JP2010041991A - Method for producing mannosyl erythritol lipid - Google Patents
Method for producing mannosyl erythritol lipid Download PDFInfo
- Publication number
- JP2010041991A JP2010041991A JP2008322881A JP2008322881A JP2010041991A JP 2010041991 A JP2010041991 A JP 2010041991A JP 2008322881 A JP2008322881 A JP 2008322881A JP 2008322881 A JP2008322881 A JP 2008322881A JP 2010041991 A JP2010041991 A JP 2010041991A
- Authority
- JP
- Japan
- Prior art keywords
- mel
- genus
- culture
- microorganism
- general formula
- 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.)
- Granted
Links
- 238000004519 manufacturing process Methods 0.000 title claims abstract description 87
- -1 mannosyl erythritol lipid Chemical class 0.000 title claims abstract description 24
- 244000005700 microbiome Species 0.000 claims abstract description 95
- 241000196324 Embryophyta Species 0.000 claims abstract description 56
- 238000012258 culturing Methods 0.000 claims abstract description 29
- 241000221566 Ustilago Species 0.000 claims abstract description 18
- 235000004347 Perilla Nutrition 0.000 claims abstract description 17
- 241000229722 Perilla <angiosperm> Species 0.000 claims abstract description 13
- 241000219198 Brassica Species 0.000 claims abstract description 11
- 235000011331 Brassica Nutrition 0.000 claims abstract description 10
- 241000339490 Brachyachne Species 0.000 claims abstract description 7
- 241000220225 Malus Species 0.000 claims abstract description 7
- 241000209051 Saccharum Species 0.000 claims abstract description 7
- OKTJSMMVPCPJKN-UHFFFAOYSA-N Carbon Chemical compound [C] OKTJSMMVPCPJKN-UHFFFAOYSA-N 0.000 claims description 31
- 229910052799 carbon Inorganic materials 0.000 claims description 31
- 239000003921 oil Substances 0.000 claims description 23
- 239000003925 fat Substances 0.000 claims description 22
- 239000002244 precipitate Substances 0.000 claims description 17
- 235000000346 sugar Nutrition 0.000 claims description 10
- BFNBIHQBYMNNAN-UHFFFAOYSA-N ammonium sulfate Chemical compound N.N.OS(O)(=O)=O BFNBIHQBYMNNAN-UHFFFAOYSA-N 0.000 claims description 8
- 229910052921 ammonium sulfate Inorganic materials 0.000 claims description 8
- 235000011130 ammonium sulphate Nutrition 0.000 claims description 8
- 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 7
- 125000004432 carbon atom Chemical group C* 0.000 claims description 2
- 125000005313 fatty acid group Chemical group 0.000 claims description 2
- 125000004435 hydrogen atom Chemical group [H]* 0.000 claims description 2
- GZQKNULLWNGMCW-PWQABINMSA-N lipid A (E. coli) Chemical compound O1[C@H](CO)[C@@H](OP(O)(O)=O)[C@H](OC(=O)C[C@@H](CCCCCCCCCCC)OC(=O)CCCCCCCCCCCCC)[C@@H](NC(=O)C[C@@H](CCCCCCCCCCC)OC(=O)CCCCCCCCCCC)[C@@H]1OC[C@@H]1[C@@H](O)[C@H](OC(=O)C[C@H](O)CCCCCCCCCCC)[C@@H](NC(=O)C[C@H](O)CCCCCCCCCCC)[C@@H](OP(O)(O)=O)O1 GZQKNULLWNGMCW-PWQABINMSA-N 0.000 claims description 2
- 238000000034 method Methods 0.000 abstract description 44
- 235000013305 food Nutrition 0.000 abstract description 15
- 239000002609 medium Substances 0.000 description 41
- XEKOWRVHYACXOJ-UHFFFAOYSA-N Ethyl acetate Chemical compound CCOC(C)=O XEKOWRVHYACXOJ-UHFFFAOYSA-N 0.000 description 36
- 238000004809 thin layer chromatography Methods 0.000 description 28
- CSNNHWWHGAXBCP-UHFFFAOYSA-L Magnesium sulfate Chemical compound [Mg+2].[O-][S+2]([O-])([O-])[O-] CSNNHWWHGAXBCP-UHFFFAOYSA-L 0.000 description 22
- 235000019198 oils Nutrition 0.000 description 22
- 229930006000 Sucrose Natural products 0.000 description 21
- 239000005720 sucrose Substances 0.000 description 21
- CZMRCDWAGMRECN-UGDNZRGBSA-N Sucrose Chemical compound O[C@H]1[C@H](O)[C@@H](CO)O[C@@]1(CO)O[C@@H]1[C@H](O)[C@@H](O)[C@H](O)[C@@H](CO)O1 CZMRCDWAGMRECN-UGDNZRGBSA-N 0.000 description 20
- 239000000203 mixture Substances 0.000 description 20
- VWDWKYIASSYTQR-UHFFFAOYSA-N sodium nitrate Chemical compound [Na+].[O-][N+]([O-])=O VWDWKYIASSYTQR-UHFFFAOYSA-N 0.000 description 20
- 235000019197 fats Nutrition 0.000 description 19
- 238000004128 high performance liquid chromatography Methods 0.000 description 18
- 239000002024 ethyl acetate extract Substances 0.000 description 16
- 239000003549 soybean oil Substances 0.000 description 15
- 235000012424 soybean oil Nutrition 0.000 description 15
- HEDRZPFGACZZDS-UHFFFAOYSA-N Chloroform Chemical compound ClC(Cl)Cl HEDRZPFGACZZDS-UHFFFAOYSA-N 0.000 description 14
- 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 14
- 239000008103 glucose Substances 0.000 description 14
- 235000011430 Malus pumila Nutrition 0.000 description 13
- 235000015103 Malus silvestris Nutrition 0.000 description 13
- 240000000111 Saccharum officinarum Species 0.000 description 13
- 229940041514 candida albicans extract Drugs 0.000 description 13
- 239000012138 yeast extract Substances 0.000 description 13
- CSCPPACGZOOCGX-UHFFFAOYSA-N Acetone Chemical compound CC(C)=O CSCPPACGZOOCGX-UHFFFAOYSA-N 0.000 description 12
- 241000893045 Pseudozyma Species 0.000 description 12
- 239000004094 surface-active agent Substances 0.000 description 12
- 229910052943 magnesium sulfate Inorganic materials 0.000 description 11
- 235000019341 magnesium sulphate Nutrition 0.000 description 11
- 229910000402 monopotassium phosphate Inorganic materials 0.000 description 11
- 235000019796 monopotassium phosphate Nutrition 0.000 description 11
- 235000014113 dietary fatty acids Nutrition 0.000 description 10
- 235000018927 edible plant Nutrition 0.000 description 10
- 239000000194 fatty acid Substances 0.000 description 10
- 229930195729 fatty acid Natural products 0.000 description 10
- 150000004665 fatty acids Chemical class 0.000 description 10
- 239000004006 olive oil Substances 0.000 description 10
- 235000008390 olive oil Nutrition 0.000 description 10
- PJNZPQUBCPKICU-UHFFFAOYSA-N phosphoric acid;potassium Chemical compound [K].OP(O)(O)=O PJNZPQUBCPKICU-UHFFFAOYSA-N 0.000 description 10
- 239000002994 raw material Substances 0.000 description 10
- 239000004317 sodium nitrate Substances 0.000 description 10
- 235000010344 sodium nitrate Nutrition 0.000 description 10
- 239000000126 substance Substances 0.000 description 10
- OKKJLVBELUTLKV-UHFFFAOYSA-N Methanol Chemical compound OC OKKJLVBELUTLKV-UHFFFAOYSA-N 0.000 description 9
- 239000003876 biosurfactant Substances 0.000 description 9
- 238000002955 isolation Methods 0.000 description 9
- IJGRMHOSHXDMSA-UHFFFAOYSA-N Atomic nitrogen Chemical compound N#N IJGRMHOSHXDMSA-UHFFFAOYSA-N 0.000 description 8
- 241000894006 Bacteria Species 0.000 description 8
- 244000081841 Malus domestica Species 0.000 description 8
- 244000070406 Malus silvestris Species 0.000 description 8
- 240000004808 Saccharomyces cerevisiae Species 0.000 description 8
- 235000007201 Saccharum officinarum Nutrition 0.000 description 8
- WQZGKKKJIJFFOK-VFUOTHLCSA-N beta-D-glucose Chemical compound OC[C@H]1O[C@@H](O)[C@H](O)[C@@H](O)[C@@H]1O WQZGKKKJIJFFOK-VFUOTHLCSA-N 0.000 description 8
- 230000015572 biosynthetic process Effects 0.000 description 8
- 230000000694 effects Effects 0.000 description 8
- 239000007788 liquid Substances 0.000 description 8
- 239000000523 sample Substances 0.000 description 8
- 241000893447 Ustilago cynodontis Species 0.000 description 7
- 240000008042 Zea mays Species 0.000 description 7
- 235000002017 Zea mays subsp mays Nutrition 0.000 description 7
- 238000004458 analytical method Methods 0.000 description 7
- 238000000746 purification Methods 0.000 description 7
- 235000015112 vegetable and seed oil Nutrition 0.000 description 7
- 239000008158 vegetable oil Substances 0.000 description 7
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 description 7
- PEDCQBHIVMGVHV-UHFFFAOYSA-N Glycerine Chemical compound OCC(O)CO PEDCQBHIVMGVHV-UHFFFAOYSA-N 0.000 description 6
- 229930186217 Glycolipid Natural products 0.000 description 6
- 241001465754 Metazoa Species 0.000 description 6
- FAPWRFPIFSIZLT-UHFFFAOYSA-M Sodium chloride Chemical compound [Na+].[Cl-] FAPWRFPIFSIZLT-UHFFFAOYSA-M 0.000 description 6
- 235000005824 Zea mays ssp. parviglumis Nutrition 0.000 description 6
- 150000001720 carbohydrates Chemical class 0.000 description 6
- 235000005822 corn Nutrition 0.000 description 6
- 238000001514 detection method Methods 0.000 description 6
- 238000000605 extraction Methods 0.000 description 6
- 239000004973 liquid crystal related substance Substances 0.000 description 6
- 235000014593 oils and fats Nutrition 0.000 description 6
- 238000012916 structural analysis Methods 0.000 description 6
- 244000026811 Brassica nipposinica Species 0.000 description 5
- 235000007294 Brassica nipposinica Nutrition 0.000 description 5
- 230000027455 binding Effects 0.000 description 5
- 238000011218 seed culture Methods 0.000 description 5
- 235000002639 sodium chloride Nutrition 0.000 description 5
- 239000006228 supernatant Substances 0.000 description 5
- 238000003786 synthesis reaction Methods 0.000 description 5
- HVCOBJNICQPDBP-UHFFFAOYSA-N 3-[3-[3,5-dihydroxy-6-methyl-4-(3,4,5-trihydroxy-6-methyloxan-2-yl)oxyoxan-2-yl]oxydecanoyloxy]decanoic acid;hydrate Chemical compound O.OC1C(OC(CC(=O)OC(CCCCCCC)CC(O)=O)CCCCCCC)OC(C)C(O)C1OC1C(O)C(O)C(O)C(C)O1 HVCOBJNICQPDBP-UHFFFAOYSA-N 0.000 description 4
- 241000950571 Acianthera trichophora Species 0.000 description 4
- 241000222120 Candida <Saccharomycetales> Species 0.000 description 4
- 244000052363 Cynodon dactylon Species 0.000 description 4
- 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 4
- 244000124853 Perilla frutescens Species 0.000 description 4
- 244000301083 Ustilago maydis Species 0.000 description 4
- 238000005119 centrifugation Methods 0.000 description 4
- 230000000052 comparative effect Effects 0.000 description 4
- 238000012136 culture method Methods 0.000 description 4
- 238000011161 development Methods 0.000 description 4
- 230000018109 developmental process Effects 0.000 description 4
- 238000010586 diagram Methods 0.000 description 4
- 239000001963 growth medium Substances 0.000 description 4
- 229910052757 nitrogen Inorganic materials 0.000 description 4
- 235000015097 nutrients Nutrition 0.000 description 4
- 239000003960 organic solvent Substances 0.000 description 4
- 230000000704 physical effect Effects 0.000 description 4
- BASFCYQUMIYNBI-UHFFFAOYSA-N platinum Chemical compound [Pt] BASFCYQUMIYNBI-UHFFFAOYSA-N 0.000 description 4
- 230000008569 process Effects 0.000 description 4
- 241000894007 species Species 0.000 description 4
- 229920001817 Agar Polymers 0.000 description 3
- 240000008100 Brassica rapa Species 0.000 description 3
- 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 3
- LFQSCWFLJHTTHZ-UHFFFAOYSA-N Ethanol Chemical compound CCO LFQSCWFLJHTTHZ-UHFFFAOYSA-N 0.000 description 3
- 241000233866 Fungi Species 0.000 description 3
- 241000282575 Gorilla Species 0.000 description 3
- 108091023242 Internal transcribed spacer Proteins 0.000 description 3
- 244000134336 Malus baccata Species 0.000 description 3
- 229930195725 Mannitol Natural products 0.000 description 3
- 239000008272 agar Substances 0.000 description 3
- 230000008901 benefit Effects 0.000 description 3
- 239000003795 chemical substances by application Substances 0.000 description 3
- 238000012790 confirmation Methods 0.000 description 3
- 238000004132 cross linking Methods 0.000 description 3
- 125000001495 ethyl group Chemical group [H]C([H])([H])C([H])([H])* 0.000 description 3
- 239000000284 extract Substances 0.000 description 3
- 239000011521 glass Substances 0.000 description 3
- 150000004676 glycans Chemical class 0.000 description 3
- 230000001939 inductive effect Effects 0.000 description 3
- 239000000594 mannitol Substances 0.000 description 3
- 235000010355 mannitol Nutrition 0.000 description 3
- 238000005259 measurement Methods 0.000 description 3
- 230000000813 microbial effect Effects 0.000 description 3
- 150000002772 monosaccharides Chemical class 0.000 description 3
- 150000007524 organic acids Chemical class 0.000 description 3
- 229920001223 polyethylene glycol Polymers 0.000 description 3
- 229920001282 polysaccharide Polymers 0.000 description 3
- 239000005017 polysaccharide Substances 0.000 description 3
- 238000001556 precipitation Methods 0.000 description 3
- 238000010898 silica gel chromatography Methods 0.000 description 3
- 239000011780 sodium chloride Substances 0.000 description 3
- 239000002904 solvent Substances 0.000 description 3
- 235000013555 soy sauce Nutrition 0.000 description 3
- 239000003760 tallow Substances 0.000 description 3
- JUUBMADBGZQVFT-KHPPLWFESA-N (z)-2-methyloctadec-9-enoic acid Chemical compound CCCCCCCC\C=C/CCCCCCC(C)C(O)=O JUUBMADBGZQVFT-KHPPLWFESA-N 0.000 description 2
- OWEGMIWEEQEYGQ-UHFFFAOYSA-N 100676-05-9 Natural products OC1C(O)C(O)C(CO)OC1OCC1C(O)C(O)C(O)C(OC2C(OC(O)C(O)C2O)CO)O1 OWEGMIWEEQEYGQ-UHFFFAOYSA-N 0.000 description 2
- GUBGYTABKSRVRQ-XLOQQCSPSA-N Alpha-Lactose Chemical compound O[C@@H]1[C@@H](O)[C@@H](O)[C@@H](CO)O[C@H]1O[C@@H]1[C@@H](CO)O[C@H](O)[C@H](O)[C@H]1O GUBGYTABKSRVRQ-XLOQQCSPSA-N 0.000 description 2
- QGZKDVFQNNGYKY-UHFFFAOYSA-N Ammonia Chemical compound N QGZKDVFQNNGYKY-UHFFFAOYSA-N 0.000 description 2
- NLXLAEXVIDQMFP-UHFFFAOYSA-N Ammonia chloride Chemical compound [NH4+].[Cl-] NLXLAEXVIDQMFP-UHFFFAOYSA-N 0.000 description 2
- VTYYLEPIZMXCLO-UHFFFAOYSA-L Calcium carbonate Chemical compound [Ca+2].[O-]C([O-])=O VTYYLEPIZMXCLO-UHFFFAOYSA-L 0.000 description 2
- 108020004414 DNA Proteins 0.000 description 2
- 239000004386 Erythritol Substances 0.000 description 2
- UNXHWFMMPAWVPI-UHFFFAOYSA-N Erythritol Natural products OCC(O)C(O)CO UNXHWFMMPAWVPI-UHFFFAOYSA-N 0.000 description 2
- 229930091371 Fructose Natural products 0.000 description 2
- 239000005715 Fructose Substances 0.000 description 2
- RFSUNEUAIZKAJO-ARQDHWQXSA-N Fructose Chemical compound OC[C@H]1O[C@](O)(CO)[C@@H](O)[C@@H]1O RFSUNEUAIZKAJO-ARQDHWQXSA-N 0.000 description 2
- VZCYOOQTPOCHFL-OWOJBTEDSA-N Fumaric acid Chemical compound OC(=O)\C=C\C(O)=O VZCYOOQTPOCHFL-OWOJBTEDSA-N 0.000 description 2
- 102000003886 Glycoproteins Human genes 0.000 description 2
- 108090000288 Glycoproteins Proteins 0.000 description 2
- VEXZGXHMUGYJMC-UHFFFAOYSA-N Hydrochloric acid Chemical compound Cl VEXZGXHMUGYJMC-UHFFFAOYSA-N 0.000 description 2
- GUBGYTABKSRVRQ-QKKXKWKRSA-N Lactose Natural products OC[C@H]1O[C@@H](O[C@H]2[C@H](O)[C@@H](O)C(O)O[C@@H]2CO)[C@H](O)[C@@H](O)[C@H]1O GUBGYTABKSRVRQ-QKKXKWKRSA-N 0.000 description 2
- GUBGYTABKSRVRQ-PICCSMPSSA-N Maltose Natural products O[C@@H]1[C@@H](O)[C@H](O)[C@@H](CO)O[C@@H]1O[C@@H]1[C@@H](CO)OC(O)[C@H](O)[C@H]1O GUBGYTABKSRVRQ-PICCSMPSSA-N 0.000 description 2
- 241001661345 Moesziomyces antarcticus Species 0.000 description 2
- 206010028980 Neoplasm Diseases 0.000 description 2
- 235000019483 Peanut oil Nutrition 0.000 description 2
- 239000001888 Peptone Substances 0.000 description 2
- 108010080698 Peptones Proteins 0.000 description 2
- ZLMJMSJWJFRBEC-UHFFFAOYSA-N Potassium Chemical compound [K] ZLMJMSJWJFRBEC-UHFFFAOYSA-N 0.000 description 2
- VYPSYNLAJGMNEJ-UHFFFAOYSA-N Silicium dioxide Chemical compound O=[Si]=O VYPSYNLAJGMNEJ-UHFFFAOYSA-N 0.000 description 2
- 241000226724 Sporisorium scitamineum Species 0.000 description 2
- 229920002472 Starch Polymers 0.000 description 2
- 235000015919 Ustilago maydis Nutrition 0.000 description 2
- 241000952807 Ustilago tragana Species 0.000 description 2
- 238000005273 aeration Methods 0.000 description 2
- 230000003266 anti-allergic effect Effects 0.000 description 2
- 230000003110 anti-inflammatory effect Effects 0.000 description 2
- 230000000845 anti-microbial effect Effects 0.000 description 2
- 230000006907 apoptotic process Effects 0.000 description 2
- 230000001580 bacterial effect Effects 0.000 description 2
- GUBGYTABKSRVRQ-QUYVBRFLSA-N beta-maltose Chemical compound OC[C@H]1O[C@H](O[C@H]2[C@H](O)[C@@H](O)[C@H](O)O[C@@H]2CO)[C@H](O)[C@@H](O)[C@@H]1O GUBGYTABKSRVRQ-QUYVBRFLSA-N 0.000 description 2
- 201000011510 cancer Diseases 0.000 description 2
- 235000014633 carbohydrates Nutrition 0.000 description 2
- 230000024245 cell differentiation Effects 0.000 description 2
- 230000010261 cell growth Effects 0.000 description 2
- 235000013339 cereals Nutrition 0.000 description 2
- 239000003153 chemical reaction reagent Substances 0.000 description 2
- 239000006059 cover glass Substances 0.000 description 2
- 239000003814 drug Substances 0.000 description 2
- 235000013399 edible fruits Nutrition 0.000 description 2
- 238000005516 engineering process Methods 0.000 description 2
- 230000007613 environmental effect Effects 0.000 description 2
- UNXHWFMMPAWVPI-ZXZARUISSA-N erythritol Chemical compound OC[C@H](O)[C@H](O)CO UNXHWFMMPAWVPI-ZXZARUISSA-N 0.000 description 2
- 235000019414 erythritol Nutrition 0.000 description 2
- 229940009714 erythritol Drugs 0.000 description 2
- 235000011389 fruit/vegetable juice Nutrition 0.000 description 2
- 230000002401 inhibitory effect Effects 0.000 description 2
- JVTAAEKCZFNVCJ-UHFFFAOYSA-N lactic acid Chemical compound CC(O)C(O)=O JVTAAEKCZFNVCJ-UHFFFAOYSA-N 0.000 description 2
- 239000008101 lactose Substances 0.000 description 2
- 150000002632 lipids Chemical group 0.000 description 2
- 239000013028 medium composition Substances 0.000 description 2
- HEBKCHPVOIAQTA-UHFFFAOYSA-N meso ribitol Natural products OCC(O)C(O)C(O)CO HEBKCHPVOIAQTA-UHFFFAOYSA-N 0.000 description 2
- 210000000653 nervous system Anatomy 0.000 description 2
- 229920001542 oligosaccharide Polymers 0.000 description 2
- 150000002482 oligosaccharides Chemical class 0.000 description 2
- 235000005985 organic acids Nutrition 0.000 description 2
- 239000000312 peanut oil Substances 0.000 description 2
- 235000019319 peptone Nutrition 0.000 description 2
- 230000001766 physiological effect Effects 0.000 description 2
- 229910052697 platinum Inorganic materials 0.000 description 2
- 229920002401 polyacrylamide Polymers 0.000 description 2
- 229920000642 polymer Polymers 0.000 description 2
- 239000011591 potassium Substances 0.000 description 2
- 229910052700 potassium Inorganic materials 0.000 description 2
- LWIHDJKSTIGBAC-UHFFFAOYSA-K potassium phosphate Substances [K+].[K+].[K+].[O-]P([O-])([O-])=O LWIHDJKSTIGBAC-UHFFFAOYSA-K 0.000 description 2
- 238000002360 preparation method Methods 0.000 description 2
- 239000003755 preservative agent Substances 0.000 description 2
- 239000000047 product Substances 0.000 description 2
- 108090000623 proteins and genes Proteins 0.000 description 2
- 238000011084 recovery Methods 0.000 description 2
- 150000003839 salts Chemical class 0.000 description 2
- 239000000741 silica gel Substances 0.000 description 2
- 229910002027 silica gel Inorganic materials 0.000 description 2
- 239000007787 solid Substances 0.000 description 2
- 239000008107 starch Substances 0.000 description 2
- 235000019698 starch Nutrition 0.000 description 2
- 238000003756 stirring Methods 0.000 description 2
