JP4853909B2 - Method for producing glycolipid - Google Patents
Method for producing glycolipid Download PDFInfo
- Publication number
- JP4853909B2 JP4853909B2 JP2006219166A JP2006219166A JP4853909B2 JP 4853909 B2 JP4853909 B2 JP 4853909B2 JP 2006219166 A JP2006219166 A JP 2006219166A JP 2006219166 A JP2006219166 A JP 2006219166A JP 4853909 B2 JP4853909 B2 JP 4853909B2
- Authority
- JP
- Japan
- Prior art keywords
- mel
- glycolipid
- producing
- triacyl
- biosurfactant
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
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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 title claims description 56
- 229930186217 Glycolipid Natural products 0.000 title claims description 56
- 238000004519 manufacturing process Methods 0.000 title claims description 54
- 239000003876 biosurfactant Substances 0.000 claims description 56
- 150000004665 fatty acids Chemical class 0.000 claims description 44
- 239000003921 oil Substances 0.000 claims description 43
- 235000014113 dietary fatty acids Nutrition 0.000 claims description 42
- 239000000194 fatty acid Substances 0.000 claims description 42
- 229930195729 fatty acid Natural products 0.000 claims description 42
- 239000000047 product Substances 0.000 claims description 24
- 239000000203 mixture Substances 0.000 claims description 22
- 150000002148 esters Chemical class 0.000 claims description 20
- 239000006228 supernatant Substances 0.000 claims description 20
- 239000002244 precipitate Substances 0.000 claims description 18
- 125000002887 hydroxy group Chemical group [H]O* 0.000 claims description 16
- 238000005886 esterification reaction Methods 0.000 claims description 14
- 244000005700 microbiome Species 0.000 claims description 14
- 238000005809 transesterification reaction Methods 0.000 claims description 14
- 239000003960 organic solvent Substances 0.000 claims description 13
- 150000002430 hydrocarbons Chemical group 0.000 claims description 11
- 125000000311 mannosyl group Chemical group C1([C@@H](O)[C@@H](O)[C@H](O)[C@H](O1)CO)* 0.000 claims description 10
- 125000004432 carbon atom Chemical group C* 0.000 claims description 8
- 125000004430 oxygen atom Chemical group O* 0.000 claims description 8
- 108090001060 Lipase Proteins 0.000 claims description 7
- 239000004367 Lipase Substances 0.000 claims description 7
- 102000004882 Lipase Human genes 0.000 claims description 7
- 238000004108 freeze drying Methods 0.000 claims description 7
- 239000001963 growth medium Substances 0.000 claims description 7
- 235000019421 lipase Nutrition 0.000 claims description 7
- 125000002777 acetyl group Chemical group [H]C([H])([H])C(*)=O 0.000 claims description 6
- 230000002209 hydrophobic effect Effects 0.000 claims description 6
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- 101710098556 Lipase A Proteins 0.000 claims 1
- 101710099648 Lysosomal acid lipase/cholesteryl ester hydrolase Proteins 0.000 claims 1
- 102100026001 Lysosomal acid lipase/cholesteryl ester hydrolase Human genes 0.000 claims 1
- CSCPPACGZOOCGX-UHFFFAOYSA-N Acetone Chemical compound CC(C)=O CSCPPACGZOOCGX-UHFFFAOYSA-N 0.000 description 53
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- VLKZOEOYAKHREP-UHFFFAOYSA-N n-Hexane Chemical compound CCCCCC VLKZOEOYAKHREP-UHFFFAOYSA-N 0.000 description 24
- 238000006243 chemical reaction Methods 0.000 description 22
- -1 fatty acid ester Chemical class 0.000 description 20
- UNXHWFMMPAWVPI-ZXZARUISSA-N erythritol Chemical compound OC[C@H](O)[C@H](O)CO UNXHWFMMPAWVPI-ZXZARUISSA-N 0.000 description 17
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- 238000004458 analytical method Methods 0.000 description 11
- 239000002808 molecular sieve Substances 0.000 description 10
- URGAHOPLAPQHLN-UHFFFAOYSA-N sodium aluminosilicate Chemical compound [Na+].[Al+3].[O-][Si]([O-])=O.[O-][Si]([O-])=O URGAHOPLAPQHLN-UHFFFAOYSA-N 0.000 description 10
- FRPZMMHWLSIFAZ-UHFFFAOYSA-N 10-undecenoic acid Chemical compound OC(=O)CCCCCCCCC=C FRPZMMHWLSIFAZ-UHFFFAOYSA-N 0.000 description 9
- WEVYAHXRMPXWCK-UHFFFAOYSA-N Acetonitrile Chemical compound CC#N WEVYAHXRMPXWCK-UHFFFAOYSA-N 0.000 description 9
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- VYPSYNLAJGMNEJ-UHFFFAOYSA-N Silicium dioxide Chemical compound O=[Si]=O VYPSYNLAJGMNEJ-UHFFFAOYSA-N 0.000 description 7
- 239000000741 silica gel Substances 0.000 description 7
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- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 description 7
- OKKJLVBELUTLKV-UHFFFAOYSA-N Methanol Chemical compound OC OKKJLVBELUTLKV-UHFFFAOYSA-N 0.000 description 6
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- FBPFZTCFMRRESA-KVTDHHQDSA-N D-Mannitol Chemical compound OC[C@@H](O)[C@@H](O)[C@H](O)[C@H](O)CO FBPFZTCFMRRESA-KVTDHHQDSA-N 0.000 description 5
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- 229940041514 candida albicans extract Drugs 0.000 description 5
- KHAVLLBUVKBTBG-UHFFFAOYSA-N caproleic acid Natural products OC(=O)CCCCCCCC=C KHAVLLBUVKBTBG-UHFFFAOYSA-N 0.000 description 5
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- 238000012258 culturing Methods 0.000 description 5
- 235000020778 linoleic acid Nutrition 0.000 description 5
- OYHQOLUKZRVURQ-IXWMQOLASA-N linoleic acid Natural products CCCCC\C=C/C\C=C\CCCCCCCC(O)=O OYHQOLUKZRVURQ-IXWMQOLASA-N 0.000 description 5
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- ZQPPMHVWECSIRJ-KTKRTIGZSA-N oleic acid Chemical compound CCCCCCCC\C=C/CCCCCCCC(O)=O ZQPPMHVWECSIRJ-KTKRTIGZSA-N 0.000 description 5
- 239000002904 solvent Substances 0.000 description 5
- 229960002703 undecylenic acid Drugs 0.000 description 5
- 239000012138 yeast extract Substances 0.000 description 5
- WRIDQFICGBMAFQ-UHFFFAOYSA-N (E)-8-Octadecenoic acid Natural products CCCCCCCCCC=CCCCCCCC(O)=O WRIDQFICGBMAFQ-UHFFFAOYSA-N 0.000 description 4
- LQJBNNIYVWPHFW-UHFFFAOYSA-N 20:1omega9c fatty acid Natural products CCCCCCCCCCC=CCCCCCCCC(O)=O LQJBNNIYVWPHFW-UHFFFAOYSA-N 0.000 description 4
- QSBYPNXLFMSGKH-UHFFFAOYSA-N 9-Heptadecensaeure Natural products CCCCCCCC=CCCCCCCCC(O)=O QSBYPNXLFMSGKH-UHFFFAOYSA-N 0.000 description 4
- IAZDPXIOMUYVGZ-WFGJKAKNSA-N Dimethyl sulfoxide Chemical compound [2H]C([2H])([2H])S(=O)C([2H])([2H])[2H] IAZDPXIOMUYVGZ-WFGJKAKNSA-N 0.000 description 4
- 102000004190 Enzymes Human genes 0.000 description 4
- 108090000790 Enzymes Proteins 0.000 description 4
- FLIACVVOZYBSBS-UHFFFAOYSA-N Methyl palmitate Chemical compound CCCCCCCCCCCCCCCC(=O)OC FLIACVVOZYBSBS-UHFFFAOYSA-N 0.000 description 4
- HPEUJPJOZXNMSJ-UHFFFAOYSA-N Methyl stearate Chemical compound CCCCCCCCCCCCCCCCCC(=O)OC HPEUJPJOZXNMSJ-UHFFFAOYSA-N 0.000 description 4
- 108010084311 Novozyme 435 Proteins 0.000 description 4
- 239000005642 Oleic acid Substances 0.000 description 4
- ZQPPMHVWECSIRJ-UHFFFAOYSA-N Oleic acid Natural products CCCCCCCCC=CCCCCCCCC(O)=O ZQPPMHVWECSIRJ-UHFFFAOYSA-N 0.000 description 4
- 235000021355 Stearic acid Nutrition 0.000 description 4
- WYURNTSHIVDZCO-UHFFFAOYSA-N Tetrahydrofuran Chemical compound C1CCOC1 WYURNTSHIVDZCO-UHFFFAOYSA-N 0.000 description 4
- 239000010495 camellia oil Substances 0.000 description 4
- 239000000287 crude extract Substances 0.000 description 4
- POULHZVOKOAJMA-UHFFFAOYSA-N dodecanoic acid Chemical compound CCCCCCCCCCCC(O)=O POULHZVOKOAJMA-UHFFFAOYSA-N 0.000 description 4
- 230000002255 enzymatic effect Effects 0.000 description 4
- IPCSVZSSVZVIGE-UHFFFAOYSA-N hexadecanoic acid Chemical compound CCCCCCCCCCCCCCCC(O)=O IPCSVZSSVZVIGE-UHFFFAOYSA-N 0.000 description 4
- QXJSBBXBKPUZAA-UHFFFAOYSA-N isooleic acid Natural products CCCCCCCC=CCCCCCCCCC(O)=O QXJSBBXBKPUZAA-UHFFFAOYSA-N 0.000 description 4
- 239000002609 medium Substances 0.000 description 4
- UQDUPQYQJKYHQI-UHFFFAOYSA-N methyl laurate Chemical compound CCCCCCCCCCCC(=O)OC UQDUPQYQJKYHQI-UHFFFAOYSA-N 0.000 description 4
- ZAZKJZBWRNNLDS-UHFFFAOYSA-N methyl tetradecanoate Chemical compound CCCCCCCCCCCCCC(=O)OC ZAZKJZBWRNNLDS-UHFFFAOYSA-N 0.000 description 4
- QIQXTHQIDYTFRH-UHFFFAOYSA-N octadecanoic acid Chemical compound CCCCCCCCCCCCCCCCCC(O)=O QIQXTHQIDYTFRH-UHFFFAOYSA-N 0.000 description 4
- OQCDKBAXFALNLD-UHFFFAOYSA-N octadecanoic acid Natural products CCCCCCCC(C)CCCCCCCCC(O)=O OQCDKBAXFALNLD-UHFFFAOYSA-N 0.000 description 4
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- 239000004094 surface-active agent Substances 0.000 description 4
- 235000015112 vegetable and seed oil Nutrition 0.000 description 4
- WTTJVINHCBCLGX-UHFFFAOYSA-N (9trans,12cis)-methyl linoleate Natural products CCCCCC=CCC=CCCCCCCCC(=O)OC WTTJVINHCBCLGX-UHFFFAOYSA-N 0.000 description 3
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- LNJCGNRKWOHFFV-UHFFFAOYSA-N 3-(2-hydroxyethylsulfanyl)propanenitrile Chemical compound OCCSCCC#N LNJCGNRKWOHFFV-UHFFFAOYSA-N 0.000 description 3
- 241000894006 Bacteria Species 0.000 description 3
- UHOVQNZJYSORNB-UHFFFAOYSA-N Benzene Chemical compound C1=CC=CC=C1 UHOVQNZJYSORNB-UHFFFAOYSA-N 0.000 description 3
- RTZKZFJDLAIYFH-UHFFFAOYSA-N Diethyl ether Chemical compound CCOCC RTZKZFJDLAIYFH-UHFFFAOYSA-N 0.000 description 3
- IAZDPXIOMUYVGZ-UHFFFAOYSA-N Dimethylsulphoxide Chemical compound CS(C)=O IAZDPXIOMUYVGZ-UHFFFAOYSA-N 0.000 description 3
- 108090000371 Esterases Proteins 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
- OYHQOLUKZRVURQ-HZJYTTRNSA-N Linoleic acid Chemical compound CCCCC\C=C/C\C=C/CCCCCCCC(O)=O OYHQOLUKZRVURQ-HZJYTTRNSA-N 0.000 description 3
- PKIXXJPMNDDDOS-UHFFFAOYSA-N Methyl linoleate Natural products CCCCC=CCCC=CCCCCCCCC(=O)OC PKIXXJPMNDDDOS-UHFFFAOYSA-N 0.000 description 3
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- DNIAPMSPPWPWGF-UHFFFAOYSA-N Propylene glycol Chemical compound CC(O)CO DNIAPMSPPWPWGF-UHFFFAOYSA-N 0.000 description 3
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- 125000002252 acyl group Chemical group 0.000 description 3
- 150000001721 carbon Chemical group 0.000 description 3
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- YRHYCMZPEVDGFQ-UHFFFAOYSA-N methyl decanoate Chemical compound CCCCCCCCCC(=O)OC YRHYCMZPEVDGFQ-UHFFFAOYSA-N 0.000 description 2
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- XNLICIUVMPYHGG-UHFFFAOYSA-N pentan-2-one Chemical compound CCCC(C)=O XNLICIUVMPYHGG-UHFFFAOYSA-N 0.000 description 2
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Description
本発明は、糖脂質の製造方法に関するものである。より詳細には、微生物が生産する糖脂質型バイオサーファクタントであるマンノシルエリスリトールリピッドのエリスリトールの水酸基に脂肪酸エステルを有するトリアシル体糖脂質の製造方法に関するものである。 The present invention relates to a method for producing a glycolipid. More specifically, the present invention relates to a method for producing a triacyl glycolipid having a fatty acid ester at the hydroxyl group of erythritol of mannosyl erythritol lipid, which is a glycolipid type biosurfactant produced by a microorganism.
