JPH046356B2 - - Google Patents
Info
- Publication number
- JPH046356B2 JPH046356B2 JP61099446A JP9944686A JPH046356B2 JP H046356 B2 JPH046356 B2 JP H046356B2 JP 61099446 A JP61099446 A JP 61099446A JP 9944686 A JP9944686 A JP 9944686A JP H046356 B2 JPH046356 B2 JP H046356B2
- Authority
- JP
- Japan
- Prior art keywords
- hyaluronic acid
- culture
- streptococcus
- glucose
- producing
- 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 76
- 229920002674 hyaluronan Polymers 0.000 claims description 76
- 229960003160 hyaluronic acid Drugs 0.000 claims description 76
- 241000894006 Bacteria Species 0.000 claims description 27
- 238000004519 manufacturing process Methods 0.000 claims description 19
- 241000194017 Streptococcus Species 0.000 claims description 15
- 238000012258 culturing Methods 0.000 claims description 14
- 241000120569 Streptococcus equi subsp. zooepidemicus Species 0.000 claims description 13
- 238000000034 method Methods 0.000 claims description 13
- 108010003272 Hyaluronate lyase Proteins 0.000 claims description 10
- 102000001974 Hyaluronidases Human genes 0.000 claims description 10
- 229960002773 hyaluronidase Drugs 0.000 claims description 10
- 230000002949 hemolytic effect Effects 0.000 claims description 8
- 239000000243 solution Substances 0.000 description 22
- 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 17
- 239000008103 glucose Substances 0.000 description 17
- 239000002609 medium Substances 0.000 description 15
- 230000001580 bacterial effect Effects 0.000 description 8
- 239000000203 mixture Substances 0.000 description 8
- 239000000126 substance 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
- 229920001817 Agar Polymers 0.000 description 5
- OKTJSMMVPCPJKN-UHFFFAOYSA-N Carbon Chemical compound [C] OKTJSMMVPCPJKN-UHFFFAOYSA-N 0.000 description 5
- 239000008272 agar Substances 0.000 description 5
- 229910052799 carbon Inorganic materials 0.000 description 5
- 238000011282 treatment Methods 0.000 description 5
- IJGRMHOSHXDMSA-UHFFFAOYSA-N Atomic nitrogen Chemical compound N#N IJGRMHOSHXDMSA-UHFFFAOYSA-N 0.000 description 4
- 108010006464 Hemolysin Proteins Proteins 0.000 description 4
- 239000000872 buffer Substances 0.000 description 4
- 238000010586 diagram Methods 0.000 description 4
- 239000003228 hemolysin Substances 0.000 description 4
- JVTAAEKCZFNVCJ-UHFFFAOYSA-N lactic acid Chemical compound CC(O)C(O)=O JVTAAEKCZFNVCJ-UHFFFAOYSA-N 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
- 230000012010 growth Effects 0.000 description 3
- 239000012528 membrane Substances 0.000 description 3
- 230000035772 mutation Effects 0.000 description 3
- 210000001519 tissue Anatomy 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
- 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
- 241000283690 Bos taurus Species 0.000 description 2
- 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 2
- 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 2
- PEDCQBHIVMGVHV-UHFFFAOYSA-N Glycerine Chemical compound OCC(O)CO PEDCQBHIVMGVHV-UHFFFAOYSA-N 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
- 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
- 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
- 229920002472 Starch Polymers 0.000 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 description 2
- 229930006000 Sucrose Natural products 0.000 description 2
- 238000000862 absorption spectrum Methods 0.000 description 2
- 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 2
- 230000003698 anagen phase Effects 0.000 description 2
- 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 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
- 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
- -1 etc.) Substances 0.000 description 2
- 238000004108 freeze drying Methods 0.000 description 2
- 229930182830 galactose Natural products 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
- 239000008101 lactose Substances 0.000 description 2
- 239000007788 liquid Substances 0.000 description 2
- 230000000813 microbial effect Effects 0.000 description 2
- 244000005700 microbiome Species 0.000 description 2
- 210000002850 nasal mucosa Anatomy 0.000 description 2
- 229910052757 nitrogen Inorganic materials 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