- 238000003860 storage Methods 0.000 description 2
- 238000012360 testing method Methods 0.000 description 2
- VZCYOOQTPOCHFL-UHFFFAOYSA-N trans-butenedioic acid Natural products OC(=O)C=CC(O)=O VZCYOOQTPOCHFL-UHFFFAOYSA-N 0.000 description 2
- 238000011282 treatment Methods 0.000 description 2
- 235000019871 vegetable fat Nutrition 0.000 description 2
- BJEPYKJPYRNKOW-REOHCLBHSA-N (S)-malic acid Chemical compound OC(=O)[C@@H](O)CC(O)=O BJEPYKJPYRNKOW-REOHCLBHSA-N 0.000 description 1
- 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
- PAWQVTBBRAZDMG-UHFFFAOYSA-N 2-(3-bromo-2-fluorophenyl)acetic acid Chemical compound OC(=O)CC1=CC=CC(Br)=C1F PAWQVTBBRAZDMG-UHFFFAOYSA-N 0.000 description 1
- MOMFXATYAINJML-UHFFFAOYSA-N 2-Acetylthiazole Chemical group CC(=O)C1=NC=CS1 MOMFXATYAINJML-UHFFFAOYSA-N 0.000 description 1
- FWMNVWWHGCHHJJ-SKKKGAJSSA-N 4-amino-1-[(2r)-6-amino-2-[[(2r)-2-[[(2r)-2-[[(2r)-2-amino-3-phenylpropanoyl]amino]-3-phenylpropanoyl]amino]-4-methylpentanoyl]amino]hexanoyl]piperidine-4-carboxylic acid Chemical compound C([C@H](C(=O)N[C@H](CC(C)C)C(=O)N[C@H](CCCCN)C(=O)N1CCC(N)(CC1)C(O)=O)NC(=O)[C@H](N)CC=1C=CC=CC=1)C1=CC=CC=C1 FWMNVWWHGCHHJJ-SKKKGAJSSA-N 0.000 description 1
- FHVDTGUDJYJELY-UHFFFAOYSA-N 6-{[2-carboxy-4,5-dihydroxy-6-(phosphanyloxy)oxan-3-yl]oxy}-4,5-dihydroxy-3-phosphanyloxane-2-carboxylic acid Chemical compound O1C(C(O)=O)C(P)C(O)C(O)C1OC1C(C(O)=O)OC(OP)C(O)C1O FHVDTGUDJYJELY-UHFFFAOYSA-N 0.000 description 1
- 241000251468 Actinopterygii Species 0.000 description 1
- 229920000936 Agarose Polymers 0.000 description 1
- 239000004254 Ammonium phosphate Substances 0.000 description 1
- 244000063299 Bacillus subtilis Species 0.000 description 1
- 235000014469 Bacillus subtilis Nutrition 0.000 description 1
- 240000002791 Brassica napus Species 0.000 description 1
- 235000011292 Brassica rapa Nutrition 0.000 description 1
- 235000004977 Brassica sinapistrum Nutrition 0.000 description 1
- 241000448187 Ciboria shiraiana Species 0.000 description 1
- 102000008186 Collagen Human genes 0.000 description 1
- 108010035532 Collagen Proteins 0.000 description 1
- 241000722863 Cortaderia jubata Species 0.000 description 1
- FBPFZTCFMRRESA-FSIIMWSLSA-N D-Glucitol Natural products OC[C@H](O)[C@H](O)[C@@H](O)[C@H](O)CO FBPFZTCFMRRESA-FSIIMWSLSA-N 0.000 description 1
- HEBKCHPVOIAQTA-QWWZWVQMSA-N D-arabinitol Chemical compound OC[C@@H](O)C(O)[C@H](O)CO HEBKCHPVOIAQTA-QWWZWVQMSA-N 0.000 description 1
- FBPFZTCFMRRESA-JGWLITMVSA-N D-glucitol Chemical compound OC[C@H](O)[C@@H](O)[C@H](O)[C@H](O)CO FBPFZTCFMRRESA-JGWLITMVSA-N 0.000 description 1
- 235000017896 Digitaria Nutrition 0.000 description 1
- 241001303487 Digitaria <clam> Species 0.000 description 1
- 241000255581 Drosophila <fruit fly, genus> Species 0.000 description 1
- 240000008620 Fagopyrum esculentum Species 0.000 description 1
- 235000009419 Fagopyrum esculentum Nutrition 0.000 description 1
- 239000004606 Fillers/Extenders Substances 0.000 description 1
- 244000194101 Ginkgo biloba Species 0.000 description 1
- 235000008100 Ginkgo biloba Nutrition 0.000 description 1
- SXRSQZLOMIGNAQ-UHFFFAOYSA-N Glutaraldehyde Chemical compound O=CCCCC=O SXRSQZLOMIGNAQ-UHFFFAOYSA-N 0.000 description 1
- SQUHHTBVTRBESD-UHFFFAOYSA-N Hexa-Ac-myo-Inositol Natural products CC(=O)OC1C(OC(C)=O)C(OC(C)=O)C(OC(C)=O)C(OC(C)=O)C1OC(C)=O SQUHHTBVTRBESD-UHFFFAOYSA-N 0.000 description 1
- 241001539176 Hime Species 0.000 description 1
- 241000209219 Hordeum Species 0.000 description 1
- 240000005979 Hordeum vulgare Species 0.000 description 1
- 235000007340 Hordeum vulgare Nutrition 0.000 description 1
- 239000004976 Lyotropic liquid crystal Substances 0.000 description 1
- 235000005087 Malus prunifolia Nutrition 0.000 description 1
- 235000010654 Melissa officinalis Nutrition 0.000 description 1
- 244000062730 Melissa officinalis Species 0.000 description 1
- 240000003433 Miscanthus floridulus Species 0.000 description 1
- 241001661343 Moesziomyces aphidis Species 0.000 description 1
- 241001661347 Moesziomyces rugulosus Species 0.000 description 1
- 241000998584 Nuda Species 0.000 description 1
- 235000002791 Panax Nutrition 0.000 description 1
- 241000208343 Panax Species 0.000 description 1
- 241000745988 Phyllostachys Species 0.000 description 1
- 239000004793 Polystyrene Substances 0.000 description 1
- OFOBLEOULBTSOW-UHFFFAOYSA-N Propanedioic acid Natural products OC(=O)CC(O)=O OFOBLEOULBTSOW-UHFFFAOYSA-N 0.000 description 1
- 241000222180 Pseudozyma tsukubaensis Species 0.000 description 1
- 235000019484 Rapeseed oil Nutrition 0.000 description 1
- JVWLUVNSQYXYBE-UHFFFAOYSA-N Ribitol Natural products OCC(C)C(O)C(O)CO JVWLUVNSQYXYBE-UHFFFAOYSA-N 0.000 description 1
- 241000235343 Saccharomycetales Species 0.000 description 1
- 240000004155 Saccharum barberi Species 0.000 description 1
- 244000130271 Saccharum robustum Species 0.000 description 1
- 235000019485 Safflower oil Nutrition 0.000 description 1
- 240000003829 Sorghum propinquum Species 0.000 description 1
- 235000011684 Sorghum saccharatum Nutrition 0.000 description 1
- 241000016362 Ustilago shanxiensis Species 0.000 description 1
- 241000743436 Ustilago sp. Species 0.000 description 1
- 235000016383 Zea mays subsp huehuetenangensis Nutrition 0.000 description 1
- SMEGJBVQLJJKKX-HOTMZDKISA-N [(2R,3S,4S,5R,6R)-5-acetyloxy-3,4,6-trihydroxyoxan-2-yl]methyl acetate Chemical compound CC(=O)OC[C@@H]1[C@H]([C@@H]([C@H]([C@@H](O1)O)OC(=O)C)O)O SMEGJBVQLJJKKX-HOTMZDKISA-N 0.000 description 1
- AVMNFQHJOOYCAP-UHFFFAOYSA-N acetic acid;propanoic acid Chemical compound CC(O)=O.CCC(O)=O AVMNFQHJOOYCAP-UHFFFAOYSA-N 0.000 description 1
- 125000002777 acetyl group Chemical group [H]C([H])([H])C(*)=O 0.000 description 1
- 229940081735 acetylcellulose Drugs 0.000 description 1
- 239000004480 active ingredient Substances 0.000 description 1
- 239000013543 active substance Substances 0.000 description 1
- 125000002252 acyl group Chemical group 0.000 description 1
- 238000013019 agitation Methods 0.000 description 1
- 150000001298 alcohols Chemical class 0.000 description 1
- 229940072056 alginate Drugs 0.000 description 1
- 235000010443 alginic acid Nutrition 0.000 description 1
- 229920000615 alginic acid Polymers 0.000 description 1
- WQZGKKKJIJFFOK-PHYPRBDBSA-N alpha-D-galactose Chemical compound OC[C@H]1O[C@H](O)[C@H](O)[C@@H](O)[C@H]1O WQZGKKKJIJFFOK-PHYPRBDBSA-N 0.000 description 1
- BJEPYKJPYRNKOW-UHFFFAOYSA-N alpha-hydroxysuccinic acid Natural products OC(=O)C(O)CC(O)=O BJEPYKJPYRNKOW-UHFFFAOYSA-N 0.000 description 1
- 229910021529 ammonia Inorganic materials 0.000 description 1
- 235000019270 ammonium chloride Nutrition 0.000 description 1
- 229910000148 ammonium phosphate Inorganic materials 0.000 description 1
- 235000019289 ammonium phosphates Nutrition 0.000 description 1
- 150000003863 ammonium salts Chemical class 0.000 description 1
- 230000003321 amplification Effects 0.000 description 1
- RJGDLRCDCYRQOQ-UHFFFAOYSA-N anthrone Chemical compound C1=CC=C2C(=O)C3=CC=CC=C3CC2=C1 RJGDLRCDCYRQOQ-UHFFFAOYSA-N 0.000 description 1
- 239000002246 antineoplastic agent Substances 0.000 description 1
- QVGXLLKOCUKJST-UHFFFAOYSA-N atomic oxygen Chemical compound [O] QVGXLLKOCUKJST-UHFFFAOYSA-N 0.000 description 1
- 235000015278 beef Nutrition 0.000 description 1
- 230000001588 bifunctional effect Effects 0.000 description 1
- 239000011230 binding agent Substances 0.000 description 1
- 230000005540 biological transmission Effects 0.000 description 1
- 235000008429 bread Nutrition 0.000 description 1
- 238000013124 brewing process Methods 0.000 description 1
- 239000000872 buffer Substances 0.000 description 1
- 239000006172 buffering agent Substances 0.000 description 1
- 239000006227 byproduct Substances 0.000 description 1
- 229910000019 calcium carbonate Inorganic materials 0.000 description 1
- 238000011088 calibration curve Methods 0.000 description 1
- 239000002775 capsule Substances 0.000 description 1
- 239000000679 carrageenan Substances 0.000 description 1
- 235000010418 carrageenan Nutrition 0.000 description 1
- 229920001525 carrageenan Polymers 0.000 description 1
- 229940113118 carrageenan Drugs 0.000 description 1
- 238000004113 cell culture Methods 0.000 description 1
- 150000001773 cellobioses Chemical class 0.000 description 1
- 229920002301 cellulose acetate Polymers 0.000 description 1
- 229920001436 collagen Polymers 0.000 description 1
- 238000004440 column chromatography Methods 0.000 description 1
- 150000001875 compounds Chemical class 0.000 description 1
- 239000000470 constituent Substances 0.000 description 1
- 238000007796 conventional method Methods 0.000 description 1
- 229910000365 copper sulfate Inorganic materials 0.000 description 1
- ARUVKPQLZAKDPS-UHFFFAOYSA-L copper(II) sulfate Chemical compound [Cu+2].[O-][S+2]([O-])([O-])[O-] ARUVKPQLZAKDPS-UHFFFAOYSA-L 0.000 description 1
- 235000005687 corn oil Nutrition 0.000 description 1
- 239000002285 corn oil Substances 0.000 description 1
- 239000002537 cosmetic Substances 0.000 description 1
- 235000012343 cottonseed oil Nutrition 0.000 description 1
- 239000002385 cottonseed oil Substances 0.000 description 1
- 238000000354 decomposition reaction Methods 0.000 description 1
- MNNHAPBLZZVQHP-UHFFFAOYSA-N diammonium hydrogen phosphate Chemical compound [NH4+].[NH4+].OP([O-])([O-])=O MNNHAPBLZZVQHP-UHFFFAOYSA-N 0.000 description 1
- ZPWVASYFFYYZEW-UHFFFAOYSA-L dipotassium hydrogen phosphate Chemical compound [K+].[K+].OP([O-])([O-])=O ZPWVASYFFYYZEW-UHFFFAOYSA-L 0.000 description 1
- 229910000396 dipotassium phosphate Inorganic materials 0.000 description 1
- 235000019797 dipotassium phosphate Nutrition 0.000 description 1
- 238000007598 dipping method Methods 0.000 description 1
- 239000007884 disintegrant Substances 0.000 description 1
- 239000002270 dispersing agent Substances 0.000 description 1
- 239000003480 eluent Substances 0.000 description 1
- 238000003912 environmental pollution Methods 0.000 description 1
- 238000002474 experimental method Methods 0.000 description 1
- 239000011790 ferrous sulphate Substances 0.000 description 1
- 235000003891 ferrous sulphate Nutrition 0.000 description 1
- 239000000796 flavoring agent Substances 0.000 description 1
- 235000013355 food flavoring agent Nutrition 0.000 description 1
- 238000009472 formulation Methods 0.000 description 1
- 239000012634 fragment Substances 0.000 description 1
- 239000001530 fumaric acid Substances 0.000 description 1
- 235000013376 functional food Nutrition 0.000 description 1
- 229930182830 galactose Natural products 0.000 description 1
- 239000007789 gas Substances 0.000 description 1
- 238000002290 gas chromatography-mass spectrometry Methods 0.000 description 1
- 239000000499 gel Substances 0.000 description 1
- 125000002791 glucosyl group Chemical group C1([C@H](O)[C@@H](O)[C@H](O)[C@H](O1)CO)* 0.000 description 1
- 230000012010 growth Effects 0.000 description 1
- 239000002035 hexane extract Substances 0.000 description 1
- 230000002209 hydrophobic effect Effects 0.000 description 1
- 125000001165 hydrophobic group Chemical group 0.000 description 1
- 210000000987 immune system Anatomy 0.000 description 1
- 239000012535 impurity Substances 0.000 description 1
- 238000001727 in vivo Methods 0.000 description 1
- 230000006698 induction Effects 0.000 description 1
- CDAISMWEOUEBRE-GPIVLXJGSA-N inositol Chemical compound O[C@H]1[C@H](O)[C@@H](O)[C@H](O)[C@H](O)[C@@H]1O CDAISMWEOUEBRE-GPIVLXJGSA-N 0.000 description 1
- 229960000367 inositol Drugs 0.000 description 1
- 229940030980 inova Drugs 0.000 description 1
- BAUYGSIQEAFULO-UHFFFAOYSA-L iron(2+) sulfate (anhydrous) Chemical compound [Fe+2].[O-]S([O-])(=O)=O BAUYGSIQEAFULO-UHFFFAOYSA-L 0.000 description 1
- 229910000359 iron(II) sulfate Inorganic materials 0.000 description 1
- 239000004310 lactic acid Substances 0.000 description 1
- 235000014655 lactic acid Nutrition 0.000 description 1
- 239000000787 lecithin Substances 0.000 description 1
- 235000010445 lecithin Nutrition 0.000 description 1
- 229940067606 lecithin Drugs 0.000 description 1
- 208000032839 leukemia Diseases 0.000 description 1
- 239000000865 liniment Substances 0.000 description 1
- 238000009630 liquid culture Methods 0.000 description 1
- 231100000053 low toxicity Toxicity 0.000 description 1
- 238000000464 low-speed centrifugation Methods 0.000 description 1
- 239000000314 lubricant Substances 0.000 description 1
- 230000002535 lyotropic effect Effects 0.000 description 1
- GVALZJMUIHGIMD-UHFFFAOYSA-H magnesium phosphate Chemical compound [Mg+2].[Mg+2].[Mg+2].[O-]P([O-])([O-])=O.[O-]P([O-])([O-])=O GVALZJMUIHGIMD-UHFFFAOYSA-H 0.000 description 1
- 239000004137 magnesium phosphate Substances 0.000 description 1
- 229910000157 magnesium phosphate Inorganic materials 0.000 description 1
- 229960002261 magnesium phosphate Drugs 0.000 description 1
- 235000010994 magnesium phosphates Nutrition 0.000 description 1
- 235000009973 maize Nutrition 0.000 description 1
- VZCYOOQTPOCHFL-UPHRSURJSA-N maleic acid Chemical compound OC(=O)\C=C/C(O)=O VZCYOOQTPOCHFL-UPHRSURJSA-N 0.000 description 1
- 239000011976 maleic acid Substances 0.000 description 1
- 239000001630 malic acid Substances 0.000 description 1
- 235000011090 malic acid Nutrition 0.000 description 1
- 229940099596 manganese sulfate Drugs 0.000 description 1
- 239000011702 manganese sulphate Substances 0.000 description 1
- 235000007079 manganese sulphate Nutrition 0.000 description 1
- SQQMAOCOWKFBNP-UHFFFAOYSA-L manganese(II) sulfate Chemical compound [Mn+2].[O-]S([O-])(=O)=O SQQMAOCOWKFBNP-UHFFFAOYSA-L 0.000 description 1
- 125000000311 mannosyl group Chemical group C1([C@@H](O)[C@@H](O)[C@H](O)[C@H](O1)CO)* 0.000 description 1
- 235000013372 meat Nutrition 0.000 description 1
- 239000012528 membrane Substances 0.000 description 1
- 229910044991 metal oxide Inorganic materials 0.000 description 1
- 150000004706 metal oxides Chemical class 0.000 description 1
- 239000000693 micelle Substances 0.000 description 1
- 239000013586 microbial product Substances 0.000 description 1
- 238000009629 microbiological culture Methods 0.000 description 1
- 150000007522 mineralic acids Chemical class 0.000 description 1
- 238000012986 modification Methods 0.000 description 1
- 230000004048 modification Effects 0.000 description 1
- 235000013557 nattō Nutrition 0.000 description 1
- QJGQUHMNIGDVPM-UHFFFAOYSA-N nitrogen group Chemical group [N] QJGQUHMNIGDVPM-UHFFFAOYSA-N 0.000 description 1
- 238000003199 nucleic acid amplification method Methods 0.000 description 1
- 230000003287 optical effect Effects 0.000 description 1
- 230000003647 oxidation Effects 0.000 description 1
- 238000007254 oxidation reaction Methods 0.000 description 1
- 239000001301 oxygen Substances 0.000 description 1
- 229910052760 oxygen Inorganic materials 0.000 description 1
- 230000007918 pathogenicity Effects 0.000 description 1
- 230000037361 pathway Effects 0.000 description 1
- 239000000546 pharmaceutical excipient Substances 0.000 description 1
- 150000003904 phospholipids Chemical class 0.000 description 1
- 230000003863 physical function Effects 0.000 description 1
- 230000035790 physiological processes and functions Effects 0.000 description 1
- 230000003032 phytopathogenic effect Effects 0.000 description 1
- 229920002223 polystyrene Polymers 0.000 description 1
- 229920002635 polyurethane Polymers 0.000 description 1
- 239000004814 polyurethane Substances 0.000 description 1
- 235000015277 pork Nutrition 0.000 description 1
- 239000005373 porous glass Substances 0.000 description 1
- ZDHURYWHEBEGHO-UHFFFAOYSA-N potassiopotassium Chemical compound [K].[K] ZDHURYWHEBEGHO-UHFFFAOYSA-N 0.000 description 1
- GNSKLFRGEWLPPA-UHFFFAOYSA-M potassium dihydrogen phosphate Chemical compound [K+].OP(O)([O-])=O GNSKLFRGEWLPPA-UHFFFAOYSA-M 0.000 description 1
- 230000001376 precipitating effect Effects 0.000 description 1
- 108090000765 processed proteins & peptides Proteins 0.000 description 1
- BDERNNFJNOPAEC-UHFFFAOYSA-N propan-1-ol Chemical compound CCCO BDERNNFJNOPAEC-UHFFFAOYSA-N 0.000 description 1
- 239000012521 purified sample Substances 0.000 description 1
- 238000011002 quantification Methods 0.000 description 1
- 239000001397 quillaja saponaria molina bark Substances 0.000 description 1
- 238000011160 research Methods 0.000 description 1
- HEBKCHPVOIAQTA-ZXFHETKHSA-N ribitol Chemical compound OC[C@H](O)[C@H](O)[C@H](O)CO HEBKCHPVOIAQTA-ZXFHETKHSA-N 0.000 description 1
- 235000005713 safflower oil Nutrition 0.000 description 1
- 239000003813 safflower oil Substances 0.000 description 1
- 229930182490 saponin Natural products 0.000 description 1
- 150000007949 saponins Chemical class 0.000 description 1
- 238000013341 scale-up Methods 0.000 description 1
- CDAISMWEOUEBRE-UHFFFAOYSA-N scyllo-inosotol Natural products OC1C(O)C(O)C(O)C(O)C1O CDAISMWEOUEBRE-UHFFFAOYSA-N 0.000 description 1
- 238000000926 separation method Methods 0.000 description 1
- 239000008159 sesame oil Substances 0.000 description 1
- 235000011803 sesame oil Nutrition 0.000 description 1
- 235000021391 short chain fatty acids Nutrition 0.000 description 1
- 150000003396 sophoroses Chemical class 0.000 description 1
- 239000000600 sorbitol Substances 0.000 description 1
- 238000001179 sorption measurement Methods 0.000 description 1
- 239000003381 stabilizer Substances 0.000 description 1
- 125000000185 sucrose group Chemical group 0.000 description 1
- 150000005846 sugar alcohols Chemical class 0.000 description 1
- 150000008163 sugars Chemical class 0.000 description 1
- 239000012134 supernatant fraction Substances 0.000 description 1
- 239000000725 suspension Substances 0.000 description 1
- 229920001059 synthetic polymer Polymers 0.000 description 1
- 239000010913 used oil Substances 0.000 description 1
- 239000000080 wetting agent Substances 0.000 description 1
- 239000007222 ypd medium Substances 0.000 description 1
- UHVMMEOXYDMDKI-JKYCWFKZSA-L zinc;1-(5-cyanopyridin-2-yl)-3-[(1s,2s)-2-(6-fluoro-2-hydroxy-3-propanoylphenyl)cyclopropyl]urea;diacetate Chemical compound [Zn+2].CC([O-])=O.CC([O-])=O.CCC(=O)C1=CC=C(F)C([C@H]2[C@H](C2)NC(=O)NC=2N=CC(=CC=2)C#N)=C1O UHVMMEOXYDMDKI-JKYCWFKZSA-L 0.000 description 1
Landscapes
- Preparation Of Compounds By Using Micro-Organisms (AREA)
Abstract
Description
本発明は、食用植物や日常的に接触する植物由来の微生物によりバイオサーファクタントの一種であるマンノシルエリスリトールリピッドを製造する方法に関するものである。より具体的には、食用植物や日常的に接触する植物由来の微生物を用いたマンノシルエリスリトールリピッドのうちの1種類を選択的に製造する方法に関するものである。 The present invention relates to a method for producing mannosyl erythritol lipid, which is a kind of biosurfactant, using microorganisms derived from edible plants and plants that come into daily contact. More specifically, the present invention relates to a method for selectively producing one type of mannosyl erythritol lipid using an edible plant or a microorganism derived from a plant that is in daily contact.
