNIYNRP-UHFFFAOYSA-N Hydrogen Chemical compound [H][H] UFHFLCQGNIYNRP-UHFFFAOYSA-N 0.000 claims description 8
- 125000002777 acetyl group Chemical group [H]C([H])([H])C(*)=O 0.000 claims description 8
- 125000001931 aliphatic group Chemical group 0.000 claims description 8
- 125000004432 carbon atom Chemical group C* 0.000 claims description 8
- 241000893045 Pseudozyma Species 0.000 claims description 7
- 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 compound 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 claims description 5
- 229920006395 saturated elastomer Polymers 0.000 claims description 3
- 229930186217 Glycolipid Natural products 0.000 description 72
- 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 65
- CSCPPACGZOOCGX-UHFFFAOYSA-N Acetone Chemical compound CC(C)=O CSCPPACGZOOCGX-UHFFFAOYSA-N 0.000 description 30
- HEDRZPFGACZZDS-UHFFFAOYSA-N Chloroform Chemical compound ClC(Cl)Cl HEDRZPFGACZZDS-UHFFFAOYSA-N 0.000 description 30
- 235000014113 dietary fatty acids Nutrition 0.000 description 26
- 239000000194 fatty acid Substances 0.000 description 26
- 229930195729 fatty acid Natural products 0.000 description 26
- 150000004665 fatty acids Chemical group 0.000 description 25
- 239000000203 mixture Substances 0.000 description 22
- 238000004809 thin layer chromatography Methods 0.000 description 20
- XEKOWRVHYACXOJ-UHFFFAOYSA-N Ethyl acetate Chemical compound CCOC(C)=O XEKOWRVHYACXOJ-UHFFFAOYSA-N 0.000 description 18
- 238000004458 analytical method Methods 0.000 description 17
- 235000000346 sugar Nutrition 0.000 description 17
- 239000002609 medium Substances 0.000 description 16
- 239000003876 biosurfactant Substances 0.000 description 15
- CSNNHWWHGAXBCP-UHFFFAOYSA-L Magnesium sulfate Chemical compound [Mg+2].[O-][S+2]([O-])([O-])[O-] CSNNHWWHGAXBCP-UHFFFAOYSA-L 0.000 description 14
- 241001183303 Moesziomyces parantarcticus Species 0.000 description 14
- VWDWKYIASSYTQR-UHFFFAOYSA-N sodium nitrate Chemical compound [Na+].[O-][N+]([O-])=O VWDWKYIASSYTQR-UHFFFAOYSA-N 0.000 description 14
- 239000000126 substance Substances 0.000 description 14
- 238000012258 culturing Methods 0.000 description 13
- QTBSBXVTEAMEQO-UHFFFAOYSA-N Acetic acid Chemical compound CC(O)=O QTBSBXVTEAMEQO-UHFFFAOYSA-N 0.000 description 12
- OKKJLVBELUTLKV-UHFFFAOYSA-N Methanol Chemical compound OC OKKJLVBELUTLKV-UHFFFAOYSA-N 0.000 description 12
- UNXHWFMMPAWVPI-ZXZARUISSA-N erythritol Chemical group OC[C@H](O)[C@H](O)CO UNXHWFMMPAWVPI-ZXZARUISSA-N 0.000 description 12
- 235000019198 oils Nutrition 0.000 description 12
- UNXHWFMMPAWVPI-UHFFFAOYSA-N Erythritol Natural products OCC(O)C(O)CO UNXHWFMMPAWVPI-UHFFFAOYSA-N 0.000 description 10
- 150000001875 compounds Chemical class 0.000 description 10
- 239000007864 aqueous solution Substances 0.000 description 9
- 235000019197 fats Nutrition 0.000 description 9
- 239000004386 Erythritol Substances 0.000 description 8
- 229940041514 candida albicans extract Drugs 0.000 description 8
- 230000000694 effects Effects 0.000 description 8
- 229940009714 erythritol Drugs 0.000 description 8
- 235000019414 erythritol Nutrition 0.000 description 8
- 239000003549 soybean oil Substances 0.000 description 8
- 235000012424 soybean oil Nutrition 0.000 description 8
- 239000004094 surface-active agent Substances 0.000 description 8
- 239000012138 yeast extract Substances 0.000 description 8
- OKTJSMMVPCPJKN-UHFFFAOYSA-N Carbon Chemical compound [C] OKTJSMMVPCPJKN-UHFFFAOYSA-N 0.000 description 7
- WQZGKKKJIJFFOK-QTVWNMPRSA-N D-mannopyranose Chemical compound OC[C@H]1OC(O)[C@@H](O)[C@@H](O)[C@@H]1O WQZGKKKJIJFFOK-QTVWNMPRSA-N 0.000 description 7
- 229910052799 carbon Inorganic materials 0.000 description 7
- 230000002209 hydrophobic effect Effects 0.000 description 7
- 229910052943 magnesium sulfate Inorganic materials 0.000 description 7
- 235000019341 magnesium sulphate Nutrition 0.000 description 7
- 229910000402 monopotassium phosphate Inorganic materials 0.000 description 7
- 235000019796 monopotassium phosphate Nutrition 0.000 description 7
- PJNZPQUBCPKICU-UHFFFAOYSA-N phosphoric acid;potassium Chemical compound [K].OP(O)(O)=O PJNZPQUBCPKICU-UHFFFAOYSA-N 0.000 description 7
- 239000004317 sodium nitrate Substances 0.000 description 7
- 235000010344 sodium nitrate Nutrition 0.000 description 7
- 239000002904 solvent Substances 0.000 description 7
- 241000894006 Bacteria Species 0.000 description 6
- 240000004808 Saccharomyces cerevisiae Species 0.000 description 6
- 239000002024 ethyl acetate extract Substances 0.000 description 6
- 125000002887 hydroxy group Chemical group [H]O* 0.000 description 6
- 239000007788 liquid Substances 0.000 description 6
- VLKZOEOYAKHREP-UHFFFAOYSA-N n-Hexane Chemical compound CCCCCC VLKZOEOYAKHREP-UHFFFAOYSA-N 0.000 description 6
- 239000000243 solution Substances 0.000 description 6
- 238000001228 spectrum Methods 0.000 description 6
- 241000222120 Candida <Saccharomycetales> Species 0.000 description 5
- RJGDLRCDCYRQOQ-UHFFFAOYSA-N anthrone Chemical compound C1=CC=C2C(=O)C3=CC=CC=C3CC2=C1 RJGDLRCDCYRQOQ-UHFFFAOYSA-N 0.000 description 5
- 239000003153 chemical reaction reagent Substances 0.000 description 5
- 238000003929 heteronuclear multiple quantum coherence Methods 0.000 description 5
- 238000000425 proton nuclear magnetic resonance spectrum Methods 0.000 description 5
- 238000001542 size-exclusion chromatography Methods 0.000 description 5
- 235000015112 vegetable and seed oil Nutrition 0.000 description 5
- 241001661345 Moesziomyces antarcticus Species 0.000 description 4
- 210000004027 cell Anatomy 0.000 description 4
- 238000004440 column chromatography Methods 0.000 description 4
- 230000007613 environmental effect Effects 0.000 description 4
- 238000004128 high performance liquid chromatography Methods 0.000 description 4
- 230000001965 increasing effect Effects 0.000 description 4
- 238000000034 method Methods 0.000 description 4
- BASFCYQUMIYNBI-UHFFFAOYSA-N platinum Chemical compound [Pt] BASFCYQUMIYNBI-UHFFFAOYSA-N 0.000 description 4
- 238000002953 preparative HPLC Methods 0.000 description 4
- 238000000746 purification Methods 0.000 description 4
- 150000004670 unsaturated fatty acids Chemical group 0.000 description 4
- 239000008158 vegetable oil Substances 0.000 description 4
- LFQSCWFLJHTTHZ-UHFFFAOYSA-N Ethanol Chemical compound CCO LFQSCWFLJHTTHZ-UHFFFAOYSA-N 0.000 description 3
- WQZGKKKJIJFFOK-GASJEMHNSA-N Glucose Natural products OC[C@H]1OC(O)[C@H](O)[C@@H](O)[C@@H]1O WQZGKKKJIJFFOK-GASJEMHNSA-N 0.000 description 3
- 241001661347 Moesziomyces rugulosus Species 0.000 description 3
- 125000002915 carbonyl group Chemical group [*:2]C([*:1])=O 0.000 description 3
- 238000004113 cell culture Methods 0.000 description 3
- 230000008859 change Effects 0.000 description 3
- 230000000052 comparative effect Effects 0.000 description 3
- 238000002290 gas chromatography-mass spectrometry Methods 0.000 description 3
- 239000008103 glucose Substances 0.000 description 3
- 238000001052 heteronuclear multiple bond coherence spectrum Methods 0.000 description 3
- 125000000311 mannosyl group Chemical group C1([C@@H](O)[C@@H](O)[C@H](O)[C@H](O1)CO)* 0.000 description 3
- 238000002156 mixing Methods 0.000 description 3
- 238000002360 preparation method Methods 0.000 description 3
- 238000011218 seed culture Methods 0.000 description 3
- 238000012916 structural analysis Methods 0.000 description 3
- UFTFJSFQGQCHQW-UHFFFAOYSA-N triformin Chemical compound O=COCC(OC=O)COC=O UFTFJSFQGQCHQW-UHFFFAOYSA-N 0.000 description 3
- HDTRYLNUVZCQOY-UHFFFAOYSA-N α-D-glucopyranosyl-α-D-glucopyranoside Natural products OC1C(O)C(O)C(CO)OC1OC1C(O)C(O)C(O)C(CO)O1 HDTRYLNUVZCQOY-UHFFFAOYSA-N 0.000 description 2
- WRIDQFICGBMAFQ-UHFFFAOYSA-N (E)-8-Octadecenoic acid Natural products CCCCCCCCCC=CCCCCCCC(O)=O WRIDQFICGBMAFQ-UHFFFAOYSA-N 0.000 description 2
- LQJBNNIYVWPHFW-UHFFFAOYSA-N 20:1omega9c fatty acid Natural products CCCCCCCCCCC=CCCCCCCCC(O)=O LQJBNNIYVWPHFW-UHFFFAOYSA-N 0.000 description 2
