JPH044868B2 - - Google Patents
Info
- Publication number
- JPH044868B2 JPH044868B2 JP61099447A JP9944786A JPH044868B2 JP H044868 B2 JPH044868 B2 JP H044868B2 JP 61099447 A JP61099447 A JP 61099447A JP 9944786 A JP9944786 A JP 9944786A JP H044868 B2 JPH044868 B2 JP H044868B2
- Authority
- JP
- Japan
- Prior art keywords
- hyaluronic acid
- negative
- resistance
- culture
- streptococcus
- 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.)
- Expired - Lifetime
Links
- KIUKXJAPPMFGSW-DNGZLQJQSA-N (2S,3S,4S,5R,6R)-6-[(2S,3R,4R,5S,6R)-3-Acetamido-2-[(2S,3S,4R,5R,6R)-6-[(2R,3R,4R,5S,6R)-3-acetamido-2,5-dihydroxy-6-(hydroxymethyl)oxan-4-yl]oxy-2-carboxy-4,5-dihydroxyoxan-3-yl]oxy-5-hydroxy-6-(hydroxymethyl)oxan-4-yl]oxy-3,4,5-trihydroxyoxane-2-carboxylic acid Chemical compound CC(=O)N[C@H]1[C@H](O)O[C@H](CO)[C@@H](O)[C@@H]1O[C@H]1[C@H](O)[C@@H](O)[C@H](O[C@H]2[C@@H]([C@@H](O[C@H]3[C@@H]([C@@H](O)[C@H](O)[C@H](O3)C(O)=O)O)[C@H](O)[C@@H](CO)O2)NC(C)=O)[C@@H](C(O)=O)O1 KIUKXJAPPMFGSW-DNGZLQJQSA-N 0.000 claims description 70
- 229920002674 hyaluronan Polymers 0.000 claims description 70
- 229960003160 hyaluronic acid Drugs 0.000 claims description 70
- 241000120569 Streptococcus equi subsp. zooepidemicus Species 0.000 claims description 20
- 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 claims description 18
- 239000008103 glucose Substances 0.000 claims description 18
- 238000004519 manufacturing process Methods 0.000 claims description 12
- 108010003272 Hyaluronate lyase Proteins 0.000 claims description 9
- 102000001974 Hyaluronidases Human genes 0.000 claims description 9
- 229960002773 hyaluronidase Drugs 0.000 claims description 9
- 230000012010 growth Effects 0.000 claims description 5
- PEDCQBHIVMGVHV-UHFFFAOYSA-N Glycerine Chemical compound OCC(O)CO PEDCQBHIVMGVHV-UHFFFAOYSA-N 0.000 claims description 4
- 229920002472 Starch Polymers 0.000 claims description 4
- 239000008107 starch Substances 0.000 claims description 4
- 235000019698 starch Nutrition 0.000 claims description 4
- 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 claims description 3
- 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 claims description 3
- 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 claims description 3
- 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 claims description 3
- 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 claims description 3
- 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 claims description 3
- 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 claims description 3
- 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 claims description 3
- 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 claims description 3
- 229930182830 galactose Natural products 0.000 claims description 3
- 239000008101 lactose Substances 0.000 claims description 3
- 239000000600 sorbitol Substances 0.000 claims description 3
- HDTRYLNUVZCQOY-UHFFFAOYSA-N α-D-glucopyranosyl-α-D-glucopyranoside Natural products OC1C(O)C(O)C(CO)OC1OC1C(O)C(O)C(O)C(CO)O1 HDTRYLNUVZCQOY-UHFFFAOYSA-N 0.000 claims description 2
- PLXMOAALOJOTIY-FPTXNFDTSA-N Aesculin Natural products OC[C@@H]1[C@@H](O)[C@H](O)[C@@H](O)[C@H](O)[C@H]1Oc2cc3C=CC(=O)Oc3cc2O PLXMOAALOJOTIY-FPTXNFDTSA-N 0.000 claims description 2
- 239000004475 Arginine Substances 0.000 claims description 2
- 108010001478 Bacitracin Proteins 0.000 claims description 2
- 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 claims description 2
- 108010010803 Gelatin Proteins 0.000 claims description 2
- 238000003794 Gram staining Methods 0.000 claims description 2
- 229930195725 Mannitol Natural products 0.000 claims description 2
- NGFMICBWJRZIBI-JZRPKSSGSA-N Salicin Natural products O([C@H]1[C@H](O)[C@@H](O)[C@@H](O)[C@H](CO)O1)c1c(CO)cccc1 NGFMICBWJRZIBI-JZRPKSSGSA-N 0.000 claims description 2
- 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 claims description 2
- 229930006000 Sucrose Natural products 0.000 claims description 2
- HDTRYLNUVZCQOY-WSWWMNSNSA-N Trehalose Natural products O[C@@H]1[C@@H](O)[C@@H](O)[C@@H](CO)O[C@@H]1O[C@@H]1[C@H](O)[C@@H](O)[C@@H](O)[C@@H](CO)O1 HDTRYLNUVZCQOY-WSWWMNSNSA-N 0.000 claims description 2
- HDTRYLNUVZCQOY-LIZSDCNHSA-N alpha,alpha-trehalose Chemical compound O[C@@H]1[C@@H](O)[C@H](O)[C@@H](CO)O[C@@H]1O[C@@H]1[C@H](O)[C@@H](O)[C@H](O)[C@@H](CO)O1 HDTRYLNUVZCQOY-LIZSDCNHSA-N 0.000 claims description 2
- NGFMICBWJRZIBI-UHFFFAOYSA-N alpha-salicin Natural products OC1C(O)C(O)C(CO)OC1OC1=CC=CC=C1CO NGFMICBWJRZIBI-UHFFFAOYSA-N 0.000 claims description 2
- PYMYPHUHKUWMLA-WDCZJNDASA-N arabinose Chemical compound OC[C@@H](O)[C@@H](O)[C@H](O)C=O PYMYPHUHKUWMLA-WDCZJNDASA-N 0.000 claims description 2
- PYMYPHUHKUWMLA-UHFFFAOYSA-N arabinose Natural products OCC(O)C(O)C(O)C=O PYMYPHUHKUWMLA-UHFFFAOYSA-N 0.000 claims description 2
- ODKSFYDXXFIFQN-UHFFFAOYSA-N arginine Natural products OC(=O)C(N)CCCNC(N)=N ODKSFYDXXFIFQN-UHFFFAOYSA-N 0.000 claims description 2
- 229960003071 bacitracin Drugs 0.000 claims description 2
- 229930184125 bacitracin Natural products 0.000 claims description 2
- CLKOFPXJLQSYAH-ABRJDSQDSA-N bacitracin A Chemical compound C1SC([C@@H](N)[C@@H](C)CC)=N[C@@H]1C(=O)N[C@@H](CC(C)C)C(=O)N[C@H](CCC(O)=O)C(=O)N[C@@H]([C@@H](C)CC)C(=O)N[C@@H]1C(=O)N[C@H](CCCN)C(=O)N[C@@H]([C@@H](C)CC)C(=O)N[C@H](CC=2C=CC=CC=2)C(=O)N[C@@H](CC=2N=CNC=2)C(=O)N[C@H](CC(O)=O)C(=O)N[C@@H](CC(N)=O)C(=O)NCCCC1 CLKOFPXJLQSYAH-ABRJDSQDSA-N 0.000 claims description 2
- SRBFZHDQGSBBOR-UHFFFAOYSA-N beta-D-Pyranose-Lyxose Natural products OC1COC(O)C(O)C1O SRBFZHDQGSBBOR-UHFFFAOYSA-N 0.000 claims description 2
- 210000000941 bile Anatomy 0.000 claims description 2
- XHCADAYNFIFUHF-TVKJYDDYSA-N esculin Chemical compound O[C@@H]1[C@@H](O)[C@H](O)[C@@H](CO)O[C@H]1OC(C(=C1)O)=CC2=C1OC(=O)C=C2 XHCADAYNFIFUHF-TVKJYDDYSA-N 0.000 claims description 2
- 239000008273 gelatin Substances 0.000 claims description 2
- 229920000159 gelatin Polymers 0.000 claims description 2
- 235000019322 gelatine Nutrition 0.000 claims description 2
- 235000011852 gelatine desserts Nutrition 0.000 claims description 2
- 235000011187 glycerol Nutrition 0.000 claims description 2
- 239000000594 mannitol Substances 0.000 claims description 2
- 235000010355 mannitol Nutrition 0.000 claims description 2
- CXKWCBBOMKCUKX-UHFFFAOYSA-M methylene blue Chemical compound [Cl-].C1=CC(N(C)C)=CC2=[S+]C3=CC(N(C)C)=CC=C3N=C21 CXKWCBBOMKCUKX-UHFFFAOYSA-M 0.000 claims description 2
- 229960000907 methylthioninium chloride Drugs 0.000 claims description 2