糖脂質は、脂質に1〜数十個の単糖が結合した物質であり、生体内において細胞間の情報伝達に関与し、神経系及び免疫系の機能維持にも重要な役割を果たしていることなどが明らかにされつつある。また、糖脂質は、糖の性質に由来する親水性と脂質の性質に由来する親油性の二つの性質を合わせ持つ両親媒性物質であり、このような性質を有する両親媒性物質は界面活性物質と呼ばれている。石油化学工業が隆盛となるまでは、レシチン、サポニン等の生体成分由来の界面活性剤(バイオサーファクタント)が利用されていた。 Glycolipids are substances in which one to several tens of monosaccharides are bound to lipids, are involved in information transmission between cells in vivo, and play an important role in maintaining the functions of the nervous system and immune system. Etc. are being revealed. Glycolipids are amphiphilic substances that have both hydrophilic properties derived from the properties of sugars and lipophilic properties derived from the properties of lipids. It is called a substance. Until the petrochemical industry prospered, surfactants (biosurfactants) derived from biological components such as lecithin and saponin were used.
近年、石油化学工業の発展により合成界面活性剤が開発され、その生産量が飛躍的に増加し、日常生活には無くてはならない物質となっている。現在、一般に用いられている合成界面活性剤の種類は、細かな構造の違いまで考慮すると、数千種類にものぼる。親水基・疎水基の種類が異なるものから、それぞれのドメインの組成が同じでも分子量や立体構造の違いによって親水性疎水性バランス(HLB)の異なる同族体など、あらゆる化合物が開発されており、これらを単一種類で用いるだけでなく、混合して用いることで、幅広い産業分野において要求される性能に対応している。しかしながら、このような合成界面活性剤の使用量の拡大に伴って環境汚染が広がり、社会問題が生じている。従って、安全性が高く、環境に対する負荷を低減できる生分解性の高い界面活性剤の開発が望まれている。 In recent years, synthetic surfactants have been developed due to the development of the petrochemical industry, and their production has increased dramatically, making them indispensable for daily life. At present, there are several thousand types of synthetic surfactants that are generally used in consideration of fine structural differences. A variety of compounds have been developed from different types of hydrophilic groups and hydrophobic groups, including homologues with different hydrophilic and hydrophobic balance (HLB) due to differences in molecular weight and steric structure even if the composition of each domain is the same. In addition to being used in a single kind, it can be used in combination to meet the performance required in a wide range of industrial fields. However, as the amount of such synthetic surfactants used increases, environmental pollution spreads and social problems arise. Therefore, development of a highly biodegradable surfactant that has high safety and can reduce the burden on the environment is desired.
微生物などが生産する界面活性物質であるバイオサーファクントは、生分解性が高く、低毒性で環境に優しく、新規な生理機能を持つといわれている。このことから、食品、化粧品、医薬品、化学工業、環境分野等にこれらのバイオサーファクタントを幅広く応用することは、環境調和型の社会を実現し、高機能な製品を提供する上で極めて有意義である。微生物由来のバイオサーファクタントは、糖脂質系、アシルペプタイド系、リン脂質系、脂肪酸系及び高分子系の界面活性物質の5つに分類されている。なかでも、糖脂質系の界面活性物質については最もよく研究されており、酵母が生産する糖脂質系の界面活性物質としてカンジダ属酵母によるソホロースリピッド(特許文献1参照)やマンノシルエリスリトールリピッド(非特許文献1から6参照)などが知られている。 Biosurfactants, which are surface-active substances produced by microorganisms, are said to have high biodegradability, low toxicity, environmental friendliness, and novel physiological functions. Therefore, the wide application of these biosurfactants to food, cosmetics, pharmaceuticals, chemical industry, environmental fields, etc. is extremely meaningful in realizing an environmentally conscious society and providing highly functional products. . Biosurfactants derived from microorganisms are classified into five types: surfactants of glycolipid type, acyl peptide type, phospholipid type, fatty acid type and polymer type. Among them, glycolipid-based surfactants have been most studied, and as glycolipid-based surfactants produced by yeast, sophorose lipids produced by Candida spp. (See Patent Document 1) and mannosyl erythritol lipids (non- Patent Documents 1 to 6) are known.
マンノシルエリスリトールリピッド(mannosyl erythritol lipid、以下、「MEL」と略記することもある)は、界面活性能以外にも抗微生物活性、白血病細胞、神経系細胞などの細胞分化誘導活性、糖タンパク質結合活性、抗炎症・抗アレルギー活性、アポトーシス誘導活性、癌細胞増殖抑制活性など多様な機能が知られている。 Mannosyl erythritol lipid (hereinafter sometimes abbreviated as “MEL”) has antimicrobial activity, cell differentiation induction activity such as leukemia cells and nervous system cells, glycoprotein binding activity, Various functions such as anti-inflammatory / anti-allergic activity, apoptosis-inducing activity, and cancer cell growth-inhibiting activity are known.
MELはマンノースにエリスリトールと0〜2個の脂肪酸と2個のアセチル基が結合した糖脂質であり、アセチル基の結合位置と数によって、4種類のMEL(MEL‐A、MEL‐B、MEL‐C、MEL‐D)が存在することが知られている。これまで説明したようにMELの生産については多くの微生物菌株の発見、及び培養技術の改良が成されている。 MEL is a glycolipid in which erythritol, 0 to 2 fatty acids and 2 acetyl groups are bound to mannose. Depending on the binding position and number of acetyl groups, MEL (MEL-A, MEL-B, MEL-) C, MEL-D) is known to exist. As explained so far, many microorganism strains have been discovered and culture techniques have been improved for MEL production.
MELの生産方法については、これまで酵母を利用する方法等、多くの報告がされており、生産量向上を目的として菌株の選択、培養条件、培地組成が検討されている。酵母によるMEL生産に関する報告としては、たとえば、Candida sp. B‐7株を用いて5質量%の大豆油から5日間で35g/L(生産速度:0.3g/L/h、原料収率:70質量%)のMELを生産した例(非特許文献1及び2参照)、Candia(Pseudozyma) antarctica T‐34を用いて8質量%の大豆油から8日間で38g/L(生産速度:0.2g/L/h、原料収率:48質量%)のMELを生産した例(非特許文献3及び4参照)、P. antarctica T‐34を用いて6日間隔で計3回の逐次流加により24日後に25質量%のピーナッツ油から110g/L(生産速度:0.2g/L/h、原料収率:44質量%)のMELを生産した例(非特許文献5参照)、Candida sp. SY‐16株を用いて10質量%の植物油脂から回分培養法により200時間で50g/L(生産速度:0.25g/L/h、原料収率:50質量%)のMELを生産し、また、20質量%の植物油から流加培養法により200時間で120g/L(生産速度:0.6g/L/h、原料収率:50質量%)のMELを生産した例(非特許文献6参照)、Pseudozyma aphidisを用いて80質量%の植物油脂から流加培養法により24時間で13.9g/L(生産速度:0.57g/L/h、原料収率:92質量%)のMELを生産した例(非特許文献7参照)などがある。また、醤油醸造工程において副産物として生産されるしょうゆ油を原料としてP. antarctica T‐34を用いて7日間で8質量%のしょうゆ油から17g/L(生産速度:0.1g/L/h、原料収率:21質量%)のMELの生産が可能であることも報告されている(特許文献2参照)。また、Ustilago maydisがMEL‐A、MEL‐B、MEL‐Cの混合物を生産することが知られている(非特許文献14参照)。 Many methods for producing MEL have been reported so far, such as a method using yeast, and selection of strains, culture conditions, and medium composition have been studied for the purpose of improving the production amount. As a report on MEL production by yeast, for example, Candida sp. Example of producing 35 g / L (production rate: 0.3 g / L / h, raw material yield: 70 mass%) of MEL from 5 mass% soybean oil using B-7 strain in 5 days (non-patent literature) 1 and 2), 38 g / L (production rate: 0.2 g / L / h, raw material yield: 48 mass%) in 8 days from 8 mass% soybean oil using Candia (Pseudozyma) antarctica T-34 (See Non-Patent Documents 3 and 4), p. 110 g / L (production rate: 0.2 g / L / h, raw material yield: 44 mass) from 25% by mass of peanut oil after 24 days by continuous feeding three times at an interval of 6 days using an Antarctica T-34 %) Of MEL (see Non-Patent Document 5), Candida sp. Using SY-16 strain, 50 g / L (production rate: 0.25 g / L / h, raw material yield: 50 mass%) of MEL is produced from 10 mass% of vegetable oils and fats by batch culture in 200 hours. Moreover, the example which produced 120 g / L (production rate: 0.6 g / L / h, raw material yield: 50 mass%) of MEL from 20 mass% vegetable oil by the fed-batch culture method in 200 hours (nonpatent literature 6) MEL of 13.9 g / L (production rate: 0.57 g / L / h, raw material yield: 92% by mass) in 24 hours by fed-batch culture method using 80% by mass of vegetable oil using Pseudozyma aphidis (See Non-Patent Document 7). In addition, soy sauce oil produced as a by-product in the soy sauce brewing process is used as a raw material. It is also possible to produce 17 g / L (production rate: 0.1 g / L / h, raw material yield: 21% by mass) of MEL from 8% by mass soy sauce oil in 7 days using antitica T-34 It has been reported (see Patent Document 2). It is also known that Ustilago maydis produces a mixture of MEL-A, MEL-B, and MEL-C (see Non-Patent Document 14).
また、シュードザイマ(Pseudozyma)属の酵母を用いて、MEL‐Aと、MEL‐Bと、MEL‐Cとを選択的に量産する方法が報告されている(例えば、非特許文献8参照)。このように、マンノシルエリスリトールリピッドのバリエーションは大きく拡充され、実用化に向けた研究が展開されている。 In addition, a method for selectively mass-producing MEL-A, MEL-B, and MEL-C using yeast belonging to the genus Pseudozyma has been reported (for example, see Non-Patent Document 8). Thus, the variation of mannosyl erythritol lipid has been greatly expanded, and research for practical application is being developed.
MEL‐Aは、これまで説明した微生物の他にも、Pseudozyma rugulosa、Pseudozyma parantarctica、Pseudozyma fujiformataを用いて量産可能であることが報告されている(例えば、非特許文献9参照)。 In addition to the microorganisms described so far, MEL-A has been reported to be mass-produced using Pseudozyma rugulosa, Pseudozyma parantactica, and Pseudozyma fujiformata (see, for example, Non-Patent Document 9).
MEL‐BについてはKlutzmanomycetous sp.I‐11(特許文献3参照)、Candida sp.SY‐16(非特許文献6参照)、Pseudozyma tsukubaensisを用いて量産可能であることが知られている。(非特許文献10)。 Regarding MEL-B, Klutzmanomycetous sp. I-11 (see Patent Document 3), Candida sp. It is known that mass production is possible using SY-16 (see Non-Patent Document 6) and Pseudozyma tsukubaensis. (Non-Patent Document 10).
MEL‐Cについては、Pseudozyma hubeiensis(非特許文献11参照)、Pseudozyma graminicola(非特許文献12参照)、Pseudozyma shanxiensis(非特許文献13参照)を用いて量産可能であることが報告されている。 About MEL-C, it is reported that mass production is possible using Pseudozyma hubiensis (refer nonpatent literature 11), Pseudozyma gramicola (refer nonpatent literature 12), and Pseudozyma shanxiensis (refer nonpatent literature 13).
このように、多くのMEL生産微生物の発見に伴って、製造可能なMELの構造のバリエーションは拡大し、MELの製造技術は大きく向上してきている。
ところで、これらMEL生産微生物は様々な自然環境中から単離されたものであるため、人体に対する病原性は知られていないが、その安全性に対する知見はほとんどない。特に、微生物生産物を食品用途等へ展開するためには、使用する微生物の単離源が重要視される。例えば、酒やパンの製造に関わる出芽酵母、乳酸菌や枯草菌(納豆菌)などは、食品分野で活躍する微生物として一般的によく知られている。これらの微生物は全て食品中から容易に単離可能な微生物である。 By the way, since these MEL-producing microorganisms have been isolated from various natural environments, their pathogenicity to the human body is not known, but little is known about their safety. In particular, in order to develop a microbial product for food use or the like, the isolation source of the microorganism to be used is regarded as important. For example, budding yeast, lactic acid bacteria, Bacillus subtilis (natto), and the like that are involved in the production of sake and bread are generally well known as microorganisms that play an active role in the food field. These microorganisms are all microorganisms that can be easily isolated from food.
しかしながら、上述したPseudozyma属の単離源は不明であるため、これらに由来するMELは食用に適しているとは言い難い。 However, since the isolation source of the above-mentioned Pseudozyma genus is unknown, it is difficult to say that MEL derived from these is suitable for food.
そこで、食用植物又は日常的に接触する植物群から単離した微生物を用いたMEL製造技術を開発することで用途範囲は拡大し、かつ安全性の面で大きな利点を得られることが期待される。 Therefore, by developing MEL production technology using microorganisms isolated from edible plants or plants that come into daily contact, it is expected that the range of applications will be expanded and great advantages will be obtained in terms of safety. .
また、ウスチラゴ メイディス(U. maydis)はトウモロコシに感染する植物病原菌であり、U. maydisが感染したトウモロコシは、ウイトラコーチェという名で一般的に食されている。特に、メキシコでは伝統的な高級食材として食されている。しかし、U. maydisはMEL‐A、MEL‐B、MEL‐Cだけでなく、セロビオースリピッドも同時に生産するため、それぞれの成分を選択的に生産することは実用上、不可能である。しなしながら、MEL‐A、MEL‐B、MEL‐Cはそれぞれ物性が異なるため、それぞれを選択的に製造することができれば、新たな食品用途への利用が期待される。 U. maydis is a phytopathogenic fungus that infects corn. Maize infected with maydis is commonly eaten under the name of Vitracoche. Especially in Mexico, it is eaten as a traditional luxury food. However, U. Since maydis produces not only MEL-A, MEL-B, and MEL-C, but also cellobiose lipid at the same time, it is practically impossible to selectively produce each component. However, since MEL-A, MEL-B, and MEL-C have different physical properties, if they can be selectively manufactured, they are expected to be used for new food applications.
以上の状況から、日常的に接触する安全性の高いことが判明している植物群又は食用植物群からMEL生産微生物を単離し、さらにMEL‐A、MEL‐B、MEL‐Cのいずれかを選択的に生産できる方法が開発されれば、MELの用途拡大に多大な影響を与え、ひいてはバイオサーファクタントの普及に繋がるものと期待される。 From the above situation, isolate the MEL-producing microorganism from the group of plants or edible plants that are known to be highly safe in daily contact, and then select one of MEL-A, MEL-B, and MEL-C. If a method capable of selective production is developed, it is expected to have a great influence on the expansion of the use of MEL and eventually lead to the spread of biosurfactants.
本発明はこのような事情に鑑みてなされたものであり、その目的は、安全性が高く、食品用途への展開が可能なマンノシルエリスリトールリピッドを製造し、かつMEL‐A、MEL‐B又はMEL‐Cを選択的に製造する技術を提供することにある。 The present invention has been made in view of such circumstances, and the object thereof is to manufacture mannosyl erythritol lipid which is highly safe and can be developed for food use, and MEL-A, MEL-B or MEL. -To provide a technique for selectively producing C.
本発明者は、上記課題を解決するため鋭意検討を重ねた結果、食用植物又は日常的にヒト又は動物が接触する(物理的に接触したり、動物種によっては口に含んだりする)植物から単離されたウスチラゴ(Ustilago)属に属する微生物を培養することで、MEL‐A、MEL‐B、MEL‐Cのいずれかを選択的に生産することが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-mentioned problems, the present inventor has found that from an edible plant or a plant that comes into contact with humans or animals on a daily basis (physical contact, or depending on the animal species, it is contained in the mouth). To cultivate an isolated microorganism belonging to the genus Ustylago, one of MEL-A, MEL-B, and MEL-C can be selectively produced, and the present invention is completed. It came.
