JPH06228200A - Production of complement c3c - Google Patents
Production of complement c3cInfo
- Publication number
- JPH06228200A JPH06228200A JP5032518A JP3251893A JPH06228200A JP H06228200 A JPH06228200 A JP H06228200A JP 5032518 A JP5032518 A JP 5032518A JP 3251893 A JP3251893 A JP 3251893A JP H06228200 A JPH06228200 A JP H06228200A
- Authority
- JP
- Japan
- Prior art keywords
- complement
- solution
- group
- milk
- exchanger
- 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.)
- Pending
Links
- 108010078010 Complement C3c Proteins 0.000 title claims abstract description 111
- 238000004519 manufacturing process Methods 0.000 title claims description 10
- 150000001768 cations Chemical class 0.000 claims abstract description 19
- 150000001450 anions Chemical class 0.000 claims abstract description 18
- 239000002994 raw material Substances 0.000 claims abstract description 11
- 238000000034 method Methods 0.000 claims description 27
- 235000013365 dairy product Nutrition 0.000 claims description 7
- 238000005349 anion exchange Methods 0.000 claims 1
- 238000005341 cation exchange Methods 0.000 claims 1
- 235000013336 milk Nutrition 0.000 abstract description 20
- 239000008267 milk Substances 0.000 abstract description 20
- 210000004080 milk Anatomy 0.000 abstract description 20
- 244000052616 bacterial pathogen Species 0.000 abstract description 18
- 239000002537 cosmetic Substances 0.000 abstract description 6
- 239000003814 drug Substances 0.000 abstract description 6
- 235000013305 food Nutrition 0.000 abstract description 6
- 229940079593 drug Drugs 0.000 abstract description 3
- 239000000243 solution Substances 0.000 description 39
- 210000004027 cell Anatomy 0.000 description 27
- 230000001717 pathogenic effect Effects 0.000 description 24
- 241000588724 Escherichia coli Species 0.000 description 21
- FAPWRFPIFSIZLT-UHFFFAOYSA-M Sodium chloride Chemical compound [Na+].[Cl-] FAPWRFPIFSIZLT-UHFFFAOYSA-M 0.000 description 20
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 description 20
- 239000007853 buffer solution Substances 0.000 description 19
- LOKCTEFSRHRXRJ-UHFFFAOYSA-I dipotassium trisodium dihydrogen phosphate hydrogen phosphate dichloride Chemical compound P(=O)(O)(O)[O-].[K+].P(=O)(O)([O-])[O-].[Na+].[Na+].[Cl-].[K+].[Cl-].[Na+] LOKCTEFSRHRXRJ-UHFFFAOYSA-I 0.000 description 17
- 238000011282 treatment Methods 0.000 description 17
- 239000000872 buffer Substances 0.000 description 15
- 239000002953 phosphate buffered saline Substances 0.000 description 15
- WCUXLLCKKVVCTQ-UHFFFAOYSA-M Potassium chloride Chemical compound [Cl-].[K+] WCUXLLCKKVVCTQ-UHFFFAOYSA-M 0.000 description 14
- 238000010828 elution Methods 0.000 description 13
- 239000000463 material Substances 0.000 description 13
- RAXXELZNTBOGNW-UHFFFAOYSA-N imidazole Natural products C1=CNC=N1 RAXXELZNTBOGNW-UHFFFAOYSA-N 0.000 description 12
- 208000015181 infectious disease Diseases 0.000 description 10
- 239000011780 sodium chloride Substances 0.000 description 10
- 238000005406 washing Methods 0.000 description 10
- 102000007544 Whey Proteins Human genes 0.000 description 9
- 108010046377 Whey Proteins Proteins 0.000 description 9
- 208000002925 dental caries Diseases 0.000 description 9
- 230000000694 effects Effects 0.000 description 9
- 239000012528 membrane Substances 0.000 description 9
- 239000000203 mixture Substances 0.000 description 9
- 244000052769 pathogen Species 0.000 description 9
- 239000008363 phosphate buffer Substances 0.000 description 9
- 239000000843 powder Substances 0.000 description 9
- JDIIGWSSTNUWGK-UHFFFAOYSA-N 1h-imidazol-3-ium;chloride Chemical compound [Cl-].[NH2+]1C=CN=C1 JDIIGWSSTNUWGK-UHFFFAOYSA-N 0.000 description 8
- 108010059712 Pronase Proteins 0.000 description 8
- UIIMBOGNXHQVGW-UHFFFAOYSA-M Sodium bicarbonate Chemical compound [Na+].OC([O-])=O UIIMBOGNXHQVGW-UHFFFAOYSA-M 0.000 description 8
- 230000002401 inhibitory effect Effects 0.000 description 8
- 238000012360 testing method Methods 0.000 description 8
- PEDCQBHIVMGVHV-UHFFFAOYSA-N Glycerine Chemical compound OCC(O)CO PEDCQBHIVMGVHV-UHFFFAOYSA-N 0.000 description 7
- 239000007788 liquid Substances 0.000 description 7
- 239000001103 potassium chloride Substances 0.000 description 7
- 235000011164 potassium chloride Nutrition 0.000 description 7
- 238000000108 ultra-filtration Methods 0.000 description 7
- 239000004793 Polystyrene Substances 0.000 description 6
- 238000009472 formulation Methods 0.000 description 6
- 239000002609 medium Substances 0.000 description 6
- 229920002223 polystyrene Polymers 0.000 description 6
- 241000283690 Bos taurus Species 0.000 description 5
- 241000699666 Mus <mouse, genus> Species 0.000 description 5
- 239000008351 acetate buffer Substances 0.000 description 5
- 239000007979 citrate buffer Substances 0.000 description 5
- 230000000295 complement effect Effects 0.000 description 5
- 238000001962 electrophoresis Methods 0.000 description 5
- 150000007524 organic acids Chemical class 0.000 description 5
- 150000003839 salts Chemical class 0.000 description 5
- 235000021119 whey protein Nutrition 0.000 description 5
- 108091003079 Bovine Serum Albumin Proteins 0.000 description 4
- LFQSCWFLJHTTHZ-UHFFFAOYSA-N Ethanol Chemical compound CCO LFQSCWFLJHTTHZ-UHFFFAOYSA-N 0.000 description 4
- DHMQDGOQFOQNFH-UHFFFAOYSA-N Glycine Chemical compound NCC(O)=O DHMQDGOQFOQNFH-UHFFFAOYSA-N 0.000 description 4
- 101000901154 Homo sapiens Complement C3 Proteins 0.000 description 4
- 108060003951 Immunoglobulin Proteins 0.000 description 4
- 241000699670 Mus sp. Species 0.000 description 4
- 239000005862 Whey Substances 0.000 description 4
- 238000005119 centrifugation Methods 0.000 description 4