- NGFMICBWJRZIBI-UJPOAAIJSA-N salicin Chemical compound O[C@@H]1[C@@H](O)[C@H](O)[C@@H](CO)O[C@H]1OC1=CC=CC=C1CO NGFMICBWJRZIBI-UJPOAAIJSA-N 0.000 claims description 2
- 229940120668 salicin Drugs 0.000 claims description 2
- 150000003839 salts Chemical class 0.000 claims description 2
- 239000005720 sucrose Substances 0.000 claims description 2
- WNBCMONIPIJTSB-BGNCJLHMSA-N Cichoriin Natural products O([C@H]1[C@H](O)[C@@H](O)[C@@H](O)[C@@H](CO)O1)c1c(O)cc2c(OC(=O)C=C2)c1 WNBCMONIPIJTSB-BGNCJLHMSA-N 0.000 claims 1
- 206010018910 Haemolysis Diseases 0.000 claims 1
- 230000015556 catabolic process Effects 0.000 claims 1
- 238000006731 degradation reaction Methods 0.000 claims 1
- 229940093496 esculin Drugs 0.000 claims 1
- AWRMZKLXZLNBBK-UHFFFAOYSA-N esculin Natural products OC1OC(COc2cc3C=CC(=O)Oc3cc2O)C(O)C(O)C1O AWRMZKLXZLNBBK-UHFFFAOYSA-N 0.000 claims 1
- 230000008588 hemolysis Effects 0.000 claims 1
- ZBCAZEFVTIBZJS-UHFFFAOYSA-M sodium;2-benzamidoacetate Chemical compound [Na+].[O-]C(=O)CNC(=O)C1=CC=CC=C1 ZBCAZEFVTIBZJS-UHFFFAOYSA-M 0.000 claims 1
- 239000000243 solution Substances 0.000 description 22
- 241000894006 Bacteria Species 0.000 description 21
- 238000000034 method Methods 0.000 description 13
- 238000012258 culturing Methods 0.000 description 12
- 239000002609 medium Substances 0.000 description 12
- 241000194017 Streptococcus Species 0.000 description 8
- 230000001580 bacterial effect Effects 0.000 description 8
- 239000000203 mixture Substances 0.000 description 8
- HEMHJVSKTPXQMS-UHFFFAOYSA-M Sodium hydroxide Chemical compound [OH-].[Na+] HEMHJVSKTPXQMS-UHFFFAOYSA-M 0.000 description 7
- 239000007864 aqueous solution Substances 0.000 description 6
- 238000003756 stirring Methods 0.000 description 6
- 239000000126 substance Substances 0.000 description 6
- 229920001817 Agar Polymers 0.000 description 5
- OKTJSMMVPCPJKN-UHFFFAOYSA-N Carbon Chemical compound [C] OKTJSMMVPCPJKN-UHFFFAOYSA-N 0.000 description 5
- 108010006464 Hemolysin Proteins Proteins 0.000 description 5
- 239000008272 agar Substances 0.000 description 5
- 229910052799 carbon Inorganic materials 0.000 description 5
- 239000003228 hemolysin Substances 0.000 description 5
- 230000002949 hemolytic effect Effects 0.000 description 5
- IJGRMHOSHXDMSA-UHFFFAOYSA-N Atomic nitrogen Chemical compound N#N IJGRMHOSHXDMSA-UHFFFAOYSA-N 0.000 description 4
- 239000000872 buffer Substances 0.000 description 4
- 238000010586 diagram Methods 0.000 description 4
- -1 etc.) Substances 0.000 description 4
- JVTAAEKCZFNVCJ-UHFFFAOYSA-N lactic acid Chemical compound CC(O)C(O)=O JVTAAEKCZFNVCJ-UHFFFAOYSA-N 0.000 description 4
- 238000000746 purification Methods 0.000 description 4
- 238000000108 ultra-filtration Methods 0.000 description 4
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 description 4
- KWYUFKZDYYNOTN-UHFFFAOYSA-M Potassium hydroxide Chemical compound [OH-].[K+] KWYUFKZDYYNOTN-UHFFFAOYSA-M 0.000 description 3
- 108010011834 Streptolysins Proteins 0.000 description 3
- 238000005273 aeration Methods 0.000 description 3
- 238000005119 centrifugation Methods 0.000 description 3
- 239000002537 cosmetic Substances 0.000 description 3
- 239000003814 drug Substances 0.000 description 3
- 238000005516 engineering process Methods 0.000 description 3
- 238000000605 extraction Methods 0.000 description 3
- 239000012528 membrane Substances 0.000 description 3
- 244000005700 microbiome Species 0.000 description 3
- 238000000926 separation method Methods 0.000 description 3
- 210000001519 tissue Anatomy 0.000 description 3
- 238000011282 treatment Methods 0.000 description 3
- POSZUTFLHGNLHX-KSBRXOFISA-N tris maleate Chemical compound OCC(N)(CO)CO.OCC(N)(CO)CO.OC(=O)\C=C/C(O)=O POSZUTFLHGNLHX-KSBRXOFISA-N 0.000 description 3
- 238000005406 washing Methods 0.000 description 3
- 241000283690 Bos taurus Species 0.000 description 2
- XLYOFNOQVPJJNP-ZSJDYOACSA-N Heavy water Chemical compound [2H]O[2H] XLYOFNOQVPJJNP-ZSJDYOACSA-N 0.000 description 2
- DGAQECJNVWCQMB-PUAWFVPOSA-M Ilexoside XXIX Chemical compound C[C@@H]1CC[C@@]2(CC[C@@]3(C(=CC[C@H]4[C@]3(CC[C@@H]5[C@@]4(CC[C@@H](C5(C)C)OS(=O)(=O)[O-])C)C)[C@@H]2[C@]1(C)O)C)C(=O)O[C@H]6[C@@H]([C@H]([C@@H]([C@H](O6)CO)O)O)O.[Na+] DGAQECJNVWCQMB-PUAWFVPOSA-M 0.000 description 2
- XEEYBQQBJWHFJM-UHFFFAOYSA-N Iron Chemical compound [Fe] XEEYBQQBJWHFJM-UHFFFAOYSA-N 0.000 description 2
- QIAFMBKCNZACKA-UHFFFAOYSA-N N-benzoylglycine Chemical compound OC(=O)CNC(=O)C1=CC=CC=C1 QIAFMBKCNZACKA-UHFFFAOYSA-N 0.000 description 2
- 241000606856 Pasteurella multocida Species 0.000 description 2
- 238000000862 absorption spectrum Methods 0.000 description 2
- 230000003698 anagen phase Effects 0.000 description 2
- 239000008280 blood Substances 0.000 description 2
- 210000004369 blood Anatomy 0.000 description 2
- 239000007853 buffer solution Substances 0.000 description 2
- 229940041514 candida albicans extract Drugs 0.000 description 2
- 238000000354 decomposition reaction Methods 0.000 description 2
- 238000010790 dilution Methods 0.000 description 2
- 239000012895 dilution Substances 0.000 description 2
- 230000000694 effects Effects 0.000 description 2
- 238000004108 freeze drying Methods 0.000 description 2
- 239000012510 hollow fiber Substances 0.000 description 2
- 239000004310 lactic acid Substances 0.000 description 2
- 235000014655 lactic acid Nutrition 0.000 description 2
- 230000000813 microbial effect Effects 0.000 description 2
- 230000035772 mutation Effects 0.000 description 2
- 210000002850 nasal mucosa Anatomy 0.000 description 2
- 229910052757 nitrogen Inorganic materials 0.000 description 2
- 239000006916 nutrient agar Substances 0.000 description 2
- 239000003960 organic solvent Substances 0.000 description 2
- 229940051027 pasteurella multocida Drugs 0.000 description 2
- 239000002504 physiological saline solution Substances 0.000 description 2
- 238000001556 precipitation Methods 0.000 description 2
- 238000011160 research Methods 0.000 description 2
- 239000011734 sodium Substances 0.000 description 2
- 229910052708 sodium Inorganic materials 0.000 description 2
- 230000001954 sterilising effect Effects 0.000 description 2
- 238000004659 sterilization and disinfection Methods 0.000 description 2
- YNJBWRMUSHSURL-UHFFFAOYSA-N trichloroacetic acid Chemical compound OC(=O)C(Cl)(Cl)Cl YNJBWRMUSHSURL-UHFFFAOYSA-N 0.000 description 2
- LWIHDJKSTIGBAC-UHFFFAOYSA-K tripotassium phosphate Chemical compound [K+].[K+].[K+].[O-]P([O-])([O-])=O LWIHDJKSTIGBAC-UHFFFAOYSA-K 0.000 description 2
- 239000012138 yeast extract Substances 0.000 description 2