また、当該微生物が生産するMEL‐A、MEL‐B及びMEL‐Cの化学構造を調べると、シュードザイマ(Pseudozyma)属の微生物が生産するMELと同様の構造を有しており、その物性・機能はシュードザイマ(Pseudozyma)属の微生物が生産するMELと同様であった。 In addition, when the chemical structure of MEL-A, MEL-B and MEL-C produced by the microorganism is examined, it has the same structure as MEL produced by a microorganism belonging to the genus Pseudozyma. Was similar to MEL produced by microorganisms of the genus Pseudozyma.
つまり、本発明者らは、ウスチラゴ(Ustilago)属に属するMEL生産菌の単離源として、食用植物又は日常的にヒト又は動物が接触する植物の中から特定の植物を見出し、かつこれらのMEL生産菌を用いれば、従来報告されているMELと同様の物性・機能を有するMEL‐A、MEL‐B、MEL‐Cのいずれかを選択的に生産することができることを見出した。本発明はかかる知見により完成されたものである。 That is, the present inventors have found a specific plant from among edible plants or plants that humans or animals are in daily contact with as an isolation source of MEL-producing bacteria belonging to the genus Ustylago, and these MELs It has been found that by using a production bacterium, any of MEL-A, MEL-B, and MEL-C having the same physical properties and functions as MEL reported heretofore can be selectively produced. The present invention has been completed based on such findings.
すなわち、本発明は以下の発明を包含する。 That is, the present invention includes the following inventions.
本発明に係る下記一般式(1) The following general formula (1) according to the present invention
(上記一般式(1)においてR1〜R5は、それぞれ独立して、水素原子又は炭素数1〜18の脂肪酸基を表す)
で表される構造を有するマンノシルエリスリトールリピッドの製造方法は、サトウキビ(Saccharum)属、リンゴ(Malus)属、シソ属(Perilla)属、アブラナ(Brassica)属及びギョウギシバ(Cynodon)属の植物のいずれかから単離されたウスチラゴ(Ustilago)属の微生物を培養する培養工程を含むことを特徴としている。
(In the general formula (1), R 1 to R 5 each independently represents a hydrogen atom or a fatty acid group having 1 to 18 carbon atoms)
A method for producing a mannosyl erythritol lipid having a structure represented by any one of the following plants: It comprises a culture step of culturing a microorganism belonging to the genus Ustilago isolated from
また、本発明に係る下記一般式(2) Further, the following general formula (2) according to the present invention:
(上記一般式(2)においてm、nは互いに独立して1〜16の整数である)
で表される構造を有するマンノシルエリスリトールリピッド‐Bの製造方法は、サトウキビ(Saccharum)属の植物から単離されたウスチラゴ(Ustilago)属の微生物を培養する培養工程を含むことを特徴としている。
(In the above general formula (2), m and n are each independently an integer of 1 to 16)
The method for producing mannosyl erythritol lipid-B having the structure represented by the formula is characterized by comprising a culture step of culturing a microorganism of the genus Ustilago isolated from a plant of the genus Saccharum.
さらに、本発明に係るMEL‐Bの製造方法では、上記微生物がウスチラゴ サイタミネア(Ustilago scitaminea)であることがより好ましい。 Furthermore, in the method for producing MEL-B according to the present invention, it is more preferable that the microorganism is Ustyago scitaminea.
さらに、本発明に係るMEL‐Bの製造方法では、上記培養工程にて用いる培地は糖を炭素源とし油脂の含有量が0〜5重量%以下であることがより好ましい。 Furthermore, in the method for producing MEL-B according to the present invention, it is more preferable that the medium used in the culturing step contains sugar as a carbon source and the fat content is 0 to 5% by weight or less.
また、本発明に係る下記一般式(3) Further, the following general formula (3) according to the present invention:
(上記一般式(3)においてm、nは互いに独立して1〜16の整数である)
で表される構造を有するマンノシルエリスリトールリピッド‐Cの製造方法は、ギョウギシバ(Cynodon)属の植物から単離されたウスチラゴ(Ustilago)属の微生物を培養する培養工程を含むことを特徴としている。
(In the general formula (3), m and n are each independently an integer of 1 to 16)
The method for producing mannosyl erythritol lipid-C having the structure represented by the formula is characterized by comprising a culture step of culturing a microorganism of the genus Ustilago isolated from a plant of the genus Cynodon.
さらに、本発明に係るMEL‐Cの製造方法では、上記微生物がウスチラゴ シノドンティス(Ustilago cynodontis)であることがより好ましい。 Furthermore, in the method for producing MEL-C according to the present invention, it is more preferable that the microorganism is Ustyago synonodontis (Ustilago cynodontis).
また、本発明に係る下記一般式(4) Further, the following general formula (4) according to the present invention:
(上記一般式(4)においてm、nは互いに独立して1〜16の整数である)
で表される構造を有するマンノシルエリスリトールリピッド‐Aの製造方法は、リンゴ(Malus)属、シソ属(Perilla)属及びアブラナ(Brassica)属のいずれかの植物から単離されたウスチラゴ(Ustilago)属の微生物を培養する培養工程を含むことを特徴としている。
(In the above general formula (4), m and n are each independently an integer of 1 to 16)
A method for producing mannosylerythritol lipid-A having a structure represented by the formula: genus Ustylago isolated from any plant of the genus Apple (Malus), Perilla (Brassilla) and Brassica It includes a culture step of culturing the microorganisms.
また、本発明に係る製造方法は、上記培養工程にて得られた培養液から菌体を取り除き、硫酸アンモニウムを添加して沈殿物を得る濃縮工程をさらに含むことがより好ましい。 Moreover, it is more preferable that the production method according to the present invention further includes a concentration step of removing cells from the culture solution obtained in the culture step and adding ammonium sulfate to obtain a precipitate.
本発明に係る製造方法は、上述のようにサトウキビ(Saccharum)属、リンゴ(Malus)属、シソ属(Perilla)属、アブラナ(Brassica)属及びギョウギシバ(Cynodon)属の植物のいずれかから単離されたウスチラゴ(Ustilago)属の微生物を培養する工程を含んでいるので、安全性が高く、食品用途等への展開が可能なマンノシルエリスリトールリピッドを製造し、かつMEL‐A、MEL‐B又はMEL‐Cを選択的に製造することができるという効果を奏する。 As described above, the production method according to the present invention is isolated from any plant of the genus Saccharum, the genus Apple, the genus Perilla, the genus Brassica, and the genus Cynodon. A mannosyl erythritol lipid which is highly safe and can be developed for food use and the like, and includes MEL-A, MEL-B or MEL, because it includes a step of culturing microorganisms belonging to the genus Ustilago -There is an effect that C can be selectively produced.
また、本発明によれば、これまでに報告されたMEL‐A、MEL‐B、MEL‐Cの3種類のうちのいずれかを選択的に生産可能なシュードザイマ(Pseudozyma)属の微生物、及び、上記3種類のうちのいずれかを選択的に生産することができないウスチラゴ メイディス(U. maydis)とは異なり、食用植物又は日常的に接触する植物を単離源とする微生物を使用して、かつ、3種類のMELのいずれかを選択的に生産できる。つまり、本発明により、安全性が高い上記3種類のいずれかのMELを、新たな食品用途に展開することが可能となった。 According to the present invention, a microorganism belonging to the genus Pseudozyma that can selectively produce any one of the three types of MEL-A, MEL-B, and MEL-C reported so far, and Unlike U. maydis, which cannot selectively produce any one of the above three types, using microorganisms that are isolated from edible plants or plants that come into daily contact, and Any one of the three types of MEL can be selectively produced. That is, according to the present invention, any one of the above three types of MEL having high safety can be developed for new food applications.
特に、本発明にて生産するMEL‐B及びMEL‐CはMEL‐Aに比べて水溶性が高く、期待される応用範囲がより広いことから、MELの構造・機能的バリエーションの拡充に貢献するだけでなく、用途展開の効率化にも貢献できるという更なる効果を奏する。 In particular, MEL-B and MEL-C produced in the present invention are higher in water solubility than MEL-A and expected to have a wider range of applications, contributing to the expansion of MEL structural and functional variations. In addition, it has the further effect that it can contribute to the efficiency of application development.
以下、本発明を詳細に説明する。 Hereinafter, the present invention will be described in detail.
本発明に係る上記一般式(1)で表される構造を有するマンノシルエリスリトールリピッドの製造方法は、サトウキビ(Saccharum)属、リンゴ(Malus)属、シソ属(Perilla)属、アブラナ(Brassica)属及びギョウギシバ(Cynodon)属の植物のいずれかから単離されたウスチラゴ(Ustilago)属の微生物を培養する工程を含めばよい。 The method for producing a mannosyl erythritol lipid having the structure represented by the above general formula (1) according to the present invention includes the genus Saccharum, the genus Apple, the genus Perilla, the genus Brassica, and A step of culturing a microorganism of the genus Ustilago isolated from any plant of the genus Cynodon may be included.
サトウキビ属、リンゴ属、シソ属、アブラナ属及びギョウギシバ属の植物は、いずれも食用植物であるか、又は日常的に接触する植物である。よって、これらの植物から単離されたウスチラゴ属の微生物は安全性が高いといえる。なお、本明細書において「植物から単離される」とは、植物中、植物の表面、植物の根圏などの植物から単離されることを意味し、具体的には植物中等から、ここに生息する菌を取得することを意味する。 Sugarcane, apple genus, perilla genus, rapeseed genus and sorghum genus are all edible plants or plants that come into daily contact. Therefore, it can be said that the microorganisms of the genus Ustylago isolated from these plants are highly safe. In the present specification, the term “isolated from a plant” means being isolated from a plant such as a plant, the surface of the plant, or the rhizosphere of the plant. It means to get the fungus to do.
サトウキビ属の植物の具体例としては特に限定されないが、サッカリウム オフィシナルム(S. officinarum)、サッカリウム スポンタネウム(S. spontaneum)、サッカリウム ロブスツム(S. robustum)、サッカリウム バーバリ(S. barberi)、サッカリウム サイネンセ(S. sinense)、サッカリウム エヂュール(S. edule)等が挙げられる。中でもサッカリウム オフィシナルムが好ましい。 Specific examples of sugarcane plants include, but are not limited to, S. officinarum, S. spotanem, S. robustum, S. barberi , S. potassium senense, S. potassium edule, and the like. Among them, the potassium potassium officinalum is preferable.
なお、ウスチラゴ属の微生物は感染する宿主の種類によって分類されているため、サトウキビから単離されたウスチラゴ属の微生物は全てUstilago scitamineaである。同様に、後述するバミューダグラスから単離されたウスチラゴ属の微生物は全てUstilago cynodontisである。 Since microorganisms belonging to the genus Ustylag are classified according to the type of host to be infected, all microorganisms belonging to the genus Ustyago isolated from sugar cane are Ustilago scitaminea. Similarly, all the microorganisms of the genus Ustylago isolated from the Bermuda glass described later are Ustilago cynodontis.
リンゴ属の植物の具体例としては特に限定されないが、リンゴ(M. domestica)、クラブアップル(M. pumila)、ヒメリンゴ(M. baccata)等が挙げられる。中でもヒメリンゴ(M. baccata)が好ましい。 Although it does not specifically limit as a specific example of a plant of an apple genus, an apple (M. domestica), crab apple (M. pumila), a hime apple (M. baccata) etc. are mentioned. Of these, M. baccata is preferable.
シソ属の植物の具体例としては特に限定されないが、シソ(Perilla frutescens)が好ましい。 Although it does not specifically limit as a specific example of the plant of Perilla genus, Perilla frucescens is preferable.
アブラナ属の植物の具体例としては特に限定されないが、ミズナ(B. rapa)、チンゲンサイ(B. chingensai)、ハクサイ(B. hakusai)、カラシナ(B. karashina)、コマツナ(B. komatsuna)等が挙げられる。中でも、ミズナ(B. rapa L. var. nipposinica)が好ましい。 Specific examples of Brassica plants are not particularly limited, but examples include Mizuna (B. rapa), B. chinensai, B. khassai, B. karasina, B. komatsusuna and the like. Can be mentioned. Among them, Mizuna (B. rapa L. var. Nipposica) is preferable.
ギョウギシバ属の植物の具体例としては特に限定されないが、バミューダグラス(ギョウギシバ、C. dactylon)、アフリカギョウギシバ(C. transvaalensis)、マクニスバーミューダグラス(C. mabennisii Hurcombe)、オニギョウギシバ(C. plectostachys)、キノドン インコンプレツス(C. incompletus)、等が挙げられる。中でもバミューダグラスが好ましい。 Specific examples of the plant belonging to the genus Gorga are not particularly limited, but include Bermudagrass (C. dactylon), African ginkgo biloba (C. tranvaalensis), Macnis vermudgrass (C. mabennisii Hurcombe), Onigyo chy p. , Quinodon incompress (C. incompletetus), and the like. Of these, Bermuda glass is preferable.
また、これらの植物から単離されたウスチラゴ属の微生物はMEL‐A、MEL‐B、MEL‐Cの3種のMELのうちのいずれかを選択的に生産することができる。 In addition, microorganisms belonging to the genus Ustillago isolated from these plants can selectively produce any one of three types of MELs, MEL-A, MEL-B, and MEL-C.
本明細書において「選択的に生産する」とは、生産する対象物が複数種類想定し得る場合、そのうちの一つの対象物を他の対象物に比べて多く生産ことを意味する。具体的には、生産する上記一般式(1)で表されるMELの全量に対して、85重量%以上を占める割合にて、3種のMELのうちの1種を製造することを意味する。 In this specification, “selectively producing” means that when a plurality of types of objects to be produced can be assumed, one of the objects is produced more than other objects. Specifically, it means that one of the three types of MEL is produced at a ratio of 85% by weight or more with respect to the total amount of MEL represented by the general formula (1) to be produced. .
〔1.植物からの単離方法〕
上述した各植物からウスチラゴ属の微生物を単離する方法としては特に限定されない。従来公知の方法を適用してもよい。例えば、植物を破砕して水又は適切な緩衝液に懸濁させ、懸濁液を栄養塩固形培地に塗布し、形成されたコロニーをMEL合成確認用培地に植えて、MEL形成能を有する菌株を単離した上で、従来公知の同定方法によりウスチラゴ属であることを確認してもよい。また、植物を破砕して、栄養塩液体培地に混合して培養した後、MELの生産が確認できた液体培地を栄養塩固形培地に塗布して、形成されたコロニーを合成確認用培地に植えて、MEL形成能を確認した上で、ウスチラゴ属か否かを同定してもよい。MEL合成確認用培地としてはMELの合成を確認できるものであれば特に限定されないが、例えば、培地に大豆油等の油脂を混合しておけばよい。当該油脂が乳化するか否かでMELの形成を確認することができる。
[1. (Isolation method from plant)
It does not specifically limit as a method of isolating the microorganism of the genus Ustyago from each plant mentioned above. A conventionally known method may be applied. For example, the plant is crushed and suspended in water or an appropriate buffer, the suspension is applied to a nutrient solid medium, the formed colonies are planted in a medium for MEL synthesis confirmation, and a strain having MEL forming ability And may be confirmed to be of the genus Ustilago by a conventionally known identification method. In addition, the plant is crushed, mixed in a nutrient liquid medium and cultured, and then a liquid medium that has been confirmed to be produced by MEL is applied to the nutrient solid medium, and the formed colonies are planted in the medium for confirmation of synthesis. Then, after confirming the ability to form MEL, it may be identified whether or not it belongs to the genus Ustilago. The medium for confirming MEL synthesis is not particularly limited as long as MEL synthesis can be confirmed. For example, oil such as soybean oil may be mixed in the medium. The formation of MEL can be confirmed by whether or not the oil or fat is emulsified.
〔2.本発明に係るMEL‐Bの製造方法に用いる微生物〕
本発明に係る上記一般式(2)で表される構造を有するMEL‐Bの製造方法は、サトウキビ(Saccharum)属の植物から単離されたウスチラゴ(Ustilago)属の微生物を培養する培養工程を含めばよい。
[2. Microorganism used in the method for producing MEL-B according to the present invention]
The method for producing MEL-B having the structure represented by the general formula (2) according to the present invention comprises a culturing step of culturing a microorganism belonging to the genus Ustilago isolated from a plant belonging to the genus Saccharum. Include it.
つまり、本発明に係るMEL‐Bの製造方法では、これまでの説明で例示した植物のうち、サトウキビ属の植物から単離されたウスチラゴ属の微生物を用いるとよい。 That is, in the method for producing MEL-B according to the present invention, among the plants exemplified in the above description, a microorganism belonging to the genus Ustyago isolated from a sugar cane plant may be used.
サトウキビ族の植物から単離されたウスチラゴ属の微生物の具体例としては、特に限定されないが、U. scitamineaが挙げられる。この微生物はマンノシルアルジトールリピッド生産微生物として知られていなかったものであるが、本発明者等により、シュードザイマ アンタクチカ等の既知のマンノシルアルジトールリピッド生産菌と同等の生産性を有することが初めて確認されたものである。しかも、ウスチラゴ サイタミネアはサトウキビから分離された微生物であり、マンノシルアルジトールリピッドの中のMEL‐Bのみを選択的に生産することができる。 Specific examples of the microorganism of the genus Ustylago isolated from sugarcane plants are not particularly limited. scitaminea. Although this microorganism was not known as a mannosyl alditol lipid-producing microorganism, the present inventors have confirmed for the first time that it has the same productivity as known mannosyl alditol lipid-producing bacteria such as pseudozyma antactica. It is a thing. Moreover, Ustyago cythaminea is a microorganism isolated from sugarcane and can selectively produce only MEL-B in mannosyl alditol lipids.
U. scitamineaは従来公知の方法でサトウキビ属の植物から単離して得ればよいが、NBRC32730株を用いてもよい。NBRC32730株を用いれば極めて高効率にMEL‐Bを選択的に生産することができる。また、U. scitamineaを培養する際は、炭素源として油脂を用いずに、糖を用いればよい点で有利である。即ち、従来公知のMEL生産菌では炭素源として油脂を含む培地で培養する必要があった。これでは、培養後に油脂を除去しMELを精製する必要があったが、これには煩雑な作業を要した。しかし、糖を炭素源として、油脂を含まず(含んだとしても炭素源の5重量%以下)であれば、容易に精製できるので、高純度のMEL‐Bを簡便に製造することができる。 U. Although scitaminea may be obtained by isolation from a sugarcane plant by a conventionally known method, NBRC32730 strain may be used. If NBRC32730 strain is used, MEL-B can be selectively produced with extremely high efficiency. In addition, U.S. When cultivating scitamine, it is advantageous in that sugar may be used without using fats and oils as a carbon source. That is, conventionally known MEL-producing bacteria have to be cultured in a medium containing fats and oils as a carbon source. In this case, it was necessary to remove the fats and oils and purify the MEL after culturing, but this required complicated work. However, since sugar can be used as a carbon source and oil and fat are not included (even if it is included, 5% by weight or less of the carbon source), it can be easily purified, so that high-purity MEL-B can be easily produced.
〔3.本発明に係るMEL‐Cの製造方法に用いる微生物〕
本発明に係る上記一般式(3)で表される構造を有するMEL‐Cの製造方法は、ギョウギシバ(Cynodon)属の植物から単離されたウスチラゴ(Ustilago)属の微生物を培養する培養工程を含めばよい。
[3. Microorganisms used in the method for producing MEL-C according to the present invention]
The method for producing MEL-C having the structure represented by the general formula (3) according to the present invention includes a cultivation step of culturing a microorganism belonging to the genus Ustilago isolated from a plant belonging to the genus Cynodon. Include it.
つまり、本発明に係るMEL‐Cの製造方法では、これまでの説明で例示した植物のうち、ギョウギシバ属の植物から単離されたウスチラゴ属の微生物を用いるとよい。ギョウギシバ属の植物から単離されたウスチラゴ属の微生物の具体例としては、特に限定されないが、ウスチラゴ シノドンティス(U. cynodontis)が挙げられる。ウスチラゴ シノドンティスはバミューダグラスから分離された微生物であり、MELのうちのMEL‐Cを選択的に生産することができる。なお、バミューダグラスは、グラウンドや庭に敷き詰められるシバであり、安全かつ日常的に接触頻度の高い植物である。 That is, in the method for producing MEL-C according to the present invention, among the plants exemplified in the above description, a microorganism belonging to the genus Ustyago, which is isolated from a plant belonging to the genus Gorilla, may be used. Specific examples of the microorganism belonging to the genus Ustyago isolated from a plant belonging to the genus Gyolia include, but are not limited to, U. cynodontis. Ustyago Shinodontis is a microorganism isolated from Bermuda grass and can selectively produce MEL-C of MEL. Bermuda grass is a plant that is spread on the ground and in the garden, and is a plant that is safe and has a high daily contact frequency.