- QSBYPNXLFMSGKH-UHFFFAOYSA-N 9-Heptadecensaeure Natural products CCCCCCCC=CCCCCCCCC(O)=O QSBYPNXLFMSGKH-UHFFFAOYSA-N 0.000 description 2
- VHUUQVKOLVNVRT-UHFFFAOYSA-N Ammonium hydroxide Chemical compound [NH4+].[OH-] VHUUQVKOLVNVRT-UHFFFAOYSA-N 0.000 description 2
- IJGRMHOSHXDMSA-UHFFFAOYSA-N Atomic nitrogen Chemical compound N#N IJGRMHOSHXDMSA-UHFFFAOYSA-N 0.000 description 2
- UNXHWFMMPAWVPI-QWWZWVQMSA-N D-Threitol Natural products OC[C@@H](O)[C@H](O)CO UNXHWFMMPAWVPI-QWWZWVQMSA-N 0.000 description 2
- IAZDPXIOMUYVGZ-UHFFFAOYSA-N Dimethylsulphoxide Chemical compound CS(C)=O IAZDPXIOMUYVGZ-UHFFFAOYSA-N 0.000 description 2
- 238000005481 NMR spectroscopy Methods 0.000 description 2
- 241000187654 Nocardia Species 0.000 description 2
- 239000005642 Oleic acid Substances 0.000 description 2
- ZQPPMHVWECSIRJ-UHFFFAOYSA-N Oleic acid Natural products CCCCCCCCC=CCCCCCCCC(O)=O ZQPPMHVWECSIRJ-UHFFFAOYSA-N 0.000 description 2
- 241000589516 Pseudomonas Species 0.000 description 2
- 241000589517 Pseudomonas aeruginosa Species 0.000 description 2
- VYPSYNLAJGMNEJ-UHFFFAOYSA-N Silicium dioxide Chemical compound O=[Si]=O VYPSYNLAJGMNEJ-UHFFFAOYSA-N 0.000 description 2
- WQDUMFSSJAZKTM-UHFFFAOYSA-N Sodium methoxide Chemical compound [Na+].[O-]C WQDUMFSSJAZKTM-UHFFFAOYSA-N 0.000 description 2
- QAOWNCQODCNURD-UHFFFAOYSA-L Sulfate Chemical compound [O-]S([O-])(=O)=O QAOWNCQODCNURD-UHFFFAOYSA-L 0.000 description 2
- HDTRYLNUVZCQOY-WSWWMNSNSA-N Trehalose 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 HDTRYLNUVZCQOY-WSWWMNSNSA-N 0.000 description 2
- QGZKDVFQNNGYKY-UHFFFAOYSA-N ammonia Natural products N QGZKDVFQNNGYKY-UHFFFAOYSA-N 0.000 description 2
- 238000006243 chemical reaction Methods 0.000 description 2
- 239000002537 cosmetic Substances 0.000 description 2
- 238000011161 development Methods 0.000 description 2
- 238000004945 emulsification Methods 0.000 description 2
- 238000005516 engineering process Methods 0.000 description 2
- 238000000605 extraction Methods 0.000 description 2
- 235000019387 fatty acid methyl ester Nutrition 0.000 description 2
- 235000013305 food Nutrition 0.000 description 2
- 238000003919 heteronuclear multiple bond coherence Methods 0.000 description 2
- QXJSBBXBKPUZAA-UHFFFAOYSA-N isooleic acid Natural products CCCCCCCC=CCCCCCCCCC(O)=O QXJSBBXBKPUZAA-UHFFFAOYSA-N 0.000 description 2
- 150000004668 long chain fatty acids Chemical class 0.000 description 2
- 231100000053 low toxicity Toxicity 0.000 description 2
- 238000001840 matrix-assisted laser desorption--ionisation time-of-flight mass spectrometry Methods 0.000 description 2
- 239000011259 mixed solution Substances 0.000 description 2
- 238000000655 nuclear magnetic resonance spectrum Methods 0.000 description 2
- ZQPPMHVWECSIRJ-KTKRTIGZSA-N oleic acid Chemical compound CCCCCCCC\C=C/CCCCCCCC(O)=O ZQPPMHVWECSIRJ-KTKRTIGZSA-N 0.000 description 2
- 229910052697 platinum Inorganic materials 0.000 description 2
- 239000013587 production medium Substances 0.000 description 2
- 239000002994 raw material Substances 0.000 description 2
- 150000004671 saturated fatty acids Chemical class 0.000 description 2
- 235000003441 saturated fatty acids Nutrition 0.000 description 2
- 239000000741 silica gel Substances 0.000 description 2
- 229910002027 silica gel Inorganic materials 0.000 description 2
- 238000010898 silica gel chromatography Methods 0.000 description 2
- 238000005063 solubilization Methods 0.000 description 2
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- 238000003756 stirring Methods 0.000 description 2
- 238000012360 testing method Methods 0.000 description 2
- 235000021122 unsaturated fatty acids Nutrition 0.000 description 2
- 235000019871 vegetable fat Nutrition 0.000 description 2
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 description 2
- IIZPXYDJLKNOIY-JXPKJXOSSA-N 1-palmitoyl-2-arachidonoyl-sn-glycero-3-phosphocholine Chemical compound CCCCCCCCCCCCCCCC(=O)OC[C@H](COP([O-])(=O)OCC[N+](C)(C)C)OC(=O)CCC\C=C/C\C=C/C\C=C/C\C=C/CCCCC IIZPXYDJLKNOIY-JXPKJXOSSA-N 0.000 description 1
- 238000001644 13C nuclear magnetic resonance spectroscopy Methods 0.000 description 1
- 238000005160 1H NMR spectroscopy Methods 0.000 description 1
- JAHNSTQSQJOJLO-UHFFFAOYSA-N 2-(3-fluorophenyl)-1h-imidazole Chemical compound FC1=CC=CC(C=2NC=CN=2)=C1 JAHNSTQSQJOJLO-UHFFFAOYSA-N 0.000 description 1
- 238000005084 2D-nuclear magnetic resonance Methods 0.000 description 1
- 241000186216 Corynebacterium Species 0.000 description 1
- IAZDPXIOMUYVGZ-WFGJKAKNSA-N Dimethyl sulfoxide Chemical compound [2H]C([2H])([2H])S(=O)C([2H])([2H])[2H] IAZDPXIOMUYVGZ-WFGJKAKNSA-N 0.000 description 1
- 235000010654 Melissa officinalis Nutrition 0.000 description 1
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- 241000316848 Rhodococcus <scale insect> Species 0.000 description 1
- 241000187561 Rhodococcus erythropolis Species 0.000 description 1
- 235000019485 Safflower oil Nutrition 0.000 description 1
- 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 1
- HEDRZPFGACZZDS-MICDWDOJSA-N Trichloro(2H)methane Chemical class [2H]C(Cl)(Cl)Cl HEDRZPFGACZZDS-MICDWDOJSA-N 0.000 description 1
- 241000204066 Tsukamurella Species 0.000 description 1
- 241000221566 Ustilago Species 0.000 description 1
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- 239000013543 active substance Substances 0.000 description 1
- 239000003513 alkali Substances 0.000 description 1
- 239000003242 anti bacterial agent Substances 0.000 description 1
- HIWPGCMGAMJNRG-UHFFFAOYSA-N beta-sophorose Natural products OC1C(O)C(CO)OC(O)C1OC1C(O)C(O)C(O)C(CO)O1 HIWPGCMGAMJNRG-UHFFFAOYSA-N 0.000 description 1
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- 210000002421 cell wall Anatomy 0.000 description 1
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- FUKUFMFMCZIRNT-UHFFFAOYSA-N hydron;methanol;chloride Chemical compound Cl.OC FUKUFMFMCZIRNT-UHFFFAOYSA-N 0.000 description 1
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- 238000002955 isolation Methods 0.000 description 1
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- 235000010445 lecithin Nutrition 0.000 description 1
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Landscapes
- Preparation Of Compounds By Using Micro-Organisms (AREA)
- Saccharide Compounds (AREA)
Description
本発明は、マンノースとエリスリトールを構成要素とするマンノシルエリスリトールリピッドにおいて、エリスリトール部分に脂肪酸が結合した新規マンノシルエリスリトールリピッド及びその製造方法に関するものである。
The present invention relates to a novel mannosyl erythritol lipid in which a fatty acid is bonded to an erythritol moiety in a mannosyl erythritol lipid having mannose and erythritol as constituents, and a method for producing the same.
糖脂質は、脂質に１〜１０数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・免疫系の機能維持にも重要な役割を果たしていること等が明らかにされつつある。一方で、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）を利用してきたが、石油化学工業の発展により合成界面活性剤が開発され、界面活性剤の生産量は飛躍的に増加し、日常生活には無くてはならない物質となった。しかしながら、その使用量の拡大につれて環境汚染が広がってきた。そこで、安全性が高く、環境に対する負荷を低減するために生分解性の高い界面活性物質の開発が望まれていた。 Glycolipids are substances in which 1 to 10 monosaccharides are bound to lipids, are involved in the transmission of information between cells in vivo, and play an important role in maintaining the functions of the nervous system and immune system. Etc. are being revealed. On the other hand, glycolipids are amphipathic substances that have both hydrophilic properties derived from the properties of sugars and lipophilic properties derived from the properties of lipids. being called. Until the petrochemical industry prospered, surfactants derived from biological components such as lecithin and saponin (biosurfactants) have been used, but synthetic surfactants have been developed due to the development of the petrochemical industry. Production has increased dramatically, making it an indispensable substance in daily life. However, environmental pollution has spread as the amount of use has increased. Therefore, it has been desired to develop a highly biodegradable surfactant in order to reduce safety and the burden on the environment.