- 238000011033 desalting Methods 0.000 description 4
- 238000001035 drying Methods 0.000 description 4
- MHMNJMPURVTYEJ-UHFFFAOYSA-N fluorescein-5-isothiocyanate Chemical compound O1C(=O)C2=CC(N=C=S)=CC=C2C21C1=CC=C(O)C=C1OC1=CC(O)=CC=C21 MHMNJMPURVTYEJ-UHFFFAOYSA-N 0.000 description 4
- 102000018358 immunoglobulin Human genes 0.000 description 4
- 230000005764 inhibitory process Effects 0.000 description 4
- 239000008055 phosphate buffer solution Substances 0.000 description 4
- BASFCYQUMIYNBI-UHFFFAOYSA-N platinum Chemical compound [Pt] BASFCYQUMIYNBI-UHFFFAOYSA-N 0.000 description 4
- 235000017557 sodium bicarbonate Nutrition 0.000 description 4
- 229910000030 sodium bicarbonate Inorganic materials 0.000 description 4
- 238000001179 sorption measurement Methods 0.000 description 4
- 239000000126 substance Substances 0.000 description 4
- 239000006228 supernatant Substances 0.000 description 4
- 125000000022 2-aminoethyl group Chemical group [H]C([*])([H])C([H])([H])N([H])[H] 0.000 description 3
- BTBUEUYNUDRHOZ-UHFFFAOYSA-N Borate Chemical compound [O-]B([O-])[O-] BTBUEUYNUDRHOZ-UHFFFAOYSA-N 0.000 description 3
- 102100022133 Complement C3 Human genes 0.000 description 3
- 241001465754 Metazoa Species 0.000 description 3
- HEMHJVSKTPXQMS-UHFFFAOYSA-M Sodium hydroxide Chemical compound [OH-].[Na+] HEMHJVSKTPXQMS-UHFFFAOYSA-M 0.000 description 3
- 239000007983 Tris buffer Substances 0.000 description 3
- 108090000631 Trypsin Proteins 0.000 description 3
- 102000004142 Trypsin Human genes 0.000 description 3
- 230000009471 action Effects 0.000 description 3
- 229940098773 bovine serum albumin Drugs 0.000 description 3
- BECPQYXYKAMYBN-UHFFFAOYSA-N casein, tech. Chemical compound NCCCCC(C(O)=O)N=C(O)C(CC(O)=O)N=C(O)C(CCC(O)=N)N=C(O)C(CC(C)C)N=C(O)C(CCC(O)=O)N=C(O)C(CC(O)=O)N=C(O)C(CCC(O)=O)N=C(O)C(C(C)O)N=C(O)C(CCC(O)=N)N=C(O)C(CCC(O)=N)N=C(O)C(CCC(O)=N)N=C(O)C(CCC(O)=O)N=C(O)C(CCC(O)=O)N=C(O)C(COP(O)(O)=O)N=C(O)C(CCC(O)=N)N=C(O)C(N)CC1=CC=CC=C1 BECPQYXYKAMYBN-UHFFFAOYSA-N 0.000 description 3
- 235000013351 cheese Nutrition 0.000 description 3
- KRKNYBCHXYNGOX-UHFFFAOYSA-N citric acid Chemical compound OC(=O)CC(O)(C(O)=O)CC(O)=O KRKNYBCHXYNGOX-UHFFFAOYSA-N 0.000 description 3
- 230000004154 complement system Effects 0.000 description 3
- 238000013329 compounding Methods 0.000 description 3
- 238000007796 conventional method Methods 0.000 description 3
- 230000008260 defense mechanism Effects 0.000 description 3
- 239000008367 deionised water Substances 0.000 description 3
- 229910021641 deionized water Inorganic materials 0.000 description 3
- 238000004925 denaturation Methods 0.000 description 3
- 230000036425 denaturation Effects 0.000 description 3
- 238000000502 dialysis Methods 0.000 description 3
- 235000019441 ethanol Nutrition 0.000 description 3
- 238000001914 filtration Methods 0.000 description 3
- 235000011187 glycerol Nutrition 0.000 description 3
- 230000000968 intestinal effect Effects 0.000 description 3
- VZCYOOQTPOCHFL-UPHRSURJSA-N maleic acid Chemical compound OC(=O)\C=C/C(O)=O VZCYOOQTPOCHFL-UPHRSURJSA-N 0.000 description 3
- 229920000642 polymer Polymers 0.000 description 3
- 239000000047 product Substances 0.000 description 3
- 235000020183 skimmed milk Nutrition 0.000 description 3
- -1 sulfopropyl group Chemical group 0.000 description 3
- VZCYOOQTPOCHFL-UHFFFAOYSA-N trans-butenedioic acid Natural products OC(=O)C=CC(O)=O VZCYOOQTPOCHFL-UHFFFAOYSA-N 0.000 description 3
- 239000012588 trypsin Substances 0.000 description 3
- 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 2
- HRPVXLWXLXDGHG-UHFFFAOYSA-N Acrylamide Chemical compound NC(=O)C=C HRPVXLWXLXDGHG-UHFFFAOYSA-N 0.000 description 2
- 229920001817 Agar Polymers 0.000 description 2
- CIWBSHSKHKDKBQ-JLAZNSOCSA-N Ascorbic acid Chemical compound OC[C@H](O)[C@H]1OC(=O)C(O)=C1O CIWBSHSKHKDKBQ-JLAZNSOCSA-N 0.000 description 2
- 241000894006 Bacteria Species 0.000 description 2
- VTYYLEPIZMXCLO-UHFFFAOYSA-L Calcium carbonate Chemical compound [Ca+2].[O-]C([O-])=O VTYYLEPIZMXCLO-UHFFFAOYSA-L 0.000 description 2
- 108010076119 Caseins Proteins 0.000 description 2
- 102000011632 Caseins Human genes 0.000 description 2
- 229920001661 Chitosan Polymers 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
- RTZKZFJDLAIYFH-UHFFFAOYSA-N Diethyl ether Chemical compound CCOCC RTZKZFJDLAIYFH-UHFFFAOYSA-N 0.000 description 2
- 239000006144 Dulbecco’s modified Eagle's medium Substances 0.000 description 2
- 239000004471 Glycine Substances 0.000 description 2
- VEXZGXHMUGYJMC-UHFFFAOYSA-N Hydrochloric acid Chemical compound Cl VEXZGXHMUGYJMC-UHFFFAOYSA-N 0.000 description 2
- 229920000168 Microcrystalline cellulose Polymers 0.000 description 2
- 241000700157 Rattus norvegicus Species 0.000 description 2
- 229920005654 Sephadex Polymers 0.000 description 2
- 239000012507 Sephadex™ Substances 0.000 description 2
- 229920002684 Sepharose Polymers 0.000 description 2
- DBMJMQXJHONAFJ-UHFFFAOYSA-M Sodium laurylsulphate Chemical compound [Na+].CCCCCCCCCCCCOS([O-])(=O)=O DBMJMQXJHONAFJ-UHFFFAOYSA-M 0.000 description 2
- 241000194019 Streptococcus mutans Species 0.000 description 2
- 229930006000 Sucrose Natural products 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
- 239000012505 Superdex™ Substances 0.000 description 2
- 101710120037 Toxin CcdB Proteins 0.000 description 2
- 235000010724 Wisteria floribunda Nutrition 0.000 description 2
- 238000002835 absorbance Methods 0.000 description 2
- 150000001413 amino acids Chemical group 0.000 description 2
- 239000003242 anti bacterial agent Substances 0.000 description 2
- 229940088710 antibiotic agent Drugs 0.000 description 2