ウスチラゴ シノドンティスは従来公知の方法でギョウギシバ属の植物から単離して得ればよいが、NBRC7530株を用いてもよい。NBRC7530株を用いれば極めて高効率にMEL‐Cを選択的に生産することができる。 The ustillago sinodontis may be obtained by isolation from a plant belonging to the genus Gorilla by a conventionally known method, but the NBRC7530 strain may be used. If NBRC7530 strain is used, MEL-C can be selectively produced with extremely high efficiency.
〔4.本発明に係るMEL‐Aの製造方法に用いる微生物〕
本発明に係る一般式(4)で表される構造を有するMEL‐Aの製造方法は、リンゴ(Malus)属、シソ属(Perilla)属及びアブラナ(Brassica)属のいずれかの植物から単離されたウスチラゴ(Ustilago)属の微生物を培養する培養工程を含めばよい。
[4. Microorganisms used in MEL-A production method according to the present invention]
The method for producing MEL-A having the structure represented by the general formula (4) according to the present invention is isolated from any plant of the genus Apple (Malus), the genus Perilla and the genus Brassica. A culture step of culturing a microorganism belonging to the genus Ustilago can be included.
つまり、本発明に係るMEL‐Aの製造方法では、これまでの説明で例示した植物のうち、リンゴ属、シソ属及びアブラナ属のいずれかの植物から単離されたウスチラゴ属の微生物を用いればよい。当該微生物は、従来公知の方法でリンゴ属、シソ属及びアブラナ属の植物から単離して得ればよい。 That is, in the method for producing MEL-A according to the present invention, among the plants exemplified in the above description, a microorganism of the genus Ustyago isolated from any one of the genus Apple, Perilla and Brassica is used. Good. The microorganism may be obtained by isolation from a plant of the genus Apple, Perilla and Brassica by a conventionally known method.
〔5.培養工程〕
上述した本発明に係るMELの製造方法、MEL‐Aの製造方法、MEL‐Bの製造方法、MEL‐Cの製造方法(なお、説明の便宜のため、これらの製造方法の総称として、単に「本発明に係る製造方法」と表記することもある)における、培養工程については、従来公知の方法でそれぞれの発明におけるウスチラゴ属の微生物を培養すればよい。
[5. (Culture process)
The above-described MEL manufacturing method, MEL-A manufacturing method, MEL-B manufacturing method, MEL-C manufacturing method according to the present invention described above (for convenience of explanation, these manufacturing methods are simply named as “generic name”. As for the culturing step in the “production method according to the present invention”), the microorganism of the genus Ustylago in each invention may be cultured by a conventionally known method.
培養に用いる培地としては、ウスチラゴ属がMELを生産する条件で培養可能なものであれば特に限定されない。ウスチラゴ属を培養すると通常MELを生産するので、一般にウスチラゴ属等の酵母の培養に用いられる培地を用いればよい。例えばYPD培地(例えば水1Lにイーストエキストラクト10g、ポリペプトン20g及びグルコース20gを溶解させて作製する)等の酵母に対して一般に用いられる培地を使用することができる。 The medium used for the culture is not particularly limited as long as it can be cultured under the conditions in which the genus Ustylago produces MEL. When cultivating the genus Ustylag, MEL is usually produced, and therefore, a medium generally used for culturing yeasts such as the genus Ustylag may be used. For example, a medium generally used for yeast such as YPD medium (for example, prepared by dissolving 10 g of yeast extract, 20 g of polypeptone and 20 g of glucose in 1 L of water) can be used.
また、培養に用いる培地には油脂を添加してもよい。油脂を添加する場合、培地中の油脂の含有量としては、特に限定されるものではないが、0〜8重量%以下が好ましく、0〜5重量%以下がさらに好ましい。油脂は培養開始時にその全量を添加しても、培養中に連続的または間欠的に添加してもよい。油脂の具体的な種類としては、植物油脂又は動物油脂等のいずれでもよく、目的に応じて適宜選定することができる。植物油脂としては、大豆油、菜種油、コーン油、ピーナッツ油、綿実油、ベニバナ油、ごま油、オリーブ油、バーム油等が挙げられ、これらの中でも、大豆油が好ましい。動物油脂としては、牛脂、豚脂、魚脂等が挙げられる。これらは、1種を単独で又は2種以上を適宜混合して用いてもよい。 Moreover, you may add fats and oils to the culture medium used for culture | cultivation. When fats and oils are added, the content of fats and oils in the medium is not particularly limited, but is preferably 0 to 8% by weight or less, and more preferably 0 to 5% by weight or less. The total amount of the oil / fat may be added at the start of the culture, or may be added continuously or intermittently during the culture. As a specific kind of fats and oils, any of vegetable oils and animal fats and the like may be used, and can be appropriately selected according to the purpose. Examples of vegetable oils include soybean oil, rapeseed oil, corn oil, peanut oil, cottonseed oil, safflower oil, sesame oil, olive oil, and balm oil. Among these, soybean oil is preferable. Examples of animal fats include beef tallow, pork tallow and fish tallow. You may use these individually by 1 type or in mixture of 2 or more types as appropriate.
培地に添加する油脂類以外の他の成分としては、当該技術分野で通常用いられる炭素源、窒素源、無機塩類、及び必要な栄養源等が用いられる。炭素源としては、該微生物が資化し得るものであればよく、炭水化物(グルコース、マンノース、グリセロール、マンニトール等の単糖、ショ糖、麦芽糖、乳糖等のオリゴ糖、デンプン等)、有機酸(酢酸、プロピオン酸、マレイン酸、フマル酸、リンゴ酸等)、アルコール類(エタノール、プロパノール等)が用いられる。窒素源としては、塩化アンモニウム、硫酸アンモニウム、硝酸アンモニウム、リン酸アンモニウム等の無機酸若しくは有機酸のアンモニウム塩、又はペプトン、酵母エキス、麦芽エキス、肉エキス、コーンスチープリカー等の含窒素化合物が用いられる。無機塩類としては、リン酸第一カリウム、リン酸第二カリウム、リン酸マグネシウム、硫酸マグネシウム、塩化ナトリウム、硫酸第一鉄、硫酸マンガン、硫酸銅、炭酸カルシウム等が用いられる。また、上記の炭水化物、有機酸、窒素源を含む植物体の搾汁(例えば、サトウキビ、トウモロコシ、穀類、果実など)も炭素源および窒素源を含む成分として使用可能である。 As other components other than fats and oils added to the medium, carbon sources, nitrogen sources, inorganic salts, necessary nutrient sources and the like that are usually used in the technical field are used. Any carbon source may be used as long as it can be assimilated by the microorganism, and includes carbohydrates (monosaccharides such as glucose, mannose, glycerol, and mannitol, oligosaccharides such as sucrose, maltose, and lactose, starch), organic acids (acetic acid) Propionic acid, maleic acid, fumaric acid, malic acid, etc.) and alcohols (ethanol, propanol, etc.) are used. As the nitrogen source, ammonium salts of inorganic acids or organic acids such as ammonium chloride, ammonium sulfate, ammonium nitrate, and ammonium phosphate, or nitrogen-containing compounds such as peptone, yeast extract, malt extract, meat extract, corn steep liquor and the like are used. As the inorganic salts, monopotassium phosphate, dipotassium phosphate, magnesium phosphate, magnesium sulfate, sodium chloride, ferrous sulfate, manganese sulfate, copper sulfate, calcium carbonate and the like are used. Moreover, the juice (for example, sugarcane, corn, cereals, fruit, etc.) of the plant body containing said carbohydrate, organic acid, and a nitrogen source can also be used as a component containing a carbon source and a nitrogen source.
ウスチラゴ サイタミネアを培養する場合、油脂を添加しなくても好適にMEL‐Bを生産することができる。培地に油脂を含ませない場合、炭素源としては糖を用いることが好ましい。糖としては、グルコース、マンノース、グリセロール、マンニトール等の単糖、ショ糖、麦芽糖、乳糖等のオリゴ糖、デンプン等の多糖が単独であるいは二種以上を組み合わせて用いられる。より好ましくは、スクロース、グルコース、フルクトース、マンノースのいずれかが単独で用いられる。培地中の糖の含有量としては、5〜20重量%が好ましい。油脂類を添加しない場合、培養後、MELの精製が容易に行なえるという利点等がある。なお、当該利点から、ウスチラゴ サイタミネアを培養する場合、油脂を含有しない培地で培養することが好ましいが、油脂を培地に含有させる場合であっても5重量%以下とすることが、容易に精製できることから好ましい。つまり、炭素源として糖を用いる場合、油脂の含有量は0〜8重量%以下であることが精製の観点から好ましく、0〜5重量%以下がさらに好ましい。 When cultivating ustillago cythaminea, MEL-B can be suitably produced without the addition of fats and oils. When oils and fats are not included in the medium, it is preferable to use sugar as the carbon source. As the sugar, monosaccharides such as glucose, mannose, glycerol and mannitol, oligosaccharides such as sucrose, maltose and lactose, and polysaccharides such as starch may be used alone or in combination of two or more. More preferably, any of sucrose, glucose, fructose and mannose is used alone. The content of sugar in the medium is preferably 5 to 20% by weight. When oils and fats are not added, there is an advantage that MEL can be easily purified after culturing. In addition, from the said advantage, when cultivating ustillago cythaminea, it is preferable to culture in a medium that does not contain fats and oils, but even when fats and oils are contained in the medium, it can be easily purified to 5% by weight or less. To preferred. That is, when sugar is used as the carbon source, the fat content is preferably 0 to 8% by weight or less from the viewpoint of purification, and more preferably 0 to 5% by weight or less.
培養温度は、20〜30℃、好ましくは22〜28℃である。培養日数としては、MELの生産量に応じて適宜設定すればよいが、3〜20日間が好ましい。培養方法としては、振盪培養、通気撹拌培養等の公知の一般的な微生物の培養方法を適用することができる。良好な微生物の生育及びMELの生産のためには、培養液に酸素を供給することが好ましく、そのためには、ジャーファメンターを用いる場合では、空気を通気しながら撹拌するか、振とう培養を行なえばよい。 The culture temperature is 20-30 ° C, preferably 22-28 ° C. The culture days may be appropriately set according to the production amount of MEL, but 3 to 20 days is preferable. As a culture method, known general microorganism culture methods such as shaking culture and aeration stirring culture can be applied. For good growth of microorganisms and production of MEL, it is preferable to supply oxygen to the culture solution. For this purpose, when using a jar fermenter, agitation with air or shaking culture is recommended. Just do it.
本発明において、例えば、前記ウスチラゴ サイタミネア(Ustilago scitaminea)又はウスチラゴ シノドントィス(Ustilago cynodontia)を用いてMEL‐B又はMEL‐Cを生産する場合、増殖能の高い菌株を用いた方が、単位培養液及び単位時間当たりのマンノシルエリスリトールリピッド生産量が高くなるため好ましい。そのため、まず前培養によって菌体を活性化させ、これを本培養の培地に接種して培養を行なうことが好ましい。例えば、種培養、本培養及びMEL生産培養の順にスケールアップしていくことが好ましい。これらの培養における、具体的な培地組成、培養条件を例示すると以下のとおりである。 In the present invention, for example, when producing MEL-B or MEL-C using the above-mentioned Ustyago scitamine or Ustyago cynodontia, the unit culture solution and This is preferable because the production amount of mannosyl erythritol lipid per unit time becomes high. For this reason, it is preferable that the cells are first activated by preculture and inoculated into the medium for main culture. For example, it is preferable to scale up in the order of seed culture, main culture, and MEL production culture. Examples of specific medium composition and culture conditions in these cultures are as follows.
グルコース10〜50g/L、酵母エキス1〜2g/L、硝酸ナトリウム1〜5g/L、リン酸2水素カリウム0.1〜2g/L、及び硫酸マグネシウム0.1〜1g/Lの組成の液体培地2mLが入った試験管に、保存菌株を1白金耳接種し、22〜28℃で1〜3日振とう培養を行なう(種培養)。次に、20〜300g/L、好ましくは50〜100g/Lの植物油脂等の油脂類を加えた上記と同じ組成の液体培地50〜100mLの入った坂口フラスコに上記の種培養を行った培養液を接種して、22〜28℃で3〜7日間振とう培養を行なう(本培養)。さらに、上記の本培養と同じ組成の液体培地1〜2Lが入ったジャーファメンターに上記の本培養を行った培養液を接種して、22〜28℃で1〜2L/分の通気速度と600〜1000rpmの撹拌速度で培養する(MEL生産培養)。このときの培養温度は22〜28℃、培養時間は、7〜28日間である。この培養においては、培養途中から油脂類を培養容器中に流下させて、培地中の油脂類濃度を50〜100g/Lに保持することが好ましい。 Liquid with composition of glucose 10-50 g / L, yeast extract 1-2 g / L, sodium nitrate 1-5 g / L, potassium dihydrogen phosphate 0.1-2 g / L, and magnesium sulfate 0.1-1 g / L A test tube containing 2 mL of the medium is inoculated with 1 platinum loop of the stock strain and cultured with shaking at 22 to 28 ° C. for 1 to 3 days (seed culture). Next, the culture which performed said seed culture to the Sakaguchi flask containing 50-100 mL of liquid culture medium of the same composition as the above which added oils and fats, such as vegetable oils and fats of 20-300 g / L, preferably 50-100 g / L The solution is inoculated and shake-cultured at 22-28 ° C. for 3-7 days (main culture). Further, a jar fermenter containing 1 to 2 L of liquid medium having the same composition as that of the main culture is inoculated with the culture solution obtained by performing the main culture, and an aeration rate of 1 to 2 L / min at 22 to 28 ° C. Culturing is performed at a stirring speed of 600 to 1000 rpm (MEL production culture). The culture temperature at this time is 22 to 28 ° C., and the culture time is 7 to 28 days. In this culture, it is preferable that oils and fats flow down into the culture vessel from the middle of the culture to maintain the oils and fats concentration in the medium at 50 to 100 g / L.
微生物菌株の培地への使用量は、例えば、菌体を接種する場合、培地1Lあたり、10〜200mL、好ましくは50〜100mLの種培養液あるいは本培養液中に含まれる量であればよい。 The amount of the microorganism strain used in the medium may be, for example, an amount contained in 10 to 200 mL, preferably 50 to 100 mL of the seed culture solution or the main culture solution, per 1 L of the medium when inoculating the cells.
本発明に係る製造方法における、培養工程は、培養液中の所望のMEL生成量が最高に達した時点で終了させることができるように、培養液中のMELをガスクロマトグラフィー、高速液体クロマトグラフィー、薄層クロマトグラフィー等の周知の方法により測定しながら行なうことが好ましい。 In the production method according to the present invention, the culturing step can be terminated when the desired MEL production amount in the culture solution reaches the maximum, so that the MEL in the culture solution is gas chromatographed or high performance liquid chromatography. It is preferable to carry out the measurement while measuring by a known method such as thin layer chromatography.
培養液中に蓄積されたMELは、培養終了後、培養液を抽出することによって取得できる。 MEL accumulated in the culture solution can be obtained by extracting the culture solution after completion of the culture.
抽出は、酢酸エチル等の有機溶媒を用いて行なう。抽出は当該分野において通常行われる方法に従って行なえばよく、例えば、培養液1Lあたり0.5〜1.5L程度の有機溶媒を用いて1〜数回抽出操作を行ない、酢酸エチル層を合わせて溶媒を留去する。 Extraction is performed using an organic solvent such as ethyl acetate. Extraction may be carried out according to a method usually performed in the art. For example, the extraction operation is performed once or several times using about 0.5 to 1.5 L of organic solvent per 1 L of the culture solution, and the ethyl acetate layer is combined with the solvent. Is distilled off.
酢酸エチル抽出物に含まれるMELを分画するには、シリカゲルカラムクロマトグラフィーによる分離精製を行なう。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を溶出液として用い、当該抽出物中の成分を分離精製する。クロロホルムのみを流した後、クロロホルムとアセトンの混合液で当該抽出物中の成分を溶出する。この時、クロロホルムとアセトンの混合液中におけるアセトンの割合は、段階的に上げていく。例えば、9:1から始め、次に、8:2あるいは7:3としていく。各溶出液は収集し、TLCにて精製度を確認する。 In order to fractionate MEL contained in the ethyl acetate extract, separation and purification by silica gel column chromatography is performed. The ethyl acetate extract is applied to a silica gel column, and components in the extract are separated and purified using chloroform and acetone as eluents. After flowing only chloroform, the components in the extract are eluted with a mixture of chloroform and acetone. At this time, the ratio of acetone in the mixed liquid of chloroform and acetone is increased stepwise. For example, start from 9: 1, then 8: 2 or 7: 3. Each eluate is collected and the degree of purification is confirmed by TLC.
また、本発明に係る製造方法においては、抽出する前に濃縮を行なってもよい。濃縮方法としては特に限定されないが、上記培養工程にて得られた培養液から菌体を取り除き、硫酸アンモニウムを添加して沈殿物を得る濃縮工程を行なうことが好ましい。この沈殿物中にMELが大量に濃縮されているので、当該沈殿物を上述の抽出に供する等、有機溶剤を用いてMEL‐Bを回収すれば、高純度なMEL‐Bを容易に回収できる。特に、工場等の大規模スケールの場合、濃縮技術は保管及び運搬のみでなく有機溶剤の使用量を削減するという観点からも重要な技術である。この濃縮工程によればMELを容易に回収することができる。 In the production method according to the present invention, concentration may be performed before extraction. Although it does not specifically limit as a concentration method, It is preferable to perform the concentration process which removes a microbial cell from the culture solution obtained at the said culture | cultivation process, and adds ammonium sulfate, and obtains a deposit. Since a large amount of MEL is concentrated in the precipitate, high-purity MEL-B can be easily recovered by recovering MEL-B using an organic solvent, such as subjecting the precipitate to the above-described extraction. . In particular, in the case of a large scale such as a factory, the concentration technique is an important technique not only from storage and transportation but also from the viewpoint of reducing the amount of organic solvent used. According to this concentration step, MEL can be easily recovered.
添加する硫酸アンモニウムの濃度としては、特に限定されないが、例えば、培養液に対して10質量%以上、30質量%以下にするとよい。 Although it does not specifically limit as a density | concentration of the ammonium sulfate to add, For example, it is good to set it as 10 to 30 mass% with respect to a culture solution.
上記培養において、微生物の形態は、特に限定されず、微生物の菌体、菌体処理物(例えば、菌体破砕物)などをいう。 In the above culture, the form of the microorganism is not particularly limited, and refers to a microorganism cell, a treated cell of the microorganism (for example, a disrupted cell).
微生物の菌体または菌体処理物は固定化して用いることもできる。固定化法としては、従来公知の担体結合法、架橋化法、包括法などの方法が挙げられる。担体結合法では、担体に菌体を固定化させるが、固定化は物理的吸着、イオン結合、共有結合のいずれであってもよい。担体としては多糖(アセチルセルロース、アガロース)、無機物質(多孔質ガラス、金属酸化物)、合成高分子(ポリアクリルアミド、ポリスチレン)等が用いられる。架橋化法では、グルタルアルデヒド等の二官能性試薬を用いて菌体同士を架橋、結合させることによって固定化する。また、包括法では、多糖(アルギン酸、カラギーナン)、ポリアクリルアミド、コラーゲン、ポリウレタン等の高分子ゲルの格子や半透膜カプセルに菌体を包み込むことによって固定化する。 The microbial cells or the processed microbial cells can be immobilized and used. Examples of the immobilization method include conventionally known methods such as a carrier binding method, a crosslinking method, and a comprehensive method. In the carrier binding method, the cells are immobilized on the carrier, and the immobilization may be any of physical adsorption, ionic bond, and covalent bond. As the carrier, polysaccharides (acetylcellulose, agarose), inorganic substances (porous glass, metal oxide), synthetic polymers (polyacrylamide, polystyrene) and the like are used. In the crosslinking method, the cells are immobilized by crosslinking and bonding with each other using a bifunctional reagent such as glutaraldehyde. In the inclusion method, the cells are immobilized by wrapping the cells in a lattice of a polymer gel such as polysaccharide (alginate, carrageenan), polyacrylamide, collagen, polyurethane, or a semipermeable membrane capsule.
〔6.MEL‐A、MEL‐B、MEL‐Cのいずれかを主成分とするマンノシルエリスリトールリピッドの用途〕
上述した本発明に係る製造方法によれば、MEL‐A、MEL‐B、MEL‐Cのいずれかを選択的に製造することができる。これにより、MEL‐A、MEL‐B、MEL‐Cのいずれかを主成分とするMELを得ることができるともいえる。なお、主成分とは、MEL‐A、B、Cのうちの1種において、他の2種の量に比べて高い含有量で含まれているものを意味する。
[6. Uses of mannosyl erythritol lipid mainly comprising any of MEL-A, MEL-B and MEL-C)
According to the manufacturing method according to the present invention described above, any one of MEL-A, MEL-B, and MEL-C can be selectively manufactured. Thereby, it can be said that MEL which has any of MEL-A, MEL-B, and MEL-C as a main component can be obtained. The main component means that one of MEL-A, B, and C contains a higher content than the other two.