- NWUYHJFMYQTDRP-UHFFFAOYSA-N 1,2-bis(ethenyl)benzene;1-ethenyl-2-ethylbenzene;styrene Chemical compound C=CC1=CC=CC=C1.CCC1=CC=CC=C1C=C.C=CC1=CC=CC=C1C=C NWUYHJFMYQTDRP-UHFFFAOYSA-N 0.000 description 1
- VHUUQVKOLVNVRT-UHFFFAOYSA-N Ammonium hydroxide Chemical compound [NH4+].[OH-] VHUUQVKOLVNVRT-UHFFFAOYSA-N 0.000 description 1
- LSNNMFCWUKXFEE-UHFFFAOYSA-M Bisulfite Chemical compound OS([O-])=O LSNNMFCWUKXFEE-UHFFFAOYSA-M 0.000 description 1
- OYPRJOBELJOOCE-UHFFFAOYSA-N Calcium Chemical compound [Ca] OYPRJOBELJOOCE-UHFFFAOYSA-N 0.000 description 1
- UXVMQQNJUSDDNG-UHFFFAOYSA-L Calcium chloride Chemical compound [Cl-].[Cl-].[Ca+2] UXVMQQNJUSDDNG-UHFFFAOYSA-L 0.000 description 1
- 241000194032 Enterococcus faecalis Species 0.000 description 1
- 108090000790 Enzymes Proteins 0.000 description 1
- 102000004190 Enzymes Human genes 0.000 description 1
- 229930091371 Fructose Natural products 0.000 description 1
- 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 1
- 239000005715 Fructose Substances 0.000 description 1
- IAJILQKETJEXLJ-UHFFFAOYSA-N Galacturonsaeure Natural products O=CC(O)C(O)C(O)C(O)C(O)=O IAJILQKETJEXLJ-UHFFFAOYSA-N 0.000 description 1
- 241000287828 Gallus gallus Species 0.000 description 1
- 239000004472 Lysine Substances 0.000 description 1
- KDXKERNSBIXSRK-UHFFFAOYSA-N Lysine Natural products NCCCCC(N)C(O)=O KDXKERNSBIXSRK-UHFFFAOYSA-N 0.000 description 1
- FYYHWMGAXLPEAU-UHFFFAOYSA-N Magnesium Chemical compound [Mg] FYYHWMGAXLPEAU-UHFFFAOYSA-N 0.000 description 1
- 241001465754 Metazoa Species 0.000 description 1
- OVRNDRQMDRJTHS-UHFFFAOYSA-N N-acelyl-D-glucosamine Natural products CC(=O)NC1C(O)OC(CO)C(O)C1O OVRNDRQMDRJTHS-UHFFFAOYSA-N 0.000 description 1
- OVRNDRQMDRJTHS-FMDGEEDCSA-N N-acetyl-beta-D-glucosamine Chemical compound CC(=O)N[C@H]1[C@H](O)O[C@H](CO)[C@@H](O)[C@@H]1O OVRNDRQMDRJTHS-FMDGEEDCSA-N 0.000 description 1
- VZUNGTLZRAYYDE-UHFFFAOYSA-N N-methyl-N'-nitro-N-nitrosoguanidine Chemical compound O=NN(C)C(=N)N[N+]([O-])=O VZUNGTLZRAYYDE-UHFFFAOYSA-N 0.000 description 1
- 241000283973 Oryctolagus cuniculus Species 0.000 description 1
- 229910019142 PO4 Inorganic materials 0.000 description 1
- 241000606860 Pasteurella Species 0.000 description 1
- 239000001888 Peptone Substances 0.000 description 1
- 108010080698 Peptones Proteins 0.000 description 1
- 235000008331 Pinus X rigitaeda Nutrition 0.000 description 1
- 235000011613 Pinus brutia Nutrition 0.000 description 1
- 241000018646 Pinus brutia Species 0.000 description 1
- ZLMJMSJWJFRBEC-UHFFFAOYSA-N Potassium Chemical compound [K] ZLMJMSJWJFRBEC-UHFFFAOYSA-N 0.000 description 1
- ATUOYWHBWRKTHZ-UHFFFAOYSA-N Propane Chemical compound CCC ATUOYWHBWRKTHZ-UHFFFAOYSA-N 0.000 description 1
- 240000004808 Saccharomyces cerevisiae Species 0.000 description 1
- 235000014680 Saccharomyces cerevisiae Nutrition 0.000 description 1
- VYPSYNLAJGMNEJ-UHFFFAOYSA-N Silicium dioxide Chemical compound O=[Si]=O VYPSYNLAJGMNEJ-UHFFFAOYSA-N 0.000 description 1
- 229920002385 Sodium hyaluronate Polymers 0.000 description 1
- 241000194042 Streptococcus dysgalactiae Species 0.000 description 1
- 241000264435 Streptococcus dysgalactiae subsp. equisimilis Species 0.000 description 1
- 241000194048 Streptococcus equi Species 0.000 description 1
- 241000193996 Streptococcus pyogenes Species 0.000 description 1
- AEMOLEFTQBMNLQ-WAXACMCWSA-N alpha-D-glucuronic acid Chemical compound O[C@H]1O[C@H](C(O)=O)[C@@H](O)[C@H](O)[C@H]1O AEMOLEFTQBMNLQ-WAXACMCWSA-N 0.000 description 1
- 150000001413 amino acids Chemical class 0.000 description 1
- QGZKDVFQNNGYKY-UHFFFAOYSA-N ammonia Natural products N QGZKDVFQNNGYKY-UHFFFAOYSA-N 0.000 description 1
- 238000004458 analytical method Methods 0.000 description 1
- 239000002518 antifoaming agent Substances 0.000 description 1
- 206010003246 arthritis Diseases 0.000 description 1
- 244000052616 bacterial pathogen Species 0.000 description 1
- 230000000721 bacterilogical effect Effects 0.000 description 1
- 239000006161 blood agar Substances 0.000 description 1
- 239000011575 calcium Substances 0.000 description 1
- 229910052791 calcium Inorganic materials 0.000 description 1
- 239000001110 calcium chloride Substances 0.000 description 1
- 229910001628 calcium chloride Inorganic materials 0.000 description 1
- 238000001460 carbon-13 nuclear magnetic resonance spectrum Methods 0.000 description 1
- 150000004649 carbonic acid derivatives Chemical class 0.000 description 1
- 229920002301 cellulose acetate Polymers 0.000 description 1
- YMKDRGPMQRFJGP-UHFFFAOYSA-M cetylpyridinium chloride Chemical compound [Cl-].CCCCCCCCCCCCCCCC[N+]1=CC=CC=C1 YMKDRGPMQRFJGP-UHFFFAOYSA-M 0.000 description 1
- 229960001927 cetylpyridinium chloride Drugs 0.000 description 1
- 239000013043 chemical agent Substances 0.000 description 1
- 239000002962 chemical mutagen Substances 0.000 description 1
- 150000001805 chlorine compounds Chemical class 0.000 description 1
- 210000002808 connective tissue Anatomy 0.000 description 1
- 238000011109 contamination Methods 0.000 description 1
- 238000007796 conventional method Methods 0.000 description 1
- 230000000593 degrading effect Effects 0.000 description 1
- 238000000502 dialysis Methods 0.000 description 1
- 229940079593 drug Drugs 0.000 description 1
- 238000001962 electrophoresis Methods 0.000 description 1
- 229940088598 enzyme Drugs 0.000 description 1
- 239000000284 extract Substances 0.000 description 1
- 239000003889 eye drop Substances 0.000 description 1
- 229940012356 eye drops Drugs 0.000 description 1
- 239000012530 fluid Substances 0.000 description 1
- 150000004676 glycans Chemical class 0.000 description 1
- 230000003394 haemopoietic effect Effects 0.000 description 1
- 208000015181 infectious disease Diseases 0.000 description 1
- 238000011081 inoculation Methods 0.000 description 1
- 229910010272 inorganic material Inorganic materials 0.000 description 1
- 239000011147 inorganic material Substances 0.000 description 1
- 229910017053 inorganic salt Inorganic materials 0.000 description 1
- 239000003456 ion exchange resin Substances 0.000 description 1
- 229920003303 ion-exchange polymer Polymers 0.000 description 1
- 229910052742 iron Inorganic materials 0.000 description 1
- 239000007788 liquid Substances 0.000 description 1
- 239000011777 magnesium Substances 0.000 description 1
- 229910052749 magnesium Inorganic materials 0.000 description 1
- WRUGWIBCXHJTDG-UHFFFAOYSA-L magnesium sulfate heptahydrate Chemical compound O.O.O.O.O.O.O.[Mg+2].[O-]S([O-])(=O)=O WRUGWIBCXHJTDG-UHFFFAOYSA-L 0.000 description 1