- 235000013361 beverage Nutrition 0.000 description 2
- 230000003115 biocidal effect Effects 0.000 description 2
- 239000005018 casein Substances 0.000 description 2
- 235000021240 caseins Nutrition 0.000 description 2
- 239000006285 cell suspension Substances 0.000 description 2
- 239000001913 cellulose Substances 0.000 description 2
- 229920002678 cellulose Polymers 0.000 description 2
- 235000010980 cellulose Nutrition 0.000 description 2
- 239000012141 concentrate Substances 0.000 description 2
- 235000008504 concentrate Nutrition 0.000 description 2
- 230000007850 degeneration Effects 0.000 description 2
- XTDYIOOONNVFMA-UHFFFAOYSA-N dimethyl pentanedioate Chemical compound COC(=O)CCCC(=O)OC XTDYIOOONNVFMA-UHFFFAOYSA-N 0.000 description 2
- 238000000909 electrodialysis Methods 0.000 description 2
- OVBPIULPVIDEAO-LBPRGKRZSA-N folic acid Chemical compound C=1N=C2NC(N)=NC(=O)C2=NC=1CNC1=CC=C(C(=O)N[C@@H](CCC(O)=O)C(O)=O)C=C1 OVBPIULPVIDEAO-LBPRGKRZSA-N 0.000 description 2
- 238000004108 freeze drying Methods 0.000 description 2
- 210000001035 gastrointestinal tract Anatomy 0.000 description 2
- 238000002523 gelfiltration Methods 0.000 description 2
- 150000004676 glycans Chemical class 0.000 description 2
- 229940072221 immunoglobulins Drugs 0.000 description 2
- 239000012535 impurity Substances 0.000 description 2
- 239000003456 ion exchange resin Substances 0.000 description 2
- 229920003303 ion-exchange polymer Polymers 0.000 description 2
- 239000006210 lotion Substances 0.000 description 2
- 244000005700 microbiome Species 0.000 description 2
- 235000019813 microcrystalline cellulose Nutrition 0.000 description 2
- 239000008108 microcrystalline cellulose Substances 0.000 description 2
- 229940016286 microcrystalline cellulose Drugs 0.000 description 2
- 238000002156 mixing Methods 0.000 description 2
- 229910052697 platinum Inorganic materials 0.000 description 2
- 229920001282 polysaccharide Polymers 0.000 description 2
- 239000005017 polysaccharide Substances 0.000 description 2
- 230000008569 process Effects 0.000 description 2
- 238000003672 processing method Methods 0.000 description 2
- 239000011347 resin Substances 0.000 description 2
- 229920005989 resin Polymers 0.000 description 2
- 238000001223 reverse osmosis Methods 0.000 description 2
- 235000019333 sodium laurylsulphate Nutrition 0.000 description 2
- 239000000600 sorbitol Substances 0.000 description 2
- 125000006850 spacer group Chemical group 0.000 description 2
- 238000001694 spray drying Methods 0.000 description 2
- 239000005720 sucrose Substances 0.000 description 2
- 239000000725 suspension Substances 0.000 description 2
- XPFJYKARVSSRHE-UHFFFAOYSA-K trisodium;2-hydroxypropane-1,2,3-tricarboxylate;2-hydroxypropane-1,2,3-tricarboxylic acid Chemical compound [Na+].[Na+].[Na+].OC(=O)CC(O)(C(O)=O)CC(O)=O.[O-]C(=O)CC(O)(CC([O-])=O)C([O-])=O XPFJYKARVSSRHE-UHFFFAOYSA-K 0.000 description 2
- FTLYMKDSHNWQKD-UHFFFAOYSA-N (2,4,5-trichlorophenyl)boronic acid Chemical compound OB(O)C1=CC(Cl)=C(Cl)C=C1Cl FTLYMKDSHNWQKD-UHFFFAOYSA-N 0.000 description 1
- 206010000050 Abdominal adhesions Diseases 0.000 description 1
- 208000002874 Acne Vulgaris Diseases 0.000 description 1
- 241000283707 Capra Species 0.000 description 1
- 208000035473 Communicable disease Diseases 0.000 description 1
- 108010028780 Complement C3 Proteins 0.000 description 1
- 108010077840 Complement C3a Proteins 0.000 description 1
- 229920000742 Cotton Polymers 0.000 description 1
- ZAKOWWREFLAJOT-CEFNRUSXSA-N D-alpha-tocopherylacetate Chemical compound CC(=O)OC1=C(C)C(C)=C2O[C@@](CCC[C@H](C)CCC[C@H](C)CCCC(C)C)(C)CCC2=C1C ZAKOWWREFLAJOT-CEFNRUSXSA-N 0.000 description 1
- ZZZCUOFIHGPKAK-UHFFFAOYSA-N D-erythro-ascorbic acid Natural products OCC1OC(=O)C(O)=C1O ZZZCUOFIHGPKAK-UHFFFAOYSA-N 0.000 description 1
- 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 1
- 102000003886 Glycoproteins Human genes 0.000 description 1
- 108090000288 Glycoproteins Proteins 0.000 description 1
- 241000282412 Homo Species 0.000 description 1
- 108010063045 Lactoferrin Proteins 0.000 description 1
- 102000010445 Lactoferrin Human genes 0.000 description 1
- 108010023244 Lactoperoxidase Proteins 0.000 description 1
- 102000045576 Lactoperoxidases Human genes 0.000 description 1
- 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 1
- 241000124008 Mammalia Species 0.000 description 1
- 240000004658 Medicago sativa Species 0.000 description 1
- 235000017587 Medicago sativa ssp. sativa Nutrition 0.000 description 1
- OVBPIULPVIDEAO-UHFFFAOYSA-N N-Pteroyl-L-glutaminsaeure Natural products C=1N=C2NC(N)=NC(=O)C2=NC=1CNC1=CC=C(C(=O)NC(CCC(O)=O)C(O)=O)C=C1 OVBPIULPVIDEAO-UHFFFAOYSA-N 0.000 description 1
- 241001494479 Pecora Species 0.000 description 1
- 108091005804 Peptidases Proteins 0.000 description 1
- 229920003171 Poly (ethylene oxide) Polymers 0.000 description 1
- OFOBLEOULBTSOW-UHFFFAOYSA-N Propanedioic acid Natural products OC(=O)CC(O)=O OFOBLEOULBTSOW-UHFFFAOYSA-N 0.000 description 1
- 239000004365 Protease Substances 0.000 description 1
- 239000012564 Q sepharose fast flow resin Substances 0.000 description 1
- 241000700159 Rattus Species 0.000 description 1
- 102100037486 Reverse transcriptase/ribonuclease H Human genes 0.000 description 1
- AUNGANRZJHBGPY-SCRDCRAPSA-N Riboflavin Chemical compound OC[C@@H](O)[C@@H](O)[C@@H](O)CN1C=2C=C(C)C(C)=CC=2N=C2C1=NC(=O)NC2=O AUNGANRZJHBGPY-SCRDCRAPSA-N 0.000 description 1
- 240000004808 Saccharomyces cerevisiae Species 0.000 description 1
- 235000014680 Saccharomyces cerevisiae Nutrition 0.000 description 1
- 229930003270 Vitamin B Natural products 0.000 description 1
- 229930003268 Vitamin C Natural products 0.000 description 1