上記の方法により得られたマンノシルエリスリトールリピッドの主成分であるMEL‐A、MEL‐B又はMEL−Cの構造は、シュードザイマ属の酵母が生産する既知のMELと同じである。これらMELは、温度範囲が室温から70℃まで、濃度範囲が0.0001%から90%までの幅広い条件下でリオトロピック液晶を形成することが可能である。リオトロピック液晶は、高濃度の水溶性成分と油溶性成分の双方を内包安定化できるという性質を有している。従って、当該マンノシルエリスリトールリピッドは、食品等の種々の組成物に含有させることにより、その界面活性作用により主成分の分散性、製品の酸化安定性向上をはかることができる。食品の形態は特に限定されないが、マンノシルエリスリトールリピッドの生理活性を活用した機能性食品・動物飼料などが挙げられる。 The structure of MEL-A, MEL-B, or MEL-C, which is the main component of mannosyl erythritol lipid obtained by the above method, is the same as the known MEL produced by Pseudozyma yeast. These MELs can form lyotropic liquid crystals under a wide range of temperature ranges from room temperature to 70 ° C. and concentration ranges from 0.0001% to 90%. The lyotropic liquid crystal has the property that it can stabilize both the high-concentration water-soluble component and the oil-soluble component. Therefore, when the mannosyl erythritol lipid is contained in various compositions such as foods, it is possible to improve the dispersibility of the main component and the oxidation stability of the product due to its surface activity. The form of the food is not particularly limited, and examples thereof include functional foods and animal feeds utilizing the physiological activity of mannosyl erythritol lipid.
また前記のとおり、マンノシルエリスリトールリピッドは界面活性能以外にも抗微生物活性、細胞分化誘導活性、糖タンパク質結合活性、抗炎症・抗アレルギー活性、アポトーシス誘導活性、癌細胞増殖抑制活性など種々の生理活性作用を有する。従って、MEL、公知の種々の方法にて各種製剤形態に調製し、それ自体を有効成分とする医薬(例えば、抗癌剤など)として用いることができる。各種製剤は、製剤上通常用いられる賦形剤、増量剤、結合剤、湿潤剤、崩壊剤、潤滑剤、界面活性剤、分散剤、緩衝剤、保存剤、溶解補助剤、防腐剤、矯味矯臭剤、無痛化剤、安定化剤、等張化剤等などを適宜選択し、常法により製造することができる。 As described above, mannosyl erythritol lipid has a variety of physiological activities such as antimicrobial activity, cell differentiation-inducing activity, glycoprotein binding activity, anti-inflammatory / anti-allergic activity, apoptosis-inducing activity, and cancer cell growth-inhibiting activity in addition to its surface activity. Has an effect. Therefore, it can be prepared in various preparation forms by various known methods such as MEL and used as a medicament (for example, an anticancer agent) containing itself as an active ingredient. Various preparations include excipients, extenders, binders, wetting agents, disintegrants, lubricants, surfactants, dispersants, buffering agents, preservatives, solubilizers, preservatives, flavoring agents that are commonly used in the formulation. Agents, soothing agents, stabilizers, tonicity agents and the like can be appropriately selected and produced by conventional methods.
以下に実施例を示し、本発明の実施の形態についてさらに詳しく説明する。もちろん、本発明は以下の実施例に限定されるものではなく、細部については様々な態様が可能であることはいうまでもない。さらに、本発明は上述した実施形態に限定されるものではなく、請求項に示した範囲で種々の変更が可能であり、それぞれ開示された技術的手段を適宜組み合わせて得られる実施形態についても本発明の技術的範囲に含まれる。また、本明細書中に記載された文献の全てが参考として援用される。 Examples will be shown below, and the embodiments of the present invention will be described in more detail. Of course, the present invention is not limited to the following examples, and it goes without saying that various aspects are possible in detail. Further, the present invention is not limited to the above-described embodiments, and various modifications can be made within the scope shown in the claims, and the present invention is also applied to the embodiments obtained by appropriately combining the disclosed technical means. It is included in the technical scope of the invention. Moreover, all the literatures described in this specification are used as reference.
〔実施例1〕
(1:MEL生産菌の探索)
ウスチラゴ属はトウモロコシ、サトウキビ、シバ、ススキといった植物体中に生息する糸状菌であり、人類が生活の中で日常的に接触してきた微生物群である。ウスチラゴ属に属する微生物は500種以上存在するが、そのうちトウモロコシから分離されるUstilago maydisのみがバイオサーファクタント生産菌として知られていた。U. maydisがMEL生産能を有することは知られているが、MELのうち、MEL‐A、MEL‐B及びMEL‐Cを混合物として生産するので、MEL‐A、MEL‐B又はMEL‐Cのいずれかを選択的に製造することはできない。そこで、全くバイオサーファクタント生産能は知られていないが、食用植物あるいは日常的に接触頻度の高い植物から単離された微生物に着目し、油脂含有培地中で培養し、それらの中からバイオサーファクタント生産微生物の探索を試みた。
[Example 1]
(1: Search for MEL-producing bacteria)
The genus Ustilago is a filamentous fungus that inhabits plants such as corn, sugarcane, buckwheat, and pampas grass, and is a group of microorganisms that humans have come into contact with on a daily basis. There are more than 500 types of microorganisms belonging to the genus Ustylago, of which only Ustilago maydis isolated from corn was known as a biosurfactant producing bacterium. U. It is known that maydis has the ability to produce MEL, but among MEL, MEL-A, MEL-B and MEL-C are produced as a mixture, so any of MEL-A, MEL-B or MEL-C Cannot be selectively manufactured. Therefore, although biosurfactant production ability is not known at all, paying attention to microorganisms isolated from edible plants or plants with high daily contact frequency, cultivate in oil-containing medium and produce biosurfactant from them I tried to search for microorganisms.
本実施例では、サトウキビ属及びギョウギシバ属の植物から単離されたウスチラゴ属の微生物を用いた。具体的にはサトウキビ属由来のウスチラゴ属の微生物としてU. scitaminea NBRC32730を用いた。また、ギョウギシバ属由来のウスチラゴ属の微生物として、U. cynodontis NBRC7530及びNBRC9727を用いた。 In this example, a microorganism of the genus Ustylago isolated from a plant of the genus Sugarcane and the genus Drosophila was used. Specifically, as a microorganism of the genus Ustyago derived from the sugarcane genus, U. scitamine NBRC32730 was used. In addition, as a microorganism of the genus Ustylago derived from the genus Gorilla, U. cynodontis NBRC7530 and NBRC9727 were used.
(2:U. scitaminea及びU. cynodontisの培養)
(2−a:種培養)
保存培地(麦芽エキス3g/L、酵母エキス3g/L、ペプトン5g/L、グルコース10g/L、寒天30g/L)に保存しておいた上記のU. cynodontisとU. scitamineaを、グルコース20g/L、酵母エキス1g/L、硝酸ナトリウム1g/L、リン酸2水素カリウム0.5g/L、及び硫酸マグネシウム0.5g/Lの組成の液体培地2mLが入った試験管に1白金耳接種し25℃で振とう培養を2日間行なった。
(2: Cultivation of U. spitaminea and U. cynodontis)
(2-a: seed culture)
The above-mentioned U.S. stored in a storage medium (malt extract 3 g / L, yeast extract 3 g / L, peptone 5 g / L, glucose 10 g / L, agar 30 g / L). cynodontis and U.S. A test tube containing 2 mL of liquid medium having a composition of 20 g / L glucose, 1 g / L yeast extract, 1 g / L sodium nitrate, 0.5 g / L potassium dihydrogen phosphate, and 0.5 g / L magnesium sulfate. 1 platinum ear was inoculated and cultured at 25 ° C. with shaking for 2 days.
(2−b:本培養)
上記2−aで得られた菌体培養液を、40g/Lの大豆油と酵母エキス1g/L、硝酸ナトリウム1g/L、リン酸2水素カリウム0.5g/L、及び硫酸マグネシウム0.5g/Lの組成の液体培地20mLが入った坂口フラスコに接種し、振とう培養を25℃にて7日間行なった。
(2-b: Main culture)
The bacterial cell culture solution obtained in 2-a above was prepared by using 40 g / L soybean oil and yeast extract 1 g / L, sodium nitrate 1 g / L, potassium dihydrogen phosphate 0.5 g / L, and magnesium sulfate 0.5 g. A Sakaguchi flask containing 20 mL of a liquid medium having a composition of / L was inoculated, and shaking culture was performed at 25 ° C. for 7 days.
それぞれの微生物を培養して得た培養液の酢酸エチル抽出液を薄層クロマトグラフィー分析した。この結果を図1に示す。図1は、U. scitaminea及びU. cynodontisの培養液を薄層クロマトグラフィーに供した結果を示す。薄層クロマトグラフィーについては、Silica gel 60F(Wako社製)を用い、展開溶媒としてはクロロホルム:メタノール:7Nアンモニア=65:15:2とした。検出は、展開後のプレートにアンスロン試薬を吹き付け、110℃で5分間加熱した。 The ethyl acetate extract of the culture solution obtained by culturing each microorganism was analyzed by thin layer chromatography. The result is shown in FIG. FIG. scitamine and U.S. The result of having used the culture solution of cynodontis for the thin layer chromatography is shown. For thin layer chromatography, Silica gel 60F (manufactured by Wako) was used, and the developing solvent was chloroform: methanol: 7N ammonia = 65: 15: 2. For detection, an anthrone reagent was sprayed on the developed plate and heated at 110 ° C. for 5 minutes.
なお、比較のためMELの標準サンプル及びU. maydisNBRC5346及びNBRC6907から得たMELを薄層クロマトグラフィーに供した。標準サンプルとしてはシュードザイマ アンタクチカ(Pseudozyma antarctica)JCM10317株を、2−a及びここに記載した方法と同じ方法で種培養及び本培養して、原料油脂等の不純物を取り除いて得た。U. maydisNBRC5346及びNBRC6907由来のMELについても、同様にして得た。これらの標準サンプル、NBRC5346及びNBRC6907由来のMELを薄層クロマトグラフィーに供した。図1のレーンSが標準サンプルを薄層クロマトグラフィーに供した結果を示している。 For comparison, a standard sample of MEL and U.I. MELs obtained from maydis NBRC5346 and NBRC6907 were subjected to thin layer chromatography. As a standard sample, Pseudozyma antarctica (Pseudozyma antarctica) JCM10317 strain was seed-cultured and main-cultured by the same method as 2-a and the method described here to remove impurities such as raw material fats and oils. U. The MELs derived from maydisNBRC5346 and NBRC6907 were obtained in the same manner. MELs derived from these standard samples, NBRC5346 and NBRC6907 were subjected to thin layer chromatography. Lane S in FIG. 1 shows the result of subjecting the standard sample to thin layer chromatography.
図1に示すように、両株ともMELを生産した。U. scitamineaはMEL‐Bを選択的に生産したことが確認できた(図1、レーンA)。また、U. cynodontisはMEL‐Cを選択的に生産したことが確認できた(図1、レーンB1及びB2)。以上の結果から、U. cynodontisはMEL‐Cのみを選択的に生産し、また、U. scitamineaはMEL‐Bのみを選択的に生産することは明らかである。 As shown in FIG. 1, both strains produced MEL. U. It was confirmed that scitamine selectively produced MEL-B (FIG. 1, lane A). In addition, U.S. It was confirmed that cynodontis selectively produced MEL-C (FIG. 1, lanes B1 and B2). From the above results, U.S. cynodontis selectively produces only MEL-C, and It is clear that scitaminea selectively produces only MEL-B.
(3:U. scitaminea及びU. cynodontisにより生産されるMELの構造解析)
(3‐a:精製標品の高速液体クロマトグラフィーによる分析)
上記2−a及び2−bと同じ方法でU. scitaminea及びU. cynodoの培養を行ない、培養終了後の培養液を採取し、酢酸エチルで抽出し、酢酸エチル可溶分中のMELを高速液体クロマトグラフィー(HPLC)を用いて検出した。結果を図2に示す。図2はU. scitaminea及びU. cynodontisが生産するMELを高速液体クロマトグラフィーで分析した結果を示す図である。なお、図2の各サンプルの結果の上に記載されている「MEL‐A:B:C」は、培養液中に含まれていたMEL‐A、MEL‐B、MEL‐Cの重量比である。また、比較のため、上記2−bで用いた標準サンプル及びU. maydis NBRC5346から得たMELを同様にHPLCに供して検出した。高速液体クロマトグラフィーによる検出には、DP‐8020(TOSOH社製)を用いた。カラムとしては、Inertsil SIL 100A 5μm、4.6×250mm(GL science社製)を用い、クロロホルムとメタノールを100:0〜0:100のグラジエントをかけて1mL/minの流速で流した。
(3: Structural analysis of MEL produced by U. spitaminea and U. cynodontis)
(3-a: Analysis of purified sample by high performance liquid chromatography)
In the same manner as 2-a and 2-b above, U.S. scitamine and U.S. Cynodo was cultured, the culture solution after completion of the culture was collected, extracted with ethyl acetate, and MEL in the ethyl acetate-soluble component was detected using high performance liquid chromatography (HPLC). The results are shown in FIG. FIG. scitamine and U.S. It is a figure which shows the result of having analyzed MEL which cynodontis produces by the high performance liquid chromatography. Note that “MEL-A: B: C” described above the results of each sample in FIG. 2 is the weight ratio of MEL-A, MEL-B, and MEL-C contained in the culture solution. is there. For comparison, the standard sample used in 2-b and the U.S. MEL obtained from maydis NBRC5346 was similarly subjected to HPLC for detection. DP-8020 (manufactured by TOSOH) was used for detection by high performance liquid chromatography. As the column, Inertsil SIL 100A 5 μm, 4.6 × 250 mm (manufactured by GL science) was used, and chloroform and methanol were applied at a flow rate of 1 mL / min with a gradient of 100: 0 to 0: 100.
なお、U. scitamineaについては、大豆油の代わりにグルコース100g/Lを培地に加えて培養することで得られたMEL‐BもHPLCによる検出に供した。 In addition, U.S. For scitamine, MEL-B obtained by adding 100 g / L of glucose to the medium instead of soybean oil and culturing was also subjected to detection by HPLC.
図2に示すように、U. scitamineaから得られたMELのピークは、JCM10317株とは異なった。また、U. scitamineaから得られたMELのメインピークは、JCM10317株から得たMEL中のMEL‐Bとのピークと一致した。U. scitamineaは、高いMEL‐B生産能を有し、一週間で約9.6g/LのMEL‐Bを生産した。なお、MEL−Bおよび後述のMEL−Cの生産量は、精製したMEL−BおよびMEL−Cを用いて作成した検量線を用い、HPLCの分析結果から定量した。 As shown in FIG. The peak of MEL obtained from scitamine was different from that of JCM10317 strain. In addition, U.S. The main peak of MEL obtained from scitaminea coincided with the peak of MEL-B in MEL obtained from JCM10317 strain. U. scitaminea had high MEL-B production capacity and produced about 9.6 g / L MEL-B in one week. The production amounts of MEL-B and MEL-C described below were quantified from the HPLC analysis results using a calibration curve prepared using purified MEL-B and MEL-C.
また、図2に示すように、U. cynodontisから得られたMELのピークはJCM10317株から得たMELのピークとは異なった。また、U. cunodontisから得たMELのメインピークは、JCM10317株から得たMEL中のMEL‐Cのピークと一致した。U. cynodontisは、高いMEL‐C生産能を有し、一週間で約1.4g/LのMEL‐Cを生産した。 As shown in FIG. The MEL peak obtained from cynodontis was different from the MEL peak obtained from the JCM10317 strain. In addition, U.S. The main peak of MEL obtained from Cunodontis coincided with the peak of MEL-C in MEL obtained from JCM10317 strain. U. cynodontis had high MEL-C production capacity and produced about 1.4 g / L MEL-C in one week.
なお、U. maydis NBRC5346については、MEL‐A:MEL‐B:MEL‐C:CLsの重量比は21:0:9:23であり、MEL中の7割程度しかMEL‐Aを生産せず、MELの3種類のうちの1種類を選択的に製造するものではないことが分かった。また、MELの生産の際、セロビオースリピッド(CLs)を大量に混合することが示された。 In addition, U.S. For Maydis NBRC5346, the weight ratio of MEL-A: MEL-B: MEL-C: CLs is 21: 0: 9: 23, and only about 70% of MEL is produced. It was found that one of the types was not selectively produced. It was also shown that cellobiose lipids (CLs) were mixed in large quantities during MEL production.
(3‐b:NMRによる構造解析)
U. citamineaの培養液の酢酸エチル可溶分から、主成分であるMEL‐Bをシリカゲルカラムクロマトグラフィーによって精製分離した後、1Hと13CNMRによる構造解析を行なった。結果を表1に示す。また、標準的なMELの構造解析結果の例としてP. antarctica JCM 10317株が生産するMELの分析結果も表1に示す。表1によれば、U. scitamineaが生産する物質は典型的なMEL‐Bの構造を有することが確認できた。なお、NMRによる測定ついては、MEL精製標品重クロロホルム中に溶解し、30℃で、Varian INOVA(400MHz)を用いて行なった。
(3-b: Structural analysis by NMR)
U. The main component MEL-B was purified and separated by silica gel column chromatography from the ethyl acetate-soluble content of the culture solution of citamine, and then structural analysis was performed by 1 H and 13 C NMR. The results are shown in Table 1. Further, as an example of a standard MEL structural analysis result, P.I. Table 1 also shows the results of analysis of MEL produced by the Antarctica JCM 10317 strain. According to Table 1, U.S. It was confirmed that the substance produced by scitamine has a typical MEL-B structure. In addition, about the measurement by NMR, it melt | dissolved in the MEL refinement | purification sample heavy chloroform, and performed it at 30 degreeC using Varian INOVA (400MHz).
また、U. cynodontisの培養液の酢酸エチル可溶分から、主成分であるMEL‐Cをシリカゲルカラムクロマトグラフィーによって精製分離した後、1H及び13C NMRによる構造解析を行った。結果を下記表2に示す。また、標準的なMELの構造解析結果の例としてP. antarctica JCM 10317株が生産するMELの分析結果も表2に示す。表2によれば、U. scitamineaが生産する物質は典型的なMEL‐Bの構造を有することが確認できた。 In addition, U.S. The main component MEL-C was purified and separated by silica gel column chromatography from the ethyl acetate-soluble content of the culture solution of cynodontis, and then structural analysis was performed by 1 H and 13 C NMR. The results are shown in Table 2 below. Further, as an example of a standard MEL structural analysis result, P.I. Table 2 also shows the results of analysis of MEL produced by the Antarctica JCM 10317 strain. According to Table 2, U.I. It was confirmed that the substance produced by scitamine has a typical MEL-B structure.
〔実施例2〕
(1:微生物の単離)
本実施例では、アブラナ(Brassica)属(ミズナ)、リンゴ(Malus)属及びシソ属(Perilla)属の植物からそれぞれウスチラゴ(Ustilago)属の微生物を単離した。
[Example 2]
(1: Isolation of microorganisms)
In this example, microorganisms belonging to the genus Ustilago were isolated from plants belonging to the genus Brassica (Mizuna), the genus Apple (Malus) and the genus Perilla.
具体的にはヒメリンゴ(M. baccata)、シソ(Perilla frutescens)、ミズナ(Brassica rapa L. var. nipposinica)の切片を培養液(40g/Lの大豆油と酵母エキス1g/L、硝酸ナトリウム1g/L、リン酸2水素カリウム0.5g/L、及び硫酸マグネシウム0.5g/L)中に浸し、25℃で3〜7日間振とう培養し、培地中の大豆油の乳化を指標として、バイオサーファクタント生産性を判定した。生育してきた微生物を、寒天培地(40g/Lのグルコースと酵母エキス1g/L、硝酸ナトリウム1g/L、リン酸2水素カリウム0.5g/L、硫酸マグネシウム0.5g/L、及び寒天30g/L)上で単離した後、さらに、得られた微生物を25℃で3〜7日間振とう培養し(培養液:40g/Lの大豆油と酵母エキス1g/L、硝酸ナトリウム1g/L、リン酸2水素カリウム0.5g/L、及び硫酸マグネシウム0.5g/L)、培養液中に分泌生産されたMELを薄層クロマトグラフィーにて検出することで、MEL生産微生物を単離した。 Specifically, slices of Japanese apple (M. baccata), perilla (Frulacescens) and Mizuna (Brassica rapa L. var. Nipposinica) were cultured in a culture solution (40 g / L soybean oil and yeast extract 1 g / L, sodium nitrate 1 g / L). L, potassium dihydrogen phosphate 0.5 g / L, and magnesium sulfate 0.5 g / L), and cultured with shaking at 25 ° C. for 3 to 7 days. Surfactant productivity was determined. Growing microorganisms were added to agar medium (40 g / L glucose and yeast extract 1 g / L, sodium nitrate 1 g / L, potassium dihydrogen phosphate 0.5 g / L, magnesium sulfate 0.5 g / L, and agar 30 g / L. L) After the above isolation, the obtained microorganism was further cultured with shaking at 25 ° C. for 3 to 7 days (culture solution: 40 g / L soybean oil and yeast extract 1 g / L, sodium nitrate 1 g / L, The MEL-producing microorganism was isolated by detecting MEL secreted and produced in the culture broth by 0.5 g / L potassium dihydrogen phosphate and 0.5 g / L magnesium sulfate) by thin layer chromatography.