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。それゆえ、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することが期待されており、そのためには、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数種類と少ない。 Biosurfactants such as glycolipids are said to have high biodegradability, low toxicity, environmental friendliness, and novel physiological functions. Therefore, it is expected to be widely spread in the food industry, the cosmetic industry, the pharmaceutical industry, the chemical industry, the environmental field, etc. For that purpose, many types of biosurfactants are required. However, the number of biosurfactants discovered to date is as small as 20 or so.
大豆油を炭素源とする選択培地を用いて生産菌の探索を行いＣａｎｄｉｄａ ａｎｔａｒｃｔｉｃａ Ｔ−３４株がマンノシルエリスリトールリピッド（ＭＥＬ）を大量に生産することが見出されている（非特許文献１）。マンノシルエリスリトールリピッド（ＭＥＬ）は酵母が作る天然系の界面活性剤であり種々の生理作用が報告されている（非特許文献２）。また最近ではエリスリトールがマンニトールに代わったマンノシルマンニトールリピッド（ＭＭＬ）が見出されている（特許文献１）。バイオサーファクタントの用途としては、抗炎症剤及び抗アレルギー剤（特許文献２）、養毛・育毛剤（特許文献３）としての有用性や、抗菌作用（特許文献４）や表面張力低下作用（特許文献５）が知られている。
生分解性が高く、低毒性で環境に優しい、糖脂質型バイオサーファクタントのトリアシル体を、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、安価で容易に製造する方法を提供することを目的とする。 Glycolipid type biosurfactant triacyl derivative with high biodegradability, low toxicity and environmental friendliness is widely used in food industry, cosmetic industry, pharmaceutical industry, chemical industry, environmental field, etc. It aims to provide a way to do.
本発明者らは、上記課題を解決するために鋭意検討した結果、ＭＥＬ生産微生物の培養液中に存在するＭＥＬ、脂肪および加水分解酵素を利用することにより、高効率かつ安価にトリアシルＭＥＬを製造できることを見出し、本発明を完成させるに至った。 As a result of intensive studies to solve the above problems, the present inventors produce triacyl MEL with high efficiency and low cost by utilizing MEL, fat and hydrolase present in the culture solution of MEL-producing microorganisms. The present inventors have found that the present invention can be accomplished and have completed the present invention.
すなわち本発明は、以下の発明を提供するものである。 That is, the present invention provides the following inventions.
（１）エステル化反応またはエステル交換反応により、糖脂質型バイオサーファクタントに脂肪酸または脂肪酸誘導体を導入することを特徴とする糖脂質型バイオサーファクタントのトリアシル体製造方法。 (1) A method for producing a triacyl derivative of a glycolipid type biosurfactant, wherein a fatty acid or a fatty acid derivative is introduced into the glycolipid type biosurfactant by esterification reaction or transesterification reaction.
（２）糖脂質型バイオサーファクタントが、マンノシルエリスリトールリピッド（ＭＥＬ）またはマンノシルマンニトールリピッド（ＭＭＬ）であることを特徴とする（１）に記載の糖脂質型バイオサーファクタントのトリアシル体製造方法。 (2) The method for producing a triacyl derivative of a glycolipid type biosurfactant according to (1), wherein the glycolipid type biosurfactant is mannosyl erythritol lipid (MEL) or mannosyl mannitol lipid (MML).
（３）糖脂質型バイオサーファクタントのトリアシル体が、式（Ｉ） (3) A triacyl derivative of a glycolipid type biosurfactant is represented by the formula (I)
（式中、Ｒ１、Ｒ２およびＲ３はそれぞれ独立して炭化水素基または酸素原子含有炭化水素基を示し、マンノースの４位および６位の水酸基のいずれか一方、あるいは両方の水素原子がアセチル基に置換されていてもよい。）で表される糖脂質であることを特徴とする（２）に記載の糖脂質型バイオサーファクタントのトリアシル体製造方法。 (In the formula, R 1, R 2 and R 3 each independently represent a hydrocarbon group or an oxygen atom-containing hydrocarbon group, and one or both of the hydroxyl groups at the 4- and 6-positions of mannose are acetyl groups. The method for producing a triacyl derivative of a glycolipid type biosurfactant according to (2), wherein the glycolipid is a glycolipid represented by (2).
（４）上記式（Ｉ）のＲ１、Ｒ２およびＲ３の炭素数が６〜２０であることを特徴とする（３）に記載の糖脂質型バイオサーファクタントのトリアシル体製造方法。 (4) The method for producing a triacyl derivative of a glycolipid biosurfactant according to (3), wherein R1, R2 and R3 of the formula (I) have 6 to 20 carbon atoms.
（５）上記脂肪酸または脂肪酸誘導体が、油類、高級脂肪酸または合成エステル由来であることを特徴とする（１）〜（４）のいずれかに記載の糖脂質型バイオサーファクタントのトリアシル体製造方法。 (5) The method for producing a triacyl derivative of a glycolipid type biosurfactant according to any one of (1) to (4), wherein the fatty acid or fatty acid derivative is derived from oils, higher fatty acids or synthetic esters.
（６）疎水性環境下において、以下の（ａ）、（ｂ）および（ｃ）を混和することにより上記エステル化反応またはエステル交換反応が進行することを特徴とする（５）に記載の糖脂質型バイオサーファクタントのトリアシル体製造方法。
（ａ）糖脂質型バイオサーファクタント；
（ｂ）油類、高級脂肪酸および合成エステルから選択される１種または２種以上の混合物；
（ｃ）加水分解酵素。
(6) The sugar according to (5), wherein the esterification reaction or transesterification reaction proceeds by mixing the following (a), (b) and (c) in a hydrophobic environment: A method for producing a triacyl derivative of a lipid type biosurfactant.
(A) a glycolipid type biosurfactant;
(B) one or a mixture of two or more selected from oils, higher fatty acids and synthetic esters;
(C) Hydrolytic enzyme.
（７）糖脂質型バイオサーファクタント生産微生物を培養している培養液であって、油類、高級脂肪酸および合成エステルから選択される１種または２種以上の混合物が添加された培養液の凍結乾燥物中に含有される上記（ａ）、（ｂ）および（ｃ）を用いることを特徴とする（６）に記載の糖脂質型バイオサーファクタントのトリアシル体製造方法。 (7) Freeze-drying of a culture solution in which a microorganism producing a glycolipid biosurfactant is cultured, to which one or a mixture selected from oils, higher fatty acids and synthetic esters is added The method for producing a triacyl derivative of a glycolipid biosurfactant according to (6), wherein the above (a), (b) and (c) contained in a product are used.
（８）糖脂質型バイオサーファクタント生産微生物を培養している培養液であって、油類、高級脂肪酸および合成エステルから選択される１種または２種以上の混合物が添加された培養液中に生成する沈殿物に含有される上記（ａ）および（ｂ）を用いることを特徴とする（６）に記載の糖脂質型バイオサーファクタントのトリアシル体製造方法。 (8) A culture solution in which a microorganism producing a glycolipid biosurfactant is cultured, and produced in a culture solution to which one or a mixture selected from oils, higher fatty acids and synthetic esters is added (6) The method for producing a triacyl derivative of a glycolipid type biosurfactant according to (6), wherein the above (a) and (b) contained in a precipitate to be used are used.
（９）糖脂質型バイオサーファクタント生産微生物を培養している培養液であって、油類、高級脂肪酸および合成エステルから選択される１種または２種以上の混合物が添加された培養液の上清の凍結乾燥物に含有される上記（ｃ）を用いることを特徴とする（６）に記載の糖脂質型バイオサーファクタントのトリアシル体製造方法。 (9) A culture medium in which a microorganism producing a glycolipid biosurfactant is cultured, and the supernatant of the culture liquid to which one or a mixture selected from oils, higher fatty acids and synthetic esters is added The method for producing a triacyl derivative of a glycolipid type biosurfactant according to (6), wherein the above (c) contained in the lyophilized product is used.