- 238000000746 purification Methods 0.000 description 2
- 239000011734 sodium Substances 0.000 description 2
- 229910052708 sodium Inorganic materials 0.000 description 2
- 239000000600 sorbitol Substances 0.000 description 2
- 239000008107 starch Substances 0.000 description 2
- 235000019698 starch Nutrition 0.000 description 2
- 230000001954 sterilising effect Effects 0.000 description 2
- 238000004659 sterilization and disinfection Methods 0.000 description 2
- 239000005720 sucrose Substances 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
- HDTRYLNUVZCQOY-UHFFFAOYSA-N α-D-glucopyranosyl-α-D-glucopyranoside Natural products OC1C(O)C(O)C(CO)OC1OC1C(O)C(O)C(O)C(CO)O1 HDTRYLNUVZCQOY-UHFFFAOYSA-N 0.000 description 1
- 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
- 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 description 1
- VHUUQVKOLVNVRT-UHFFFAOYSA-N Ammonium hydroxide Chemical compound [NH4+].[OH-] VHUUQVKOLVNVRT-UHFFFAOYSA-N 0.000 description 1
- 239000004475 Arginine Substances 0.000 description 1
- 108010001478 Bacitracin Proteins 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
- 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 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
- 108010010803 Gelatin Proteins 0.000 description 1
- 238000003794 Gram staining Methods 0.000 description 1
- 206010018910 Haemolysis Diseases 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
- 229930195725 Mannitol Natural products 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
- 240000004808 Saccharomyces cerevisiae Species 0.000 description 1
- 235000014680 Saccharomyces cerevisiae Nutrition 0.000 description 1
- 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 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
- HDTRYLNUVZCQOY-WSWWMNSNSA-N Trehalose Natural products O[C@@H]1[C@@H](O)[C@@H](O)[C@@H](CO)O[C@@H]1O[C@@H]1[C@H](O)[C@@H](O)[C@@H](O)[C@@H](CO)O1 HDTRYLNUVZCQOY-WSWWMNSNSA-N 0.000 description 1
- XXFCIFBVVVYKSU-UHFFFAOYSA-N [S].CCC Chemical compound [S].CCC XXFCIFBVVVYKSU-UHFFFAOYSA-N 0.000 description 1
- 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 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
- NGFMICBWJRZIBI-UHFFFAOYSA-N alpha-salicin Natural products OC1C(O)C(O)C(CO)OC1OC1=CC=CC=C1CO NGFMICBWJRZIBI-UHFFFAOYSA-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
- PYMYPHUHKUWMLA-WDCZJNDASA-N arabinose Chemical compound OC[C@@H](O)[C@@H](O)[C@H](O)C=O PYMYPHUHKUWMLA-WDCZJNDASA-N 0.000 description 1
- PYMYPHUHKUWMLA-UHFFFAOYSA-N arabinose Natural products OCC(O)C(O)C(O)C=O PYMYPHUHKUWMLA-UHFFFAOYSA-N 0.000 description 1
- ODKSFYDXXFIFQN-UHFFFAOYSA-N arginine Natural products OC(=O)C(N)CCCNC(N)=N ODKSFYDXXFIFQN-UHFFFAOYSA-N 0.000 description 1
- 206010003246 arthritis Diseases 0.000 description 1
- 229960003071 bacitracin Drugs 0.000 description 1
- 229930184125 bacitracin Natural products 0.000 description 1
- 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 description 1
- 244000052616 bacterial pathogen Species 0.000 description 1
- 230000000721 bacterilogical effect Effects 0.000 description 1
- SRBFZHDQGSBBOR-UHFFFAOYSA-N beta-D-Pyranose-Lyxose Natural products OC1COC(O)C(O)C1O SRBFZHDQGSBBOR-UHFFFAOYSA-N 0.000 description 1
- 210000000941 bile Anatomy 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
- 239000003795 chemical substances by application 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
- 238000001962 electrophoresis Methods 0.000 description 1
- 229940088598 enzyme Drugs 0.000 description 1
- 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 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
- 239000008273 gelatin Substances 0.000 description 1
- 229920000159 gelatin Polymers 0.000 description 1
- 235000019322 gelatine Nutrition 0.000 description 1
- 235000011852 gelatine desserts Nutrition 0.000 description 1
- 150000004676 glycans Chemical class 0.000 description 1
- 235000011187 glycerol Nutrition 0.000 description 1
- 239000001963 growth medium Substances 0.000 description 1
- 230000008588 hemolysis Effects 0.000 description 1
- 238000011534 incubation Methods 0.000 description 1
- 208000015181 infectious disease Diseases 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