- 229940061634 magnesium sulfate heptahydrate Drugs 0.000 description 1
- 239000000463 material Substances 0.000 description 1
- 239000011159 matrix material Substances 0.000 description 1
- 238000000691 measurement method Methods 0.000 description 1
- 235000013372 meat Nutrition 0.000 description 1
- 210000004379 membrane Anatomy 0.000 description 1
- 238000005374 membrane filtration Methods 0.000 description 1
- 239000011785 micronutrient Substances 0.000 description 1
- 235000013369 micronutrients Nutrition 0.000 description 1
- 229950006780 n-acetylglucosamine Drugs 0.000 description 1
- 150000002823 nitrates Chemical class 0.000 description 1
- 238000000655 nuclear magnetic resonance spectrum Methods 0.000 description 1
- 235000015097 nutrients Nutrition 0.000 description 1
- 150000007524 organic acids Chemical class 0.000 description 1
- 235000005985 organic acids Nutrition 0.000 description 1
- 239000011368 organic material Substances 0.000 description 1
- 235000019319 peptone Nutrition 0.000 description 1
- 235000021317 phosphate Nutrition 0.000 description 1
- 150000003013 phosphoric acid derivatives Chemical class 0.000 description 1
- 229920000642 polymer Polymers 0.000 description 1
- 229920001282 polysaccharide Polymers 0.000 description 1
- 239000005017 polysaccharide Substances 0.000 description 1
- 239000011148 porous material Substances 0.000 description 1
- 239000011591 potassium Substances 0.000 description 1
- 229910052700 potassium Inorganic materials 0.000 description 1
- 229910000160 potassium phosphate Inorganic materials 0.000 description 1
- 235000011009 potassium phosphates Nutrition 0.000 description 1
- 239000002994 raw material Substances 0.000 description 1
- 230000035939 shock Effects 0.000 description 1
- 239000000741 silica gel Substances 0.000 description 1
- 229910002027 silica gel Inorganic materials 0.000 description 1
- 229940010747 sodium hyaluronate Drugs 0.000 description 1
- YWIVKILSMZOHHF-QJZPQSOGSA-N sodium;(2s,3s,4s,5r,6r)-6-[(2s,3r,4r,5s,6r)-3-acetamido-2-[(2s,3s,4r,5r,6r)-6-[(2r,3r,4r,5s,6r)-3-acetamido-2,5-dihydroxy-6-(hydroxymethyl)oxan-4-yl]oxy-2-carboxy-4,5-dihydroxyoxan-3-yl]oxy-5-hydroxy-6-(hydroxymethyl)oxan-4-yl]oxy-3,4,5-trihydroxyoxane-2- Chemical compound [Na+].CC(=O)N[C@H]1[C@H](O)O[C@H](CO)[C@@H](O)[C@@H]1O[C@H]1[C@H](O)[C@@H](O)[C@H](O[C@H]2[C@@H]([C@@H](O[C@H]3[C@@H]([C@@H](O)[C@H](O)[C@H](O3)C(O)=O)O)[C@H](O)[C@@H](CO)O2)NC(C)=O)[C@@H](C(O)=O)O1 YWIVKILSMZOHHF-QJZPQSOGSA-N 0.000 description 1
- 238000001179 sorption measurement Methods 0.000 description 1
- 239000007921 spray Substances 0.000 description 1
- 238000001694 spray drying Methods 0.000 description 1
- 229940115920 streptococcus dysgalactiae Drugs 0.000 description 1
- 150000003467 sulfuric acid derivatives Chemical class 0.000 description 1
- 239000010409 thin film Substances 0.000 description 1
- 238000004809 thin layer chromatography Methods 0.000 description 1
- 238000012546 transfer Methods 0.000 description 1
- 210000003954 umbilical cord Anatomy 0.000 description 1
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- 230000001018 virulence Effects 0.000 description 1
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- 239000003357 wound healing promoting agent Substances 0.000 description 1
Landscapes
- Preparation Of Compounds By Using Micro-Organisms (AREA)
- Micro-Organisms Or Cultivation Processes Thereof (AREA)
Description
産業上の利用分野
本発明はヒアルロン酸生産能を有し、ヒアルロ
ニダーゼ非生産性でかつ非溶血性を示す新規なス
トレプトコツカス・ズーエピデミカス
（Streptococcus zooepidemicus）に関する。よ
り具体的には、本発明は溶血素を含まずかつ高分
子量のヒアルロン酸を効率よく生産する能力を有
するストレプトコツカス・ズーエピデミカスの変
異株に関する。
従来の技術
ヒアルロン酸は今日では動物体の結合組織のあ
らゆる部分に存在することが認められており、工
業的には鶏のトサカや臍帯等の生体組織から抽出
法によつて得られ、その機能は細胞間に水を保持
し、又組織内にゼリー様マトリツクスを形成して
細胞を保持したり、細胞間の物質移動を制御した
り、外からの物理的シヨツクあるいは細菌等の感
染を防ぐことが挙げられている。このような機能
を利用してヒアルロン酸は医薬品（関節炎治療
薬、眼薬、創傷治癒剤等）、化粧品等に使用され
ている。
しかしながら生体組織からの抽出によるヒアル
ロン酸の製造は、分離精製の複雑性のため大量生
産がむつかしく極めて高価である。そしてこのこ
とがヒアルロン酸の用途開発の道を閉ざしてい
る。
微生物によるヒアルロン酸の生産についてはス
トレプトコツカス属細菌のうちの、ランスフイー
ルド（Lancefield）血清群のＡ、ＣおよびＤ型
菌、例えばストレプトコツカス・ピオゲネス
（Streptococcus pyogenes）、ストレプトコツカ
ス・ズーエピデミカス（Streptococcus
zooepidemicus）、ストレプトコツカス・エクイ
（Streptococcus equi）、ストレプトコツカス・エ
クイシミリス（Streptococcus equisimilis）、ス
トレプトコツカス・デイスガラクチイエ
（Streptococcus dysgalactiae）およびストレプ
トコツカス・フエカリス・バー・ザイモゲネス
（Streptococcus faecalis var.zymogenes）そし
てパスツレラ・マルトシダ（Pasteurella
multocida）等がヒアルロン酸を生成することが
既に知られており、例えばケンドール等（F.E.
Kendall et al.，J.Biol.Chem.，118，61，
1937）、ピアース等（W.A.Pierce et al.，J.
Baact.，63，301，1952）、マツクレナン（A.P.
MacLennan，J.Gen.Microbiol.，14，134−142，
1956；J.Gen.Microbiol.，15，485−491，1956）、
ホルムストレーム等（B.Holmstro¨m et al.，
Appl.Microbiol.，15，1409−1413，1967）、ウー
ルコツク（J.B.Woolcock，J.Gen.Microbiol.，
85，372−375，1974）、キエム等（E.Kjems et
al.，Acta Path.Microbiol.Scand.Sect.B，84，
162−164，1976）、バーガン等（T.Bergan et
al.，Acta Path.Microbiol.Scand.，75，97−
103，1969）そしてシフオネリ（J.A.Cifonelli，
Carbohyd.Res.，14，272−276，1970）によつて
既に報告されている。これらの報告はヒアルロン
酸の大量生産を目的としたものではなく、炭素源
としてグルコースを１−1.5％用いて培養したも
ので、そのヒアルロン酸生産量は0.5−0.6ｇ／
以下であり、対糖収率は６％以下であつた。マツ
クレナンは上記の報告の中でストレプトコツカス
属のランスフイールド血清群Ｃ型菌の一種につい
て好気条件による培養はヒアルロン酸の生産を促
進する可能性があることを報告している。上記の
ヒアルロン酸を生産する微生物のうち、ストレプ
トコツカス属のランスフイールド血清群Ａ型菌や
パスツレラは人に対する病原菌として知られ、実
際大量培養するには不適である。
工業的にストレプトコツカス属のヒアルロン酸
生産菌を培養して、その培養液からヒアルロン酸
を抽出し、精製する方法が特開昭58−56692号に
開示されている。この方法はストレプトコツカス
属のランスフイールド血清群Ａ、Ｃ型菌を培養し
てヒアルロン酸を大量に得る方法で、炭素源とし
てグルコースを倍地に８％添加して培養し、４
ｇ／のヒアルロン酸を得ている。この場合のヒ
アルロン酸の対糖収率は５％であり、グルコース
添加量を１％から８％と変化させても変わつてい
ない。したがつてこの対糖収率（５％）は既報告
におけるヒアルロン酸生産菌の対糖収率とほとん
どかわりはない。この他にストレプトコツカス属
細菌を使用してヒアルロン酸を得る方法として、
特開昭60−500597、特開昭60−133894、特開昭61
−15698が有るが、得られるヒアルロン酸が低分
子量であつたり、収率が低いなどの問題点が存在
する。又いずれも、ヒアルロン酸生産菌株がスト
レプトリジン（可溶性溶血素）を生成し、β−溶
血性を示す事が知られている。この様な菌を大量
に培養してヒアルロン酸を生産しようとする場
合、該溶血素がヒアルロン酸生産物へ混入するお
それがあり、かかるヒアルロン酸を化粧品や医薬
品に配合することは好ましくない。
この欠点を改良する為に、化学変異剤による変
異処理によつて、ストレプトリジン生成能を欠如
させたヒアルロン酸生産菌株を培養することによ
つてヒアルロン酸を得る方法が特開昭60−251898
に開示されている。この中には、グルコースを６
％添加することにより3.6ｇ／のヒアルロン酸
が得られたことが記載されており、この時のヒア
ルロン酸の対糖収率は６％であり、やはり対糖収
率の観点から生産性の低いものである。
発明が解決しようとする問題点
上記問題点に鑑み、溶血素（ストレプトリジ
ン）を生成せず、高いヒアルロン酸生産能を有す
る微生物を創製することを目的として鋭意研究の
結果、自然界から単離したヒアルロン酸生産能を
有するストレプトコツカス・ズーエピデミカスか
ら変異処理によつて得た溶血性をしめさなくなつ
た菌株を、再度変異処理することにより、ヒアル
ロニダーゼ生成能を欠如し、高分子量ヒアルロン
酸の生産性が極めて高い新規な菌株を得ることに
成功した。
本発明はかかる知見に基づいて完成されたもの
であり、したがつて本発明は溶血素を含まずかつ
高分子量のヒアルロン酸を効率良く生産する能力
を有するストレプトコツカス・ズーエピデミカス
の変異株を提供するものである。
問題点を解決するための手段
(1) 変異株の取得
本発明者らは、本発明の目的を達成するべ
く、次の方法により新規変異株を取得した。ま
ず牛鼻粘膜よりヒアルロニダーゼ（ヒアルロン
酸分解酵素）の強い生成能を有しかつヒアルロ
ン酸を生産するランスフイールド血清群Ｃ型に
属するストレプトコツカス・ズーエピデミカス
（本菌の同定は、バージエイズ・マニユアル・
オブ・デターミネイテイブ・バクテリオロジイ
ー第８版、1974によつた）を得た。この菌株は
マツクレナン（MacLennan，J.Gen.