（１０）上記加水分解酵素が、リパーゼ、プロテアーゼおよびエステラーゼから選択される少なくとも１種以上であることを特徴とする（６）に記載の糖脂質型バイオサーファクタントのトリアシル体製造方法。 (10) The method for producing a triacyl derivative of a glycolipid biosurfactant according to (6), wherein the hydrolase is at least one selected from lipase, protease and esterase.
（１１）上記エステル化反応またはエステル交換反応が、有機溶媒中で進行することを特徴とする（６）に記載の糖脂質型バイオサーファクタントのトリアシル体製造方法。 (11) The method for producing a triacyl derivative of a glycolipid type biosurfactant according to (6), wherein the esterification reaction or transesterification reaction proceeds in an organic solvent.
本発明により、糖脂質型バイオサーファクタントのトリアシル体を高効率かつ安価に製造することができる。また、糖脂質型バイオサーファクタントに任意の脂肪酸が結合したトリアシル体を容易に製造することができる。 According to the present invention, a triacyl body of a glycolipid type biosurfactant can be produced with high efficiency and at low cost. In addition, a triacyl derivative in which an arbitrary fatty acid is bound to a glycolipid type biosurfactant can be easily produced.
本発明に係る糖脂質型バイオサーファクタントのトリアシル体製造方法（以下「本発明に係る製造方法」と記載する）は、エステル化反応またはエステル交換反応により、糖脂質型バイオサーファクタントに脂肪酸または脂肪酸誘導体を導入するものであればよい。 The method for producing a triacyl derivative of a glycolipid type biosurfactant according to the present invention (hereinafter referred to as “production method according to the present invention”) is a method in which a fatty acid or a fatty acid derivative is added to a glycolipid type biosurfactant by esterification or transesterification. Anything can be introduced.
「バイオサーファクタント」とは生物によって生み出される界面活性能力や乳化能力を有する物質の総称であり、優れた界面活性や，高い生分解性を示すばかりでなく，様々な生理作用を有していることから合成界面活性剤とは異なる挙動・機能を発現する可能性がある。 “Biosurfactant” is a general term for substances having surface-active ability and emulsifying ability produced by living organisms, and not only exhibits excellent surface activity and high biodegradability, but also has various physiological functions. Therefore, there is a possibility of developing different behaviors and functions from synthetic surfactants.
「糖脂質型バイオサーファクタント」とは、微生物によって生産される天然系の界面活性剤のうち、糖質と脂肪酸部分からなるサーファクタントである。マンノシルエリスリトールリピッド（ＭＥＬ）、マンノシルマンニトールリピッド（ＭＭＬ）、ソホロリピッド、ラムノリピッド、トレハロースリピッドなどが知られている。いずれも生分解性が高く環境に負荷が少ない、生体に対しても安全性の高い素材である。なかでも、本発明に係る製造方法に用いられる糖脂質型バイオサーファクタントとしては、ＭＥＬまたはＭＭＬが好適である。 “Glycolipid type biosurfactant” is a surfactant composed of a carbohydrate and a fatty acid part among natural surfactants produced by microorganisms. Mannosyl erythritol lipid (MEL), mannosyl mannitol lipid (MML), sophorolipid, rhamnolipid, trehalose lipid and the like are known. All of them are highly biodegradable, have a low environmental impact, and are highly safe for living organisms. Among these, MEL or MML is preferable as the glycolipid type biosurfactant used in the production method according to the present invention.
ＭＥＬは、マンノースの４位および６位のアセチル基の有無からＭＥＬ−Ａ、ＭＥＬ−Ｂ、ＭＥＬ−ＣおよびＭＥＬ−Ｄの４種類が知られている。式（ＩＩ）にＭＥＬ−Ａの構造を示す。式（ＩＩ）中、Ｒ１およびＲ２は炭化水素基を示す。すなわち、ＭＥＬ−Ａは、式（ＩＩ）中、マンノースの２位、３位に炭素数５〜１９のアルカノイル基を有し、マンノースの４位、６位にアセチル基を有する化合物である。なお、ＭＥＬ−Ｂは式（ＩＩ）においてマンノースの４位のＡｃがＨであり、ＭＥＬ−Ｃは式（ＩＩ）においてマンノースの６位のＡｃがＨであり、ＭＥＬ−Ｄは式（ＩＩ）においてマンノースの４位および６位のＡｃがいずれもＨである。 Four types of MELs are known, MEL-A, MEL-B, MEL-C and MEL-D, depending on the presence or absence of acetyl groups at the 4th and 6th positions of mannose. Formula (II) shows the structure of MEL-A. In formula (II), R1 and R2 represent a hydrocarbon group. That is, MEL-A is a compound having an alkanoyl group having 5 to 19 carbon atoms at the 2nd and 3rd positions of mannose and the acetyl group at the 4th and 6th positions of mannose in the formula (II). In MEL-B, Ac at the 4-position of mannose is H in formula (II), MEL-C is H at 6-position of mannose in formula (II), and MEL-D is represented by formula (II). In the mannose, Ac at positions 4 and 6 is H.
ＭＭＬは上記式（ＩＩ）において、エリスリトールが炭素数の２個多いマンニトールに置換された構造を有する。 MML has a structure in which erythritol is substituted with mannitol having two carbon atoms in the above formula (II).
「糖脂質型バイオサーファクタントのトリアシル体を製造する」とは、例えばＭＥＬのエリスリトール部の水酸基やＭＭＬのマンニトール部の水酸基に脂肪酸または脂肪酸誘導体を導入し、３つの脂肪酸側鎖を有する糖脂質を製造することであり、「糖脂質型バイオサーファクタントのトリアシル化」と同義である。 “Manufacturing a triacyl derivative of a glycolipid biosurfactant” means, for example, introducing a fatty acid or a fatty acid derivative into the hydroxyl group of the erythritol part of MEL or the hydroxyl group of the mannitol part of MML to produce a glycolipid having three fatty acid side chains. It is synonymous with “triacylation of glycolipid biosurfactant”.
上記ＭＥＬのエリスリトール部の水酸基やＭＭＬのマンニトール部の水酸基への脂肪酸または脂肪酸誘導体の導入は、エステル化反応またはエステル交換反応により行われる。ただし、製造された糖脂質型バイオサーファクタントのトリアシル体の脂肪酸側鎖がＭＥＬのエリスリトール部の水酸基やＭＭＬのマンニトール部の水酸基とエステル結合していれば、その反応がエステル化反応であるかエステル交換反応であるかが特定される必要はない。 The fatty acid or the fatty acid derivative is introduced into the hydroxyl group of the erythritol part of MEL or the hydroxyl group of the mannitol part of MML by an esterification reaction or a transesterification reaction. However, if the fatty acid side chain of the triacyl body of the manufactured glycolipid type biosurfactant is ester-bonded with the hydroxyl group of the erythritol part of MEL or the hydroxyl group of the mannitol part of MML, the reaction is an esterification reaction or transesterification. It is not necessary to specify whether it is a reaction.
導入される脂肪酸は特に限定されず、飽和脂肪酸でも不飽和脂肪酸でもよい。不飽和脂肪酸の場合、複数の二重結合を有していてもよい。炭素鎖の炭素数は特に限定されないが、６〜２０が好ましい。また、炭素鎖は直鎖状であってもよく分岐鎖状であってもよい。脂肪酸誘導体としては、酸素原子を含む脂肪酸を挙げることができる。含まれる酸素原子の数および位置は限定されない。 The fatty acid to be introduced is not particularly limited, and may be a saturated fatty acid or an unsaturated fatty acid. In the case of an unsaturated fatty acid, it may have a plurality of double bonds. Although carbon number of a carbon chain is not specifically limited, 6-20 are preferable. The carbon chain may be linear or branched. Examples of fatty acid derivatives include fatty acids containing oxygen atoms. The number and position of oxygen atoms contained are not limited.
以下、ＭＥＬを例として、本発明に係る製造方法について説明するが、これに限定されるものではない。 Hereinafter, although the manufacturing method which concerns on this invention is demonstrated taking MEL as an example, it is not limited to this.
本発明に係る製造方法により製造されるＭＥＬのトリアシル体は、式（Ｉ）で表される構造を有する。 The triacyl form of MEL produced by the production method according to the present invention has a structure represented by the formula (I).
式（Ｉ）中、Ｒ１、Ｒ２およびＲ３はそれぞれ独立して炭化水素基または酸素原子含有炭化水素基を示し、マンノースの４位および６位の水酸基のいずれか一方、あるいは両方の水素原子がアセチル基に置換されていてもよい。炭化水素基は飽和結合のみを有していてもよく、不飽和結合を有していてもよい。不飽和結合を有している場合、複数の二重結合を有していてもよい。炭素鎖は直鎖状であってもよく分岐鎖状であってもよい。また、酸素原子含有炭化水素基の場合、含まれる酸素原子の数および位置は限定されない。 In formula (I), R 1, R 2 and R 3 each independently represent a hydrocarbon group or an oxygen atom-containing hydrocarbon group, and one or both of the hydroxyl groups at the 4-position and 6-position of mannose are acetylated It may be substituted with a group. The hydrocarbon group may have only a saturated bond or may have an unsaturated bond. When it has an unsaturated bond, it may have a plurality of double bonds. The carbon chain may be linear or branched. In the case of an oxygen atom-containing hydrocarbon group, the number and position of oxygen atoms contained are not limited.
式（Ｉ）中、Ｒ１、Ｒ２は炭素数が６〜２０であることが好ましく、脂肪族アシル基（ＲＣＯ−）としてマンノースの２位および３位の水酸基とエステル結合をしており、残りの水酸基にはアセチル基がエステル結合していてもよい。Ｒ３は炭素数が６〜２０であることが好ましく、脂肪族アシル基（ＲＣＯ−）としてエリスリトールの一級水酸基とエステル結合をしている。 In the formula (I), R1 and R2 preferably have 6 to 20 carbon atoms, and have an ester bond with the hydroxyl groups at the 2nd and 3rd positions of mannose as the aliphatic acyl group (RCO-), An acetyl group may be ester-bonded to the hydroxyl group. R3 preferably has 6 to 20 carbon atoms and has an ester bond with a primary hydroxyl group of erythritol as an aliphatic acyl group (RCO-).
本発明に係る製造方法において、ＭＥＬのエリスリトール部の水酸基に導入される脂肪酸または脂肪酸誘導体は油類、高級脂肪酸または合成エステル由来であることが好ましい。 In the production method according to the present invention, the fatty acid or fatty acid derivative introduced into the hydroxyl group of the erythritol part of MEL is preferably derived from oils, higher fatty acids or synthetic esters.