現在、微生物が生産する界面活性物質としては、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化合物系の５つに分類されている。この内の糖脂質系の界面活性剤については、最もよく研究され、細菌及び酵母による多くの種類の物質が報告されている。 Currently, surfactants produced by microorganisms are classified into five groups: glycolipids, acylpeptides, phospholipids, fatty acids, and polymer compounds. Of these, the glycolipid-based surfactants have been studied the most, and many types of substances from bacteria and yeast have been reported.
ラムノリピッドは、結核菌の抗生物質としてＰｓｅｕｄｏｍｏｎａｓ ａｅｒｕｇｉｎｏｓａ（シュードモナス アエルジノーサ）（緑膿菌）の培養液から最初に発見された（非特許文献１参照）。以来、これまでにＰｓｅｕｄｏｍｏｎａｓ（シュードモナス）属の細菌から４種類の同族体が報告され、当初は数ｇ／Ｌ程度の生産量であったが、現在では１００ｇ／Ｌ以上の生産を可能にしている（非特許文献２参照）。 Rhamnolipid was first discovered from a culture solution of Pseudomonas aeruginosa (Pseudomonas aeruginosa) as an antibiotic of Mycobacterium tuberculosis (see Non-Patent Document 1). Since then, four homologs have been reported from bacteria of the genus Pseudomonas (Pseudomonas), and the initial production was about several g / L, but now it is possible to produce over 100 g / L. (Refer nonpatent literature 2).
ソホロースリピッドは、Ｃａｎｄｉｄａ（以前はＴｏｒｕｌｏｐｓｉｓ） ｂｏｍｂｉｃｏｌａ（キャンデダ ボンビコーラ）の培養液から発見された（非特許文献３参照）。これ以外に、Ｃａｎｄｉｄａ ａｐｉｃｏｌａも生産し、脂肪酸の結合様式によりラクトン型と線状型の２種類が報告されている。現在では、３００ｇ／Ｌ以上の生産を可能にしている（非特許文献４及び５参照）。 Sophorose lipid was discovered from the culture solution of Candida (formerly Torulopsis) bombicola (Candida Bonbicola) (see Non-Patent Document 3). In addition to this, Candida apicola is also produced, and two types, lactone type and linear type, have been reported depending on the fatty acid binding mode. At present, production of 300 g / L or more is enabled (see Non-Patent Documents 4 and 5).
トレハロースリピッドは、Ｃｏｒｙｎｅｂａｃｔｅｒｉｕｍ（コリネバクテリウム）等の細胞表層物質として発見された。類似の物質が、Ｍｙｃｏｂａｃｔｅｒｉｕｍ（マイコバクテリウム）、Ｎｏｃａｒｄｉａ（ノカルディア）、Ｒｏｄｏｃｏｃｃｕｓ（ロドコッカス）属細菌からも報告されている（非特許文献６参照）。一般に、細胞壁に結合しているために生産量は低いが、Ｒｏｄｏｃｏｃｃｕｓ ｅｒｙｔｈｒｏｐｏｌｉｓ（ロドコッカス エリスロポリス）を窒素制限下で培養を行うとサクシノイルトレハロースリピッドを３２ｇ／Ｌ生産することが報告されている（非特許文献７参照）。 Trehalose lipid has been discovered as a cell surface material such as Corynebacterium. Similar substances have also been reported from bacteria belonging to the genus Mycobacterium (Nocodia), Nocardia (Nocardia), and Rhodococcus (see Non-Patent Document 6). In general, production is low due to binding to the cell wall, but it has been reported that when Rhodococcus erythropolis is cultured under nitrogen restriction, 32 g / L of succinoyl trehalose lipid is produced (non-nose). (See Patent Document 7).
マンノシルエリスリトールリピッドは、Ｕｓｔｉｌａｇｏ ｎｕｄａ（ウスチラゴ ヌーダ）とＳｈｉｚｏｎｅｌｌａ ｍｅｌａｎｏｇｒａｍｍａ（シゾネラ メラノグラマ）から発見された（非特許文献８及び９参照）。その後、イタコン酸生産の変異株であるＣａｎｄｉｄａ属酵母（特許文献１及び非特許文献１０参照）、Ｃａｎｄｉｄａ ａｎｔａｒｃｔｉｃａ（キャンデダ アンダークチカ）（非特許文献１１及び１２参照）、Ｋｕｒｔｚｍａｎｏｍｙｃｅｓ（クルツマノマイセス）属（非特許文献１３参照）等の酵母が生産することを報告している。現在では、長時間の連続培養・生産を行うことで３００ｇ／Ｌ以上の生産を可能にしている。 Mannosyl erythritol lipid was discovered from Ustilago nuda (Ustyago Nuda) and Shizonella melanogramma (see Non-Patent Documents 8 and 9). Subsequently, Candida genus yeast (see Patent Document 1 and Non-patent Document 10), Candida antarctica (see Candida Andalucica) (see Non-Patent Documents 11 and 12), and Kurtzmanomices (Kurzmanomyces) genus (mutants of itaconic acid production) It is reported that yeast such as Non-Patent Document 13) produces. At present, it is possible to produce 300 g / L or more by performing continuous culture and production for a long time.
セロビオースリピッドは、Ｕｓｔｉｌａｇｏ ｍａｙｄｉｓ（ウスチラゴ マイディス）により１５ｇ／Ｌ（非特許文献１４及び１５参照）、オリゴ糖リピッドは、Ｔｓｕｋａｍｕｒｅｌｌａ（ツカムレラ）属の酵母により３０ｇ／Ｌ生産される（非特許文献１６参照）ことが報告されている。 Cellobiose lipid is produced at 15 g / L (see Non-Patent Documents 14 and 15) by Ustilago maydis (Ustilago Mydis), and oligosaccharide lipid is produced at 30 g / L by yeast belonging to the genus Tsukamurella (see Non-Patent Document 16). It has been reported.
糖脂質等のバイオサーファクタントは、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、食品工業、化粧品工業、医薬品工業、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適用することは、持続可能社会の実現と高機能製品の提供という、両面を兼ね備えており極めて有意義である。 Biosurfactants such as glycolipids are said to have high biodegradability, low toxicity, environmental friendliness, and novel physiological functions. For this reason, the wide application of these biosurfactants in the food industry, cosmetic industry, pharmaceutical industry, chemical industry, environmental field, etc. has both the realization of a sustainable society and the provision of high-performance products. Meaningful.
現在、一般的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見ると数百、数千種類に上る。親水基・親油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、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、ありと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、数種混合して用いることで、上記のような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。 At present, there are hundreds or thousands of types of synthetic surfactants that are generally used, even if there are small differences in structure. Different types of hydrophilic groups and lipophilic groups have been developed, such as 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, These are used not only in a single type, but also in a mixture of several types, thereby meeting the performance required in a wide range of industrial fields as described above.
したがって、これらのバイオサーファクタントの幅広い普及を図るためには、多くの種類のバイオサーファクタントが必要である。しかしながら、現在までに発見されているバイオサーファクタントの種類は２０数種類と少なく、新規な構造を有するバイオサーファクタントの発見が望まれている。さらに、構造・組成に多くのバリエーションを持たせ、これらを組み合わせて機能を緻密に制御することが極めて重要である。 Therefore, many types of biosurfactants are required to widely spread these biosurfactants. However, there are as few as 20 types of biosurfactants discovered so far, and the discovery of biosurfactants having a novel structure is desired. Furthermore, it is extremely important to give many variations to the structure and composition, and to combine these to precisely control the function.
上記事情に鑑み,本発明は、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糖脂質等のバイオサーファクタントを、食品工業、化粧品工業、医薬品工業、化学工業、環境分野等に広く普及をはかるため、従来のマンノシルエリスリトールリピッドとは親油基の結合位置、結合数が異なる、新規構造のマンノシルエリスリトールリピッド及びその製造方法を提供することを目的とするものである。
界面活性剤の高機能化の手段として、一般的にはHLBの調節や他成分との混合等を施すことで乳化・可溶化、分散化、洗浄能の向上が図られている。糖脂質型バイオサーファクタントの場合では、親油基である脂肪酸由来ドメインの結合数や結合位置、鎖長等の違いによって界面活性の幅広い制御が可能となる。例えば、脂質ドメインの結合数、鎖長の増加は油溶性の増大に繋がり、乳化・可溶化技術の精密化をもたらす。また、結合位置の違いは自己集合能を大きく変化させ、それに応じた相挙動の変化に伴って液晶化技術など従来の界面制御技術をより高度化することも可能となる。
これらの構造改変は化学的な手法を用いても理論上では可能であるが、糖脂質の場合では反応を位置・立体選択的に制御することが極めて困難であり、また糖骨格を維持したまま構造改変を行うためには多段階の複雑な保護・脱保護反応を必要とする。これに対して、微生物生産では精巧な生合成経路を経由するため、位置・立体構造が完全に制御された特殊構造を維持し、かつHLB等の性質の異なる同族体を、一段階のステップのみで製造する方法を提供でき、バイオサーファクタントのバリエーションの拡張に繋がる極めて有効な手段と成り得る。
In view of the above circumstances, the present invention provides biosurfactants such as glycolipids having high degradability, low toxicity, environmental friendliness, and novel physiological functions in the food industry, cosmetic industry, pharmaceutical industry, chemical industry, environmental field, etc. Therefore, it is an object of the present invention to provide a mannosyl erythritol lipid having a novel structure in which the lipophilic group has a different bonding position and number of bonds from the conventional mannosyl erythritol lipid and a method for producing the same.
In general, as a means for enhancing the functionality of a surfactant, emulsification / solubilization, dispersion, and improvement of cleaning ability are achieved by adjusting HLB, mixing with other components, and the like. In the case of a glycolipid type biosurfactant, a wide range of surface activity can be controlled depending on the number of bonds, position, chain length, etc. of fatty acid-derived domains that are lipophilic groups. For example, the increase in the number of lipid domains bonded and the chain length lead to an increase in oil solubility, resulting in refinement of emulsification / solubilization techniques. In addition, the difference in the bonding position greatly changes the self-assembly ability, and it is possible to further enhance the conventional interface control technology such as liquid crystal technology in accordance with the change in the phase behavior accordingly.