Microbiol.，14，134−142，1956）が指摘した
ように好気条件においてヒアルロン酸を良く生
産し、炭素源としてグルコースを用いた場合、
４％のグルコース添加によつて２ｇ／のヒア
ルロン酸を生産した（ヒアルロン酸の対糖収率
は５％）。そしてこの時得られたヒアルロン酸
の分子量は30−60万であつた。この菌株を常法
（細菌・フアージ遺伝実験法、淡白質核酸酵素
別冊、共立出版1972）によつて紫外線や化学剤
（Ｎ−メチル−N′−ニトロ−Ｎ−ニトロソグア
ニジン（NTG）、エチルメタンスルフオン酸
等）で処理して、この処理菌体を血液寒天倍地
に混釈してまき、溶血性を示さない集落を採取
し、次にこの菌体を再び変異処理した後、ヒア
ルロン酸を含有した栄養寒天倍地上に塗布し、
ヒアルロン酸を分解しない集落を採取すること
によつてストレプトコツカス・ズーエピデミカ
スの変異株１株を得た（後記実施例１参照）。
(2) 菌学的性質
後記実施例１で得たストレプトコツカス・ズ
ーエピデミカスの変異株（以下本菌という）
は、トツド・ヒユーイツト・ブロス（Todd
Hewitt broth）寒天倍地上で極めて強い粘性
を有する透明な集落を形成し、非溶血性（β−
溶血性：陰性）、ヒアルロニダーゼ非生産性、
ランスフイールド血清群Ｃ型に属する連鎖状球
菌であり、本菌の菌学的性質は下記の通りであ
る。
(a) グラム染色性：陽性
(b) 10℃増殖性：陰性
(c) 45℃増殖性：陰性
(d) 0.1％メチレンブルー抵抗性：陰性
(e) 6.5％食塩抵抗性：陰性
(f) 40％胆汁抵抗性：陰性
(g) バシトラシン抵抗性：陽性
(h) PH9.6抵抗性：陰性
(i) 60℃、30分抵抗性：陰性
(j) ゼラチン分解性：陰性
(k) 澱粉分解性：陽性
(l) 馬尿酸ソーダ分解性：陰性
(m) エスクリン分解性：弱陽性
(n) アルギニン分解性：陽性
(o) 糖醗酵性：グルコース、ガラクトース、
シユークロース、ラクトース、マルトース、
ソルビトールおよびサリシンは陽性、
グリセリン、マンニトール、トレハロース
およびアラビノースは陰性。
本発明者らは本菌の菌学的性質から、本菌を
ストレプトコツカス・ズーエピデミカス
YIT2030と命名し、工業技術院微生物工業技
術研究所に微工研条寄第1305号として寄託され
ている。
(3) 本菌によるヒアルロン酸の製造
本菌を培養してヒアルロン酸を得る培地は、
炭素源、有機無機窒素源、無機塩およびその他
必要に応じて有機微量栄養素を含有するもので
あることが好ましい。炭素源としては、グレコ
ース、ガラクトース、シユークロース、ラクト
ース、フラクトース、マルトース、ソルビトー
ル、澱粉加水分解物等の糖分を含むものが好ま
しく、他には有機酸や脂肪族アルコール等でも
よい。窒素源としては有機無機一般的な材料で
よく、各種肉エキス、アミノ酸混合物、ペプト
ン、酵母エキス等が好ましい。更に、ナトリウ
ム、カリウム、カルシウム、マグネシウム、鉄
等の塩化物、硫酸塩、燐酸塩、硝酸塩、炭酸塩
そしてビタミンなどが必要に応じて添加されう
る。
培養は好気的条件が必須であり、培養液の粘
度の上昇に応じ撹拌速度を上げるのが良いが過
度の撹拌は好ましくない。培養温度は菌の増殖
が行われる25−38℃で行うことが一般的であ
る。更に培養時、本菌が乳酸を生成しその乳酸
によつて菌の増殖ならびにヒアルロン酸の生産
が抑制されることから、乳酸の中和の為にアル
カリ水溶液を添加して、PH６−８の範囲内に調
整することが必要である。この時使用するアル
カリ水溶液は水酸化ナトリウム、水酸化カリウ
ムの水溶液やアンモニア水でよい。
本菌は高分子量のヒアルロン酸（分子量200
−300万）を極めて高い収率、生産率で生産す
る菌株であるが、炭素源としてグルコースを用
いると特に良い結果がえられる。糖の添加量３
％以下では対糖収率14−15％であり、それ以上
の添加量では若干対糖収率は減少する傾向にあ
つた。糖の添加を６％にすると（参考例１参
照）、培養液の粘性は36℃で8000センチポアズ
（cp）となり、ほとんど培養液は流動性がなく
なり撹拌速度を上げても影響なく培養の限界と
なつた。
第１表に培地中のグルコースの添加量を変え
て培養したときのヒアルロン酸生産量を示し
た。即ち生成ヒアルロン酸を常法により精製し
た結果、グルコース１％添加時には対糖収率15
％で1.5ｇ／、６％添加時には対糖収率11％
で6.7ｇ／のヒアルロン酸が得られた。
INDUSTRIAL APPLICATION FIELD The present invention relates to a novel Streptococcus zooepidemicus that has the ability to produce hyaluronic acid, does not produce hyaluronidase, and is non-hemolytic. More specifically, the present invention relates to a mutant strain of Streptococcus zooepidemicus that does not contain hemolysin and has the ability to efficiently produce high molecular weight hyaluronic acid. Conventional technology Today, hyaluronic acid is recognized to exist in all parts of the connective tissue of animals, and is industrially obtained from biological tissues such as chicken crests and umbilical cords by extraction methods. It retains water between cells, forms a jelly-like matrix within tissues to retain cells, controls material transfer between cells, and prevents physical shock or infection from outside by bacteria. are listed. Taking advantage of these functions, hyaluronic acid is used in medicines (arthritis treatments, eye drops, wound healing agents, etc.), cosmetics, and the like. However, the production of hyaluronic acid by extraction from living tissues is difficult and extremely expensive to mass produce due to the complexity of separation and purification. This has closed off the path to developing new uses for hyaluronic acid. Regarding the production of hyaluronic acid by microorganisms, bacteria of the genus Streptococcus, type A, C, and D of the Lancefield serogroup, such as Streptococcus pyogenes, Streptococcus zooepidemicus ( Streptococcus
zooepidemicus), Streptococcus equi, Streptococcus equisimilis, Streptococcus dysgalactiae and Streptococcus faecalis var. zymogenes) and Pasteurella multocida (Pasteurella
multocida) etc., are already known to produce hyaluronic acid; for example, Kendall et al. (FE
Kendall et al., J.Biol.Chem., 118, 61,
1937), WAPierce et al., J.
Baact., 63, 301, 1952), Pine Crenan (AP
MacLennan, J. Gen. Microbiol., 14, 134-142,
1956; J.Gen.Microbiol., 15, 485-491, 1956),
B. Holmström et al.
Appl.Microbiol., 15, 1409-1413, 1967), JBWoolcock, J.Gen.Microbiol.
85, 372-375, 1974), E. Kjems et al.
al., Acta Path.Microbiol.Scand.Sect.B, 84,
162-164, 1976), T. Bergan et al.
al., Acta Path.Microbiol.Scand., 75, 97−
103, 1969) and JACifonelli,
Carbohyd. Res., 14, 272-276, 1970). These reports were not aimed at mass production of hyaluronic acid, but were cultured using 1-1.5% glucose as a carbon source, and the amount of hyaluronic acid produced was 0.5-0.6g/
The yield based on sugar was 6% or less. In the above report, Matsukrennan reported that culturing a type of Lancefield serogroup C bacterium of the genus Streptococcus under aerobic conditions may promote the production of hyaluronic acid. Among the above-mentioned microorganisms that produce hyaluronic acid, Lancefield serogroup A bacteria of the genus Streptococcus and Pasteurella are known as pathogenic bacteria to humans, and are actually unsuitable for mass culture. JP-A-58-56692 discloses a method for industrially culturing hyaluronic acid-producing bacteria of the genus Streptococcus and extracting and purifying hyaluronic acid from the culture solution. This method is a method to obtain a large amount of hyaluronic acid by culturing Lancefield serogroup A and C bacteria of the genus Streptococcus.
g/g of hyaluronic acid is obtained. In this case, the yield of hyaluronic acid based on sugar was 5%, and remained unchanged even when the amount of glucose added was changed from 1% to 8%. Therefore, this sugar yield (5%) is almost the same as the sugar yield of hyaluronic acid producing bacteria in previous reports. In addition, there is a method for obtaining hyaluronic acid using Streptococcus bacteria.
JP-A-60-500597, JP-A-60-133894, JP-A-61
-15698, but there are problems such as the hyaluronic acid obtained has a low molecular weight and the yield is low. Furthermore, it is known that the hyaluronic acid-producing bacterial strains in both cases produce streptolysin (soluble hemolysin) and exhibit β-hemolytic properties. When attempting to produce hyaluronic acid by culturing a large amount of such bacteria, there is a risk that the hemolysin may be mixed into the hyaluronic acid product, and it is not preferable to incorporate such hyaluronic acid into cosmetics or pharmaceuticals. In order to improve this drawback, a method for obtaining hyaluronic acid by culturing a hyaluronic acid-producing strain that lacks the ability to produce streptolysin through mutation treatment with a chemical mutagen was proposed in Japanese Patent Application Laid-Open No. 60-251898.
has been disclosed. This contains 6 glucose
It is stated that 3.6g/of hyaluronic acid was obtained by adding 3.6% of hyaluronic acid, and the yield of hyaluronic acid based on sugar was 6%, which is still low productivity from the viewpoint of sugar yield. It is something. Problems to be Solved by the Invention In view of the above problems, as a result of intensive research aimed at creating a microorganism that does not produce hemolysin (streptolysin) and has a high ability to produce hyaluronic acid, the present invention has been isolated from nature. By mutating a strain that no longer exhibits hemolytic properties obtained through mutation from Streptococcus zooepidemicus that has the ability to produce hyaluronic acid, the strain loses the ability to produce hyaluronidase and produces high-molecular-weight hyaluronic acid. We succeeded in obtaining a new strain with extremely high virulence. The present invention was completed based on this knowledge, and therefore provides a mutant strain of Streptococcus zooepidemicus that does not contain hemolysin and has the ability to efficiently produce high-molecular-weight hyaluronic acid. It is something to do. Means for Solving the Problems (1) Obtaining a Mutant Strain In order to achieve the object of the present invention, the present inventors obtained a new mutant strain by the following method. First, Streptococcus zooepidemicus belongs to Lancefield serogroup C, which has a strong ability to produce hyaluronidase (hyaluronic acid degrading enzyme) and produces hyaluronic acid from bovine nasal mucosa.
of Determinative Bacteriology, 8th edition, 1974). This strain is known as MacLennan (MacLennan, J.Gen.