「油類」としては、植物油、動物油、鉱物油およびその硬化油であればよい。具体的には、アボカド油、オリーブ油、ゴマ油、ツバキ油、月見草油、タートル油、マカデミアンナッツ油、トウモロコシ油、ミンク油、ナタネ油、卵黄油、パーシック油、小麦胚芽油、サザンカ油、ヒマシ油、アマニ油、サフラワー油、綿実油、エノ油、大豆油、落花生油、茶実油、カヤ油、コメヌカ油、キリ油、ホホバ油、カカオ脂、ヤシ油、馬油、パーム油、パーム核油、牛脂、羊脂、豚脂、ラノリン、鯨ロウ、ミツロウ、カルナウバロウ、モクロウ、キャンデリラロウ、スクワラン等の動植物油およびその硬化油。流動パラフィン、ワセリン等の鉱物油、トリパルミチン酸グリセリン等の合成トリグリセリンが挙げられる。好ましくはアボカド油、オリーブ油、ゴマ油、ツバキ油、月見草油、タートル油、マカデミアンナッツ油、トウモロコシ油、ミンク油、ナタネ油、卵黄油、パーシック油、小麦胚芽油、サザンカ油、ヒマシ油、アマニ油、サフラワー油、綿実油、エノ油、大豆油、落花生油、茶実油、カヤ油、コメヌカ油、より好ましくはオリーブ油、大豆油である。 The “oils” may be vegetable oils, animal oils, mineral oils, and hardened oils thereof. Specifically, avocado oil, olive oil, sesame oil, camellia oil, evening primrose oil, turtle oil, macadamian nut oil, corn oil, mink oil, rapeseed oil, egg yolk oil, persic oil, wheat germ oil, sasanqua oil, castor oil , Linseed oil, safflower oil, cottonseed oil, eno oil, soybean oil, peanut oil, tea seed oil, kaya oil, rice bran oil, kiri oil, jojoba oil, cacao butter, palm oil, horse oil, palm oil, palm kernel oil Animal and vegetable oils such as beef tallow, sheep fat, pork tallow, lanolin, whale wax, beeswax, carnauba wax, molasses, candelilla wax, squalane and their hardened oils. Examples thereof include mineral oil such as liquid paraffin and petrolatum, and synthetic triglycerin such as glycerin tripalmitate. Preferably avocado oil, olive oil, sesame oil, camellia oil, evening primrose oil, turtle oil, macadamia nut oil, corn oil, mink oil, rapeseed oil, egg yolk oil, persic oil, wheat germ oil, southern oil, castor oil, flaxseed oil , Safflower oil, cottonseed oil, eno oil, soybean oil, peanut oil, tea seed oil, kaya oil, rice bran oil, and more preferably olive oil and soybean oil.
「高級脂肪酸」としては、例えばカプロン酸、カプリル酸、カプリン酸、ラウリン酸、ミリスチン酸、パルミチン酸、オレイン酸、リノール酸、リノレン酸、ステアリン酸、ベヘン酸、１２−ヒドロキシステアリン酸、イソステアリン酸、ウンデシン酸、トール酸、エイコサペンタエン酸、ドコサヘキサエン酸などが挙げられる。好ましくはラウリン酸、ミリスチン酸、パルミチン酸、オレイン酸、リノール酸、リノレン酸、ステアリン酸、ウンデシレン酸、より好ましくはオレイン酸、リノール酸、ウンデシレン酸である。 Examples of the “higher fatty acid” include caproic acid, caprylic acid, capric acid, lauric acid, myristic acid, palmitic acid, oleic acid, linoleic acid, linolenic acid, stearic acid, behenic acid, 12-hydroxystearic acid, isostearic acid, Examples include undecic acid, tolic acid, eicosapentaenoic acid, docosahexaenoic acid, and the like. Preferred are lauric acid, myristic acid, palmitic acid, oleic acid, linoleic acid, linolenic acid, stearic acid and undecylenic acid, and more preferred are oleic acid, linoleic acid and undecylenic acid.
「合成エステル」としては、例えば、カプロン酸メチル、カプリル酸メチル、カプリン酸メチル、ラウリン酸メチル、ミリスチン酸メチル、パルミチン酸メチル、オレイン酸メチル、リノール酸メチル、リノレン酸メチル、ステアリン酸メチル、ウンデシン酸メチル、カプロン酸エチル、カプリル酸エチル、カプリン酸エチル、ラウリン酸エチル、ミリスチン酸エチル、パルミチン酸エチル、オレイン酸エチル、リノール酸エチル、リノレン酸エチル、ステアリン酸エチル、ウンデシン酸エチル、カプロン酸ビニル、カプリル酸ビニル、カプリン酸ビニル、ラウリン酸ビニル、ミリスチン酸ビニル、パルミチン酸ビニル、オレイン酸ビニル、リノール酸ビニル、リノレン酸ビニル、ステアリン酸ビニル、ウンデシン酸ビニル、オクタン酸セチル、ミリスチン酸オクチルドデシル、ミリスチン酸イソプロピル、ミリスチン酸ミリスチル、パルミチン酸イソプロピル、ステアリン酸ブチル、ラウリン酸ヘキシル、オレンイ酸デシル、ジメチルオクタン酸、乳酸セチル、乳酸ミリスチル等が挙げられる。好ましくはラウリン酸メチル、ミリスチン酸メチル、パルミチン酸メチル、オレイン酸メチル、リノール酸メチル、リノレン酸メチル、ステアリン酸メチル、ウンデシレン酸メチル、より好ましくはオレイン酸メチル、リノール酸メチル、ウンデシレン酸メチルである。 Examples of the “synthetic ester” include methyl caproate, methyl caprylate, methyl caprate, methyl laurate, methyl myristate, methyl palmitate, methyl oleate, methyl linoleate, methyl linolenate, methyl stearate, undecine. Methyl acid, ethyl caproate, ethyl caprylate, ethyl caprate, ethyl laurate, ethyl myristate, ethyl palmitate, ethyl oleate, ethyl linoleate, ethyl linolenate, ethyl stearate, ethyl undecinate, vinyl caproate , Vinyl caprylate, vinyl caprate, vinyl laurate, vinyl myristate, vinyl palmitate, vinyl oleate, vinyl linoleate, vinyl linolenate, vinyl stearate, vinyl undecinate, cetyl octanoate , Octyldodecyl myristate, isopropyl myristate, myristyl myristate, isopropyl palmitate, butyl stearate, hexyl laurate, Oren'i acid decyl dimethyl octanoate, cetyl lactate, myristyl lactate, and the like. Preferred are methyl laurate, methyl myristate, methyl palmitate, methyl oleate, methyl linoleate, methyl linolenate, methyl stearate, methyl undecylate, more preferably methyl oleate, methyl linoleate, and methyl undecylate. .
本発明に係る製造方法は、疎水性環境下において、（ａ）糖脂質型バイオサーファクタント；（ｂ）油類、高級脂肪酸および合成エステルから選択される１種または２種以上の混合物；および（ｃ）加水分解酵素を混和することによりエステル化反応またはエステル交換反応が進行するものであればよい。 The production method according to the present invention comprises, in a hydrophobic environment, (a) a glycolipid type biosurfactant; (b) one or a mixture of two or more selected from oils, higher fatty acids and synthetic esters; and (c ) Any esterification reaction or transesterification reaction may be performed by mixing a hydrolase.
疎水性環境下は特に限定されないが、有機溶媒中において上記（ａ）、（ｂ）および（ｃ）を反応させることを挙げることができる。さらに、反応溶液中にモレキュラーシーブスを加えてもよい。有機溶媒としては、上記（ａ）、（ｂ）および（ｃ）を可溶化できるものであれば限定されない。全部を可溶化できなくても一部を可溶化できるものであればよい。また、有機溶媒は複数の有機溶媒の混合物でもよい。具体的には、例えば、メタノール、エタノール、プロパノール、ブタノール、アセトン、プロパノン、ブタノン、ペンタン−２−オン、１，２−エタンジオール、２，３−ブタンジオール、ジオキサン、アセトニトリル、２−メチル−ブタン−２−オール、第３級ブタノール、２−メチルプロパノール、４−ヒドロキシ−２−メチルペンタノン、テトラヒドロフラン、ヘキサン、ＤＭＦ、ＤＭＳＯ、ピリジン、メチルエチルケトンなどを挙げることができる。好ましくはアセトン、テトラヒドロフラン、第３級ブタノール、アセトニトリル、ジオキサン、より好ましくはアセトンである。 Although it does not specifically limit in hydrophobic environment, It can mention reacting said (a), (b) and (c) in an organic solvent. Furthermore, molecular sieves may be added to the reaction solution. The organic solvent is not limited as long as it can solubilize the above (a), (b) and (c). What is necessary is just to be able to solubilize a part even if not all can be solubilized. The organic solvent may be a mixture of a plurality of organic solvents. Specifically, for example, methanol, ethanol, propanol, butanol, acetone, propanone, butanone, pentan-2-one, 1,2-ethanediol, 2,3-butanediol, dioxane, acetonitrile, 2-methyl-butane -2-ol, tertiary butanol, 2-methylpropanol, 4-hydroxy-2-methylpentanone, tetrahydrofuran, hexane, DMF, DMSO, pyridine, methyl ethyl ketone and the like. Acetone, tetrahydrofuran, tertiary butanol, acetonitrile and dioxane are preferable, and acetone is more preferable.
また、疎水性環境は無溶媒でも構わない。 The hydrophobic environment may be solventless.
加水分解酵素としては、リパーゼ、プロテアーゼ、エステラーゼが挙げられる。これらの中から選択される少なくとも１種を用いることが好ましく、複数の加水分解酵素を用いてもよい。好ましくはリパーゼ、エステラーゼ、より好ましくはリパーゼである。 Examples of hydrolases include lipases, proteases, and esterases. It is preferable to use at least one selected from these, and a plurality of hydrolases may be used. Lipase and esterase are preferable, and lipase is more preferable.
本発明に係る製造方法においては、糖脂質型バイオサーファクタント生産微生物を培養している培養液であって、油類、高級脂肪酸および合成エステルから選択される１種または２種以上の混合物が添加された培養液の凍結乾燥物に含有される上記（ａ）、（ｂ）および（ｃ）を用いることが好ましい。 In the production method according to the present invention, a culture solution in which a microorganism producing a glycolipid biosurfactant is cultured, and one or a mixture of two or more selected from oils, higher fatty acids and synthetic esters is added. It is preferable to use the above (a), (b) and (c) contained in the freeze-dried product of the culture broth.
また、上記培養液中に生成する沈殿物に含有される上記（ａ）および（ｂ）を用い、（ｃ）を外部から添加してもよい。 Further, the above (a) and (b) contained in the precipitate generated in the culture solution may be used, and (c) may be added from the outside.
さらに、上記培養液の上清に含有される上記（ｃ）を用いることができる。 Furthermore, the above (c) contained in the supernatant of the culture solution can be used.
ＭＥＬ生産微生物としては、本発明者らはPseudozyma antarctica NBRC 10736 を用いているがこれに限定されず、Candida antarctica、Candida sp.、等を用いることができる。これらの微生物の培養により、容易にＭＥＬが得られることは当業者に周知である。 As the MEL-producing microorganism, the present inventors use Pseudozyma antarctica NBRC 10736, but are not limited thereto, and Candida antarctica, Candida sp., Etc. can be used. It is well known to those skilled in the art that MEL can be easily obtained by culturing these microorganisms.