なお、得られた微生物のITS1領域における約260bpの配列をBLAST解析により比較した結果、より具体的には、ウスチラゴ ツラガナ(Ustilago tragana)の近縁種であることが分かった。ITS1領域のPCRによる増幅は、プライマーA(配列番号1)及びプライマーB(配列番号2)を用いて行なった。PCRのテンプレートとして用いた上記微生物のゲノムDNAは、GenTLE(TaKaRa社製)を用いて抽出した。その後、増幅した遺伝子断片の塩基配列を解析した。その結果を配列番号3(ミズナ由来の微生物A1)、配列番号4(ヒメリンゴ由来の微生物B1)、配列番号5(シソ由来の微生物C1)、配列番号6(シソ由来の微生物C2)、配列番号7(シソ由来の微生物C3)に示す。得られた遺伝子配列について、DNAデータベース(DDBJ)にアクセスし、BLAST解析によって相同性検索を行ったところ、ウスチラゴ ツラガナ(Ustilago tragana)の対応するITS1領域の塩基配列と94%一致した。このことから、本実施例で同定を行なった微生物はウスチラゴ属の微生物(Ustilago sp.)であることが分かった。 In addition, as a result of comparing the sequence of about 260 bp in the ITS1 region of the obtained microorganism by BLAST analysis, more specifically, it was found to be a related species of Ustilago tragana (Ustilago tragana). Amplification of the ITS1 region by PCR was performed using primer A (SEQ ID NO: 1) and primer B (SEQ ID NO: 2). Genomic DNA of the microorganism used as a PCR template was extracted using GenTLE (TaKaRa). Thereafter, the base sequence of the amplified gene fragment was analyzed. As a result, SEQ ID NO: 3 (microorganism A1 derived from Mizuna), SEQ ID NO: 4 (microorganism B1 derived from Japanese apple), SEQ ID NO: 5 (microorganism C1 derived from perilla), SEQ ID NO: 6 (microorganism C2 derived from perilla), SEQ ID NO: 7 This is shown in (Perilla-derived microorganism C3). When the obtained gene sequence was accessed to the DNA database (DDBJ) and homology search was performed by BLAST analysis, it was 94% identical with the base sequence of the corresponding ITS1 region of Ustyago tragana. From this, it was found that the microorganism identified in this example was a microorganism belonging to the genus Ustilago (Ustilago sp.).
(2:培養及び生産されたMELの確認)
本実施例2で得た微生物を使用し、また、大豆油の代わりにオリーブ油を用いた以外は、実施例1の2−a及び2−bに記載の方法で微生物の培養を行なった。
(2: Confirmation of cultured and produced MEL)
The microorganisms were cultured by the method described in 2-a and 2-b of Example 1 except that the microorganisms obtained in Example 2 were used and olive oil was used instead of soybean oil.
次に、得られたMELを実施例1に記載の方法と同じ方法で薄層クロマトグラフィーに供した。結果を図3に示す。図3は本実施例で得たMELを薄層クロマトグラフィーに供した結果を示す図である。図3に示すように、本実施例で得た微生物はいずれもMEL‐Aを選択的に生産することが確認された。 Next, the obtained MEL was subjected to thin layer chromatography by the same method as described in Example 1. The results are shown in FIG. FIG. 3 is a diagram showing the results of subjecting the MEL obtained in this example to thin layer chromatography. As shown in FIG. 3, it was confirmed that all the microorganisms obtained in this example selectively produced MEL-A.
また、上記微生物C1から実施例1と同じ方法で得た酢酸エチルの抽出液をHPLC解析に供した。結果を図4に示す。なお、図4に記載されている「MEL‐A:B:C」は、培養液中に含まれていたMEL‐A、MEL‐B、MEL‐Cの重量比である。図4に示すように、上記のウスチラゴ ツラガナ近縁種から得られたMELのピークはJCM10317株から得たMEL−Aのピークと一致した。上記のウスチラゴ ツラガナの近縁種は、高いMEL‐A生産能を有し、一週間で約11.6g/LのMEL‐Aを生産した。なお、MEL−Aの生産量は、MEL−Aの精製標品を用いて作成した検量線を用い、HPLCの分析結果から定量した。また、MEL‐Aの化学構造は、実施例1の3−a及び3−bに記載の方法に従って行ない、その結果を表3に記載した。表3によれば、上記のウスチラゴ ツラガナ近縁種が生産する物質は典型的なMEL‐Aの構造を有することが確認できた。 In addition, an ethyl acetate extract obtained from the microorganism C1 in the same manner as in Example 1 was subjected to HPLC analysis. The results are shown in FIG. In addition, “MEL-A: B: C” described in FIG. 4 is a weight ratio of MEL-A, MEL-B, and MEL-C contained in the culture solution. As shown in FIG. 4, the MEL peak obtained from the above-mentioned species related to Ustillago tsuragana coincided with the MEL-A peak obtained from the JCM10317 strain. The above-mentioned species of Ustirago tsuragana has a high ability to produce MEL-A and produced about 11.6 g / L of MEL-A in one week. In addition, the production amount of MEL-A was quantified from the analysis result of HPLC using the analytical curve created using the refinement | purification sample of MEL-A. Further, the chemical structure of MEL-A was determined according to the method described in 3-a and 3-b of Example 1, and the results are shown in Table 3. According to Table 3, it was confirmed that the substance produced by the above-mentioned species related to Ustirago tsuragana has a typical MEL-A structure.
〔実施例3〕
本実施例では、様々な炭素源を用いたU. scitaminea NBRC32730の培養を行ないMELの生産を試みた。
Example 3
In this example, U.S. using various carbon sources. Citaminea NBRC32730 was cultured and MEL production was attempted.
培地としては、炭素源10重量%、硝酸ナトリウム0.3重量%、リン酸2水素カリウム0.03重量%、硫酸マグネシウム0.03重量%、酵母エキス0.1重量%を含み、残部を水とし、全量を10mlとした液体培地を用いた。また、培養期間を7日間、培養温度を25℃として、200rpmにて振とう培養した。 The medium includes 10% by weight of carbon source, 0.3% by weight of sodium nitrate, 0.03% by weight of potassium dihydrogen phosphate, 0.03% by weight of magnesium sulfate, and 0.1% by weight of yeast extract, with the balance being water. And a liquid medium with a total volume of 10 ml was used. Further, the culture was carried out at 200 rpm with a culture period of 7 days and a culture temperature of 25 ° C.
炭素源としては、スクロース、グルコース、フルクトース、マンノース、ガラクトース、ソルビトール、マンニトール、リビトール、アラビトール、イノシトール、エリトリトール、メチルオレイン酸、オリーブ油をそれぞれ用いた。 As the carbon source, sucrose, glucose, fructose, mannose, galactose, sorbitol, mannitol, ribitol, arabitol, inositol, erythritol, methyl oleic acid, and olive oil were used.
それぞれの炭素源を用いることで得られた培養液の酢酸エチル抽出液1μlを、実施例1に記載の方法と同じ方法で薄層クロマトグラフィーに供した。結果を図5に示す。図5は本実施例にて得た培養液の酢酸エチル抽出液を薄層クロマトグラフィーに供した結果を示す図である。図5に示すように糖を炭素源としてU. scitamineaを培養することでMEL‐Bを選択的に生産できることが示された。一方、糖アルコールを炭素源とした場合、MEL‐Bを生産しなかった。 1 μl of the ethyl acetate extract of the culture solution obtained by using each carbon source was subjected to thin layer chromatography in the same manner as described in Example 1. The results are shown in FIG. FIG. 5 shows the results of subjecting the ethyl acetate extract of the culture solution obtained in this example to thin-layer chromatography. As shown in FIG. It was shown that MEL-B can be selectively produced by culturing scitamine. On the other hand, when sugar alcohol was used as the carbon source, MEL-B was not produced.
次に、上記酢酸エチル抽出液を実施例1の記載と同じ方法でHPLC解析に供した。結果を図6に示す。なお、図6に記載されている「MEL‐A:B:C」は、培養液中に含まれていたMEL‐A、MEL‐B、MEL‐Cの重量比である。また、図6に示すように、MELのピークはJCM10317株から得たMEL−Bのピークと一致した。そのMEL−B生産量は、一週間で約9.8g/Lに達した。なお、MEL−Bの生産量は、MEL−Bの精製標品を用いて作成した検量線を用い、HPLCの分析結果から定量した。図6から明らかなように、炭素源として、スクロース等の糖質を用いた場合、メチルオレイン酸やオリーブ油を用いた場合と比較して、培地中に油脂等の混在はなく、少なくとも95%以上の純度である。この結果は、スクロース等の糖質を炭素源することでMELの精製プロセスを大幅に簡略化できることを示している。また、スクロース等の糖質を多く含む溶液(例えば、サトウキビ、トウモロコシ、穀類、果実などの搾汁)であればMEL生産のための培地と成り得ることも、この結果から容易に類推できる。このように、スクロース等の糖質を原料として用いた場合、U. scitaminea NBRC32730のMEL生産量は、従来のMEL生産微生物を凌駕するものである。 Next, the ethyl acetate extract was subjected to HPLC analysis in the same manner as described in Example 1. The results are shown in FIG. Note that “MEL-A: B: C” shown in FIG. 6 is a weight ratio of MEL-A, MEL-B, and MEL-C contained in the culture solution. Further, as shown in FIG. 6, the MEL peak coincided with the MEL-B peak obtained from the JCM10317 strain. The MEL-B production amount reached about 9.8 g / L in one week. In addition, the production amount of MEL-B was quantified from the analysis result of HPLC using the analytical curve created using the refinement | purification sample of MEL-B. As is clear from FIG. 6, when a saccharide such as sucrose is used as the carbon source, there is no mixture of fats and oils in the medium as compared with the case where methyl oleic acid or olive oil is used, and at least 95% or more. Of purity. This result shows that the purification process of MEL can be greatly simplified by using a saccharide such as sucrose as a carbon source. In addition, it can be easily inferred from this result that a solution containing a large amount of sugar such as sucrose (for example, juice from sugarcane, corn, cereals, fruits, etc.) can be a medium for MEL production. Thus, when a saccharide such as sucrose is used as a raw material, U.S. The amount of MEL produced by scitamine NBRC32730 exceeds that of conventional MEL-producing microorganisms.
〔比較例〕
本比較例では、オオムギ(Hordeum)属、マダケ(Phyllostachys)属、ススキ(Miscanthus)属、メヒシバ(Digitaria)属(アキメヒシバ)、及びヒエ(Echinochloa)属の植物からそれぞれウスチラゴ(Ustilago)属の微生物が、MELを生産するか否か確認した。
[Comparative Example]
In this comparative example, the genus Ustilago from plants of the genus Hordeum, the genus Phyllostachys, the genus Miscanthus, the genus Digitaria (Akihishiba), and the genus Echinochlo, respectively. Whether or not to produce MEL was confirmed.
具体的にはオオムギ属の植物から単離されたU. nuda NBRC6906、マダケ属の植物から単離されたU. shiraiana NBRC 30149、U. shiraiana NBRC 8809、ススキ属の植物から単離されたU. kusanoi NBRC9070、アキメヒシバから単離されたU. rabenhorstiana NBRC8995、ヒエ属の植物から単離されたU. trichophora NBRC100155、U. trichophora NBRC100156、U. trichophora NBRC100157、U. trichophora NBRC100158、U. trichophora NBRC100159、U. trichophora NBRC100160を用いた。 Specifically, it has been isolated from a plant of the genus Barley. nuda NBRC 6906, a U.S. shiraiana NBRC 30149, U.S.A. shiraiana NBRC 8809, a U.S. strain isolated from a plant of the genus Susuki. Kusanoi NBRC 9070, a U.S. rabenhorstiana NBRC8995, a U. cerevisiae isolated from a plant of the genus Panax. trichophora NBRC100155, U.S.A. trichophora NBRC100156, U.S.A. trichophora NBRC100157, U.I. trichophora NBRC100158, U.S.A. trichophora NBRC100159, U.S.A. Trichophora NBRC100160 was used.
また、比較のため実施例1にて用いたU. scitaminea NBRC32730、MEL‐A生産菌として公知のU. maydis(NBRC5346及びNBRC6907)も用いた。 For comparison, the U.I. scitamine NBRC32730, a U.K. known as MEL-A producing bacterium. Maydis (NBRC5346 and NBRC6907) were also used.
微生物の培養、培養液からMELの抽出、薄層クロマトグラフィーについては実施例2に記載の方法と同じ方法で行なった。結果を図7に示す。図7は本比較例における薄層クロマトグラフィーの結果を示す図である。 Microbial culture, MEL extraction from the culture, and thin-layer chromatography were performed in the same manner as described in Example 2. The results are shown in FIG. FIG. 7 shows the results of thin layer chromatography in this comparative example.
図7に示すように、本発明に係る製造方法で用いるU. scitaminea以外はMEL‐A、MEL‐B、MEL‐Cのうちの一つを選択的に生産することは確認できなかった。 As shown in FIG. 7, the U.S. used in the manufacturing method according to the present invention. It was not possible to confirm that one of MEL-A, MEL-B, and MEL-C was selectively produced except for scitamine.
〔実施例4〕
本実施例では、U. scitaminea に分類される様々な菌株について、実施例3と同様にして培養し、MEL生産能力があるかどうか調べた。結果を図8に示す。図8は実施例4にて得た培養液の酢酸エチル抽出液を薄層クロマトグラフィーに供した結果を示す図である。図8に示すように、本実施例にて用いたU. scitaminea は全てMEL‐B生産能力を有することが明らかとなった。
Example 4
In this embodiment, U.S. Various strains classified as scitamine were cultured in the same manner as in Example 3 and examined for their ability to produce MEL. The results are shown in FIG. FIG. 8 shows the results of subjecting the ethyl acetate extract of the culture solution obtained in Example 4 to thin layer chromatography. As shown in FIG. All of the scitamines were found to have MEL-B production capacity.
〔実施例5〕
本実施例では、U. scitaminea NBRC32730によって生産されたMEL‐Bの、回収・分離方法について検討した。
Example 5
In this embodiment, U.S. A method for recovering and separating MEL-B produced by scitamine NBRC32730 was examined.
まず、U. scitaminea NBRC32730を培養液(スクロース100g/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)中に浸し、25℃で7日間振とう培養し、得られた培養液を実験に用いた。図9に本実施例におけるMEL‐Bの回収方法及び結果を示す。 First, U.I. dipping scitamine NBRC32730 in a culture solution (sucrose 100 g / L, yeast extract 1 g / L, sodium nitrate 1 g / L, potassium dihydrogen phosphate 0.5 g / L and magnesium sulfate 0.5 g / L) at 25 ° C. The culture was shaken for 7 days, and the resulting culture was used for the experiment. FIG. 9 shows the recovery method and results of MEL-B in this example.
図9に示すように、得られた培養液に酢酸エチルを等量添加して5800gで5分間遠心分離した。遠心分離後の酢酸エチル抽出画分を画分1とした。また、培養液を5800gで5分間遠心分離すると、上層、沈殿層、これらの中間層が生じるため、当該上層、当該中間層、当該沈殿層のそれぞれを分取して、それぞれに酢酸エチルを等量(培養液と等量)添加し、5800gで5分間遠心分離した。当該上層、当該中間層、当該沈殿層のそれぞれから得られた酢酸エチル抽出画分を、画分2、3、4とした。 As shown in FIG. 9, an equal amount of ethyl acetate was added to the obtained culture broth and centrifuged at 5800 g for 5 minutes. The fraction extracted with ethyl acetate after centrifugation was designated as fraction 1. In addition, when the culture solution is centrifuged at 5800 g for 5 minutes, an upper layer, a precipitate layer, and an intermediate layer thereof are generated. Therefore, the upper layer, the intermediate layer, and the precipitate layer are separated, and ethyl acetate is added to each. An amount (equal to the culture solution) was added and centrifuged at 5800 g for 5 minutes. The ethyl acetate extract fractions obtained from each of the upper layer, the intermediate layer, and the precipitation layer were designated as fractions 2, 3, and 4.
また、上記培養液を400gで5分間遠心分離した。得られた上層および沈殿層を分取して、それぞれに酢酸エチルを等量(培養液と等量)添加し、5800gで5分間遠心分離した。当該上層、当該沈殿層のそれぞれから得られた酢酸エチル抽出画分を画分5、6とした。画分1〜6を薄層クロマトグラフィーに供してMEL‐Bの存在を確認した。 The culture broth was centrifuged at 400 g for 5 minutes. The obtained upper layer and precipitated layer were separated, and an equal amount of ethyl acetate (equivalent to the culture solution) was added to each and centrifuged at 5800 g for 5 minutes. The ethyl acetate extract fractions obtained from the upper layer and the precipitated layer were designated as fractions 5 and 6, respectively. Fractions 1-6 were subjected to thin layer chromatography to confirm the presence of MEL-B.
図9に示すように、培養液に等量の酢酸エチルを添加することで、MEL‐Bを回収することができることが示された。また、培養液を5800gで遠心分離すると、上層、中間層、沈殿層が得られ、それぞれに培養液と等量の酢酸エチルを添加することで、MEL‐Bを回収することができるが、この場合、MEL‐Bは遠心分離によって一部分が沈殿することが示された。一方、培養液を400gで遠心分離すると、上層及び沈殿層が得られ、それぞれに培養液と等量の酢酸エチルを添加してMEL‐Bを回収した結果、沈殿にはMEL‐Bが検出されず、全て上層から検出された。すなわち、培養液を400gで遠心分離することで、MEL‐Bを沈殿させることなく、菌体と完全に分離し、MEL‐B溶液を回収することが可能であることが示された。 As shown in FIG. 9, it was shown that MEL-B can be recovered by adding an equal amount of ethyl acetate to the culture solution. Moreover, when the culture solution is centrifuged at 5800 g, an upper layer, an intermediate layer, and a precipitation layer are obtained, and MEL-B can be recovered by adding the same amount of ethyl acetate as the culture solution. In some cases, MEL-B was shown to partially precipitate by centrifugation. On the other hand, when the culture broth was centrifuged at 400 g, an upper layer and a precipitate layer were obtained. MEL-B was recovered by adding the same amount of ethyl acetate as the culture broth to each, and MEL-B was detected in the precipitate. All were detected from the upper layer. That is, by centrifuging the culture solution at 400 g, it was shown that the MEL-B solution can be recovered by completely separating from the bacterial cells without precipitating MEL-B.
また、画分1〜6におけるMEL‐Bの含有量は、それぞれ、2.3g/l、0.7g/l、0.5g/l、1.8g/l、0.4g/l、1.5g/lであった。 The contents of MEL-B in fractions 1 to 6 are 2.3 g / l, 0.7 g / l, 0.5 g / l, 1.8 g / l, 0.4 g / l, and 1. It was 5 g / l.
〔実施例6〕
本実施例では実施例5において得た画分5(本実施例において以下「培養液」いう。)を用いて、MEL‐Bの濃縮方法について検討した。
Example 6
In this example, a method for concentrating MEL-B was examined using fraction 5 obtained in Example 5 (hereinafter referred to as “culture medium” in this example).
検討した条件は(a)培養液を60℃の環境下に3時間静置、(b)培養液を4℃の環境下に3時間静置、(c)培養液のpHを2に調整して、4℃の環境下に3時間静置、(d)培養液に硫酸アンモニウムを20質量%添加して、4℃の環境下に3時間静置、(e)培養液に塩化ナトリウムを10質量%添加して、4℃の環境下に3時間静置の5通りである。 The conditions examined were: (a) the culture broth was left in an environment of 60 ° C. for 3 hours, (b) the broth was left in a 4 ° C. environment for 3 hours, (c) the pH of the broth was adjusted to 2. (D) 20% by mass of ammonium sulfate was added to the culture solution and left at 4 ° C for 3 hours. (E) 10% sodium chloride was added to the culture solution. %, And left in a 4 ° C. environment for 3 hours.