Microbiol., 14, 134-142, 1956) pointed out that when hyaluronic acid is produced well under aerobic conditions and glucose is used as the carbon source,
By adding 4% glucose, 2 g/hyaluronic acid was produced (yield of hyaluronic acid based on sugar was 5%). The molecular weight of the hyaluronic acid obtained at this time was 300,000 to 600,000. This strain was treated with ultraviolet rays and chemical agents (N-methyl-N'-nitro-N-nitrosoguanidine (NTG), ethylmethane, sulfonic acid, etc.), the treated bacterial cells are mixed and spread on blood agar medium, and colonies that do not show hemolytic properties are collected.The bacterial cells are then mutated again, and then treated with hyaluronic acid. Apply nutrient agar containing nutrient agar to the ground,
A mutant strain of Streptococcus zooepidemicus was obtained by collecting colonies that did not degrade hyaluronic acid (see Example 1 below). (2) Mycological properties Mutant strain of Streptococcus zooepidemicus obtained in Example 1 (hereinafter referred to as the main strain)
Todd Huyt Broth
Hewitt broth) forms transparent colonies with extremely strong viscosity on the agar plate, and is non-hemolytic (β-
Hemolytic: negative), non-production of hyaluronidase,
This bacterium is a streptococcus belonging to Lancefield serogroup C, and its bacteriological properties are as follows. (a) Gram staining: positive (b) 10℃ growth: negative (c) 45℃ growth: negative (d) 0.1% methylene blue resistance: negative (e) 6.5% salt resistance: negative (f) 40 % Bile resistance: Negative (g) Bacitracin resistance: Positive (h) PH9.6 resistance: Negative (i) 60℃, 30 minutes resistance: Negative (j) Gelatin decomposition: Negative (k) Starch decomposition :Positive (l) Sodium hippuric acid degradability: Negative (m) Aesculin degradability: Weakly positive (n) Arginine degradability: Positive (o) Sugar fermentability: Glucose, galactose,
seuucrose, lactose, maltose,
Positive for sorbitol and salicin, negative for glycerin, mannitol, trehalose and arabinose. Based on the mycological properties of this bacterium, the present inventors identified this bacterium as Streptococcus zooepidemicus.
It was named YIT2030 and has been deposited with the Institute of Microbial Technology, Agency of Industrial Science and Technology as Microtechnology Research Article No. 1305. (3) Production of hyaluronic acid using this bacterium The medium for culturing this bacterium to produce hyaluronic acid is
It is preferable that it contains a carbon source, an organic and inorganic nitrogen source, an inorganic salt, and other organic micronutrients as necessary. As the carbon source, those containing sugar such as glucose, galactose, sucrose, lactose, fructose, maltose, sorbitol, and starch hydrolyzate are preferable, and organic acids, aliphatic alcohols, and the like may also be used. As the nitrogen source, common organic and inorganic materials may be used, and various meat extracts, amino acid mixtures, peptone, yeast extract, etc. are preferable. Furthermore, chlorides, sulfates, phosphates, nitrates, carbonates, vitamins, etc. of sodium, potassium, calcium, magnesium, iron, etc. may be added as necessary. Aerobic conditions are essential for culturing, and it is better to increase the stirring speed as the viscosity of the culture solution increases, but excessive stirring is not preferred. The culture temperature is generally 25-38°C, which is the temperature at which bacteria grow. Furthermore, during culturing, this bacterium produces lactic acid, which inhibits the growth of the bacterium and the production of hyaluronic acid. Therefore, an alkaline aqueous solution is added to neutralize the lactic acid to maintain a pH within the range of 6-8. It is necessary to adjust within The alkaline aqueous solution used at this time may be an aqueous solution of sodium hydroxide, potassium hydroxide, or aqueous ammonia. This bacterium contains high molecular weight hyaluronic acid (molecular weight 200
-3 million) at an extremely high yield and production rate, and particularly good results are obtained when glucose is used as the carbon source. Added amount of sugar 3
% or less, the sugar yield was 14-15%, and if the amount added was more than that, the sugar yield tended to decrease slightly. When the addition of sugar is 6% (see Reference Example 1), the viscosity of the culture solution becomes 8000 centipoise (cp) at 36°C, and the culture solution becomes almost non-fluid, reaching the limit of culture without any effect even if the stirring speed is increased. Summer. Table 1 shows the amount of hyaluronic acid produced when culturing was performed with varying amounts of glucose added in the medium. That is, as a result of purifying the produced hyaluronic acid using a conventional method, the sugar yield was 15 when 1% glucose was added.
%, 1.5g/, 11% sugar yield when adding 6%
6.7 g/hyaluronic acid was obtained.
【表】
次いでグルコース２％の組成の培地を使用
し、希釈率0.3（hr-1）で連続培養を行うと、操
作し易い低粘度で極めて安定に連続的に高収
率、高生産率でヒアルロン酸を生産することが
できた。ヒアルロン酸の対糖収率は15％、その
生産性は0.9ｇ／／hrであり、一日当たり
21.6ｇ／のヒアルロン酸を生産することがで
きた（参考例２参照）。
本菌は高分子物質として、ヒアルロン酸以外
の物質は培養液中に蓄積しないので、培養後、
培養液中に蓄積されたヒアルロン酸の分離、精
製は容易で、既に公知の多糖類の分離精製法を
用いればよい。
ヒアルロン酸の分離、精製法の一例を示す。
培養液を適当な粘度となるように（100センチ
ボアズ以下が好ましい）水で希釈し、トリクロ
ル酢酸にてPHを４以下にする。次いで遠心分離
あるいは膜濾過（ポアーサイズ 0.2μｍ以下）
によつて菌体を分離除去する。次ぎに溶液中に
溶解している低分子物質を、限外濾過、透析、
有機溶媒沈澱法又はイオン交換樹脂等による吸
着法などによつて除去した後、有機溶媒沈澱
法、凍結乾燥又は噴霧乾燥などの手段を用いて
ヒアルロン酸を得ることができる（参考例１参
照）。
このようにして上記培養液から抽出精製して得
たヒアルロン酸について、ヒアルロン酸標品
（Sigma社製）と対比しながら種々の検討を行つ
た結果、本品はヒアルロン酸であることを確認し
た。以下にその性質を示す。
(1) 酢酸セルロース膜を用いる電気泳動において
標品と同じ移動度を示す。
(2) 放線菌ヒアルロニダーゼ（天野製薬製）によ
つて分解を受け、その分解物をシリカゲル薄層
クロマトグラフイーにかけると、処理後の標品
分解物と同じ移動度で二つのスポツトが現れ
る。
(3) 化学組成を分析すると、Ｎ−アセチル−Ｄ−
グルコサミンとＤ−グルクロン酸がモル比１：
１で存在する。
(4) 比施光度は〔α〕20 D＝−69°である。
(5) 薄膜法による赤外吸収スペクトルは第３図の
通りで標品と同じ。
(6) 重水に溶解して測定した 13C−NMRスペク
トルは第４図の通りで標品と同じ。
(7) 分子量は粘度測定法（T.C.Laurent et al.，
Biochim.Biophys.Acta，42，476−485，1960）
による結果、200−300万であつた。
以下に実施例および参考例を示して本発明をさ
らに詳細に説明する。
実施例 １
牛鼻粘膜より採取した、β−溶血性を示し、ヒ
アルロニダーゼを生産し、かつヒアルロン酸を生
産するストレプトコツカス・ズーエピデミカスを
トツド・ヒユーイツト・ブロス培地（デイフコ
製）中、37℃で10時間培養し、対数増殖期の菌体
を遠心分離によつて集め、低温下遠心分離を繰り
返しつつ２回0.05Mトリスーマレイン酸緩衝液
（PH6.0）を用いて無菌的に洗浄した後、１×
108／mlの菌濃度となるように同緩衝液に懸濁し、
これにNTGを200μｇ／mlのなるよう添加し37℃
にて30分間振とうした。つづいて、低温下菌体を
0.05Mトリス−マレイン酸緩衝液（PH6.0）で２
回洗浄した後、トツド・ヒユーイツト・ブロス培
地に接種して37℃、18時間培養した。この培養液
を滅菌生理食塩水にて１×103／mlとなるよう希
釈し、その0.1mlを血液（ウサギ脱繊血）寒天
（20ml）に混釈してまき培養し、溶血性を示さな
い集落を採取した。この変異株の取得頻度は約４
×10-6であつた。次ぎにこの非容血性菌株を、上
と同様に、トツド・ヒユーイツト・ブロス培地に
37℃で培養し、対数増殖期の菌体を集め、0.05M
トリスーマレイン酸緩衝液（PH6.0）で洗浄後、
NTG200μｇ／mlを含む同緩衝液中で37℃、20分
間振とうした。つづいて、低温下菌体を同緩衝液
で洗浄後、限外濾過処理培地（トツド・ヒユーイ
ツト・ブロス培地からアミコン製限外濾過膜YM
−10にて高分子画分を除去したもの）に接種して
37℃、18時間培養した。この培養液を滅菌生理食
塩水にて１−５×102／mlとなるよう希釈し、そ
の0.1mlをヒアルロン酸ソーダ0.1％を含む上記限
外濾過処理培地寒天（高純度寒天）上に塗布して
37℃、20−40時間モイスチヤーチヤンバー中で培
養し、増殖した集落中の菌をレプリカ法にて採取
しておき、寒天上に10％セチルピリジニユームク
ロライド水溶液を噴霧して、約50万の菌株の中か
ら集落周囲が濁る集落を形成するヒアルロニダー
ゼ非生産変異株ストレプトコツカス・ズーエピデ
ミカスYIT2030を取得した。
なお上記ヒアルロニダーゼ生産・非生産菌の識
別法はエリカバルケ等の方法（Erika Balke et
al.，Zbl.Bakt.Hyg.A，259，194−200，1985）
を改変して行なつた。
参考例 １
バツチ培養
グルコース６％、ポリペプトン（大五栄養化学
製）1.5％、パン酵母エキス（オリエンタル酵母