上記ＭＥＬ生産微生物の培養に用いる培養液としては、酵母エキス、ペプトン等のＮ源、グルコース、フルクトース等のＣ源、および硝酸ナトリウム、リン酸水素二カリウム、硫酸マグネシウム７水塩等の無機塩類からなる一般的な組成の培養液を用いることができ、さらに油類、高級脂肪酸および合成エステルから選択される１種または２種以上の混合物が添加される。なかでも植物油脂を添加することが好ましい。植物油脂は特に限定されず、目的に応じて適宜選択することができる。例えば、大豆油、菜種油、コーン油、ピーナッツ油、綿実油、ベニバナ油、ゴマ油、オリーブ油、パーム油などが挙げられ、これらの中でも、大豆油がＭＥＬの生産効率（生産量、生産速度、および収率）を向上させることができる点で特に好ましい。これらは、１種を単独で、または２種以上を併用しても構わない。 The culture solution used for culturing the above-mentioned MEL-producing microorganism includes N sources such as yeast extract and peptone, C sources such as glucose and fructose, and inorganic salts such as sodium nitrate, dipotassium hydrogen phosphate and magnesium sulfate heptahydrate. A culture solution having a general composition can be used, and one or a mixture of two or more selected from oils, higher fatty acids and synthetic esters is added. Among these, it is preferable to add vegetable oils and fats. Vegetable fats and oils are not particularly limited, and can be appropriately selected depending on the purpose. For example, soybean oil, rapeseed oil, corn oil, peanut oil, cottonseed oil, safflower oil, sesame oil, olive oil, palm oil, etc. Among these, soybean oil is the production efficiency (production amount, production rate, and yield) of MEL. ) Is particularly preferred in that it can be improved. These may be used alone or in combination of two or more.
培養条件（培養温度、培養時間など）は、任意に設定できる。例えば、本発明者らは、Pseudozyma antarctica NBRC 10736 を３０℃、２００ｒｐｍ（攪拌回転）、０．５Ｌ／ｍｉｎ（Ａｉｒ）の条件で５Ｌ−ｊａｒを用いて１１日間培養し、大豆油を２日おきに追加添加するという条件で培養している。ただし、これに限定されるものではない。 The culture conditions (culture temperature, culture time, etc.) can be arbitrarily set. For example, the present inventors cultured Pseudozyma antarctica NBRC 10736 for 11 days using 5 L-jar under the conditions of 30 ° C., 200 rpm (stirring rotation), 0.5 L / min (Air), and soybean oil every two days. It is cultured under the condition that it is additionally added to. However, it is not limited to this.
ＭＥＬを製造するためにＭＥＬ生産微生物を上記のようにして培養した培養液（菌体を含む）を凍結乾燥すれば、上記（ａ）、（ｂ）および（ｃ）を含有する凍結乾燥物が得られる。培養液の凍結乾燥方法は特に限定されず、市販の凍結乾燥装置等を用いて公知の方法により行うことができる。培養液を凍結乾燥する理由は、加水分解酵素を触媒としてエステル化反応またはエステル交換反応を行う場合、水が存在していると加水分解反応が生じるためエステル化反応またはエステル交換反応が進行し難くなるためである。 If a culture solution (including cells) in which MEL-producing microorganisms are cultured as described above to produce MEL is freeze-dried, a freeze-dried product containing the above (a), (b) and (c) is obtained. can get. The lyophilization method of the culture solution is not particularly limited, and can be performed by a known method using a commercially available lyophilization apparatus or the like. The reason for freeze-drying the culture solution is that when an esterification reaction or transesterification reaction is performed using a hydrolase as a catalyst, the hydrolysis reaction occurs in the presence of water, so the esterification reaction or transesterification reaction does not proceed easily. It is to become.
また、培養液中に生成される沈殿物は、培養液を遠心分離し上清を除去することにより取得することができる。この沈殿物には上記（ａ）および（ｂ）が含まれている。得られた沈殿物は、そのままトリアシル体の製造に用いることができる。あるいは、沈殿物に酢酸エチル等の適当な抽出溶媒を添加し、十分攪拌後遠心分離して得られた上清をトリアシル体の製造に用いてもよい。さらに当該上清をエバポレーターで濃縮したものをトリアシル体の製造に用いてもよい。なお、遠心分離により除去した培養液上清中にも（ａ）が含まれるため、上清を酢酸エチルで抽出して得られた（ａ）をトリアシル体の製造に用いることができる。 Moreover, the precipitate produced | generated in a culture solution can be acquired by centrifuging a culture solution and removing a supernatant liquid. This precipitate contains the above (a) and (b). The obtained precipitate can be used for production of a triacyl body as it is. Alternatively, a supernatant obtained by adding an appropriate extraction solvent such as ethyl acetate to the precipitate, sufficiently stirring and centrifuging may be used for the production of the triacyl derivative. Furthermore, what concentrated the said supernatant with the evaporator may be used for manufacture of a triacyl body. In addition, since (a) is contained also in the culture-medium supernatant removed by centrifugation, (a) obtained by extracting a supernatant with ethyl acetate can be used for manufacture of a triacyl body.
また、培養液を遠心分離して上清を取得し、これを凍結乾燥することにより上記（ｃ）を含有する凍結乾燥物が得られる。培養液の凍結乾燥方法は特に限定されず、市販の凍結乾燥装置等を用いて公知の方法により行うことができる。 Further, the culture solution is centrifuged to obtain a supernatant, and this is freeze-dried to obtain a freeze-dried product containing the above (c). The lyophilization method of the culture solution is not particularly limited, and can be performed by a known method using a commercially available lyophilization apparatus or the like.
また、培養液からＭＥＬを抽出または精製し、これを上記（ａ）として用いることができる。ＭＥＬの抽出（回収）、精製方法としては特に制限はなく、目的に応じて適宜選定することができるが、例えば、培養液を遠心分離して油分を回収し、酢酸エチルエステルで抽出濃縮することにより回収できる。 Moreover, MEL is extracted or refine | purified from a culture solution, and this can be used as said (a). The MEL extraction (recovery) and purification method are not particularly limited and can be appropriately selected according to the purpose. For example, the culture solution is centrifuged to recover the oil, and extracted and concentrated with ethyl acetate. Can be recovered.
抽出溶媒としては、水、アルコール類（例えば、メタノール、無水エタノール、エタノールなどの低級アルコール、またはプロピレングリコール、１，３−ブチレングリコールなどの多価アルコール）、アセトンなどのケトン類、ジエチルエーテル、ジオキサン、アセトニトリル、酢酸エチルなどのエステル類、キシレン、ベンゼン、クロロホルムなどの有機溶媒を、単独であるいは２種類以上の混液を任意に組み合わせて使用することができ、また、各々の溶媒抽出物が組み合わされたものでも使用することができる。 Examples of the extraction solvent include water, alcohols (for example, lower alcohols such as methanol, absolute ethanol, and ethanol, or polyhydric alcohols such as propylene glycol and 1,3-butylene glycol), ketones such as acetone, diethyl ether, and dioxane. , Esters such as acetonitrile and ethyl acetate, and organic solvents such as xylene, benzene, and chloroform can be used alone or in any combination of two or more kinds, and each solvent extract is combined. Can also be used.
抽出方法は特に制限されるものはないが、通常、常温から常圧下での溶媒の沸点の範囲であればよく、抽出後は濾過またはイオン交換樹脂を用い、吸着・脱色・精製して溶液状、ペースト状、ゲル状、粉末状とすればよい。多くの場合は、そのままの状態で利用できるが、必要であれば、その効力に影響のない範囲でさらに脱臭、脱色などの精製処理を加えてもよい。脱臭・脱色等の精製処理手段としては、活性炭カラムなどを用いればよく、抽出物質により一般的に適用される通常の手段を任意に選択して行えばよい。必要に応じて、シリカゲルカラムを用いて精製することにより、純度の高いＭＥＬを得ることができる。なお、得られるＭＥＬの中に少量のトリアシルＭＥＬが含まれる。 There are no particular limitations on the extraction method, but usually it may be in the range of the boiling point of the solvent from room temperature to normal pressure. After extraction, it is filtered or adsorbed, decolored and purified using an ion exchange resin to form a solution. , Paste, gel, and powder. In many cases, it can be used as it is, but if necessary, further purification treatment such as deodorization and decolorization may be added as long as the effect is not affected. As a purification treatment means such as deodorization and decolorization, an activated carbon column or the like may be used, and a normal means generally applied depending on the extracted substance may be arbitrarily selected. If necessary, MEL having high purity can be obtained by purification using a silica gel column. A small amount of triacyl MEL is contained in the obtained MEL.
以下に、具体的なトリアシルＭＥＬ製造方法について説明するが、これらに限定されるものではない。 Although the specific triacyl MEL manufacturing method is demonstrated below, it is not limited to these.
（i）精製ＭＥＬの有機溶媒溶液に、油類、高級脂肪酸および合成エステルから選択される１種または２種以上の混合物（以下「油脂等」と記載する）並びに加水分解酵素を添加して反応させる場合
精製ＭＥＬは上述のように培養液から精製したＭＥＬを用いることができる。なお、最終的に活性炭カラムやシリカゲルカラムを用いて脱臭、脱色処理をした精製ＭＥＬに限定されず、その前段階において抽出後濾過またはイオン交換樹脂処理を施した粗精製ＭＥＬも使用できる。
(I) One or a mixture selected from oils, higher fatty acids and synthetic esters (hereinafter referred to as “oil and fat”) and a hydrolase are added to an organic solvent solution of purified MEL for reaction. In the case of using purified MEL, MEL purified from the culture solution as described above can be used. In addition, it is not limited to the refined MEL which finally deodorized and decolored using the activated carbon column or the silica gel column, The rough refined MEL which performed the filtration after the extraction or the ion exchange resin process in the previous step can also be used.
有機溶媒は特に限定されないが、アセトンが好ましい。 The organic solvent is not particularly limited, but acetone is preferable.
加水分解酵素は、市販のリパーゼ（例えば、ノボザイム４３５（ノボザイムズ社製）など）を好適に用いることができる。または、上述の培養液上清の凍結乾燥物（以下「上清凍結乾燥物」と記載する）を用いてもよい。上清凍結乾燥物を用いると加水分解酵素を購入する必要がないためコストを低く抑えることができる。 As the hydrolase, a commercially available lipase (for example, Novozyme 435 (manufactured by Novozymes) etc.) can be suitably used. Alternatively, a freeze-dried product of the above-mentioned culture supernatant (hereinafter referred to as “supernatant freeze-dried product”) may be used. When the supernatant lyophilized product is used, it is not necessary to purchase a hydrolase, so that the cost can be kept low.