- 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
- 239000000594 mannitol Substances 0.000 description 1
- 235000010355 mannitol Nutrition 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
- 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 description 1
- 229960000907 methylthioninium chloride Drugs 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
- 239000006916 nutrient agar Substances 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
- 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
- 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 description 1
- 229940120668 salicin Drugs 0.000 description 1
- 150000003839 salts Chemical class 0.000 description 1
- 238000000926 separation method Methods 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
- 241001446247 uncultured actinomycete Species 0.000 description 1
- 235000013343 vitamin Nutrition 0.000 description 1
- 239000011782 vitamin Substances 0.000 description 1
- 229940088594 vitamin Drugs 0.000 description 1
- 229930003231 vitamin Natural products 0.000 description 1
- 239000003357 wound healing promoting agent Substances 0.000 description 1
Landscapes
- Preparation Of Compounds By Using Micro-Organisms (AREA)
Description
産業上の利用分野
本発明はヒアルロン酸の製造方法に関する。よ
り詳細に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ストレプ
トコツカス属に属する細菌を培地に培養して、溶
血素を含まずかつ高分子量のヒアルロン酸を効率
よく製造する方法に関する。
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〓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、創傷治瘉剤等)、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.
Bact.、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
てグルコースを培地に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、化学異変剤による変
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
の観点からは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)を含まずかつ高分子量のヒアルロン酸を大量
に効率よく製造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属に属する細菌から変
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
生産能が極めて高い菌株を得、該菌が上記目的に
叶うものであることを見出した。
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
性を示すストレプトコツカス属に属する細菌を培
地に培養して、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製造する方法を提供するも
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
ず牛鼻粘膜よりヒルアルロニダーゼ(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、蛋白質核酸酵素
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培地
に混釈してまき、溶血性を示さない集落を採取
し、次にこの菌株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培地上に塗布し、
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(後記参考例1参照)。
(2) 菌学的性質
後記参考例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)寒天培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・スーエピデミカス
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
本菌を培養と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
ルコース、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(実施例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 method for producing hyaluronic acid. More specifically, bacteria belonging to the genus Streptococcus, which have the ability to produce hyaluronic acid but are non-hyaluronidase-producing and non-hemolytic, are cultured in a medium to efficiently produce high-molecular-weight hyaluronic acid that does not contain hemolysin. Concerning how to manufacture well. Conventional technology Today, hyaluronic acid is recognized to exist in all parts of the connective tissue of animals, and is industrially obtained from living tissues such as chicken crests and velvets by extraction methods. Its functions are to retain water between cells, to form a jelly-like matrix within tissues to retain cells, to control the movement of substances between cells, and to prevent external physical shocks and infections such as bacteria. This is mentioned. Utilizing these functions, hyaluronic acid is used in pharmaceuticals (arthritis treatments, eye drops, wound healing agents, etc.), cosmetics, and the like. However, the production of hyaluronic acid by extraction from living tissue 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.
Bact., 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 Lansfield 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 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 mutating agent was proposed in JP-A-60-251898.