- HCHKCACWOHOZIP-UHFFFAOYSA-N Zinc Chemical compound [Zn] HCHKCACWOHOZIP-UHFFFAOYSA-N 0.000 description 1
- DPXJVFZANSGRMM-UHFFFAOYSA-N acetic acid;2,3,4,5,6-pentahydroxyhexanal;sodium Chemical compound [Na].CC(O)=O.OCC(O)C(O)C(O)C(O)C=O DPXJVFZANSGRMM-UHFFFAOYSA-N 0.000 description 1
- 230000002378 acidificating effect Effects 0.000 description 1
- 206010000496 acne Diseases 0.000 description 1
- 230000004913 activation Effects 0.000 description 1
- 239000004480 active ingredient Substances 0.000 description 1
- 238000001042 affinity chromatography Methods 0.000 description 1
- 239000008272 agar Substances 0.000 description 1
- 230000000172 allergic effect Effects 0.000 description 1
- 125000000539 amino acid group Chemical group 0.000 description 1
- 208000010668 atopic eczema Diseases 0.000 description 1
- 230000001580 bacterial effect Effects 0.000 description 1
- 210000004369 blood Anatomy 0.000 description 1
- 239000008280 blood Substances 0.000 description 1
- 210000004556 brain Anatomy 0.000 description 1
- 229910000019 calcium carbonate Inorganic materials 0.000 description 1
- XAAHAAMILDNBPS-UHFFFAOYSA-L calcium hydrogenphosphate dihydrate Chemical compound O.O.[Ca+2].OP([O-])([O-])=O XAAHAAMILDNBPS-UHFFFAOYSA-L 0.000 description 1
- 239000002775 capsule Substances 0.000 description 1
- 125000003178 carboxy group Chemical group [H]OC(*)=O 0.000 description 1
- 229920003123 carboxymethyl cellulose sodium Polymers 0.000 description 1
- 125000002057 carboxymethyl group Chemical group [H]OC(=O)C([H])([H])[*] 0.000 description 1
- 229940063834 carboxymethylcellulose sodium Drugs 0.000 description 1
- 230000001013 cariogenic effect Effects 0.000 description 1
- 239000000679 carrageenan Substances 0.000 description 1
- 235000010418 carrageenan Nutrition 0.000 description 1
- 229920001525 carrageenan Polymers 0.000 description 1
- 229940113118 carrageenan Drugs 0.000 description 1
- 238000006243 chemical reaction Methods 0.000 description 1
- 239000003795 chemical substances by application Substances 0.000 description 1
- 229940112822 chewing gum Drugs 0.000 description 1
- 235000015218 chewing gum Nutrition 0.000 description 1
- 239000003086 colorant Substances 0.000 description 1
- 238000011109 contamination Methods 0.000 description 1
- 238000004132 cross linking Methods 0.000 description 1
- 238000012258 culturing Methods 0.000 description 1
- ZAKOWWREFLAJOT-UHFFFAOYSA-N d-alpha-Tocopheryl acetate Natural products CC(=O)OC1=C(C)C(C)=C2OC(CCCC(C)CCCC(C)CCCC(C)C)(C)CCC2=C1C ZAKOWWREFLAJOT-UHFFFAOYSA-N 0.000 description 1
- 238000011161 development Methods 0.000 description 1
- 235000019700 dicalcium phosphate Nutrition 0.000 description 1
- 239000003651 drinking water Substances 0.000 description 1
- 235000020188 drinking water Nutrition 0.000 description 1
- 210000003013 erythroid precursor cell Anatomy 0.000 description 1
- 230000003203 everyday effect Effects 0.000 description 1
- 230000005284 excitation Effects 0.000 description 1
- 235000013861 fat-free Nutrition 0.000 description 1
- 239000012091 fetal bovine serum Substances 0.000 description 1
- 239000000796 flavoring agent Substances 0.000 description 1
- 235000019634 flavors Nutrition 0.000 description 1
- 239000007850 fluorescent dye Substances 0.000 description 1
- 229960000304 folic acid Drugs 0.000 description 1
- 235000019152 folic acid Nutrition 0.000 description 1
- 239000011724 folic acid Substances 0.000 description 1
- 235000011194 food seasoning agent Nutrition 0.000 description 1
- 239000003205 fragrance Substances 0.000 description 1
- 238000007710 freezing Methods 0.000 description 1
- 230000008014 freezing Effects 0.000 description 1
- 239000008103 glucose Substances 0.000 description 1
- 230000036541 health Effects 0.000 description 1
- 238000010438 heat treatment Methods 0.000 description 1
- 102000057770 human C3 Human genes 0.000 description 1
- 230000007062 hydrolysis Effects 0.000 description 1
- 238000006460 hydrolysis reaction Methods 0.000 description 1
- 229940027941 immunoglobulin g Drugs 0.000 description 1
- 238000011534 incubation Methods 0.000 description 1
- 238000001802 infusion Methods 0.000 description 1
- 239000004615 ingredient Substances 0.000 description 1
- 239000003112 inhibitor Substances 0.000 description 1
- 238000002347 injection Methods 0.000 description 1
- 239000007924 injection Substances 0.000 description 1
- 229910052500 inorganic mineral Inorganic materials 0.000 description 1
- 150000002500 ions Chemical class 0.000 description 1
- 108010028463 kappa-casein glycomacropeptide Proteins 0.000 description 1
- 230000002147 killing effect Effects 0.000 description 1
- CSSYQJWUGATIHM-IKGCZBKSSA-N l-phenylalanyl-l-lysyl-l-cysteinyl-l-arginyl-l-arginyl-l-tryptophyl-l-glutaminyl-l-tryptophyl-l-arginyl-l-methionyl-l-lysyl-l-lysyl-l-leucylglycyl-l-alanyl-l-prolyl-l-seryl-l-isoleucyl-l-threonyl-l-cysteinyl-l-valyl-l-arginyl-l-arginyl-l-alanyl-l-phenylal Chemical compound C([C@H](N)C(=O)N[C@@H](CCCCN)C(=O)N[C@@H](CS)C(=O)N[C@@H](CCCNC(N)=N)C(=O)N[C@@H](CCCNC(N)=N)C(=O)N[C@@H](CC=1C2=CC=CC=C2NC=1)C(=O)N[C@@H](CCC(N)=O)C(=O)N[C@@H](CC=1C2=CC=CC=C2NC=1)C(=O)N[C@@H](CCCNC(N)=N)C(=O)N[C@@H](CCSC)C(=O)N[C@@H](CCCCN)C(=O)N[C@@H](CCCCN)C(=O)N[C@@H](CC(C)C)C(=O)NCC(=O)N[C@@H](C)C(=O)N1CCC[C@H]1C(=O)N[C@@H](CO)C(=O)N[C@@H]([C@@H](C)CC)C(=O)N[C@@H]([C@@H](C)O)C(=O)N[C@@H](CS)C(=O)N[C@@H](C(C)C)C(=O)N[C@@H](CCCNC(N)=N)C(=O)N[C@@H](CCCNC(N)=N)C(=O)N[C@@H](C)C(=O)N[C@@H](CC=1C=CC=CC=1)C(O)=O)C1=CC=CC=C1 CSSYQJWUGATIHM-IKGCZBKSSA-N 0.000 description 1
- 235000021242 lactoferrin Nutrition 0.000 description 1
- 229940078795 lactoferrin Drugs 0.000 description 1