These structural modifications are theoretically possible even if chemical methods are used, but in the case of glycolipids, it is extremely difficult to control the reaction in a position- and stereoselective manner, and the sugar skeleton is maintained. In order to modify the structure, a multi-step complex protection / deprotection reaction is required. In contrast, microbial production goes through an elaborate biosynthetic pathway, so it maintains a special structure with completely controlled position and conformation, and homologues with different properties, such as HLB, only in one step. And can be a very effective means leading to the expansion of biosurfactant variations.
本発明者は、上記課題を解決するため鋭意検討した結果、マンノシルエリスリトールリピッドを生産する微生物の培養物から様々な生産物を採取し、これらについて精密な分離・構造解析を行うことにより、これまで全く注目されていない、新規構造の糖脂質が存在することを知見した。さらに培養方法等を含む上記新規糖脂質の生産方法についても検討することにより、本発明をなすに至ったものである。 As a result of intensive studies to solve the above problems, the present inventors have collected various products from a culture of microorganisms that produce mannosylerythritol lipids, and have conducted precise separation and structural analysis on these products so far. It has been found that there is a glycolipid having a novel structure that has not attracted much attention. Furthermore, the present invention has been made by studying the production method of the above-mentioned novel glycolipid including a culture method and the like.
従って、本発明は下記発明を提供する。
〔１〕次の式（１）で表されるマンノシルエリスリトールリピッド。
〔２〕上記式中、Ｒ１で示される各置換基、及びＲ３で示される置換基が、 それぞれ炭素数６〜２０の飽和又は不飽和脂肪族アシル基からなることを特徴とする、上記〔１〕に記載のマンノシルエリスリトールリピッド。
〔３〕シュードザイマ属に属し、マンノシルエリスリトールリピッドを生産する能力を有する微生物を培養し、培養物から上記〔１〕又は〔２〕に記載の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。
〔４〕生産培地に油脂類を加えることを特徴とする上記〔３〕に記載の糖脂質の製造方法。
Accordingly, the present invention provides the following inventions.
[1] Mannosyl erythritol lipid represented by the following formula (1).
[2] In the above formula, each substituent represented by R 1 and each substituent represented by R 3 are each composed of a saturated or unsaturated aliphatic acyl group having 6 to 20 carbon atoms, The mannosyl erythritol lipid according to [1].
[3] A mannosyl erythritol characterized by culturing a microorganism belonging to the genus Pseudozyma and capable of producing mannosyl erythritol lipid, and collecting the mannosyl erythritol lipid according to [1] or [2] from the culture. Manufacturing method of lipid.
[4] The method for producing a glycolipid as described in [3] above, wherein fats and oils are added to the production medium.
本発明によれば、マンノシルエリスリトールリピッドを生産する能力を有する微生物の培養物から、新規構造のマンノシルエリスリトールリピッド（以下、ＭＥＬという場合がある。）を得ることができ、この糖脂質は従来のマンノシルエリスリトールリピッドにはみられない性質を有し、バイオサーファクタントとして有用である。一方、この新規ＭＥＬを生産する微生物としては、シュードザイマ属に属する微生物が挙げられるが、その中でも、特にシュードザイマ・パラアンタクティカ（Pseudozyma parantarctica JCM 11752 株）を使用した場合に、この新規ＭＥＬをきわめて効率よく生産できる。
According to the present invention, a mannosyl erythritol lipid having a novel structure (hereinafter sometimes referred to as MEL) can be obtained from a culture of a microorganism having an ability to produce mannosyl erythritol lipid, and this glycolipid is a conventional mannosyl. It has properties not found in erythritol lipid and is useful as a biosurfactant. On the other hand, microorganisms belonging to the genus Pseudozyma can be cited as microorganisms that produce this new MEL. Among them, especially when Pseudozyma parantarctica JCM 11752 is used, this new MEL is extremely different. Can be produced efficiently.
以下、本発明につきさらに詳しく説明する。
〈糖脂質〉
ＭＥＬはＭＥＬ生産菌の培養によって得られ、従来型ＭＥＬの化学構造の代表例は以下の式（２）に示され、４−Ｏ−β−Ｄ−マンノピラノシル−ｍｅｓｏ−エリスリトールをその基本構造とするものである。
本発明の新規ＭＥＬもこれらの点では従来型MELと同様ではあるが、本発明の新規ＭＥＬは、以下の式（１）で表され、エリスリトール部分にも脂肪酸残基が結合している点で、従来型ＭＥＬと区別される。
なお、上記式中の各置換基Ｒ１及びＲ３は、直鎖飽和脂肪族アシル基であっても同不飽和脂肪族アシル基であってもよく、また、これらが同一分子内に共存するものを含んでもよい。
Hereinafter, the present invention will be described in more detail.
<Glycolipid>
MEL is obtained by culturing MEL-producing bacteria. A typical example of the chemical structure of conventional MEL is shown in the following formula (2), and 4-O-β-D-mannopyranosyl-meso-erythritol is used as its basic structure. Is.
The novel MEL of the present invention is similar to the conventional MEL in these respects. However, the novel MEL of the present invention is represented by the following formula (1), and a fatty acid residue is also bound to the erythritol moiety. To distinguish from conventional MEL.
Each substituent R 1 and R 3 in the above formula may be a linear saturated aliphatic acyl group or the same unsaturated aliphatic acyl group, and these coexist in the same molecule. Things may be included.
本発明の新規ＭＥＬは、界面活性能を有する新規構造を有するバイオサーファクタントであり、たとえば、シュードザイマ属に属する酵母を油脂類添加培地で培養することによって得ることができる。
本発明の新規ＭＥＬは、基本的には、上記式（１）におけるこれら置換基Ｒの脂肪族アシル基の炭素数あるいは二重結合の有無等において異なる各化合物の混合物の形態で得られるが、油脂類として例えば、単一の脂肪酸 あるいは単一の脂肪酸を有するトリグリセライドを培地に含有させ、培養条件を厳密に制御すれば、単一化合物に近いＭＥＬ（例えば純度７５〜８０％）も得ることが可能である。これらはさらに分取ＨＰＬＣ等により精製すれば、単一のＭＥＬ化合物とすることもできる。
The novel MEL of the present invention is a biosurfactant having a novel structure having a surface activity, and can be obtained, for example, by culturing yeast belonging to the genus Pseudozyma in an oil and fat-added medium.
The novel MEL of the present invention is basically obtained in the form of a mixture of compounds different in the carbon number of the aliphatic acyl group of the substituent R in the above formula (1) or the presence or absence of a double bond, etc. As fats and oils, for example, a single fatty acid or triglyceride having a single fatty acid is contained in the medium, and if the culture conditions are strictly controlled, MEL close to a single compound (for example, 75 to 80% purity) can also be obtained. Is possible. If these are further purified by preparative HPLC or the like, they can be converted into single MEL compounds.
〈使用微生物〉
本発明の使用微生物については、シュードザイマ属に属し、マンノシルエリスリトールリピッドを生産する能力を有するものであれば特に制限はなく、例えばシュードザイマ・パラアンタクティカ、シュードザイマ・アンタクティカ、シュードザイマ・ルグローサ等に属する微生物が挙げられ、このうち特に好ましい微生物としては、シュードザイマ・パラアンタクティカに属する微生物が挙げることができる。このシュードザイマ・パラアンタクティカ（Pseudozyma parantarctica）に属する微生物は、例えば３３〜３７℃で培養した場合の生産性向上効果が高く、特にシュードザイマ・パラアンタクティカ（Pseudozyma parantarctica）JCM 11752株の場合、培養温度３５℃の場合に最も良好な生産性が得られる。
〈培地・培養条件〉
本発明における使用微生物の培養においては、培地に、脂肪酸、脂肪酸トリグリセリド等の脂肪酸エステル類、あるいは植物油等の油脂類を含有させるが、その他の条件については、特に制限はなく、適宜選定することができる。例えば、酵母に対して一般に用いられる培地を使用でき、このような培地として、例えば、ＹＰＤ培地（イーストイクストラクト１０ｇ、 ポリペプトン２０ｇ、及びグルコース１００ｇ）を挙げることができる。特に、シュードザイマ・パラアンタクティカ（Pseudozyma parantarctica）JCM 11752株を用いる場合は培養温度を３３〜３７℃に設定することが好ましいという知見を得ている。
本発明の使用微生物、特に前記シュードザイマ・パラアンタクティカ（Pseudozyma parantarctica JCM 11752）株を用いてマンノシルエリスリトールリピッドを生産する場合の好適な培地組成は、以下のとおりである。
酵母エキス；０．１〜２ｇ／Ｌが好ましく、１ｇ／Ｌが特に好ましい
硝酸ナトリウム；０．１〜１ｇ／Ｌが好ましく、０．５ｇ／Ｌが特に好ましい。
リン酸２水素カリウム；、０．１〜２ｇ／Ｌが好ましく、０．４ｇ／Ｌが特に好ましい。
硫酸マグネシウム；、０．１〜１ｇ／Ｌが好ましく、０．２ｇ／Ｌが特に好ましい。
油脂類；８０ｇ／Ｌ以上が好ましく、１８０ｇ／Ｌが特に好ましい。
<Used microorganism>
The microorganism used in the present invention is not particularly limited as long as it belongs to the genus Pseudozyma and has the ability to produce mannosyl erythritol lipids. Among these, particularly preferred microorganisms include microorganisms belonging to Pseudozyma paraantactica. The microorganism belonging to Pseudozyma parantarctica has a high productivity improvement effect when cultured at, for example, 33 to 37 ° C., and particularly in the case of Pseudozyma parantarctica JCM 11752, The best productivity is obtained when the culture temperature is 35 ° C.