has been disclosed. This contains 6 glucose
It is stated that 3.6 g/g of hyaluronic acid was obtained by adding 3.6% of hyaluronic acid, and the yield of hyaluronic acid to sugar was 6%. It is low. Problems to be Solved by the Invention In view of the above problems, as a result of intensive research with the aim of efficiently producing large amounts of high molecular weight hyaluronic acid that does not contain hemolysin (streptolysin), we have isolated it from nature. By mutating a strain that no longer exhibits hemolytic properties obtained through mutation treatment from a bacterium belonging to the genus Streptococcus that has the ability to produce hyaluronic acid, the strain loses the ability to produce hyaluronidase and produces high-molecular-weight hyaluronic acid. We obtained a bacterial strain with extremely high production ability, and found that this strain satisfies the above objectives. The present invention has been completed based on this knowledge, and therefore, the present invention uses bacteria belonging to the genus Streptococcus that have hyaluronic acid-producing ability, non-hyaluronidase production, and non-hemolytic properties in a culture medium. The present invention provides a method for efficiently producing hemolysin-free, high-molecular-weight hyaluronic acid by culturing. 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 the bovine nasal mucosa (the identification of this bacterium was carried out by the Verges Manual). 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 sulfur, The treated bacterial cells were plated on a blood agar medium, a colony that did not show hemolysis was collected, and this strain was then subjected to mutation treatment again to produce cells containing hyaluronic acid. Spread on nutrient agar medium,
A mutant strain of Streptococcus zooepidemicus was obtained by collecting a colony that did not degrade hyaluronic acid (see Reference Example 1 below). (2) Mycological properties Mutant strain of Streptococcus zooepidemicus obtained in Reference Example 1 below (hereinafter referred to as the main strain)
Todd Huyt Broth
It forms transparent colonies with extremely strong viscosity on agar medium (Hewitt broth), 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,
Positive for sucrose, lactose, maltose, 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 suoepidemicus.
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 producing hyaluronic acid by culturing this bacterium preferably contains a carbon source, an organic/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 preferred, and organic acids, aliphatic alcohols, and the like may also be used. As a nitrogen source, common organic and inorganic materials may be used, such as various meat extracts, amino acid mixtures,
Peptone, yeast extract, etc. are preferred. Furthermore, chlorides, sulfates, phosphates, nitrates of sodium, potassium, calcium, magnesium, iron, etc.
Carbonates, vitamins, 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, and the pH is within the range of 6-8. It is necessary to adjust within the range. 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 sugar addition was 6% (see Example 1), the viscosity of the culture solution became 8000 centipoise (cP) at 36°C, and the culture solution became almost non-fluid, reaching the limit of culture without any effect even if the stirring speed was 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.
% is 1.5g/, and when 6% is added, the sugar yield is 11%.
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(実施例2参照)。
本菌は高分子物質として、ヒアルロン酸以外の
物質を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センチポア
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(実施例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。
以下に参考例および実施例を示して本発明をさ
らに詳細に説明する。
参考例 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−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となるよう添加し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後、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。次ぎにこの非溶血性菌株を、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−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
ゼ非生産性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)
を改変して行つた。
実施例 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た培養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
(H1P30−43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。
実施例 2
連続培養
グルコース濃度を2.5%にした以外は実施例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%にした以外は実施例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一週間おこなつ
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、実施例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を高収率、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, a medium with a composition of 2% glucose was used,
When continuous culture was carried out at a dilution rate of 0.3 (hr -1 ), hyaluronic acid could be produced continuously and extremely stably at a high yield and production rate with a low viscosity that was easy to operate. The yield of hyaluronic acid based on sugar is 15%, and its productivity is 0.9g/
/hr, and 21.6 g/hr of hyaluronic acid could be produced per day (see Example 2). Since this bacterium is a polymeric substance and does not accumulate substances other than hyaluronic acid in the culture solution, it is easy to separate and purify the hyaluronic acid accumulated in the culture solution after culturing, and it is already known to separate and purify polysaccharides. Just use the law. 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 centipoise or less), and adjust the pH to 4 or less with trichloroacetic acid. Next, the bacterial cells are separated and removed by centrifugation or membrane filtration (pore size 0.2 μm or less). Next, the low molecular weight substances dissolved in the solution are removed by ultrafiltration, dialysis, organic solvent precipitation, or adsorption using an ion exchange resin, followed by organic solvent precipitation, freeze drying, or spray drying. Hyaluronic acid can be obtained using the following methods (see 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 luminosity 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 further detail with reference to Reference Examples and Examples below. Reference 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 Huyt Broth Medium (manufactured by Difco). After culturing, the cells in the logarithmic growth phase were collected by centrifugation, and washed twice aseptically 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 Todo 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 this was poured onto blood (rabbit defibrinated blood) agar (20 ml) and cultured, after which it showed hemolytic properties. We sampled villages where there were no. The acquisition frequency of this mutant strain is approximately 4
It was ×10 -6 . This non-hemolytic strain was then added to Todd Huyt's 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 Huyt Broth medium to ultrafiltration membrane YM made by Amicon) was used.