工業製）0.5％、燐酸第二カリ0.2％、硫酸マグネ
シウム７水塩0.1％、塩化カルシウム0.005％アデ
カノールLG−109（消泡剤 旭電化工業製）0.001
％の組成の培地（PH7.0）を10のジヤーフアー
メンターに５入れ、滅菌後、前培養したストレ
プトコツカス・ズーエピデミカスYIT2030を１
％接種し、6N−水酸化ナトリウム水溶液にて培
養PHを７に連続的に調節しながら37℃で42時間通
気撹拌培養した。
グルコースは別滅菌して、培養開始時に一度に
添加した。この時の培養経過を第１図に示す。培
養の経過と共に、ヒアルロン酸が蓄積し培養29時
間で、培養液の粘性は8000センチポアズ近くに達
しほとんど流動性がなくなり、培養42時間後、培
養液中のグルコースが零に達した時点で培養を終
了した。
収穫した培養液は流動性がないため、これを水
にて粘性が100センチポアズ以下となるように希
釈した。次ぎにこの溶液をトリクロル酢酸にてPH
を４以下にして、中空糸マイクロフイルターモジ
ユール（PW−103旭化成製）に通し、菌体およ
び不溶成分を除去し、更に中空糸限外濾過膜
（HIP30−43 アミコン製）に、濾過内液に水を
注加しながら通し溶液中の低分子物質を除去し
た。そしてこの溶液を凍結乾燥法によつて乾燥し
ヒアルロン酸を培養液１当たり6.7ｇ得た。
参考例 ２
連続培養
グルコース濃度を2.5％にした以外は参考例１
と同一の組成の培地を10のジヤーフアーメンタ
ーに５入れ、滅菌後（グルコースは別滅菌）、
前培養したストレプトコツカス・ズーエピデミカ
スYIT2030を１％接種し、6N−水酸化ナトリウ
ム水溶液にて培養PHを７に調節しながら、37℃で
15時間通気撹拌培養した。その後グルコース濃度
を２％にした以外は参考例１と同一の組成の培地
を、希釈率0.3（hr-1）で連続的に注加しながら、
37℃、PH７で通気撹拌連続培養を１週間おこなつ
た。
この時の培養経過を第２図に示す。培養槽外に
流出した培養液を一定時間ごとに集め、参考例１
と同様にしてヒアルロン酸を抽出精製した。この
結果、ヒアルロン酸の対糖収率は15％、その生産
性は0.9ｇ／／hrであり一日当たり21.6ｇ／
のヒアルロン酸を得ることができた。
発明の効果
本発明によるストレプトコツカス・ズーエピデ
ミカス変異株を培養することによつて、今まで報
告されたストレプトコツカス属細菌を使つたヒア
ルロン酸の製造法における収率、収量をはるかに
上回る、ストレプトリジンの混入の全く無いかつ
高分子量のヒアルロン酸の高収率、高生産率で安
価に得ることができる。この様にして製造したヒ
アルロン酸は化粧品、医薬品原料として最適なも
のである。[Table] Next, by using a medium with a composition of 2% glucose and performing continuous culture at a dilution rate of 0.3 (hr -1 ), it is possible to achieve high yields and production rates continuously with a low viscosity that is easy to operate, and extremely stably. We were able to produce hyaluronic acid. The yield of hyaluronic acid based on sugar is 15%, and its productivity is 0.9g//hr, which is
It was possible to produce 21.6 g/hyaluronic acid (see Reference Example 2). As this bacterium is a polymeric substance, substances other than hyaluronic acid do not accumulate in the culture solution, so after culturing,
Separation and purification of hyaluronic acid accumulated in the culture solution is easy, and a known separation and purification method for polysaccharides may be used. An example of a method for separating and purifying hyaluronic acid is shown.
Dilute the culture solution with water to an appropriate viscosity (preferably 100 centibore or less), and adjust the pH to 4 or less with trichloroacetic acid. Then centrifugation or membrane filtration (pore size 0.2μm or less)
The bacterial cells are separated and removed. Next, the low molecular weight substances dissolved in the solution are removed by ultrafiltration, dialysis,
After removal by organic solvent precipitation, adsorption using an ion exchange resin, etc., hyaluronic acid can be obtained using methods such as organic solvent precipitation, freeze drying, or spray drying (see Reference Example 1). As a result of performing various studies on the hyaluronic acid obtained by extraction and purification from the above culture solution and comparing it with a hyaluronic acid standard (manufactured by Sigma), it was confirmed that this product is hyaluronic acid. . Its properties are shown below. (1) Shows the same mobility as the standard sample in electrophoresis using cellulose acetate membrane. (2) When it is degraded by actinomycete hyaluronidase (manufactured by Amano Pharmaceutical) and the degraded product is subjected to silica gel thin layer chromatography, two spots appear with the same mobility as the standard degraded product after treatment. (3) Analysis of the chemical composition reveals that N-acetyl-D-
Glucosamine and D-glucuronic acid in a molar ratio of 1:
1 exists. (4) The specific illuminance is [α] 20 D = -69°. (5) The infrared absorption spectrum measured by the thin film method is shown in Figure 3 and is the same as the standard product. (6) The 13 C-NMR spectrum measured after dissolving in heavy water is shown in Figure 4 and is the same as the standard. (7) Molecular weight is determined by viscosity measurement method (TCLaurent et al.,
Biochim. Biophys. Acta, 42, 476-485, 1960)
The result was 2-3 million. The present invention will be explained in more detail by showing Examples and Reference Examples below. Example 1 Streptococcus zooepidemicus, which exhibits β-hemolytic properties, produces hyaluronidase, and produces hyaluronic acid, collected from bovine nasal mucosa, was incubated at 37°C for 10 hours in Todd Huitt broth medium (manufactured by Difco). After culturing, the cells in the logarithmic growth phase were collected by centrifugation, and washed twice with 0.05M tris-maleate buffer (PH6.0) while repeating centrifugation at low temperature. ×
Suspend in the same buffer solution to a bacterial concentration of 10 8 /ml,
Add NTG to this at 200μg/ml and hold at 37°C.
The mixture was shaken for 30 minutes. Next, the bacterial cells at low temperature were
2 with 0.05M Tris-maleate buffer (PH6.0)
After washing twice, the cells were inoculated into Todd Huyt's broth medium and cultured at 37°C for 18 hours. This culture solution was diluted with sterile physiological saline to a concentration of 1 x 10 3 /ml, and 0.1 ml of the solution was poured onto blood (definated rabbit blood) agar (20 ml) and cultured. We sampled villages where there were no. The acquisition frequency of this mutant strain is approximately 4
It was ×10 -6 . Next, this non-hematopoietic strain was transferred to Totsudo Huyt Broth medium as above.
Culture at 37℃, collect cells in logarithmic growth phase, and add 0.05M
After washing with tris-maleate buffer (PH6.0),
The mixture was shaken at 37°C for 20 minutes in the same buffer containing 200 μg/ml of NTG. Next, after washing the microbial cells at low temperature with the same buffer solution, the ultrafiltration medium (Totdo Huyts Broth medium to ultrafiltration membrane YM made by Amicon) was used.
-10 to remove the polymer fraction).
Culture was carried out at 37°C for 18 hours. This culture solution was diluted with sterile physiological saline to a concentration of 1-5 x 10 2 /ml, and 0.1 ml of the solution was applied onto the ultrafiltrated medium agar (high purity agar) containing 0.1% sodium hyaluronate. do
Culture the bacteria in a moisture chamber at 37℃ for 20-40 hours, collect the grown bacteria using the replica method, and spray a 10% cetylpyridinium chloride aqueous solution onto the agar. We obtained a non-hyaluronidase-producing mutant strain Streptococcus zooepidemicus YIT2030, which forms colonies with cloudy surroundings, from among ten thousand bacterial strains. The above method for identifying hyaluronidase-producing and non-producing bacteria is based on the method of Erika Balke et al.
al., Zbl.Bakt.Hyg.A, 259, 194-200, 1985)
I did this by modifying. Reference example 1 Batch culture Glucose 6%, Polypeptone (Daigo Nutrient Chemical) 1.5%, Baker's yeast extract (Oriental Yeast Co., Ltd.) 0.5%, Potassium phosphate 0.2%, Magnesium sulfate heptahydrate 0.1%, Calcium chloride 0.005 % Adekanol LG-109 (antifoaming agent manufactured by Asahi Denka Kogyo) 0.001
% composition of medium (PH7.0) in 10 jars, and after sterilization, 1 portion of pre-cultured Streptococcus zooepidemicus YIT2030 was added.