<Medium and culture conditions>
In culturing the microorganisms used in the present invention, the medium contains fatty acid esters such as fatty acids and fatty acid triglycerides, or fats and oils such as vegetable oils, but other conditions are not particularly limited and may be appropriately selected. it can. For example, a medium generally used for yeast can be used, and examples of such a medium include YPD medium (yeast extract 10 g, polypeptone 20 g, and glucose 100 g). In particular, when using Pseudozyma parantarctica JCM 11752 strain, it has been found that the culture temperature is preferably set to 33 to 37 ° C.
Microorganisms use of the present invention, the preferred medium composition of especially when producing mannosylerythritol lipid using the Pseudozyma para Anta click Tikka (Pseudozyma parantarctica JCM 11752) strain is as follows.
Yeast extract; preferably 0.1 to 2 g / L, particularly preferably 1 g / L, sodium nitrate; preferably 0.1 to 1 g / L, particularly preferably 0.5 g / L.
Potassium dihydrogen phosphate; 0.1 to 2 g / L is preferable, and 0.4 g / L is particularly preferable.
Magnesium sulfate; 0.1 to 1 g / L is preferable, and 0.2 g / L is particularly preferable.
Fats and oils: 80 g / L or more is preferable, and 180 g / L is particularly preferable.
本発明の新規ＭＥＬの製造方法は、特に制限はなく、目的に応じて適宜選定することができるが、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望ましい。
これらの培養における、培地、培養条件を例示すると以下のとおりである。
ａ）種培養；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、３０℃で１日間振とう培養を行う。
ｂ）本培養；所定量の植物性油脂等の油脂類と、酵母エキス１ｇ／Ｌ、硝酸ナトリウム１ ｇ／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１００ｍＬの入った坂口フラスコに接種して、３３〜３７℃で２日間培養を行う。
ｃ）マンノシルエリスリトールリピッド生産培養；所定量の植物性油脂等の油脂類と酵母エキス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、３３〜３７℃で８００ｒｐｍの撹拌速度で培養を行う。この培養においては、培養途中から植物性油脂を培養容器中に流下させて、培地中の油脂類濃度を４０〜２００ｇ／Ｌに保持することが望ましい。
The method for producing the novel MEL of the present invention is not particularly limited and can be appropriately selected according to the purpose. For example, it is desirable to scale up in the order of seed culture, main culture and mannosylerythritol lipid production culture. .
Examples of culture media and culture conditions in these cultures are as follows.
a) Seed culture: 20 g / L of glucose, 1 g / L of yeast extract, 1 g / L of sodium nitrate, 0.5 g / L of potassium dihydrogen phosphate, and 4 mL of a liquid medium having a composition of 0.5 g / L of magnesium sulfate A platinum loop is inoculated into a test tube and cultured with shaking at 30 ° C. for 1 day.
b) Main culture: a predetermined amount of fats and oils such as vegetable oil and fat, 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 100 mL of the liquid medium having the composition is inoculated and cultured at 33 to 37 ° C. for 2 days.
c) Mannosyl erythritol lipid production culture; predetermined amount of oils and fats such as vegetable 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 jar fermenter containing 1.4 L of a liquid medium having the following composition is inoculated and cultured at 33-37 ° C. at a stirring speed of 800 rpm. In this culturing, it is desirable that the vegetable fats and oils flow down into the culture vessel from the middle of the culturing to maintain the fats and oils concentration in the medium at 40 to 200 g / L.
炭素源としては油脂類であれば特に制限がなく、例えば、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。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。 The carbon source is not particularly limited as long as it is an oil or fat. Examples of vegetable oils include soybean oil, rapeseed oil, corn oil, peanut oil, cottonseed oil, safflower oil, sesame oil, olive oil, and balm oil. Of these, soybean oil is preferable. You may use these individually by 1 type or in mixture of 2 or more types as appropriate.
培養終了後、等〜４容積倍の酢酸エチルで脂質成分を抽出し、酢酸エチルを、エバポレーターを用いて留去して脂質及び糖脂質成分を回収する。この脂質成分を等量のクロロホルムに溶解し、これをシリカゲルクロマトグラフィーにかけ、クロロホルム、クロロホルム：アセトン（７０：３０）、アセトン：クロロホルム（４０：６０）、アセトンの順で溶出させる。各溶液を薄層クロマトグラフィー（ＴＬＣ）プレートにチャージし、クロロホルム：メタノール：アンモニア水＝６５：１５：２（容積比）で展開する。展開終了後、アンスロン硫酸試薬で糖脂質の存在を確認する。糖脂質の含まれる溶出液を集め、溶媒を留去して糖脂質成分を得る。 After completion of the culture, the lipid component is extracted with ethyl acetate having an equivalent volume of 4 to 4 times, and the ethyl acetate is distilled off using an evaporator to recover the lipid and glycolipid component. This lipid component is dissolved in an equal amount of chloroform, and this is subjected to silica gel chromatography, and eluted in the order of chloroform, chloroform: acetone (70:30), acetone: chloroform (40:60), and acetone. Each solution is charged on a thin layer chromatography (TLC) plate and developed with chloroform: methanol: aqueous ammonia = 65: 15: 2 (volume ratio). After completion of the development, the presence of glycolipid is confirmed with an anthrone sulfate reagent. The eluate containing the glycolipid is collected and the solvent is distilled off to obtain a glycolipid component.
得られた糖脂質成分を再度クロロホルムに溶解し、サイズ排除クロマトグラフィー（ＳＥＣ）カラムを搭載した分取ＨＰＬＣシステムを用いることで、分子量の異なるフラクションに分離、回収する。各フラクション分離物についてＴＬＣ分析を行い、原料である油脂類、脂肪酸、グリセリンエステル等を除いた糖脂質成分のみが含まれているフラクションについて、再度シリカゲルクロマトグラフィーにかけ、クロロホルム：アセトン＝８５：１５（容積比）で溶出させ、ＴＬＣで単一のバンドを示す糖脂質成分を単離することにより、本発明の新規ＭＥＬを得ることができる。 The obtained glycolipid component is dissolved again in chloroform and separated and recovered into fractions having different molecular weights by using a preparative HPLC system equipped with a size exclusion chromatography (SEC) column. TLC analysis was performed on each fraction isolate, and the fraction containing only the glycolipid component excluding the fats and oils, fatty acids, glycerin esters and the like as raw materials was subjected to silica gel chromatography again, and chloroform: acetone = 85: 15 ( The novel MEL of the present invention can be obtained by isolating a glycolipid component that is eluted at a volume ratio and shows a single band by TLC.
〈新規ＭＥＬの構造決定〉
上記により得られる糖脂質成分の構造決定は、以下のようにして行う（シュードザイマ・パラアンタクティカ（Pseudozyma parantarctica JCM 11752 株）を用いて大豆油を炭素源として培養して得られた新規ＭＥＬの構造決定手法を例にして以下説明する）。
単離した糖脂質成分は、ＴＬＣプレート上で、アンスロン硫酸試薬で青緑色に呈色することにより糖脂質成分であると判断できる。この糖脂質について、1Ｈ、13Ｃ、二次元ＮＭＲ解析を行い、得られたスペクトルと、構造既知である従来型のマンノシルエリスリトールリピッド（ＭＥＬ−Ａ〜Ｄ）（式２）のスペクトルとを比較することで、構造解析を行う。
<Structure determination of new MEL>
The structure of the glycolipid component obtained as described above is determined as follows ( Pseudozyma parantarctica JCM 11752 strain) and a novel MEL obtained by culturing soybean oil as a carbon source. This will be described below using a structure determination method as an example).
The isolated glycolipid component can be judged to be a glycolipid component by being colored blue-green with an anthrone sulfate reagent on a TLC plate. This glycolipid was analyzed by 1 H, 13 C, and two-dimensional NMR, and the obtained spectrum was compared with the spectrum of a conventional mannosyl erythritol lipid (MEL-A to D) (formula 2) having a known structure. By doing so, structural analysis is performed.
糖脂質の糖組成の決定は、アルカリ（ＮａＯＣＨ３）でケン化して得られた糖鎖のＮＭＲ解析により行う。得られた糖脂質と従来型のＭＥＬの糖鎖のＮＭＲスペクトルを比較することで、本発明のＭＥＬの糖鎖部分の構造が、従来型ＭＥＬと同じ４−Ｏ−β−Ｄ−マンノピラノシル−ｍｅｓｏ−エリスリトール構造を有し、本糖脂質が新規構造のＭＥＬであることを確認できる。
２）脂質組成の解析
一方、脂質部分の組成は、得られた糖脂質を塩酸メタノールでメタノリシスし、ヘキサンで抽出して得られた脂肪酸メチルエステルのＧＣ／ＭＳで分析することにより決定する。
2) Analysis of lipid composition On the other hand, the composition of the lipid part is determined by GC / MS analysis of fatty acid methyl ester obtained by methanolysis of the obtained glycolipid with methanolic methanol and extraction with hexane.
３）脂肪酸成分の結合部位の解析
1Ｈ−ＮＭＲスペクトルでは、一般に糖アルコール上の還元末端以外のプロトンは３．５ｐｐｍ前後にまとまって検出されるが、水酸基の水素がアシル基とエステル結合すると、そのα−プロトンのシグナルは低磁場にシフトすることが知られている。本糖脂質の1Ｈ−ＮＭＲスペクトルを、従来型ＭＥＬ（ＭＥＬ−Ａ）のものと比較し、各プロトンに由来するピークの化学シフトを帰属した後、どの水酸基に何分子アシル基が結合しているかを類推する。また、２ｐｐｍ付近に観測されるアセチル基（−ＣＯＣＨ 3）由来のシグナルを確認することで、結合しているアシル基の何分子が酢酸で、何分子が脂肪酸であるかを予測する。
3) Analysis of the binding site of fatty acid components
In the 1 H-NMR spectrum, protons other than the reducing end on the sugar alcohol are generally detected around 3.5 ppm, but when the hydroxyl hydrogen is ester-bonded to the acyl group, the α-proton signal is low in the magnetic field. It is known to shift to The 1 H-NMR spectrum of this glycolipid is compared with that of a conventional MEL (MEL-A), and after assigning the chemical shift of the peak derived from each proton, how many molecular acyl groups bind to which hydroxyl group. Infer that it is. Also, by checking the signal derived from the acetyl group (-COC H 3) observed near 2 ppm, what molecule of the acyl groups are bonded with acetic acid, what molecules to predict whether a fatty acid.