添加する油脂等は特に限定されない。ＭＥＬ製造用培養液に添加した油脂等と同一のものを添加してもよく、異なるものを添加してもよい。ここで添加した油脂等によりＭＥＬのエリスリトール部に導入される脂肪酸基が決定されるので、添加する油脂等を適宜選択することにより、所望の脂肪酸がエリスリトール部に導入されたトリアシルＭＥＬを製造することが可能となる。 The fats and oils to be added are not particularly limited. The same oil or fat added to the culture solution for MEL production may be added, or a different one may be added. Since the fatty acid group introduced into the erythritol part of MEL is determined by the added fat or the like here, a triacyl MEL in which the desired fatty acid is introduced into the erythritol part is selected by appropriately selecting the added fat or the like. Is possible.
この製造方法の場合、反応温度は１０〜１００℃、好ましくは２０〜５０℃、より好ましくは２５〜４０℃で、１日〜７日間攪拌すればよい。また、反応液にモレキュラーシーブスを添加してもよい。この製造方法により、材料として添加したＭＥＬがほぼ定量的にトリアシル体となる。この知見は当業者でも容易に予測できないことであった。 In the case of this production method, the reaction temperature is 10 to 100 ° C., preferably 20 to 50 ° C., more preferably 25 to 40 ° C., and stirring may be performed for 1 to 7 days. Further, molecular sieves may be added to the reaction solution. By this manufacturing method, MEL added as a material becomes a triacyl body almost quantitatively. This finding was not easily predicted by those skilled in the art.
（ii）上述の培養液中の沈殿物（以下「培養沈殿物」と記載する）を用いる場合
培養沈殿物中にはＭＥＬおよび油脂等が含まれている。この培養沈殿物を有機溶媒に溶解し、これに加水分解酵素を添加して反応させればよい。さらにモレキュラーシーブスを添加してもよい。
(Ii) When using the above-mentioned precipitate in the culture solution (hereinafter referred to as “culture precipitate”) The culture precipitate contains MEL, fats and oils, and the like. The culture precipitate may be dissolved in an organic solvent, and a hydrolase may be added thereto for reaction. Further, molecular sieves may be added.
有機溶媒は特に限定されないが、アセトンが好ましい。加水分解酵素は、市販のリパーゼ（例えば、ノボザイム４３５（ノボザイムズ社製）など）を好適に用いることができる。または、上記上清凍結乾燥物を用いてもよい。 The organic solvent is not particularly limited, but acetone is preferable. As the hydrolase, a commercially available lipase (for example, Novozyme 435 (manufactured by Novozymes) etc.) can be suitably used. Alternatively, the supernatant lyophilized product may be used.
この製造方法の場合、反応温度は１０〜１００℃、好ましくは２０〜５０℃、より好ましくは２５〜４０℃で、１日〜７日間攪拌すればよい。これにより、材料として添加したＭＥＬがほぼ定量的にトリアシル体となる。この知見は当業者でも容易に予測できないことであった。 In the case of this production method, the reaction temperature is 10 to 100 ° C., preferably 20 to 50 ° C., more preferably 25 to 40 ° C., and stirring may be performed for 1 to 7 days. Thereby, MEL added as a material becomes a triacyl body almost quantitatively. This finding was not easily predicted by those skilled in the art.
この製造方法において、市販の加水分解酵素を用いずに上清凍結乾燥物を用いれば、ＭＥＬを製造するための培養液のみからトリアシルＭＥＬを製造することができ、何ら他の原料を購入する必要がないため、安価にトリアシルＭＥＬを製造することが可能となる。 In this production method, if a supernatant lyophilized product is used without using a commercially available hydrolase, triacyl MEL can be produced only from the culture solution for producing MEL, and it is necessary to purchase any other raw materials. Therefore, triacyl MEL can be produced at low cost.
（iii）上述の培養液の凍結乾燥物（以下「培養液凍結乾燥物」と記載する）を用いる場合
培養液凍結乾燥物にはＭＥＬ、油脂等および加水分解酵素が含まれている。したがって、培養液凍結乾燥物を有機溶媒に溶解し、反応させればよい。さらにモレキュラーシーブスを添加してもよい。有機溶媒は特に限定されないが、アセトンが好ましい。
(Iii) When using the lyophilized product of the above-mentioned culture solution (hereinafter referred to as “cultured solution lyophilized product”) The cultured solution lyophilized product contains MEL, fats and oils, and hydrolase. Therefore, the lyophilized culture solution may be dissolved in an organic solvent and reacted. Further, molecular sieves may be added. The organic solvent is not particularly limited, but acetone is preferable.
この製造方法の場合、反応温度は１０〜１００℃、好ましくは２０〜５０℃、より好ましくは２５〜４０℃で、１日〜７日間攪拌すればよい。 In the case of this production method, the reaction temperature is 10 to 100 ° C., preferably 20 to 50 ° C., more preferably 25 to 40 ° C., and stirring may be performed for 1 to 7 days.
この製造方法は、ＭＥＬを製造するための培養液を凍結乾燥するのみで、何ら他の原料を購入する必要がない。また、培養液を上清と沈殿物とに分けてそれぞれ別の操作を行う必要もない。したがって、非常に簡便かつ低コストで効率よくトリアシルＭＥＬを製造することが可能となる。 In this production method, only the culture solution for producing MEL is freeze-dried, and no other raw materials need to be purchased. Further, it is not necessary to divide the culture solution into a supernatant and a precipitate and perform different operations for each. Therefore, it is possible to produce triacyl MEL very easily and efficiently at low cost.
以上のように、本発明に係る製造方法を用いれば、副生成物がほとんど生成しないトリアシルＭＥＬの低コスト製造が可能となる。また、便宜上ＭＥＬを例として本発明を説明したが、トリアシルＭＥＬの事例を示せば、他の糖脂質型バイオサーファクタント（例えば、ＭＭＬなど）に応用できることを、当業者は容易に理解する。 As described above, if the production method according to the present invention is used, low-cost production of a triacyl MEL that hardly produces by-products can be achieved. Further, although the present invention has been described by taking MEL as an example for convenience, those skilled in the art will easily understand that if a case of triacyl MEL is shown, it can be applied to other glycolipid type biosurfactants (for example, MML).
また、本発明に係る製造方法を用いれば、添加する油類や高級脂肪酸や合成エステルを任意に変えることにより、培養液中から得られない新規なトリアシルＭＥＬも容易にデザインできる。この点で、本発明は、糖脂質型バイオサーファクタントを応用していく上で、単品ではなくいくつかの物性が異なる材を提供する方法を示したものである。一例として挙げたトリアシルＭＥＬは、従来のＭＥＬに比べて疎水性が高く、疎水性を必要とする用途に応じたＭＥＬ類の一つとして産業に寄与するものである。 Moreover, if the manufacturing method which concerns on this invention is used, the novel triacyl MEL which cannot be obtained from a culture solution can also be designed easily by changing oils, a higher fatty acid, and synthetic ester to add arbitrarily. In this respect, the present invention shows a method for providing a material having several different physical properties instead of a single product when applying a glycolipid type biosurfactant. The triacyl MEL mentioned as an example has higher hydrophobicity than the conventional MEL, and contributes to the industry as one of MELs corresponding to applications that require hydrophobicity.
なお、発明を実施するための最良の形態の項においてなした具体的な実施態様および以下の実施例は、あくまでも、本発明の技術内容を明らかにするものであって、そのような具体例にのみ限定して狭義に解釈されるべきものではなく、当業者は、本発明の精神および添付の特許請求の範囲内で変更して実施することができる。 It should be noted that the specific embodiments and the following examples made in the section of the best mode for carrying out the invention are intended to clarify the technical contents of the present invention, and to such specific examples. It is not to be construed as limiting in any way whatsoever, and those skilled in the art can implement the invention within the spirit of the invention and the scope of the appended claims.
以下の実施例により、本発明を更に詳細に説明するが、本発明は、これらに何ら限定されるものではない。 The following examples further illustrate the present invention in detail but are not to be construed to limit the scope thereof.
〔実施例１：大豆油を原料にしたＭＥＬ―Ａ、トリアシルＭＥＬの製造〕
種菌培養はＰｓｅｕｄｏｚｙｍａ ａｎｔａｒｃｔｉｃａ ＮＢＲＣ １０７３６を種培地（２０ｍｌ／５００ｍｌ容坂口フラスコ×４本）に１ｒｏｏｐ植菌して実施した。３０℃、１８０ｒｐｍにて一晩培養した。得られた培養液を種菌とした。種培地組成は４％ Ｇｌｕｃｏｓｅ、０．３％ ＮａＮＯ３、０．０２％ ＭｇＳＯ４・Ｈ２Ｏ、０．０２％ ＫＨ２ＰＯ４、０．１％ ｙｅａｓｔ ｅｘｔｒａｃｔとした。ＭＥＬ製造培養は上記種菌１００ｍｌを生産培地２．０Ｌ（５Ｌ−ｊａｒ）に植菌し、３０℃、２００ｒｐｍ（攪拌回転）、０．５Ｌ／ｍｉｎ（Ａｉｒ）の条件で５Ｌ−ｊａｒを用いて１１日間培養し、大豆油３０ｇに等量の水を加え２日おきに追加添加をした。生産培地組成は、３％ 大豆油、０．０２％ ＭｇＳＯ４・Ｈ２Ｏ、０．０２％ ＫＨ２ＰＯ４、０．１％ ｙｅａｓｔ ｅｘｔｒａｃｔとした。得られた培養液２Ｌを５００ｍｌ容遠心管×６本に分注し、遠心分離（６５００ｒｐｍ、３０ｍｉｎ）した。上清を取り除き、沈殿（菌体）を回収した。沈殿に、５０ｍｌの酢酸エチルを加え、十分攪拌後、遠心（８５００ｒｐｍ、３０ｍｉｎ）し、沈殿と上清に分け、上清をエバポーレーターで濃縮し、粗抽出物８６ｇを得た。シリカゲルを用いて、ヘキサン：アセトン＝５：１、ヘキサン：アセトン＝１：２で溶出し、ＭＥＬ―Ａ８０ｇとトリアシルＭＥＬとしてＭＥＬのエリスリトールリノール酸エステル体（ＬＩ−ＭＥＬ（ＳＢ））３．５ｇを単離し、その構造をＮＭＲで確認した。Ｃ−ＮＭＲ解析の結果、すなわち、大豆油から製造したＭＥＬのトリアシル体のＣ−ＮＭＲ（溶媒：ＤＭＳＯ−ｄ６における各炭素原子のケミカルシフト値（ｐｐｍ）を、表１に示した。なお、表１のＥ１，Ｅ２，Ｅ３，Ｅ４，Ｍ１，Ｍ２，Ｍ３，Ｍ４，Ｍ５，Ｍ６は下記式（ＩＩＩ）のマンノシルエリスリトール骨格の炭素原子を示す。
[Example 1: Production of MEL-A and triacyl MEL from soybean oil]
Inoculum culture was performed by inoculating Pseudozyma antarctica NBRC 10736 in a seed medium (20 ml / 500 ml Sakaguchi flask × 4) for 1 loop. The cells were cultured overnight at 30 ° C. and 180 rpm. The obtained culture broth was used as an inoculum. The composition of the seed medium was 4% Glucose, 0.3% NaNO 3 , 0.02% MgSO 4 · H 2 O, 0.02% KH 2 PO 4 , and 0.1% yeast extract. In MEL production culture, 100 ml of the above inoculum was inoculated into 2.0 L (5 L-jar) of the production medium, and 5 L-jar was used under the conditions of 30 ° C., 200 rpm (stirring rotation), 0.5 L / min (Air). After culturing for a day, an equal amount of water was added to 30 g of soybean oil and added every two days. The composition of the production medium was 3% soybean oil, 0.02% MgSO 4 · H 2 O, 0.02% KH 2 PO 4 , and 0.1% yeast extract. 2 L of the obtained culture solution was dispensed into 6 x 500 ml centrifuge tubes and centrifuged (6500 rpm, 30 min). The supernatant was removed, and the precipitate (bacteria) was collected. 50 ml of ethyl acetate was added to the precipitate, and after sufficient stirring, it was centrifuged (8500 rpm, 30 min), divided into a precipitate and a supernatant, and the supernatant was concentrated with an evaporator to obtain 86 g of a crude extract. Elution with hexane: acetone = 5: 1, hexane: acetone = 1: 2 using silica gel, 80 g of MEL-A and 3.5 g of erythritol linoleate (LI-MEL (SB)) of MEL as triacyl MEL Isolated and its structure confirmed by NMR. As a result of C-NMR analysis, that is, C-NMR (chemical shift value (ppm) of each carbon atom in DMSO-d6 of a MEL triacyl derivative produced from soybean oil is shown in Table 1. E1, E2, E3, E4, M1, M2, M3, M4, M5 and M6 in 1 represent carbon atoms of a mannosylerythritol skeleton of the following formula (III).