% inoculation, and cultured with aeration and stirring at 37° C. for 42 hours while continuously controlling the culture pH to 7 with a 6N-sodium hydroxide aqueous solution. Glucose was sterilized separately and added all at once at the start of culture. The progress of the culture at this time is shown in FIG. As the culture progresses, hyaluronic acid accumulates, and after 29 hours of culture, the viscosity of the culture solution reaches nearly 8,000 centipoise, and it has almost no fluidity. After 42 hours of culture, when the glucose in the culture solution reaches zero, the culture is stopped. finished. Since the harvested culture solution had no fluidity, it was diluted with water to a viscosity of 100 centipoise or less. Next, PH this solution with trichloroacetic acid.
4 or less, pass through a hollow fiber microfilter module (PW-103 manufactured by Asahi Kasei) to remove bacterial cells and insoluble components, and then pass the filtrated liquid through a hollow fiber ultrafiltration membrane (HIP30-43 manufactured by Amicon). The low molecular weight substances in the solution were removed by adding water to the solution. This solution was then dried by freeze-drying to obtain 6.7 g of hyaluronic acid per culture solution. Reference example 2 Continuous culture Reference example 1 except that the glucose concentration was 2.5%
Pour 5 media of the same composition into 10 jar fermenters, and after sterilization (glucose is sterilized separately),
Inoculate 1% of the pre-cultured Streptococcus zooepidemicus YIT2030 and grow at 37°C while adjusting the culture pH to 7 with a 6N-sodium hydroxide aqueous solution.
Culture was carried out with aeration and stirring for 15 hours. Thereafter, a medium having the same composition as in Reference Example 1 except that the glucose concentration was changed to 2% was continuously added at a dilution rate of 0.3 (hr -1 ).
Continuous culture with aeration and stirring was performed at 37°C and pH 7 for one week. The progress of the culture at this time is shown in FIG. Collect the culture solution that has flowed out of the culture tank at regular intervals, and use it as reference example 1.
Hyaluronic acid was extracted and purified in the same manner. As a result, the yield of hyaluronic acid based on sugar was 15%, and its productivity was 0.9 g//hr, which was 21.6 g/hr/day.
of hyaluronic acid was obtained. Effects of the Invention By culturing the Streptococcus zooepidemicus mutant strain of the present invention, the yield of Streptococcus zooepidemicus far exceeds that of the previously reported method for producing hyaluronic acid using Streptococcus bacteria. High molecular weight hyaluronic acid without any lysine contamination can be obtained at high yield and production rate at low cost. Hyaluronic acid produced in this manner is optimal as a raw material for cosmetics and pharmaceuticals.
第１図は本発明の参考例１におけるヒアルロン
酸製造の培養経過を示す図であり、第２図は参考
例２における培養経過を示す図であり、第３図お
よび第４図はそれぞれ参考例１で得られたヒアル
ロン酸の赤外吸収スペクトルおよびNMRスペク
トルを示す図である。
FIG. 1 is a diagram showing the culture progress for producing hyaluronic acid in Reference Example 1 of the present invention, FIG. 2 is a diagram showing the culture progress in Reference Example 2, and FIGS. 3 and 4 are diagrams showing the culture progress in Reference Example 2. 1 is a diagram showing an infrared absorption spectrum and an NMR spectrum of hyaluronic acid obtained in Example 1.
Claims (1)
ゼ非生産性でかつ非溶血性を示すストレプトコツ
カス・ズーエピデミカス（Streptococcus
zooepidemicus）。 ２ 下記の菌学的性質を示す特許請求の範囲第１
項に記載のストレプトコツカス・ズーエピデミカ
ス (a) グラム染色性：陽性 (b) 10℃増殖性：陰性 (c) 45℃増殖性：陰性 (d) 0.1％メチレンブルー抵抗性：陰性 (e) 6.5％食塩抵抗性：陰性 (f) 40％胆汁抵抗性：陰性 (g) バシトラシン抵抗性：陽性 (h) PH9.6抵抗性：陰性 (i) 60℃，30分抵抗性：陰性 (j) ゼラチン分解性：陰性 (k) 澱粉分解性：陽性 (l) 馬尿酸ソーダ分解性：陰性 (m) エスクリン分解性：弱陽性 (n) アルギニン分解性：陽性 (o) 糖醗酵性：グルコース、ガラクトース、シ
ユークロース、ラクトース、マルトース、ソル
ビトールおよびサリシンは陽性、 グリセリン、マンニトール、トレハロースお
よびアラビノースは陰性。 ３ ストレプトコツカス・ズーエピデミカスがス
トレプトコツカス・ズーエピデミカスYIT2030
（微工研条寄第1305号）である特許請求の範囲第
１項または第２項に記載のストレプトコツカス・
ズーエピデミカス。[Scope of Claims] 1. Streptococcus zooepidemicus that has hyaluronic acid producing ability, non-hyaluronidase production, and non-hemolysis.
zooepidemicus). 2 Claim 1 showing the following mycological properties
Streptococcus zooepidemicus (a) Gram staining: positive (b) 10℃ growth: negative (c) 45℃ growth: negative (d) 0.1% methylene blue resistance: negative (e) 6.5% Salt resistance: Negative (f) 40% bile resistance: Negative (g) Bacitracin resistance: Positive (h) PH9.6 resistance: Negative (i) 60℃, 30 minutes resistance: Negative (j) Gelatin degradation Degradability of starch: negative (k) Degradability of starch: positive (l) Degradability of sodium hippurate: negative (m) Degradability of esculin: weakly positive (n) Degradability of arginine: positive (o) Sugar fermentability: glucose, galactose, sucrose , lactose, maltose, sorbitol, and salicin; negative for glycerin, mannitol, trehalose, and arabinose. 3 Streptococcus zooepidemicus is Streptococcus zooepidemicus YIT2030
(Feikoken Jokyo No. 1305).
Zooepidemicus.
Priority Applications (7)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| JP61099447A JPS62257382A (en) | 1986-05-01 | 1986-05-01 | Novel streptococcus zooepidemicus |
| US07/038,907 US5023175A (en) | 1986-05-01 | 1987-04-16 | Novel production process of hyaluronic acid and bacterium strain therefor |
| CA000535098A CA1314508C (en) | 1986-05-01 | 1987-04-21 | Production process of hyaluronic acid and bacterium strain therefor as well as cosmetic composition containing hyaluronic acid |
| AU71960/87A AU598809B2 (en) | 1986-05-01 | 1987-04-24 | Novel production process of hyaluronic acid and bacterium strain therefor as well as cosmetic composition containing hyaluronic acid |
| KR1019870003999A KR960005736B1 (en) | 1986-05-01 | 1987-04-25 | Novel production process of hyaluronic acid |
| DE3750733T DE3750733T2 (en) | 1986-05-01 | 1987-04-29 | Process for the production of hyaluronic acid, bacterial strains required therefor and cosmetic composition which contains hyaluronic acid. |
| EP87106247A EP0244757B1 (en) | 1986-05-01 | 1987-04-29 | Novel production process of hyaluronic acid and bacterium strain therefor as well as cosmetic composition containing hyaluronic acid |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| JP61099447A JPS62257382A (en) | 1986-05-01 | 1986-05-01 | Novel streptococcus zooepidemicus |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| JPS62257382A JPS62257382A (en) | 1987-11-09 |
| JPH044868B2 true JPH044868B2 (en) | 1992-01-29 |
Family
ID=14247615
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| JP61099447A Granted JPS62257382A (en) | 1986-05-01 | 1986-05-01 | Novel streptococcus zooepidemicus |
Country Status (1)
| Country | Link |
|---|---|
| JP (1) | JPS62257382A (en) |
Families Citing this family (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| JPH0713099B2 (en) | 1988-12-14 | 1995-02-15 | 工業技術院長 | Photosensitive polyvinyl alcohol derivative |
| US7575914B2 (en) | 2002-08-19 | 2009-08-18 | Kolon Life Science, Inc. | Microorganism producing hyaluronic acid and purification method of hyaluronic acid |
| JP4920552B2 (en) * | 2007-11-07 | 2012-04-18 | 株式会社ヤクルト本社 | Method for producing hyaluronic acid |
Citations (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| JPS60251898A (en) * | 1984-05-25 | 1985-12-12 | Shiseido Co Ltd | Preparation of hyaluronic acid by fermentation method |
-
1986
- 1986-05-01 JP JP61099447A patent/JPS62257382A/en active Granted
Patent Citations (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| JPS60251898A (en) * | 1984-05-25 | 1985-12-12 | Shiseido Co Ltd | Preparation of hyaluronic acid by fermentation method |
Also Published As
| Publication number | Publication date |
|---|---|
| JPS62257382A (en) | 1987-11-09 |
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Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| LAPS | Cancellation because of no payment of annual fees |