次に、ＨＭＱＣ（Heteronuclear Multiple Quantum Coherence）、ＨＭＢＣ（Heteronuclear Multiple Bond Coherence）スペクトル解析を行い、上記の１Ｈ、１３Ｃ−ＮＭＲ解析の結果を基にして糖骨格の完全帰属を行う。ＨＭＱＣスペクトルによって、マンノース及びエリスリトール上の炭素、水素の化学シフトをそれぞれ完全帰属する。また、ＨＭＢＣスペクトルにおいて、どの水酸基に酢酸と脂肪酸がエステル結合しているかを証明する。 Next, HMQC (Heteronuclear Multiple Quantum Coherence) and HMBC (Heteronuclear Multiple Bond Coherence) spectrum analysis is performed, and complete assignment of the sugar skeleton is performed based on the results of the above 1 H, 13 C-NMR analysis. The chemical shifts of carbon and hydrogen on mannose and erythritol are completely assigned by HMQC spectrum, respectively. In the HMBC spectrum, it is proved which hydroxyl group has an ester bond with acetic acid and fatty acid.
得られた糖脂質は脂質部分の脂肪酸鎖長が異なる成分の混合物として回収されるが、さらにこれは逆相カラム（ODSカラム）を用いたHPLC分析を行うことで分離できる。分離した各ピーク化合物についてMS分析を行うことで主成分の構造を決定する。 The obtained glycolipid is recovered as a mixture of components having different fatty acid chain lengths in the lipid portion, which can be further separated by performing HPLC analysis using a reverse phase column (ODS column). The structure of the main component is determined by performing MS analysis on each separated peak compound.
さらに、これらの菌体が生産する化合物群には、ＴＬＣ上で他にもいくつかのスポットが検出されており、マンノース上のアセチル基の数とその結合位置の異なる成分、エリスリトール上に結合している脂肪酸の数と結合位置の異なる成分等も存在していることが認められる。 In addition, in the compound group produced by these cells, several other spots were detected on TLC, which bound to erythritol, a component having a different number of acetyl groups on mannose and different binding positions. It is recognized that components having different numbers of fatty acids and bonding positions are also present.
本発明の新規ＭＥＬは、優れた界面活性能を有するため、バイオサーファクタントとして用いることができる。界面活性能については、下記の方法で簡易に観察することができる。 Since the novel MEL of the present invention has excellent surface activity, it can be used as a biosurfactant. The surface activity can be easily observed by the following method.
〈界面活性能の観察〉
単離した新規ＭＥＬの水溶液を疎水性のフィルム上にスポットし、その表面張力の変化を観察する。また、コントロールとして水、及び従来型ＭＥＬの水溶液をそれぞれスポットして比較する。
<Observation of surface activity>
An isolated aqueous solution of MEL is spotted on a hydrophobic film, and the change in the surface tension is observed. Further, as a control, water and an aqueous solution of conventional MEL are spotted for comparison.
実施例１
（Pseudozyma parantarctica JCM 11752株の培養）
ａ）種培養；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、３０℃で１日間振とう培養を行い、次いで、
ｂ）本培養；得られた菌体培養液を大豆油１６０ｇ／Ｌと、酵母エキス１ｇ／Ｌ、硝酸ナトリウム１ ｇ／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１００ｍＬの入った坂口フラスコに接種して、３５℃で行い、さらに
ｃ）（マンノシルエリスリトールリピッド生産培養）これを大豆油１６０ｇ／Ｌ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５００ｍＬが入った坂口フラスコに接種して、３５℃で３００ｒｐｍの撹拌速度で培養を行った。
上記ａ）の培養を１日間行った後、ｂ）の培養を、７日間行い、得られたPseudozyma parantarctica JCM 11752株の菌体培養液に等量の酢酸エチルを添加・攪拌して、酢酸エチル抽出物を得た。この酢酸エチル抽出物を薄層クロマトグラフィー（ＴＬＣ）に供し、糖脂質を青緑色に発色させるアンスロン試薬を用いて、マンノシルエリスリトールリピッド（ＭＥＬ）の生産を確認した。展開溶媒はクロロホルム：メタノール：７Ｎアンモニア水= ６５：１５：２ を用いた。比較例としてPseudozyma antarctica KM-34(FERMP-20730) 株由来のＭＥＬ精製標品を用いた。結果を図１に示す。なお、図中、左端は従来型ＭＥＬの標準であり、ＭＥＬ−Ａ、ＭＥＬ−Ｂ及びＭＥＬ−Ｃはそれぞれ先述の式（２）で表される化合物を示す。これによれば、両株とも大豆油含有培地でＭＥＬを生産しており、さらに、ＭＥＬよりＲｆ値の高い（上方）位置に糖脂質の存在を示す青緑色のスポットが生じている。
Example 1
( Pseudozyma parantarctica JCM 11752 strain culture)
a) Seed culture: 20 g / L of glucose, 1 g / L of yeast extract, 1 g / L of sodium nitrate, 0.5 g / L of potassium dihydrogen phosphate, and 4 mL of a liquid medium having a composition of 0.5 g / L of magnesium sulfate 1 platinum ear is inoculated into a test tube, and cultured with shaking at 30 ° C. for 1 day,
b) Main culture; 160 g / L soybean oil, 1 g / L yeast extract, 1 g / L sodium nitrate, 0.5 g / L potassium dihydrogen phosphate, and 0.5 g magnesium sulfate Inoculate into a Sakaguchi flask containing 100 mL of liquid medium having a composition of / L, and carry out at 35 ° C. C) (Mannosylerythritol lipid production culture) This is soybean oil 160 g / L, yeast extract 1 g / L, sodium nitrate 1 g / L, 0.5 g / L potassium dihydrogen phosphate, and 500 mL liquid medium containing 0.5 g / L magnesium sulfate were inoculated and cultured at 35 ° C. with a stirring speed of 300 rpm. .
After culturing a) for 1 day, b) is cultured for 7 days, and an equal amount of ethyl acetate is added to and stirred in the cell culture solution of the obtained Pseudozyma parantarctica JCM 11752 strain. An extract was obtained. This ethyl acetate extract was subjected to thin layer chromatography (TLC), and production of mannosyl erythritol lipid (MEL) was confirmed using an anthrone reagent that causes the glycolipid to develop blue-green color. The developing solvent used was chloroform: methanol: 7N aqueous ammonia = 65: 15: 2. As a comparative example, a purified MEL preparation derived from Pseudozyma antarctica KM-34 (FERMP-20730) strain was used. The results are shown in FIG. In the figure, the left end is a standard of conventional MEL, and MEL-A, MEL-B and MEL-C each represent a compound represented by the above formula (2). According to this, both strains produce MEL in a soybean oil-containing medium, and further, a blue-green spot indicating the presence of glycolipid is generated at a position (above) where the Rf value is higher than MEL.
実施例２
（Pseudozyma parantarctica JCM 11752株の培養）
上記ａ）の培養を１日間行った後、ｂ）の培養を、２日間行い、さらにｃ）の培養７日間行い、得られたPseudozyma parantarctica JCM 11752株の菌体培養液に等量の酢酸エチルを添加・攪拌して、酢酸エチル抽出物を得た。この酢酸エチル抽出物をＴＬＣに供し、糖脂質を青緑色に発色させるアンスロン試薬を用いて、ＭＥＬの生産を確認した。比較例としてPseudozyma antarctica KM-34(FERMP-20730) 株由来のＭＥＬ精製標品を用いた。結果を図２に示す。これによれば、両株とも大豆油含有培地でＭＥＬを生産しており、さらに、ＭＥＬよりＲｆ値の高い（上方）位置に糖脂質の存在を示す青緑色のスポットが生じている。
Example 2
( Pseudozyma parantarctica JCM 11752 strain culture)
After culturing a) for 1 day, culturing b) for 2 days, and further c) culturing for 7 days. An equal amount of ethyl acetate is added to the obtained Pseudozyma parantarctica JCM 11752 cell culture. Was added and stirred to obtain an ethyl acetate extract. This ethyl acetate extract was subjected to TLC, and MEL production was confirmed using an anthrone reagent that causes the glycolipid to develop a blue-green color. As a comparative example, a purified MEL preparation derived from Pseudozyma antarctica KM-34 (FERMP-20730) strain was used. The results are shown in FIG. According to this, both strains produce MEL in a soybean oil-containing medium, and further, a blue-green spot indicating the presence of glycolipid is generated at a position (above) where the Rf value is higher than MEL.
実施例３
（Pseudozyma rugulosa NBRC 10877株の培養）
上記ａ）の培養を１日間行った後、ｂ）の培養を、３０℃で７日間行い、得られたPseudozyma rugulosa NBRC 10877株の菌体培養液に等量の酢酸エチルを添加・攪拌して、酢酸エチル抽出物を得た。この酢酸エチル抽出物をＴＬＣに供し、糖脂質を青緑色に発色させるアンスロン試薬を用いて、ＭＥＬの生産を確認した。比較例としてPseudozyma antarctica KM-34(FERMP-20730) 株由来のＭＥＬ精製標品を用いた。結果を図３に示す。これによれば、両株とも大豆油含有培地でＭＥＬを生産しており、さらに、ＭＥＬよりＲｆ値の高い（上方）位置に糖脂質の存在を示す青緑色のスポットが生じている。
Example 3
( Pseudozyma rugulosa NBRC 10877 culture)
After culturing in a) above for 1 day, culturing in b) is carried out at 30 ° C. for 7 days, and an equal amount of ethyl acetate is added to the cell culture solution of the obtained Pseudozyma rugulosa NBRC 10877 strain and stirred. An ethyl acetate extract was obtained. This ethyl acetate extract was subjected to TLC, and MEL production was confirmed using an anthrone reagent that causes the glycolipid to develop a blue-green color. As a comparative example, a purified MEL preparation derived from Pseudozyma antarctica KM-34 (FERMP-20730) strain was used. The results are shown in FIG. According to this, both strains produce MEL in a soybean oil-containing medium, and further, a blue-green spot indicating the presence of glycolipid is generated at a position (above) where the Rf value is higher than MEL.