-10 to remove the high molecular fraction).
Cultured 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 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 modified it. 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 %, ADEKA NOL LG-109 (antifoaming agent manufactured by Asahi Denka Kogyo)
Streptococcus zooepidemicus YIT2030 was pre-cultured after sterilization by placing 0.001% composition medium (PH7.0) in 10 jar fermenters.
was inoculated at 1%, 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. Culture in which hyaluronic acid accumulated over the course of culture29
After 42 hours of incubation, the viscosity of the culture solution reached nearly 8000 centipoise and became almost non-liquid.
The culture was terminated when the glucose in the culture solution reached zero. 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 (H1P30-43 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. Example 2 Continuous culture 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 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 carried out for one week at 37°C and pH 7. The progress of the culture at this time is shown in FIG. The culture solution flowing out of the culture tank was collected at regular intervals, and the culture solution was collected in 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%, the productivity was 0.9 g/hr, and 21.6 g/hr of hyaluronic acid could be 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 completely free of 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図は本発明の実施例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図は実施
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図はそれぞれ実施例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 of hyaluronic acid production in Example 1 of the present invention, FIG. 2 is a diagram showing the culture progress in Example 2, and FIGS. 3 and 4 are diagrams showing the culture progress in 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
カス属に属する細菌を培地に培養し、培養物から
ヒアルロン酸を採取することを特徴とするヒアル
ロン酸の製造方法。 2 ストレプトコツカス属に属する細菌がストレ
プトコツカス・ズーエピデミカスである特許請求
の範囲第1項記載のヒアルロン酸の製造方法。 3 ストレプトコツカス属に属する細菌がストレ
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項も
しくは第2項に記載のヒアルロン酸の製造方法。[Scope of Claims] 1. A method comprising culturing in a medium bacteria belonging to the genus Streptococcus, which has the ability to produce hyaluronic acid, does not produce hyaluronidase, and is non-hemolytic, and collects hyaluronic acid from the culture. A method for producing hyaluronic acid. 2. The method for producing hyaluronic acid according to claim 1, wherein the bacterium belonging to the genus Streptococcus is Streptococcus zooepidemicus. 3. The method for producing hyaluronic acid according to claim 1 or 2, wherein the bacterium belonging to the genus Streptococcus is Streptococcus zooepidemicus YIT2030 (Feikoken Jokyo No. 1305).
Priority Applications (7)
Application Number | Priority Date | Filing Date | Title |
---|---|---|---|
JP61099446A JPS62257393A (en) | 1986-05-01 | 1986-05-01 | Production of hyaluronic acid |
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 |
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 |
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. |
Applications Claiming Priority (1)
Application Number | Priority Date | Filing Date | Title |
---|---|---|---|
JP61099446A JPS62257393A (en) | 1986-05-01 | 1986-05-01 | Production of hyaluronic acid |
Publications (2)
Publication Number | Publication Date |
---|---|
JPS62257393A JPS62257393A (en) | 1987-11-09 |
JPH046356B2 true JPH046356B2 (en) | 1992-02-05 |
Family
ID=14247593
Family Applications (1)
Application Number | Title | Priority Date | Filing Date |
---|---|---|---|
JP61099446A Granted JPS62257393A (en) | 1986-05-01 | 1986-05-01 | Production of hyaluronic acid |
Country Status (1)
Country | Link |
---|---|
JP (1) | JPS62257393A (en) |
Families Citing this family (3)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
US5496726A (en) * | 1993-04-16 | 1996-03-05 | Lucky Limited | Streptococcus zooepidemicus medium and process for preparing hyaluronic acid |
KR100558751B1 (en) * | 2004-10-18 | 2006-03-14 | 주식회사 태평양 | Cosmetic composition for skin miosture |
CN104055701B (en) * | 2014-06-11 | 2017-01-18 | 山东安华生物医药股份有限公司 | Anti-allergy conditioning shampoo |
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 JP61099446A patent/JPS62257393A/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 |
---|---|
JPS62257393A (en) | 1987-11-09 |
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