- 229940057428 lactoperoxidase Drugs 0.000 description 1
- 239000008101 lactose Substances 0.000 description 1
- 210000004185 liver Anatomy 0.000 description 1
- 239000011976 maleic acid Substances 0.000 description 1
- MYWUZJCMWCOHBA-VIFPVBQESA-N methamphetamine Chemical compound CN[C@@H](C)CC1=CC=CC=C1 MYWUZJCMWCOHBA-VIFPVBQESA-N 0.000 description 1
- 238000001471 micro-filtration Methods 0.000 description 1
- 235000021243 milk fat Nutrition 0.000 description 1
- 235000010755 mineral Nutrition 0.000 description 1
- 239000011707 mineral Substances 0.000 description 1
- 239000011259 mixed solution Substances 0.000 description 1
- 210000000214 mouth Anatomy 0.000 description 1
- 235000015205 orange juice Nutrition 0.000 description 1
- WPKBXFWUTXTJRH-UHFFFAOYSA-N oxocalcium dihydrate Chemical compound O.O.O=[Ca] WPKBXFWUTXTJRH-UHFFFAOYSA-N 0.000 description 1
- 239000002304 perfume Substances 0.000 description 1
- 125000002467 phosphate group Chemical group [H]OP(=O)(O[H])O[*] 0.000 description 1
- 239000002244 precipitate Substances 0.000 description 1
- 238000001556 precipitation Methods 0.000 description 1
- 238000002360 preparation method Methods 0.000 description 1
- 239000003755 preservative agent Substances 0.000 description 1
- 230000002335 preservative effect Effects 0.000 description 1
- 230000003449 preventive effect Effects 0.000 description 1
- 230000000644 propagated effect Effects 0.000 description 1
- 125000001453 quaternary ammonium group Chemical group 0.000 description 1
- 230000002829 reductive effect Effects 0.000 description 1
- 229940108461 rennet Drugs 0.000 description 1
- 108010058314 rennet Proteins 0.000 description 1
- 229940085605 saccharin sodium Drugs 0.000 description 1
- 230000003248 secreting effect Effects 0.000 description 1
- 238000000926 separation method Methods 0.000 description 1
- 239000001509 sodium citrate Substances 0.000 description 1
- NLJMYIDDQXHKNR-UHFFFAOYSA-K sodium citrate Chemical compound O.O.[Na+].[Na+].[Na+].[O-]C(=O)CC(O)(CC([O-])=O)C([O-])=O NLJMYIDDQXHKNR-UHFFFAOYSA-K 0.000 description 1
- 239000000758 substrate Substances 0.000 description 1
- 125000000020 sulfo group Chemical group O=S(=O)([*])O[H] 0.000 description 1
- 125000001174 sulfone group Chemical group 0.000 description 1
- 208000024891 symptom Diseases 0.000 description 1
- 239000006188 syrup Substances 0.000 description 1
- 235000020357 syrup Nutrition 0.000 description 1
- 229940042585 tocopherol acetate Drugs 0.000 description 1
- 229940034610 toothpaste Drugs 0.000 description 1
- 239000000606 toothpaste Substances 0.000 description 1
- PHYFQTYBJUILEZ-IUPFWZBJSA-N triolein Chemical compound CCCCCCCC\C=C/CCCCCCCC(=O)OCC(OC(=O)CCCCCCC\C=C/CCCCCCCC)COC(=O)CCCCCCC\C=C/CCCCCCCC PHYFQTYBJUILEZ-IUPFWZBJSA-N 0.000 description 1
- LENZDBCJOHFCAS-UHFFFAOYSA-N tris Chemical compound OCC(N)(CO)CO LENZDBCJOHFCAS-UHFFFAOYSA-N 0.000 description 1
- 229930003231 vitamin Natural products 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
- 235000019156 vitamin B Nutrition 0.000 description 1
- 239000011720 vitamin B Substances 0.000 description 1
- 235000019154 vitamin C Nutrition 0.000 description 1
- 239000011718 vitamin C Substances 0.000 description 1
- 150000003722 vitamin derivatives Chemical class 0.000 description 1
- 238000001262 western blot Methods 0.000 description 1
- 235000008939 whole milk Nutrition 0.000 description 1
- 235000011844 whole wheat flour Nutrition 0.000 description 1
- 239000011701 zinc Substances 0.000 description 1
- 229910052725 zinc Inorganic materials 0.000 description 1
- UHVMMEOXYDMDKI-JKYCWFKZSA-L zinc;1-(5-cyanopyridin-2-yl)-3-[(1s,2s)-2-(6-fluoro-2-hydroxy-3-propanoylphenyl)cyclopropyl]urea;diacetate Chemical compound [Zn+2].CC([O-])=O.CC([O-])=O.CCC(=O)C1=CC=C(F)C([C@H]2[C@H](C2)NC(=O)NC=2N=CC(=CC=2)C#N)=C1O UHVMMEOXYDMDKI-JKYCWFKZSA-L 0.000 description 1
Landscapes
- Peptides Or Proteins (AREA)
- Coloring Foods And Improving Nutritive Qualities (AREA)
- Medicines That Contain Protein Lipid Enzymes And Other Medicines (AREA)
- Agricultural Chemicals And Associated Chemicals (AREA)
Abstract
Description
【０００１】[0001]
【産業上の利用分野】本発明は、乳質原料から病原菌付
着阻止能を有する補体Ｃ３ｃを製造する方法に関する。
この補体Ｃ３ｃは、病原菌付着阻止能を有するので、病
原菌付着阻止能を付与する目的で飲食品、医薬、化粧
品、飼料などに添加して利用することが可能である。BACKGROUND OF THE INVENTION 1. Field of the Invention The present invention relates to a method for producing complement C3c having an ability to inhibit the attachment of pathogenic bacteria from a milk material.
Since this complement C3c has the ability to prevent the attachment of pathogenic bacteria, it can be used by adding it to foods, drinks, medicines, cosmetics, feeds, etc. for the purpose of imparting the ability to prevent the attachment of pathogenic bacteria.
【０００２】[0002]
【従来の技術】一般に病原菌は、体内への進入、標的細
胞への付着、増殖という過程を経て感染症を引き起こ
す。現在、この感染症に対する治療に用いられているも
のは主に抗生物質である。抗生物質は、細胞に付着、増
殖した病原菌を死滅させることによって病原を根絶しよ
うとするもので、病原菌の感染過程の最終段階に作用さ
せるものである。この抗生物質による感染治療は、発病
後の生体に対する治療法として非常に有効であるが、抗
生物質の性格上、様々な副作用あるいはアレルギー症状
を引起す等の問題点も多い。2. Description of the Related Art Generally, pathogenic bacteria cause infectious diseases through the processes of entering the body, adhering to target cells, and multiplying. At present, what is used for the treatment of this infection is mainly antibiotics. Antibiotics are intended to eradicate pathogens by killing the pathogens that have adhered to and propagated in cells, and act at the final stage of the infection process of pathogens. The treatment of infection with this antibiotic is very effective as a treatment method for the living body after the onset of illness, but due to the nature of the antibiotic, there are many problems such as causing various side effects or allergic symptoms.