実施例４
（Pseudozyma parantarctica JCM 11752株由来マンノシルエリスリトールリピッドの精製）
実施例２で得られたマンノシルエリスリトールリピッドの精製を、既知の精製手法によって分離した。すなわち、上記酢酸エチ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の混合液を展開溶媒とするカラムクロマトグラフィーで精製した。クロロホルムとアセトンの割合は、７０：３０で残存原料と脂肪酸を含むMEL-AよりRf値の高い物質を分離回収し、続いて４０：６０でMEL-A、-B、-Cを分離回収した。そして、それぞれの回収した画分を上記ＴＬＣに供した（図４）。これによれば、構造不明の糖脂質はクロロホルム：アセトン＝７０：３０の画分に検出できた。
Example 4
( Purification of mannosylerythritol lipid from Pseudozyma parantarctica JCM 11752)
Purification of mannosyl erythritol lipid obtained in Example 2 was separated by known purification techniques. That is, the ethyl acetate extract was applied to a silica gel column and purified by column chromatography using a mixed solution of chloroform and acetone as a developing solvent. The ratio of chloroform and acetone was 70:30, and the substances having a higher Rf value than MEL-A containing the remaining raw materials and fatty acids were separated and recovered, and then MEL-A, -B, and -C were separated and recovered at 40:60. . And each collect | recovered fraction was used for the said TLC (FIG. 4). According to this, glycolipid of unknown structure was detected in the fraction of chloroform: acetone = 70: 30.
実施例５
（構造不明糖脂質の単離・精製）
実施例４で得られた構造不明の糖脂質を含む画分を全て回収し、エバポレーターを用いて溶媒を留去した後、再度クロロホルムに溶解させ、サイズ排除クロマトグラフィー（ＳＥＣ）カラムを搭載したリサイクル分取ＨＰＬＣシステムを用いることで、分子量の異なるフラクションに分離、回収した。リサイクル回数を増やすことで大きく３つのフラクションに分離された。クロマトグラムを図５に示す。
Example 5
(Isolation and purification of glycolipid of unknown structure)
All the fractions containing glycolipids of unknown structure obtained in Example 4 were collected, the solvent was distilled off using an evaporator, then dissolved again in chloroform, and recycled with a size exclusion chromatography (SEC) column. By using a preparative HPLC system, fractions with different molecular weights were separated and collected. By increasing the number of recycles, it was largely separated into three fractions. The chromatogram is shown in FIG.
各フラクション成分を上記ＴＬＣに供した（図６）。これによれば、ＨＰＬＣ分析において最も高分子量であったフラクション（１）に構造不明の糖脂質が含有されていることが確認された。 Each fraction component was subjected to the TLC (FIG. 6). According to this, it was confirmed that the fraction (1) having the highest molecular weight in HPLC analysis contained a glycolipid of unknown structure.
さらに上記フラクション（１）を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で精製した。クロロホルムとアセトンの割合は８５：１５とし、回収したそれぞれの画分を上記ＴＬＣに供した（図７）。これによれば、フラクション（１）中には単一成分である主な２つの成分（a）、（ｂ）、及びその他の分離できない成分があることが分かる。
以降、フラクション（１）中の成分（a）の成分について詳細に説明する。
Further, the fraction (1) was applied to a silica gel column and purified by column chromatography using a mixed solution of chloroform and acetone as a developing solvent. The ratio of chloroform and acetone was 85:15, and each collected fraction was subjected to the TLC (FIG. 7). This shows that there are two main components (a) and (b), which are single components, and other components that cannot be separated in fraction (1).
Hereinafter, the component (a) in the fraction (1) will be described in detail.
実施例６
（構造不明糖脂質の構造解析）
１）糖組成の解析
実施例５で得られた糖脂質を、メタノール中でナトリウムメトキシドによって加水分解を行った。反応終了後の生成物を酢酸エチル中に再沈殿させて回収し、９０％エタノール中で再結晶操作を行うことにより糖鎖の結晶を得た。従来型ＭＥＬについても同様の操作で糖鎖を回収し、得られた各糖鎖について、重水素化ジメチルスルホキシド（DMSO-ｄ６）を溶媒として１H−ＮＭＲ解析を行った。その結果、本糖脂質と従来型のＭＥＬの糖鎖のＮＭＲスペクトルは完全に一致した。よって以上の結果から、実施例５で得られた構造不明糖脂質の糖鎖部分の構造は、従来型MELと同様４−Ｏ−β−Ｄ−マンノピラノシル−ｍｅｓｏ−エリスリトールであり、本糖脂質が新規構造のＭＥＬであることが予想された。
Example 6
(Structural analysis of glycolipids with unknown structure)
1) Analysis of sugar composition The glycolipid obtained in Example 5 was hydrolyzed with sodium methoxide in methanol. The product after completion of the reaction was recovered by reprecipitation in ethyl acetate, and a sugar chain crystal was obtained by performing a recrystallization operation in 90% ethanol. For the conventional MEL, sugar chains were collected in the same manner, and 1 H-NMR analysis was performed on each obtained sugar chain using deuterated dimethyl sulfoxide (DMSO-d6) as a solvent. As a result, the NMR spectra of the glycolipid and the conventional MEL sugar chain completely matched. Therefore, from the above results, the structure of the sugar chain part of the glycolipid of unknown structure obtained in Example 5 is 4-O-β-D-mannopyranosyl-meso-erythritol as in the conventional MEL. It was expected to be a new structure MEL.
２）脂質組成の解析
実施例５で得られた糖脂質の脂質部分の組成は、塩酸メタノール加水分解物からヘキサンで抽出して得られた脂肪酸メチルエステルのGC/MS分析により行った。分析結果とGCチャートを図８に示す。比較のために構造既知である従来型MEL（MEL-A）のチャートも示す。分析の結果、本糖脂質では、短〜中鎖の脂肪酸（C８〜C１２）の直鎖飽和及び不飽和脂肪酸が主成分として検出さる点で従来型のものとよく一致したチャートが得られたが、さらに長鎖脂肪酸（C１６、１８）が含まれていることが示された。特に、不飽和基を一つ有するC１８の脂肪酸（オレイン酸）が主成分として検出された。以上の結果より、本糖脂質では従来型のMELに対してもう１つこれらの長鎖脂肪酸がエステル結合していると予測された。
2) Analysis of lipid composition The composition of the lipid part of the glycolipid obtained in Example 5 was determined by GC / MS analysis of a fatty acid methyl ester obtained by extraction with hexane from a hydrochloric acid methanol hydrolyzate. The analysis results and GC chart are shown in FIG. For comparison, a chart of a conventional MEL (MEL-A) having a known structure is also shown. As a result of the analysis, in this glycolipid, a chart was obtained that was in good agreement with the conventional type in that linear saturated and unsaturated fatty acids of short to medium chain fatty acids (C8 to C12) were detected as the main components. Furthermore, it was shown that long chain fatty acids (C16, 18) were contained. In particular, C18 fatty acid (oleic acid) having one unsaturated group was detected as the main component. From the above results, it was predicted that in the glycolipid, another long chain fatty acid was ester-linked to the conventional MEL.
３）脂肪酸成分の結合部位の解析
実施例５で得られた糖脂質について、重クロロホルム（CDCｌ３）を溶媒とするＮＭＲ解析により構造の同定を行った。本糖脂質の1Ｈ−ＮＭＲスペクトル（図９）は、構造既知である従来型ＭＥＬ（ＭＥＬ−Ａ）のものと比較して非常によく一致しており、特にマンノース上のＨ−２’〜４’、６’のプロトンに由来するピークの化学シフトは完全に一致した（Ｈ−２’；５．５１ｐｐｍ、Ｈ−３’；５．０７、Ｈ−４’；５．２７、Ｈ−６’；４．２０ｐｐｍ）、このことから本糖脂質は従来型のMEL−Aと同様、マンノース上の４つの水酸基の全てにアシル基が結合していると同定できる。一方、本糖脂質ではさらに、アシル基とエステル結合したことで低磁場シフトしたα−プロトンのシグナルが４．３ｐｐｍ付近にも観測された。以上の結果から、本糖脂質は従来型のＭＥＬとは異なりエリスリトール上の水酸基にもアシル基が結合していることが示された。さらに、２．１０ｐｐｍと２．０３ｐｐｍに２分子のアセチル基（−ＣＯＣＨ 3）のメチルプロトンの存在を示すシグナルが観測されたことから、合計５分子のアシル基のうち、２分子が酢酸で３分子が脂肪酸であると同定できる。
3) Analysis of binding site of fatty acid component The structure of the glycolipid obtained in Example 5 was identified by NMR analysis using deuterated chloroform (CDCl 3 ) as a solvent. The 1 H-NMR spectrum (FIG. 9) of this glycolipid is very consistent with that of a conventional MEL (MEL-A) having a known structure, particularly H-2′- The chemical shifts of the peaks derived from the 4 ′ and 6 ′ protons were completely consistent (H-2 ′; 5.51 ppm, H-3 ′; 5.07, H-4 ′; 5.27, H-6). '; 4.20 ppm) From this, the glycolipid can be identified as having acyl groups bonded to all four hydroxyl groups on mannose, as in the conventional MEL-A. On the other hand, in this glycolipid, a signal of α-proton shifted by a low magnetic field due to ester bond with an acyl group was also observed in the vicinity of 4.3 ppm. From the above results, it was shown that, unlike the conventional MEL, the glycolipid has an acyl group bonded to the hydroxyl group on erythritol. Furthermore, since the signal indicative of the presence of methyl protons of the acetyl group of 2 molecules 2.10ppm and 2.03ppm (-COC H 3) was observed among the acyl groups of a total of 5 molecules, 2 molecules in acetic acid Three molecules can be identified as fatty acids.