〔実施例２：オリーブ油を原料にしたＭＥＬ―Ａ、トリアシルＭＥＬの製造〕
種菌培養はＰｓｅｕｄｏｚｙｍａ ａｎｔａｒｃｔｉｃａ ＮＢＲＣ １０７３６を種培地（２０ｍｌ／５００ｍｌ容坂口フラスコ×４本）に１ｒｏｏｐ植菌して実施した。３０℃、１８０ｒｐｍにて一晩培養した。得られた培養液を種菌とした。種培地組成は４％ Ｇｌｕｃｏｓｅ、０．３％ ＮａＮＯ３、０．０２％ ＭｇＳＯ４・Ｈ２Ｏ、０．０２％ ＫＨ２ＰＯ４、０．１％ ｙｅａｓｔ ｅｘｔｒａｃｔとした。ＭＥＬ製造培養は上記種菌１００ｍｌを生産培地２．０Ｌ（５Ｌ−ｊａｒ）に植菌し、３０℃、２００ｒｐｍ（攪拌回転）、０．５Ｌ／ｍｉｎ（Ａｉｒ）の条件で５Ｌ−ｊａｒを用いて１１日間培養し、オリーブ油３０ｇに等量の水を加え２日おきに追加添加をした。生産培地組成は、３％ オリーブ油、０．０２％ ＭｇＳＯ４・Ｈ２Ｏ、０．０２％ ＫＨ２ＰＯ４、０．１％ ｙｅａｓｔ ｅｘｔｒａｃｔとした。得られた培養液２Ｌを５００ｍｌ容遠心管×６本に分注し、遠心分離（６５００ｒｐｍ、３０ｍｉｎ）した。上清を取り除き、沈殿（菌体）を回収した。沈殿に、５０ｍｌの酢酸エチルを加え、十分攪拌後、遠心（８５００ｒｐｍ、３０ｍｉｎ）し、沈殿と上清に分け、上清をエバポレーターで濃縮し、粗抽出物７０ｇを得た。シリカゲルを用いて、ヘキサン：アセトン＝５：１、ヘキサン：アセトン＝１：２で溶出し、ＭＥＬ―Ａ６０ｇとトリアシルＭＥＬとしてＭＥＬのエリスリトールオレイン酸エステル体（ＯＬ−ＭＥＬ（ＯＬ））２．５ｇを単離し、その構造をＮＭＲで確認した。Ｃ−ＮＭＲ解析の結果、すなわち、オリーブ油から製造したＭＥＬのトリシル体のＣ−ＮＭＲ（溶媒：ＤＭＳＯ−ｄ６における各炭素原子のケミカルシフト値（ｐｐｍ）を、表２に示した。表２のＥ１，Ｅ２，Ｅ３，Ｅ４，Ｍ１，Ｍ２，Ｍ３，Ｍ４，Ｍ５，Ｍ６は下記式（ＩＶ）のマンノシルエリスリトール骨格の炭素原子を示す。
[Example 2: Production of MEL-A and triacyl MEL from olive oil]
Inoculum culture was performed by inoculating Pseudozyma antarctica NBRC 10736 in a seed medium (20 ml / 500 ml Sakaguchi flask × 4) for 1 loop. The cells were cultured overnight at 30 ° C. and 180 rpm. The obtained culture broth was used as an inoculum. The composition of the seed medium was 4% Glucose, 0.3% NaNO 3 , 0.02% MgSO 4 · H 2 O, 0.02% KH 2 PO 4 , and 0.1% yeast extract. In MEL production culture, 100 ml of the above inoculum was inoculated into 2.0 L (5 L-jar) of the production medium, and 5 L-jar was used under the conditions of 30 ° C., 200 rpm (stirring rotation), 0.5 L / min (Air). After culturing for days, an equal amount of water was added to 30 g of olive oil and added every two days. The composition of the production medium was 3% olive oil, 0.02% MgSO 4 · H 2 O, 0.02% KH 2 PO 4 , and 0.1% yeast extract. 2 L of the obtained culture solution was dispensed into 6 x 500 ml centrifuge tubes and centrifuged (6500 rpm, 30 min). The supernatant was removed, and the precipitate (bacteria) was collected. 50 ml of ethyl acetate was added to the precipitate, and after sufficient stirring, it was centrifuged (8500 rpm, 30 min), divided into a precipitate and a supernatant, and the supernatant was concentrated with an evaporator to obtain 70 g of a crude extract. Using silica gel, eluting with hexane: acetone = 5: 1, hexane: acetone = 1: 2, MEL-A 60 g and 2.5 g of erythritol oleate ester (OL-MEL (OL)) as triacyl MEL Isolated and its structure confirmed by NMR. As a result of C-NMR analysis, that is, C-NMR (chemical shift value (ppm) of each carbon atom in the solvent: DMSO-d6) of the trisyl form of MEL produced from olive oil is shown in Table 2. E1 in Table 2 , E2, E3, E4, M1, M2, M3, M4, M5 and M6 represent carbon atoms of the mannosylerythritol skeleton of the following formula (IV).
〔実施例３：培養粗抽出物への酵素添加によるトリアシルＭＥＬの合成〕
実施例１の大豆油で培養して得られた粗抽出物１．５ｇをアセトン１０ｍｌに溶かし、モレキュラーシーブス４Ａ １ｇ、ノボザイム４３５ ２００ｍｇを加え、室温（２０℃）で３日間撹拌した。反応液を濾過して酵素粒子を除去した後、エバポレーターを用いてアセトンを留去し、シリカゲルカラムを用いて酢酸エチル：ヘキサン（１：５）で流して生成物を単離した。トリアシルＭＥＬとしてＭＥＬのエリスリトールリノール酸エステル体（ＬＩ−ＭＥＬ（ＳＢ））の淡黄色油状生成物１．５ｇを得た。なお、モレキュラーシーブスを加えなくてもよいがその場合は反応液中にフリーの脂肪酸が残存するので、反応液を酢酸エチルと水で分液し、水層を除去する操作が必要である。Ｃ−ＮＭＲ解析の結果は、上記実施例１で単離したＬＩ−ＭＥＬ（ＳＢ）の値（表１参照）と同様の値であった。
[Example 3: Synthesis of triacyl MEL by adding enzyme to cultured crude extract]
1.5 g of the crude extract obtained by culturing with soybean oil of Example 1 was dissolved in 10 ml of acetone, 1 g of molecular sieves 4A and 200 mg of Novozyme 435 were added, and the mixture was stirred at room temperature (20 ° C.) for 3 days. After the reaction solution was filtered to remove enzyme particles, acetone was distilled off using an evaporator, and the product was isolated by flowing with ethyl acetate: hexane (1: 5) using a silica gel column. As a triacyl MEL, 1.5 g of a pale yellow oily product of erythritol linoleic acid ester of MEL (LI-MEL (SB)) was obtained. Although molecular sieves need not be added, in that case, since free fatty acid remains in the reaction solution, an operation of separating the reaction solution with ethyl acetate and water and removing the aqueous layer is necessary. The result of C-NMR analysis was the same as the value of LI-MEL (SB) isolated in Example 1 (see Table 1).
〔実施例４：培養液を用いたトリアシルＭＥＬの合成〕
実施例１で得られた大豆油培養液２０ｍｌをそのまま凍結乾燥後、アセトン１０ｍｌに溶かし、モレキュラーシーブス４Ａ １ｇを加え、室温（２０℃）で３日間撹拌した。エバポレーターを用いてアセトンを留去し、シリカゲルカラムを用いて酢酸エチル：ヘキサン（１：５）で流して生成物を単離した。トリアシルＭＥＬとしてＭＥＬのエリスリトールリノール酸エステル体（ＬＩ−ＭＥＬ（ＳＢ））の淡黄色油状生成物０．８ｇを得た。なお、モレキュラーシーブスを加えなくてもよいがその場合は反応液中にフリーの脂肪酸が残存するので、反応液を酢酸エチルと水で分液し、水層を除去する操作が必要である。Ｃ−ＮＭＲ解析の結果は、上記実施例１で単離したＬＩ−ＭＥＬ（ＳＢ）の値（表１参照）と同様の値であった。
[Example 4: Synthesis of triacyl MEL using culture medium]
20 ml of the soybean oil culture solution obtained in Example 1 was freeze-dried as it was, dissolved in 10 ml of acetone, 1 g of molecular sieves 4A was added, and the mixture was stirred at room temperature (20 ° C.) for 3 days. Acetone was distilled off using an evaporator, and the product was isolated by flowing with ethyl acetate: hexane (1: 5) using a silica gel column. As a triacyl MEL, 0.8 g of a pale yellow oily product of erythritol linoleic acid ester of MEL (LI-MEL (SB)) was obtained. Although molecular sieves need not be added, in that case, since free fatty acid remains in the reaction solution, an operation of separating the reaction solution with ethyl acetate and water and removing the aqueous layer is necessary. The result of C-NMR analysis was the same as the value of LI-MEL (SB) isolated in Example 1 (see Table 1).