【０００３】一方、生体は病原菌の感染に対し、感染初
期の段階において防御機構を備えている。例えば、消化
管内における分泌型ＩｇＡを主体とする免疫グロブリン
による防御機構がそれであり、これらの免疫グロブリン
は病原菌による感染初期の段階、すなわち、病原菌が標
的細胞へ付着することを阻止することによって生体を感
染から守っている。On the other hand, the living body has a defense mechanism against the infection of pathogenic bacteria at the early stage of infection. For example, it is a defense mechanism by immunoglobulins mainly composed of secretory IgA in the digestive tract, and these immunoglobulins protect the living body at an early stage of infection by pathogens, that is, by preventing the pathogens from adhering to target cells. Protects from infection.
【０００４】一般に病原菌が標的細胞に付着するに際し
ては、その標的細胞上の特定構造を受容体として認識し
ていることが知られている。したがって、これらの受容
体あるいは受容体と類似した構造を有し、病原菌との結
合能を有する物質は、病原菌表面の受容体と結合する部
位に特異的に結合し、結果的に病原菌が標的細胞上の受
容体に結合することを特異的に阻害するものと考えられ
ている。このような作用は、病原菌による感染を初期の
段階で阻止するという意味で、上述の免疫グロブリンに
よる生体防御機構に類似しているといえる。このよう
に、病原菌と特異的に結合し得る物質は、病原菌による
感染を未然に防ぎ、しかもその作用が穏やかであり、副
作用が少ない病原菌付着阻止剤になり得ると考えられ
る。そして、乳中に含まれるκ−カゼイングリコマクロ
ペプチドやラクトフェリンを有効成分とする病原菌付着
阻止剤が提案されている〔特開昭６３−２８４１３３号
公報、特開平３−２２０１３０号公報〕。It is generally known that, when a pathogenic bacterium attaches to a target cell, it recognizes a specific structure on the target cell as a receptor. Therefore, a substance having a structure similar to these receptors or a receptor and having the ability to bind to a pathogen is specifically bound to a site that binds to the receptor on the surface of the pathogen, and as a result, the pathogen is a target cell. It is believed to specifically inhibit binding to the above receptors. It can be said that such an action is similar to the above-mentioned biological defense mechanism by immunoglobulin in the sense that it blocks infection by pathogenic bacteria at an early stage. Thus, it is considered that the substance capable of specifically binding to the pathogenic bacterium can prevent the infection by the pathogenic bacterium, has a mild action, and can be a pathogen adhesion inhibitor with few side effects. Then, an agent for inhibiting the adherence of pathogenic bacteria, which contains κ-casein glycomacropeptide and lactoferrin contained in milk as active ingredients, has been proposed [JP-A-63-284133 and JP-A-3-220130].
【０００５】ところで、補体系成分として補体第３成分
（Ｃ３）が知られている。ヒトＣ３は、αおよびβ鎖が
Ｓ−Ｓ結合で架橋された構造を持つ分子量約１８０ｋＤ
ａの糖タンパク質で、補体系成分のうち血中含量が最も
高く、補体系の活性化に関与しているといわれている。
また、このＣ３の加水分解産物として、分子量約９ｋＤ
ａのＣ３ａ、分子量約１７１ｋＤａのＣ３ｂ、分子量約
１４０ｋＤａのＣ３ｃ、分子量約３０ｋＤａのＣ３ｄお
よび分子量約１２ｋＤａのＣ３ｅが知られている。By the way, a third complement component (C3) is known as a complement system component. Human C3 has a structure in which the α and β chains are cross-linked by S—S bonds and has a molecular weight of about 180 kD.
It is said that the glycoprotein of a has the highest blood content among components of the complement system and is involved in activation of the complement system.
Also, as a hydrolysis product of this C3, a molecular weight of about 9 kD
C3a having a molecular weight of about 171 kDa, C3b having a molecular weight of about 140 kDa, C3d having a molecular weight of about 30 kDa, and C3e having a molecular weight of about 12 kDa are known.
【０００６】従来、補体Ｃ３ｃを得る方法として、補体
Ｃ３をプロテアーゼ処理した後、電気泳動によって補体
Ｃ３ｃ画分を分取する方法が知られている〔Ｊａｒｍｉ
ｌａｅｔ ａｌ．，Ｊ．Ｉｍｍｕｎｏｌ．Ｍｅｔｈ．、
第８５巻、１７−２６頁、１９８５年〕。しかし、この
方法では補体Ｃ３ｃを大量に調製できないという問題が
あった。また、乳中から分取して補体Ｃ３ｃを大量に調
製する方法については、現在までのところ報告されてい
ない。As a conventional method for obtaining complement C3c, a method of treating complement C3 with a protease and then fractionating the complement C3c fraction by electrophoresis is known [Jarmi.
la et al. J. Immunol. Meth. ,
85, 17-26, 1985]. However, this method has a problem that a large amount of complement C3c cannot be prepared. In addition, a method for collecting a large amount of complement C3c by separating it from milk has not been reported so far.
【０００７】[0007]
【発明が解決しようとする課題】本発明者らは、病原菌
付着阻止能を有する補体Ｃ３ｃが乳中に比較的大量に存
在することを見出し、さらに効率よく補体Ｃ３ｃを乳中
から分離できる方法を見出して本発明を成すに至った。
したがって、本発明は乳質原料から補体Ｃ３ｃを製造す
る方法を提供することを課題とする。DISCLOSURE OF THE INVENTION The present inventors have found that a relatively large amount of complement C3c capable of inhibiting the attachment of pathogenic bacteria is present in milk, and it is possible to separate complement C3c from milk more efficiently. The inventors have found a method and completed the present invention.
Therefore, it is an object of the present invention to provide a method for producing complement C3c from a milk material.
【０００８】[0008]
【発明を解決するための手段】乳質原料から大量に補体
Ｃ３ｃを得る方法について、以下に説明する。補体Ｃ３
ｃを含む乳質原料を陽イオン交換体と接触させ、補体Ｃ
３ｃを陽イオン交換体に吸着させた後、イオン強度の異
なる溶液を用いて補体Ｃ３ｃを溶出する。A method for obtaining a large amount of complement C3c from a dairy raw material will be described below. Complement C3
A milk material containing c is brought into contact with a cation exchanger to obtain complement C.
After adsorbing 3c to the cation exchanger, the complement C3c is eluted using a solution having different ionic strength.