次に、ＨＭＱＣ（Heteronuclear Multiple Quantum Coherence）、ＨＭＢＣ（Heteronuclear Multiple Bond Coherence）スペクトル解析を行い、糖骨格の完全帰属を行った。ＨＭＱＣスペクトル（図１０）により、マンノース及びエリスリトール上の炭素、水素の化学シフトがそれぞれ完全帰属された。また、ＨＭＢＣスペクトル（図１１）において、Ｈ−４’とＨ−６’のプロトンシグナルが、アセチル基由来のカルボニル基のシグナル（１６９．６ｐｐｍと１７０．９ｐｐｍ）とそれぞれカップリングしていることが認められたことから、マンノースの４’位と６’位には酢酸がエステル結合していることが証明された。さらに、Ｈ−２’とＨ−３’、及びＨ−１（４．３ｐｐｍ）のプロトンシグナルが、脂肪酸アシル基由来のカルボニル基のシグナル（173.4ppm、172.9ppm、及び174.7ppm）とそれぞれカップリングしていることが認められたことから、マンノースの２’、３’位及びエリスリトールの１位には脂肪酸がエステル結合していることが証明された。 Next, HMQC (Heteronuclear Multiple Quantum Coherence) and HMBC (Heteronuclear Multiple Bond Coherence) spectrum analysis were performed, and complete assignment of the sugar skeleton was performed. The chemical shifts of carbon and hydrogen on mannose and erythritol were completely assigned by the HMQC spectrum (FIG. 10). Further, in the HMBC spectrum (FIG. 11), the proton signals of H-4 ′ and H-6 ′ are coupled to the signals of carbonyl groups derived from the acetyl group (169.6 ppm and 170.9 ppm), respectively. From the recognition, it was proved that acetic acid had an ester bond at the 4 'and 6' positions of mannose. In addition, proton signals of H-2 ′, H-3 ′, and H-1 (4.3 ppm) are coupled to carbonyl group signals derived from fatty acid acyl groups (173.4 ppm, 172.9 ppm, and 174.7 ppm), respectively. As a result, it was proved that a fatty acid had an ester bond at the 2 ′, 3 ′ position of mannose and the 1 position of erythritol.
以上のことから、本糖脂質の構造は、１−アルカノイル−４−（４’，６’−ジ−Ｏ−アセチル−２’，３’−ジ−Ｏ−アルカノイル−β−Ｄ−マンノピラノシル−）ｍｅｓｏ−エリスリトールと同定できた。
From the above, the structure of this glycolipid is 1-alkanoyl-4- (4 ′, 6′-di-O-acetyl-2 ′, 3′-di-O-alkanoyl-β-D-mannopyranosyl-). It was identified as meso-erythritol.
さらに、この糖脂質はＭＡＬＤＩ−ＴＯＦ／ＭＳ分析において、擬似分子イオン［Ｍ＋Ｎａ］+ ｍ／ｚ ９３５．９が検出された（図１２）。この結果と脂肪酸組成分析の結果から、本糖脂質はマンノシルエリスリトール骨格の２’位と３’位の水酸基にC８及びC１０の直鎖飽和脂肪酸が１つずつエステル結合しており、１位水酸基にオレイン酸が結合している構造の化合物が主成分であることが示唆される。
Furthermore, in the MALDI-TOF / MS analysis, a pseudomolecular ion [M + Na] + m / z 935.9 was detected from this glycolipid (FIG. 12). From this result and the result of fatty acid composition analysis, this glycolipid has an ester bond of C8 and C10 linear saturated fatty acids one by one to the 2 'and 3' hydroxyl groups of the mannosyl erythritol skeleton. It is suggested that a compound having a structure to which oleic acid is bonded is a main component.
また、フラクション（１）中の成分（ｂ）について同様の解析を行った結果、これは上記の新規MELに対して、マンノース上の４’位の水酸基に酢酸エステルが存在しない、１−アルカノイル−４−（６’−Ｏ−アセチル−２’，３’−ジ−Ｏ−アルカノイル−β−Ｄ−マンノピラノシル−）ｍｅｓｏ−エリスリトールであることが確認された。図１３にその1Ｈ−ＮＭＲスペクトルを示す。
In addition, as a result of conducting the same analysis on the component (b) in the fraction (1), it was found that 1-alkanoyl-, which does not have an acetate ester at the 4′-position hydroxyl group on mannose, was compared to the above-mentioned new MEL. It was confirmed to be 4- (6′-O-acetyl-2 ′, 3′-di-O-alkanoyl-β-D-mannopyranosyl-) meso-erythritol. FIG. 13 shows the 1 H-NMR spectrum.
これらの菌体が生産する化合物群には、上記のようにマンノース上のアセチル基の数との結合位置の異なる成分が存在しており、またTLC上にはエリスリトール上に結合している脂肪酸の数と結合位置の異なる成分と思しきスポット等も認められている。 In the group of compounds produced by these cells, there are components having different binding positions with the number of acetyl groups on mannose as described above, and on the TLC, fatty acid bound on erythritol. Spots that appear to be components having different numbers and bonding positions are also recognized.
実施例７
（界面活性能の観察）
実施例５で得られた新規ＭＥＬの希釈水溶液を作成し、疎水性のフィルム上にスポットすることで、その表面張力の変化を観察した。また、コントロールとして水、及び従来型ＭＥＬ（ＭＥＬ−Ａ）の水溶液をそれぞれスポットして比較した。これらの結果を図１４に示す。これによれば、本発明での糖脂質は水溶液の表面張力が低下しており、界面活性剤としての機能を有することが示された。また、本糖脂質の水溶液のスポットは、従来型ＭＥＬと比較するとスポットの広がりは小さくなった。本糖脂質は親油基である脂肪酸エステルの結合数が従来型ＭＥＬよりも１つ多いため、より疎水的なバイオサーファクタントであると推測されるが、本観察の結果はそれを示唆するものであった。
Example 7
(Observation of surface activity)
A dilute aqueous solution of the new MEL obtained in Example 5 was prepared and spotted on a hydrophobic film, and the change in the surface tension was observed. Further, as a control, water and an aqueous solution of conventional MEL (MEL-A) were spotted for comparison. These results are shown in FIG. According to this, it was shown that the glycolipid in the present invention has a reduced surface tension of the aqueous solution and has a function as a surfactant. Moreover, the spot spread of the aqueous solution of the glycolipid was smaller than that of the conventional MEL. This glycolipid is estimated to be a more hydrophobic biosurfactant because the number of bonds of fatty acid ester, which is a lipophilic group, is one more than that of conventional MEL, but the results of this observation suggest it. there were.
さらに、新規ＭＥＬとＭＥＬ−Ａを任意の割合で混ぜたＭＥＬ混合水溶液（０．５mg/mL）を作成し、疎水性のフィルム上にスポットすることで、その表面張力の変化を観察した（図１５）。新規ＭＥＬの混合比を増加させることで、表面張力の低下率を容易に制御できることが示された。新規ＭＥＬは従来型ＭＥＬの類似骨格を有しており、これを混合することは溶液の性質を変えることなく界面活性を調節できると推測される。したがって、他の合成界面活性剤やバイオサーファクタントを混合するよりも有効と考えられる。
Furthermore, a MEL mixed aqueous solution (0.5 mg / mL) in which new MEL and MEL-A were mixed at an arbitrary ratio was prepared, and changes in the surface tension were observed by spotting on a hydrophobic film (Fig. 15). It was shown that the reduction rate of the surface tension can be easily controlled by increasing the mixing ratio of the new MEL. The new MEL has a similar skeleton to the conventional MEL, and it is speculated that mixing this can adjust the surface activity without changing the properties of the solution. Therefore, it is considered more effective than mixing other synthetic surfactants and biosurfactants.
本発明の糖脂質は従来型ＭＥＬと比較して、より親油的でバルキーな置換基が導入されており、ＨＬＢ、相挙動等が大きく異なっている。本発明の新規ＭＥＬを従来の系に数％添加するだけで構造・組成の類似性から活性剤そのものの性質を変えることなく、乳化、可溶化、分散化能を広く調整することが可能である。糖脂質型バイオサーファクタントとして本来有する特性である分解性の高さや低毒性、環境適合性、高生理活性に加え、目的に応じて界面活性を幅広く調整できる利便性等を利用して、食品工業、化粧品工業、医薬品工業、化学工業、環境分野等に広く利用できる。
The glycolipid of the present invention is introduced with a more lipophilic and bulky substituent as compared with conventional MEL, and is greatly different in HLB, phase behavior and the like. It is possible to adjust the emulsification, solubilization, and dispersibility widely without changing the properties of the active agent itself from the similarity of structure and composition by adding a few% of the novel MEL of the present invention to the conventional system. . In addition to the high degradability, low toxicity, environmental compatibility, and high physiological activity that are inherent characteristics of glycolipid biosurfactants, the food industry, It can be widely used in the cosmetics industry, pharmaceutical industry, chemical industry, environmental fields, etc.
Claims (3)
（式中、Ｒ１は、同一でも異なっていてもよい炭素数６〜２０の脂肪族アシル基であり、Ｒ２は水素又はアセチル基、Ｒ３は水素又は炭素数６〜２０の脂肪族アシル基を表す。） Mannosyl erythritol lipid represented by the following formula (1).
Wherein R 1 is an aliphatic acyl group having 6 to 20 carbon atoms which may be the same or different, R 2 is hydrogen or acetyl group, R 3 is hydrogen or aliphatic acyl having 6 to 20 carbon atoms. Represents a group.)
A microorganism belonging to the genus Pseudozyma and capable of producing mannosyl erythritol lipid is cultured so that the concentration of fats and oils in the medium is maintained at 40 to 200 g / L, and the mannosyl erythritol according to claim 1 or 2 is obtained from the culture. A method for producing mannosyl erythritol lipid, comprising collecting the lipid.
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