〔実施例５：オレイン酸を有するトリアシルＭＥＬの酵素合成〕
（１）大豆油を原料に実施例１で得られたＭＥＬ−Ａ１．４８ｇをアセトン１０ｍｌに溶かし、オリーブ油４ｍｌ、モレキュラーシーブス４Ａ １ｇ、ノボザイム４３５ ２００ｍｇを加え、室温（２０℃）で３日間撹拌した。反応液を濾過して酵素粒子を除去した後、エバポレーターを用いてアセトンを留去し、シリカゲルカラムを用いて酢酸エチル：ヘキサン（１：５）で流して生成物を単離した。トリアシルＭＥＬとしてＭＥＬのエリスリトールオレイン酸エステル体（ＯＬ−ＭＥＬ（ＳＢ））の淡黄色油状生成物１．４ｇを得た。Ｃ−ＮＭＲ解析の結果は表１に示した。なお、モレキュラーシーブスを加えなくてもよいがその場合は反応液中にフリーの脂肪酸が残存するので、反応液を酢酸エチルと水で分液し、水層を除去する操作が必要である。
[Example 5: Enzymatic synthesis of triacyl MEL having oleic acid]
(1) MEL-A (1.48 g) obtained in Example 1 using soybean oil as a raw material was dissolved in 10 ml of acetone, 4 ml of olive oil, 1 g of molecular sieves 4A and 200 mg of Novozyme 435 were added, and the mixture was stirred at room temperature (20 ° C.) for 3 days. . After the reaction solution was filtered to remove enzyme particles, acetone was distilled off using an evaporator, and the product was isolated by flowing with ethyl acetate: hexane (1: 5) using a silica gel column. As a triacyl MEL, 1.4 g of a pale yellow oily product of MEL erythritol oleate (OL-MEL (SB)) was obtained. The results of C-NMR analysis are shown in Table 1. Although molecular sieves need not be added, in that case, since free fatty acid remains in the reaction solution, an operation of separating the reaction solution with ethyl acetate and water and removing the aqueous layer is necessary.
（２）オリーブ油を原料に実施例２で得られたＭＥＬ−Ａを用いる以外は上記（１）と同様に行った場合も、トリアシルＭＥＬとしてエリスリトールオレイン酸エステル体（ＯＬ−ＭＥＬ（ＯＬ））１．４ｇが得られた。Ｃ−ＮＭＲ解析の結果は、上記実施例２で単離したＯＬ−ＭＥＬ（ＯＬ）の値（表２参照）と同様の値であった。 (2) Erythritol oleate ester (OL-MEL (OL)) 1 as triacyl MEL when the same procedure as in (1) above is used except that olive oil is used as the raw material and MEL-A obtained in Example 2 is used. .4 g was obtained. The result of C-NMR analysis was the same as the value of OL-MEL (OL) isolated in Example 2 (see Table 2).
〔実施例６：リノール酸を有するトリアシルＭＥＬの酵素合成〕
（１）上記実施例５（１）において、オリーブ油を大豆油に変更した以外は同様の方法で反応を行い、トリアシルＭＥＬとしてエリスリトールリノール酸エステル体（ＬＩ−ＭＥＬ（ＳＢ））の淡黄色油状生成物１．３ｇを得た。Ｃ−ＮＭＲ解析の結果は、上記実施例１で単離したＬＩ−ＭＥＬ（ＳＢ）の値（表１参照）と同様の値であった。
[Example 6: Enzymatic synthesis of triacyl MEL having linoleic acid]
(1) The reaction was carried out in the same manner as in Example 5 (1) except that olive oil was changed to soybean oil, and a light yellow oily production of erythritol linoleic acid ester (LI-MEL (SB)) as triacyl MEL was produced. 1.3 g of product was obtained. The result of C-NMR analysis was the same as the value of LI-MEL (SB) isolated in Example 1 (see Table 1).
（２）上記（１）において、実施例１で得られたＭＥＬ−Ａを実施例２で得られたＭＥＬ−Ａに変更した以外は同様の方法で反応行った場合も、トリアシルＭＥＬとしてエリスリトールリノール酸エステル体（ＬＩ−ＭＥＬ（ＯＬ））１．４５ｇが得られた。Ｃ−ＮＭＲ解析の結果は表２に示した。 (2) In the above (1), erythritol linole as triacyl MEL is obtained when the reaction is carried out in the same manner except that MEL-A obtained in Example 1 is changed to MEL-A obtained in Example 2. 1.45 g of acid ester form (LI-MEL (OL)) was obtained. The results of C-NMR analysis are shown in Table 2.
〔実施例７：ウンデシレン酸を有するトリアシルＭＥＬの酵素合成〕
（１）上記実施例５（１）において、オリーブ油をウンデシレン酸に変更した以外は同様の方法で反応を行い、ウンデシレン酸エステルをエリスリトール部に有するトリアシルＭＥＬ（ＵＮＤＥ−ＭＥＬ（ＳＢ））の淡黄色油状生成物１．３５ｇを得た。Ｃ−ＮＭＲ解析の結果は表１に示した。
[Example 7: Enzymatic synthesis of triacyl MEL having undecylenic acid]
(1) The reaction in Example 5 (1) except that olive oil was changed to undecylenic acid, and the reaction was carried out in the same manner, and a light yellow of triacyl MEL (UNDE-MEL (SB)) having an undecylenic acid ester in the erythritol part. 1.35 g of oily product was obtained. The results of C-NMR analysis are shown in Table 1.
（２）上記（１）において、実施例１で得られたＭＥＬ−Ａを実施例２で得られたＭＥＬ−Ａに変更した以外は同様の方法で反応行った場合も、ウンデシレン酸エステルをエリスリトール部に有するトリアシルＭＥＬ（ＵＮＤＥ−ＭＥＬ（ＯＬ））１．２５ｇが得られた。Ｃ−ＮＭＲ解析の結果は表２に示した。 (2) In the above (1), when the reaction was carried out in the same manner except that MEL-A obtained in Example 1 was changed to MEL-A obtained in Example 2, undecylenate was converted to erythritol. 1.25 g of triacyl MEL (UNDE-MEL (OL)) in part was obtained. The results of C-NMR analysis are shown in Table 2.
〔実施例８：ステアリン酸を有するトリアシルＭＥＬの酵素合成〕
上記実施例５（２）において、オリーブ油をステアリン酸変更した以外は同様の方法で反応を行い、ステアリン酸エステルをエリスリトール部に有するトリアシルＭＥＬ（ＳＴＥ−ＭＥＬ（ＯＬ））の淡黄色油状生成物１．５ｇを得た。Ｃ−ＮＭＲ解析の結果は表２に示した。
[Example 8: Enzymatic synthesis of triacyl MEL having stearic acid]
A light yellow oily product 1 of triacyl MEL (STE-MEL (OL)) having a stearate ester in the erythritol part was reacted in Example 5 (2) except that the stearic acid was changed to olive oil. .5 g was obtained. The results of C-NMR analysis are shown in Table 2.
表１および表２より、大豆油あるいはオリーブ油を原料に製造したＭＥＬのケミカルシフト値とトリアシルＭＥＬのシフト値を比べ、Ｅ４炭素原子が低磁場シフトし隣接するＥ３，Ｅ２炭素が高磁場シフトしていることから、エリスルトールの末端の一級水酸基に脂肪酸が結合していることが確認された。 From Table 1 and Table 2, comparing the chemical shift value of MEL produced from soybean oil or olive oil with the shift value of triacyl MEL, the E4 carbon atom is shifted by a low magnetic field, and the adjacent E3 and E2 carbons are shifted by a high magnetic field. From this, it was confirmed that the fatty acid was bonded to the primary hydroxyl group at the terminal of erythritol.
本発明は、糖脂質型バイオサーファクタントのトリアシル体を低コストで製造できる技術を提供するものであり、食品産業、化粧品産業、医薬品産業、化学産業、環境分野等に広く利用可能である。 The present invention provides a technology capable of producing a triacyl derivative of a glycolipid type biosurfactant at a low cost, and can be widely used in the food industry, the cosmetics industry, the pharmaceutical industry, the chemical industry, the environmental field, and the like.
Claims (6)
上記糖脂質型バイオサーファクタントのトリアシル体が、式（Ｉ）
疎水性環境下において、以下の（ａ）、（ｂ）および（ｃ）を混和することにより上記エステル化反応またはエステル交換反応が進行するものであり、
糖脂質型バイオサーファクタント生産微生物を培養している培養液であって、油類、高級脂肪酸および合成エステルから選択される１種または２種以上の混合物が添加された培養液の凍結乾燥物に含有される以下の（ａ）、（ｂ）および（ｃ）を用いることを特徴とする糖脂質型バイオサーファクタントのトリアシル体製造方法。
（ａ）糖脂質型バイオサーファクタント；
（ｂ）油類、高級脂肪酸および合成エステルから選択される１種または２種以上の混合物；
（ｃ）リパーゼ A method for producing a triacyl derivative of a glycolipid type biosurfactant by introducing a fatty acid or a fatty acid derivative into the glycolipid type biosurfactant by an esterification reaction or a transesterification reaction ,
The triacyl derivative of the glycolipid type biosurfactant has the formula (I)
In a hydrophobic environment, the above esterification reaction or transesterification reaction proceeds by mixing the following (a), (b) and (c):
A culture medium in which microorganisms producing glycolipid type biosurfactant are cultured, which is contained in a freeze-dried culture medium to which one or a mixture selected from oils, higher fatty acids and synthetic esters is added A method for producing a triacyl derivative of a glycolipid type biosurfactant, characterized by using the following (a), (b) and (c) :
(A) a glycolipid type biosurfactant;
(B) one or a mixture of two or more selected from oils, higher fatty acids and synthetic esters;
(C) Lipase
上記糖脂質型バイオサーファクタントのトリアシル体が、式（Ｉ）
疎水性環境下において、以下の（ａ）、（ｂ）および（ｃ）を混和することにより上記エステル化反応またはエステル交換反応が進行するものであり、
糖脂質型バイオサーファクタント生産微生物を培養している培養液であって、油類、高級脂肪酸および合成エステルから選択される１種または２種以上の混合物が添加された培養液の上清の凍結乾燥物に含有される以下の（ｃ）を用いることを特徴とする糖脂質型バイオサーファクタントのトリアシル体製造方法。
（ａ）糖脂質型バイオサーファクタント；
（ｂ）油類、高級脂肪酸および合成エステルから選択される１種または２種以上の混合物；
（ｃ）リパーゼ A method for producing a triacyl derivative of a glycolipid type biosurfactant by introducing a fatty acid or a fatty acid derivative into the glycolipid type biosurfactant by an esterification reaction or a transesterification reaction,
The triacyl derivative of the glycolipid type biosurfactant has the formula (I)
In a hydrophobic environment, the above esterification reaction or transesterification reaction proceeds by mixing the following (a), (b) and (c):
Lyophilization of the supernatant of a culture medium in which microorganisms producing glycolipid type biosurfactant are cultured, to which one or a mixture selected from oils, higher fatty acids and synthetic esters is added A method for producing a triacyl derivative of a glycolipid type biosurfactant, which comprises using (c) below contained in a product:
(A) a glycolipid type biosurfactant;
(B) one or a mixture of two or more selected from oils, higher fatty acids and synthetic esters;
(C) Lipase
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