【０００９】補体Ｃ３ｃを含む乳質原料としては、ヒ
ト、ウシ、水牛、ヒツジ、ヤギ等哺乳動物由来の乳ある
いは乳清を挙げることができ、脱脂粉乳、全粉乳、ホエ
ー粉、ホエータンパク質を７５％以上含有するＷＰＣ
（ホエータンパク質濃縮物）あるいはホエータンパク質
を９０％以上含有するＷＰＩ（ホエータンパク質分離
物）を還元したものを用いることもできる。さらに、等
電点沈殿によってカゼインを除去したり、レンネットに
よってカゼインカードを除去した上清、あるいはチーズ
製造時に排出されるチーズホエーなども利用することが
できる。これらの乳質原料は、予めクラリファイヤー、
マイクロフィルトレーション、濾過などの操作によって
沈殿物を除去したものを用いることが望ましい。このよ
うな乳質原料を用いることにより、イオン交換体への吸
着効率を高めることができ、補体Ｃ３ｃの純度を高める
ことができる。なお、補体Ｃ３ｃは変性し易いので、穏
やかな熱処理条件の乳質原料を用いることが好ましい。
また、乳質原料のｐＨについては特に制限はないが、強
酸性条件あるいは強アルカリ条件下では補体Ｃ３ｃが変
性するので望ましくない。Examples of the milk material containing complement C3c include milk or whey derived from mammals such as humans, cows, buffalo, sheep and goats, and skim milk powder, whole milk powder, whey powder and whey protein can be used. % Or more WPC
(Whey protein concentrate) or WPI (whey protein isolate) containing 90% or more whey protein may be reduced. Furthermore, casein can be removed by isoelectric point precipitation, casein curd removed by rennet, or cheese whey discharged during cheese production can be used. These dairy ingredients are preliminarily clarifier,
It is desirable to use the one from which the precipitate has been removed by operations such as microfiltration and filtration. By using such a milk material, the efficiency of adsorption to the ion exchanger can be increased, and the purity of complement C3c can be increased. Since complement C3c is easily denatured, it is preferable to use a milk material under mild heat treatment conditions.
The pH of the dairy raw material is not particularly limited, but it is not desirable because the complement C3c is denatured under strongly acidic conditions or strong alkaline conditions.
【００１０】補体Ｃ３ｃの吸着に用いる陽イオン交換体
としては、架橋型の多糖類、セルロース、アクリルアミ
ド樹脂などの高分子にカルボキシル基、スルホン基、リ
ン酸基等を導入したもの、あるいは、高分子と交換基の
間にスペーサーを導入したものなどを挙げることがで
き、具体的には、カルボキシメチル基を導入したＣＭ−
セルロファインＣ−５００（生化学工業（株）製）、ス
ルホン基を導入したスルホン化キトパール（富士紡績
（株）製）、スルホプロピル基を導入したＳＰ−トヨパ
ール５５０Ｃ（東ソー（株）製）、Ｓ−セファロース
ファースト フロー（ファルマシア社製）などを例示す
ることができる。As the cation exchanger used for adsorption of complement C3c, a polymer having a carboxyl group, a sulfone group, a phosphate group or the like introduced into a polymer such as a cross-linked polysaccharide, cellulose or acrylamide resin, or a high cation exchanger Examples thereof include those in which a spacer is introduced between the molecule and the exchange group, and specifically, CM-in which a carboxymethyl group is introduced.
Cellulofine C-500 (manufactured by Seikagaku Corporation), sulfonated chitopearl (manufactured by Fuji Spinning Co., Ltd.) having a sulfo group introduced, SP-Toyopearl 550C (manufactured by Tosoh Corporation) having a sulfopropyl group introduced, S-Sepharose
First flow (Pharmacia) and the like can be exemplified.
【００１１】補体Ｃ３ｃを含む乳質原料を陽イオン交換
体と接触させる方法としては、タンク内で接触させる方
法、陽イオン交換体を充填したカラムに乳質原料を通液
する方法、回転型カラム反応器を用いる方法〔特開平２
−１３８２９５号公報〕など従来より知られている方法
を用いることができるが、大量に補体Ｃ３ｃを調製する
に際しては回転型カラム反応器を用いる方法を採用する
と特に効率的である。補体Ｃ３ｃを含む乳質原料を陽イ
オン交換体と接触させる際の温度条件については特に制
限はないが、通常４℃〜６０℃の範囲で行うことが望ま
しい。４℃未満の温度で接触を行うと乳質原料の凍結、
粘度の上昇、脂肪分離などの問題が生じる。また、６０
℃以上の温度で接触を行うと補体Ｃ３ｃが変性する危険
性が高まる。なお、１５℃以上の温度で接触を行うと微
生物の繁殖が著しくなるので、乳質原料を大量に使用す
る場合は、１５℃未満で接触を行うことが望ましい。陽
イオン交換体は乳質原料１ｋｇに対して０．１〜１００
ｇを目安に用い、１５分間〜１５時間程度、陽イオン交
換体と乳質原料を接触させる。The method for contacting the milk material containing the complement C3c with the cation exchanger includes a method of contacting in a tank, a method of passing the milk material through a column packed with the cation exchanger, and a rotary column reaction. Method using a container
No. 138295 gazette] can be used, but when a large amount of complement C3c is prepared, a method using a rotary column reactor is particularly efficient. There are no particular restrictions on the temperature conditions for contacting the dairy raw material containing complement C3c with the cation exchanger, but it is usually desirable to perform it within the range of 4 ° C to 60 ° C. Freezing of the dairy raw material when contacted at a temperature below 4 ° C
Problems such as increased viscosity and fat separation occur. Also, 60
Contacting at a temperature of ℃ or higher increases the risk of denaturation of complement C3c. If the contact is carried out at a temperature of 15 ° C or higher, the growth of microorganisms will be remarkable. Therefore, when a large amount of dairy raw material is used, it is desirable to carry out the contact at a temperature lower than 15 ° C. The cation exchanger is 0.1 to 100 for 1 kg of milk material.
Using g as a guide, the cation exchanger and the milk raw material are brought into contact with each other for about 15 minutes to 15 hours.
【００１２】陽イオン交換体に吸着させた補体Ｃ３ｃの
溶出は、次のような手順で行う。まず、好ましくは、イ
オン強度が０．１５未満でｐＨが２〜１０の溶液で補体
Ｃ３ｃを吸着させた陽イオン交換体を洗浄する。この
際、洗浄に用いる溶液のイオン強度が０．１５以上にな
ると補体Ｃ３ｃが溶出してしまう危険性が高まり、ま
た、ｐＨが２未満あるいは１０を越えると補体Ｃ３ｃが
変性する危険性が高まる。この洗浄に用いる溶液として
は、水に食塩や塩化カリウムなどの塩を溶解してイオン
強度を０．１５未満に調整した溶液、あるいは、酢酸緩
衝液、クエン酸緩衝液などの有機酸緩衝液やグリシン、
ジメチルグルタル酸緩衝液などの緩衝液、さらには、リ
ン酸緩衝液などを挙げることができる。なお、この洗浄
に先立って、水あるいは温湯で予備洗浄を行っておく
と、洗浄に使用する溶液の液量を節約することができる
ので、コスト面で有利となる。Elution of complement C3c adsorbed on the cation exchanger is carried out by the following procedure. First, preferably, the cation exchanger having adsorbed complement C3c is washed with a solution having an ionic strength of less than 0.15 and a pH of 2 to 10. At this time, if the ionic strength of the solution used for washing is 0.15 or more, the risk of complement C3c elution increases, and if the pH is less than 2 or more than 10, the risk of denaturation of complement C3c increases. Increase. The solution used for this washing is a solution in which salt such as sodium chloride or potassium chloride is dissolved in water to adjust the ionic strength to less than 0.15, or an organic acid buffer solution such as an acetate buffer solution or a citrate buffer solution. glycine,
A buffer solution such as a dimethyl glutarate buffer solution, and a phosphate buffer solution may be mentioned. It should be noted that pre-washing with water or warm water prior to this washing can save the amount of the solution used for washing, which is advantageous in terms of cost.