(a)〜(e)の処理を行なった後、8000rpmで5分間遠心分離を行なった。得られた上清と沈殿物とをわけて、それぞれに4mlの酢酸エチルを添加して混合した。酢酸エチル中のMEL‐Bを薄層クロマトグラフィー及びHPLCによって検出した。また、上清中のMEL‐Bと沈殿物中のMEL‐Bとの質量の比を算出した。この結果を、MEL‐Bを検出するまでのプロセスと共に図10に示す。図10は本実施例においてMEL‐Bを濃縮して得た結果を示す図である。なお、図10において「TLC検出結果」と「HPLC定量結果」との間に記載の「上清」は上清からMEL‐Bを検出した結果を示し、「沈殿」は沈殿物からMEL‐Bを検出した結果を示す。図10に示すように、温度、pHの調整においてMEL‐Bは沈殿せず、遠心分離後の上清及び沈殿物の両方に分布していた。一方、硫酸アンモニウムを添加することによって、MEL‐Bは効率よく沈殿し、79%のMEL‐Bを沈殿物として回収することができることが示された。また、NaClを添加した場合、MEL‐Bのほとんどは沈殿せず、上清画分として回収することができることが示された。以上の結果によれば、培養液から、低速遠心分離によって菌体の大部分を除いた後、硫酸アンモニウムを添加し、再度遠心分離することで、MEL‐Bを濃縮できることが示された。 After performing the treatments (a) to (e), centrifugation was performed at 8000 rpm for 5 minutes. The obtained supernatant was separated from the precipitate, and 4 ml of ethyl acetate was added to each and mixed. MEL-B in ethyl acetate was detected by thin layer chromatography and HPLC. Moreover, the mass ratio of MEL-B in the supernatant and MEL-B in the precipitate was calculated. This result is shown in FIG. 10 together with the process up to detection of MEL-B. FIG. 10 is a graph showing the results obtained by concentrating MEL-B in this example. In FIG. 10, “supernatant” described between “TLC detection result” and “HPLC quantification result” indicates the result of detecting MEL-B from the supernatant, and “precipitation” indicates MEL-B from the precipitate. The result of detecting is shown. As shown in FIG. 10, MEL-B did not precipitate in adjusting the temperature and pH, and was distributed in both the supernatant and the precipitate after centrifugation. On the other hand, it was shown that by adding ammonium sulfate, MEL-B precipitates efficiently and 79% of MEL-B can be recovered as a precipitate. In addition, when NaCl was added, most of MEL-B did not precipitate, indicating that it can be recovered as a supernatant fraction. According to the above results, it was shown that MEL-B can be concentrated by removing most of the cells from the culture solution by low-speed centrifugation, adding ammonium sulfate, and centrifuging again.
〔実施例7〕
本実施例では、U. scitaminea NBRC32730を、炭素源としてスクロース、グルコース又は大豆油のみを炭素源として添加した培地で培養し、MELを生産するか否か確認した。まず、それぞれの菌株を培養液(炭素源(スクロース、グルコース又は大豆油)100g/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)中に浸し、25℃で7日間振とう培養し、得られた培養液中のMELを薄層クロマトグラフィーにて検出した。図11に結果を示す。図11は本実施例で得られたMELを薄層クロマトグラフィーによって検出した結果を示す図である。
Example 7
In this embodiment, U.S. Citaminea NBRC32730 was cultured in a medium supplemented with only sucrose, glucose or soybean oil as a carbon source as a carbon source, and it was confirmed whether or not MEL was produced. First, each strain was cultured in a culture solution (carbon source (sucrose, glucose or soybean oil) 100 g / L, yeast extract 1 g / L, sodium nitrate 1 g / L, potassium dihydrogen phosphate 0.5 g / L, and magnesium sulfate 0 0.5 g / L) and cultured with shaking at 25 ° C. for 7 days, and MEL in the obtained culture broth was detected by thin layer chromatography. The results are shown in FIG. FIG. 11 is a diagram showing the results of detecting the MEL obtained in this example by thin layer chromatography.
図11に示すように、U. scitaminea NBRC32730は、スクロース又はグルコースのみを炭素源として得られた培養液中にMELを蓄積した。また、図11に示すように、スクロース又はグルコースを炭素源として培養したU. scitaminea NBRC32730により生産された主なMELは、薄層クロマトグラフィーの結果から、大豆油のみを炭素源として培養した場合と同様、MEL−Bであった。この結果から、この菌株が、スクロース等の糖類のみを炭素源としてMELを生産できる菌株であることが示された。 As shown in FIG. scitamine NBRC32730 accumulated MEL in the culture medium obtained using only sucrose or glucose as a carbon source. Moreover, as shown in FIG. 11, U. coli cultured with sucrose or glucose as a carbon source. The main MEL produced by scitamine NBRC32730 was MEL-B, as in the case of culturing using only soybean oil as a carbon source, from the results of thin layer chromatography. From this result, it was shown that this strain is a strain capable of producing MEL using only saccharides such as sucrose as a carbon source.
さらに、本実施例では、スクロース培養で得られたU. scitaminea NBRC32730の主なMELをカラムクロマトグラフィーにて均一精製した後、MELの脂肪酸組成および界面物性について検討した。MELの脂肪酸組成は、塩酸メタノール処理後のヘキサン抽出液を用い、GC−MSによって解析した。その結果を表4に示す。 Further, in this example, U.S. obtained by sucrose culture was used. The main MEL of scitamine NBRC32730 was uniformly purified by column chromatography, and then the fatty acid composition and interface physical properties of MEL were examined. The fatty acid composition of MEL was analyzed by GC-MS using a hexane extract after treatment with hydrochloric acid and methanol. The results are shown in Table 4.
表4に示されるように、スクロース由来のMEL−Bの脂肪酸組成は、C8、C10及びC12であった。一方、植物油由来のMELの脂肪酸組成は、C8、C10、C12及びC14であった。従って、炭素源が異なると、生産されるMELの脂肪酸組成が変化することが示された。 As shown in Table 4, the fatty acid composition of sucrose-derived MEL-B was C8, C10, and C12. On the other hand, the fatty acid composition of MEL derived from vegetable oil was C8, C10, C12 and C14. Therefore, it was shown that the fatty acid composition of the produced MEL changes with different carbon sources.
また、界面物性の指標である、臨界ミセル濃度(CMC)については、U. scitaminea NBRC32730のMEL−Bは2.8×10−6Mであった。このCMC測定の結果から、U. scitaminea NBRC32730由来のMELは、優れた界面活性を有する界面活性剤であることが示された。さらに、MELの液晶形成能を、顕微鏡用スライドガラスにMEL標品を塗布してカバーガラスをかぶせた後、カバーガラスの横から水を浸入させて、MELと水との接触面を光学顕微鏡下で偏光観察することにより判定した。結果を図12に示している。図12は本実施例において得られたMELの液晶形成能を観察した結果を示す図である。図12に示されるように、液晶形成能が認められた。すなわち、スクロースで生産したMELは、大豆油で生産したものと脂肪酸組成が多少異なるが、優れた界面活性を有しており、かつ液晶形成能(高度な自己集合特性)をも有する、高機能な界面活性剤として使用可能であることが確認できた。なお、脂肪酸組成の違いは微生物のMEL合成経路に依存しており、大豆油等の油脂類の場合は分解反応の中間体がMEL生合成に用いられていると考えられ、一方、スクロースの場合、脂肪酸は細胞内で合成されるため、比較的短い脂肪酸が構成成分となっていると考えられる。 Regarding the critical micelle concentration (CMC), which is an index of interface physical properties, see U.S. Pat. The MEL-B of scitamine NBRC32730 was 2.8 × 10 −6 M. From the results of this CMC measurement, U. MEL derived from scitamine NBRC32730 has been shown to be a surfactant with excellent surface activity. Furthermore, the liquid crystal forming ability of MEL was applied to the slide glass for the microscope and covered with the cover glass. Then, water was infiltrated from the side of the cover glass, and the contact surface between the MEL and water was observed under the optical microscope. This was determined by observing the polarized light. The results are shown in FIG. FIG. 12 is a diagram showing the result of observing the liquid crystal forming ability of the MEL obtained in this example. As shown in FIG. 12, liquid crystal forming ability was recognized. In other words, MEL produced with sucrose has a slightly different fatty acid composition from that produced with soybean oil, but has excellent surface activity and liquid crystal forming ability (advanced self-assembling properties). It was confirmed that it can be used as a novel surfactant. The difference in fatty acid composition depends on the MEL synthesis pathway of microorganisms, and in the case of fats and oils such as soybean oil, it is considered that the intermediate of decomposition reaction is used for MEL biosynthesis, whereas in the case of sucrose Since fatty acids are synthesized in cells, it is considered that relatively short fatty acids are constituents.
〔実施例8〕
本実施例では、スクロース又はオリーブ油を炭素源として用い、炭素源の流下培養法によってU. scitaminea NBRC32730の培養を行ない、MELの生産を試みた。
Example 8
In this example, sucrose or olive oil was used as a carbon source, and U.V. Citaminea NBRC32730 was cultured and MEL production was attempted.
培地としては、炭素源(スクロース及びオリーブ油)15〜23重量%、硝酸ナトリウム0.3重量%、リン酸2水素カリウム0.03重量%、硫酸マグネシウム0.03重量%、酵母エキス0.1重量%を含み、残部を水とし、全量を20mlとした液体培地を用いた。また、培養期間を7日間、培養温度を25℃として、200rpmにて振とう培養した。その後、炭素源のみを培養液中に添加して、7日間の培養を2回繰り返した。それぞれの培養で得られた培養液の酢酸エチル抽出液を用いて、実施例1(3−a)に記載のHPLC法によってMEL生産量を定量した。結果を図13に示す。図13は炭素源の量とMEL生産量との関係を示す図である。図13に示すように15重量%のスクロースのみを炭素源としてU. scitamineaを培養することでMEL‐Bを生産した場合、その生産量は21日間で12g/Lに達した。15重量%のスクロール及び1重量%のオリーブ油を含む培地で培養した場合、MEL−Bの生産量は25g/Lに達した。さらに、15重量%のスクロール及び2重量%のオリーブ油を含む培地で培養した場合、MEL−Bの生産量は31g/Lに達した。15重量%のスクロール及び4重量%のオリーブ油を含む培地で培養した場合、MEL−Bの生産量は37g/Lに達した。15重量%のスクロール及び8重量%のオリーブ油を含む培地で培養した場合、MEL−Bの生産量は37g/Lに達した。以上の結果から、U. scitamineaを用いたMEL−Bの培養において、スクロースのみからMEL−Bを生産可能であり、さらに油脂を添加することで生産量を向上させることができることが示された。 As a medium, carbon source (sucrose and olive oil) 15 to 23% by weight, sodium nitrate 0.3% by weight, potassium dihydrogen phosphate 0.03% by weight, magnesium sulfate 0.03% by weight, yeast extract 0.1% by weight %, And the balance was water, and the total volume was 20 ml. Further, the culture was carried out at 200 rpm with a culture period of 7 days and a culture temperature of 25 ° C. Thereafter, only the carbon source was added to the culture solution, and the 7-day culture was repeated twice. MEL production was quantified by the HPLC method described in Example 1 (3-a) using the ethyl acetate extract of the culture obtained in each culture. The results are shown in FIG. FIG. 13 is a diagram showing the relationship between the amount of carbon source and the amount of MEL production. As shown in FIG. When MEL-B was produced by culturing scitamine, the production amount reached 12 g / L in 21 days. When cultured in a medium containing 15% by weight scroll and 1% by weight olive oil, the production of MEL-B reached 25 g / L. Furthermore, when cultured in a medium containing 15% by weight scroll and 2% by weight olive oil, the production amount of MEL-B reached 31 g / L. When cultured in a medium containing 15 wt% scroll and 4 wt% olive oil, MEL-B production reached 37 g / L. When cultured in a medium containing 15% by weight scroll and 8% by weight olive oil, the production of MEL-B reached 37 g / L. From the above results, U.S. In the culture of MEL-B using scitamine, it was shown that MEL-B can be produced only from sucrose and that the production amount can be improved by adding oils and fats.
本発明に係る製造方法により得られたMELは、食用植物や日常的に接触する植物から単離された微生物を培養して得られたものであり、安全性がより高く、食品用途への展開等が期待される。また、培養に用いる微生物を適宜選択することにより、用途に応じて、親水性が異なるMEL‐A、MEL‐B、MEL‐Cをそれぞれ選択的に得ることができるので、新たな食品用途に適用することが期待される。 The MEL obtained by the production method according to the present invention is obtained by culturing microorganisms isolated from edible plants and plants that come into daily contact, and has higher safety and development for food applications. Etc. are expected. In addition, MEL-A, MEL-B, and MEL-C with different hydrophilicity can be selectively obtained depending on the application by appropriately selecting the microorganism to be used for culture, so it can be applied to new food applications. Is expected to do.
Claims (8)
で表される構造を有するマンノシルエリスリトールリピッドの製造方法。 Cultivate a microorganism of the genus Ustilago isolated from any plant of the genus Saccharum, Malus, Perilla, Brassica and Cynodon The following general formula (1) characterized by including a culture step
The manufacturing method of the mannosyl erythritol lipid which has a structure represented by these.
で表される構造を有するマンノシルエリスリトールリピッド‐Bの製造方法。 The following general formula (2), which comprises a culture step of culturing a microorganism of the genus Ustilago isolated from a plant of the genus Saccharum
The manufacturing method of the mannosyl erythritol lipid-B which has a structure represented by these.
で表される構造を有するマンノシルエリスリトールリピッド‐Cの製造方法。 The following general formula (3), comprising a culturing step of culturing a microorganism belonging to the genus Ustylago isolated from a plant belonging to the genus Cynodon
The manufacturing method of the mannosyl erythritol lipid-C which has a structure represented by these.
で表される構造を有するマンノシルエリスリトールリピッド‐Aの製造方法。 The following general formula, which comprises a culture step of culturing a microorganism of the genus Ustilago isolated from any plant of the genus Apple (Malus), the genus Perilla and the genus Brassica (4)
The manufacturing method of mannosyl erythritol lipid-A which has a structure represented by these.
Priority Applications (1)
Application Number | Priority Date | Filing Date | Title |
---|---|---|---|
JP2008322881A JP5137204B2 (en) | 2008-03-21 | 2008-12-18 | Method for producing mannosyl erythritol lipid |
Applications Claiming Priority (5)
Application Number | Priority Date | Filing Date | Title |
---|---|---|---|
JP2008074593 | 2008-03-21 | ||
JP2008074593 | 2008-03-21 | ||
JP2008187077 | 2008-07-18 | ||
JP2008187077 | 2008-07-18 | ||
JP2008322881A JP5137204B2 (en) | 2008-03-21 | 2008-12-18 | Method for producing mannosyl erythritol lipid |
Publications (2)
Publication Number | Publication Date |
---|---|
JP2010041991A true JP2010041991A (en) | 2010-02-25 |
JP5137204B2 JP5137204B2 (en) | 2013-02-06 |
Family
ID=42013924
Family Applications (1)
Application Number | Title | Priority Date | Filing Date |
---|---|---|---|
JP2008322881A Active JP5137204B2 (en) | 2008-03-21 | 2008-12-18 | Method for producing mannosyl erythritol lipid |
Country Status (1)
Country | Link |
---|---|
JP (1) | JP5137204B2 (en) |
Cited By (2)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
JP2011182740A (en) * | 2010-03-10 | 2011-09-22 | National Institute Of Advanced Industrial Science & Technology | New microorganism and method for producing sugar type biosurfactant therewith |
CN112280806A (en) * | 2019-07-23 | 2021-01-29 | 广东现代汉方科技有限公司 | Method for preparing ceramide by emulsification fermentation and application thereof |
-
2008
- 2008-12-18 JP JP2008322881A patent/JP5137204B2/en active Active
Non-Patent Citations (2)
Title |
---|
JPN6012044859; Appl. Microbiol. Biotechnol. vol.51, 1999, p.33-9 * |
JPN6012044861; Biosci. Biotechnol. Biochem. vol.73, 2009, p.788-92 * |
Cited By (2)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
JP2011182740A (en) * | 2010-03-10 | 2011-09-22 | National Institute Of Advanced Industrial Science & Technology | New microorganism and method for producing sugar type biosurfactant therewith |
CN112280806A (en) * | 2019-07-23 | 2021-01-29 | 广东现代汉方科技有限公司 | Method for preparing ceramide by emulsification fermentation and application thereof |
Also Published As
Publication number | Publication date |
---|---|
JP5137204B2 (en) | 2013-02-06 |
Similar Documents
Publication | Publication Date | Title |
---|---|---|
JP5470857B2 (en) | Microorganism having ability to produce sugar-type biosurfactant and method for producing sugar-type biosurfactant using the same | |
Ahmed et al. | Effects of various process parameters on the production of g-linolenic acid in submerged fermentation | |
JP2008247845A (en) | Method for producing acid-type sophorose lipid | |
CN1986822A (en) | Crypthecodinium connii fermenting process for producing docosahexaenoic acid grease | |
EP2110438B1 (en) | Method for increasing the content of docosahexaenoic acid in material containing oil-and-fat or oil-and-fat | |
JP4803434B2 (en) | Highly efficient production method of mannosyl erythritol lipid | |
JP4286558B2 (en) | Method for producing mannosyl erythritol lipid | |
JP4722386B2 (en) | Glycolipid and method for producing the same | |
JP2010200695A (en) | Method for producing biosurfactant | |
JP2009126820A (en) | New mannosyl erythritol lipid and method for producing the same | |
JP4978908B2 (en) | Method for producing mannosyl erythritol lipid | |
JP5622190B2 (en) | Novel microorganism and method for producing sugar-type biosurfactant using the same | |
JP5137204B2 (en) | Method for producing mannosyl erythritol lipid | |
Langer et al. | Production and modification of bioactive biosurfactants | |
JP2008245607A (en) | Yeast and use of the same | |
JP4735971B2 (en) | Mannosyl erythritol lipid production method | |
JP2010022217A (en) | Method for producing glucosylceramide | |
JPH0449396B2 (en) | ||
JP5145540B2 (en) | Mannosyl alditol lipid and method for producing the same | |
JP4654415B2 (en) | Separation and recovery method of mannosyl erythritol lipid | |
JP5565796B2 (en) | Novel microorganism and method for producing sugar-type biosurfactant using the same | |
KR101147451B1 (en) | Method for Culturing Microalgae Thraustochytrid | |
JP4982885B2 (en) | Production method of biosurfactant | |
JP2698052B2 (en) | Eicosapentaenoic acid-producing microorganism | |
JPS6314696A (en) | Production of bishomo gamma-linolenic acid |
Legal Events
Date | Code | Title | Description |
---|---|---|---|
A621 | Written request for application examination |
Free format text: JAPANESE INTERMEDIATE CODE: A621 Effective date: 20100311 |
|
A131 | Notification of reasons for refusal |
Free format text: JAPANESE INTERMEDIATE CODE: A131 Effective date: 20120828 |
|
A521 | Request for written amendment filed |
Free format text: JAPANESE INTERMEDIATE CODE: A523 Effective date: 20121001 |
|
TRDD | Decision of grant or rejection written | ||
A01 | Written decision to grant a patent or to grant a registration (utility model) |
Free format text: JAPANESE INTERMEDIATE CODE: A01 Effective date: 20121030 |
|
A01 | Written decision to grant a patent or to grant a registration (utility model) |
Free format text: JAPANESE INTERMEDIATE CODE: A01 |
|
A61 | First payment of annual fees (during grant procedure) |
Free format text: JAPANESE INTERMEDIATE CODE: A61 Effective date: 20121109 |
|
R150 | Certificate of patent or registration of utility model |
Free format text: JAPANESE INTERMEDIATE CODE: R150 Ref document number: 5137204 Country of ref document: JP Free format text: JAPANESE INTERMEDIATE CODE: R150 |
|
FPAY | Renewal fee payment (event date is renewal date of database) |
Free format text: PAYMENT UNTIL: 20151122 Year of fee payment: 3 |
|
S533 | Written request for registration of change of name |
Free format text: JAPANESE INTERMEDIATE CODE: R313533 |
|
R350 | Written notification of registration of transfer |
Free format text: JAPANESE INTERMEDIATE CODE: R350 |
|
R250 | Receipt of annual fees |
Free format text: JAPANESE INTERMEDIATE CODE: R250 |
|
R250 | Receipt of annual fees |
Free format text: JAPANESE INTERMEDIATE CODE: R250 |
|
R250 | Receipt of annual fees |
Free format text: JAPANESE INTERMEDIATE CODE: R250 |
|
R250 | Receipt of annual fees |
Free format text: JAPANESE INTERMEDIATE CODE: R250 |
|
R250 | Receipt of annual fees |
Free format text: JAPANESE INTERMEDIATE CODE: R250 |
|
R250 | Receipt of annual fees |
Free format text: JAPANESE INTERMEDIATE CODE: R250 |
|
R250 | Receipt of annual fees |
Free format text: JAPANESE INTERMEDIATE CODE: R250 |
|
R250 | Receipt of annual fees |
Free format text: JAPANESE INTERMEDIATE CODE: R250 |
|
R250 | Receipt of annual fees |
Free format text: JAPANESE INTERMEDIATE CODE: R250 |