【００１３】次に、イオン強度が０．１５以上、好まし
くは０．３〜２．０でｐＨが２〜１０、好ましくは４〜
８の溶液で補体Ｃ３ｃを陽イオン交換体から溶出させ
る。この際、溶出に用いる溶液のイオン強度が０．１５
未満では補体Ｃ３ｃの溶出量が少なくなり、また、この
溶液のｐＨが２未満あるいは１０を越えると補体Ｃ３ｃ
が変性する危険性が高まる。この溶出に用いることので
きる溶液としては、水に食塩や塩化カリウムなどの塩を
溶解してイオン強度を０．１５以上に調整した溶液、あ
るいは、クエン酸緩衝液、酢酸緩衝液、マレイン酸緩衝
液などの有機酸緩衝液、リン酸緩衝液、イミダゾール緩
衝液、重炭酸ナトリウム緩衝液、ホウ酸緩衝液、トリス
緩衝液などを挙げることができる。Next, the ionic strength is 0.15 or more, preferably 0.3 to 2.0, and the pH is 2 to 10, preferably 4 to.
Elute complement C3c from the cation exchanger with the solution of 8. At this time, the ionic strength of the solution used for elution was 0.15
When the pH is less than 2, the elution amount of complement C3c becomes small, and when the pH of this solution is less than 2 or more than 10, the complement C3c becomes less.
The risk of degeneration increases. The solution that can be used for this elution is a solution in which salt such as sodium chloride or potassium chloride is dissolved in water to adjust the ionic strength to 0.15 or more, or a citrate buffer solution, an acetate buffer solution, a maleate buffer solution. Examples thereof include organic acid buffers such as liquids, phosphate buffers, imidazole buffers, sodium bicarbonate buffers, borate buffers, and Tris buffers.
【００１４】このようにして得られた補体Ｃ３ｃ含有画
分については、必要に応じて濃縮、脱塩、乾燥などの処
理を行う。濃縮は、真空濃縮法、限外濾過法、逆浸透圧
濾過法などの処理方法を用いることができる。また、脱
塩は、限外濾過膜、透析膜、電気透析膜、あるいはイオ
ン交換樹脂、ゲル濾過用担体などを用いて行うことがで
きる。さらに、乾燥は、凍結乾燥法や噴霧乾燥法などの
処理方法を用いることができる。なお、この場合の補体
Ｃ３ｃの純度は３０％以上となる。The complement C3c-containing fraction thus obtained is optionally subjected to treatments such as concentration, desalting, and drying. For the concentration, a treatment method such as a vacuum concentration method, an ultrafiltration method or a reverse osmosis filtration method can be used. Further, desalting can be carried out using an ultrafiltration membrane, a dialysis membrane, an electrodialysis membrane, an ion exchange resin, a gel filtration carrier or the like. Further, for the drying, a processing method such as a freeze drying method or a spray drying method can be used. The purity of complement C3c in this case is 30% or more.
【００１５】このようにして得られた補体Ｃ３ｃ含有画
分を病原菌付着阻止能を有する物質として飲食品、医
薬、化粧品、飼料などに添加することもできるが、この
補体Ｃ３ｃ含有画分中には、免疫グロブリン、カゼイン
残渣、ラクトパーオキシダーゼなどの不純物が存在する
ので、さらに高純度の補体Ｃ３ｃを得るには、これらの
不純物を除去するために陰イオン交換体を用いて処理を
行うとよい。The complement C3c-containing fraction thus obtained can be added to foods and drinks, pharmaceuticals, cosmetics, feeds, etc. as a substance having an ability to inhibit the attachment of pathogenic bacteria. Contain impurities such as immunoglobulin, casein residue, and lactoperoxidase. Therefore, in order to obtain higher-purity complement C3c, treatment with an anion exchanger is performed to remove these impurities. Good.
【００１６】陰イオン交換体に接触させる際のｐＨは２
以上１０以下、好ましくはｐＨ４以上８以下に調整す
る。ｐＨが２未満の場合、あるいはｐＨが１０を越える
場合、補体Ｃ３ｃは陰イオン交換体に殆ど吸着しない
し、さらに、強アルカリ条件下で接触を行うと補体Ｃ３
ｃが変性する危険性も高まる。なお、吸着を効率的に行
うためには、補体Ｃ３ｃ含有画分のイオン強度を０．１
以下に調整しておくことが好ましい。The pH at the time of contact with the anion exchanger is 2
The pH is adjusted to 10 or more, preferably pH 4 or more and 8 or less. If the pH is less than 2 or more than 10, the complement C3c hardly adsorbs to the anion exchanger, and if contact is performed under strong alkaline conditions, complement C3c
The risk of degeneration of c also increases. For efficient adsorption, the ionic strength of the complement C3c-containing fraction should be 0.1
It is preferable to make the following adjustments.
【００１７】陰イオン交換体に接触させる補体Ｃ３ｃ含
有画分としては、陽イオン交換体処理によって得られた
補体Ｃ３ｃ含有画分を濃縮・脱塩したもの、あるいは補
体Ｃ３ｃ含有画分の乾燥粉末を水または緩衝液で溶解し
たものなどを用いる。補体Ｃ３ｃ含有画分の乾燥粉末を
溶解するのに用いることのできる緩衝液は、特に限定さ
れないが、クエン酸緩衝液、酢酸緩衝液、マレイン酸緩
衝液などの有機酸緩衝液、リン酸緩衝液、イミダゾール
緩衝液、重炭酸ナトリウム緩衝液、ホウ酸緩衝液、トリ
ス緩衝液などを挙げることができる。As the complement C3c-containing fraction to be brought into contact with the anion exchanger, the complement C3c-containing fraction obtained by the cation exchanger treatment is concentrated and desalted, or the complement C3c-containing fraction is obtained. A dry powder dissolved in water or a buffer is used. The buffer solution that can be used to dissolve the dry powder of the complement C3c-containing fraction is not particularly limited, but may be an organic acid buffer solution such as a citrate buffer solution, an acetate buffer solution, a maleate buffer solution, or a phosphate buffer solution. Liquid, imidazole buffer, sodium bicarbonate buffer, borate buffer, Tris buffer and the like.
【００１８】用いる陰イオン交換体としては、架橋型の
多糖類、セルロース、アクリルアミド樹脂などの高分子
にアミノエチル基、第４級アミノエチル基、４級アンモ
ニウム基などを導入したもの、あるいは、高分子と交換
基の間にスペーサーを導入したものなどを挙げることが
できる。具体的には、ジエチルアミノエチル基を導入し
たＤＥＡＥ−セルロファイン（生化学工業（株）製）や
第４級アミノエチル基を導入したＱＡＥ−セファデック
ス（ファルマシア社製）を用いるとよい。The anion exchanger to be used may be one obtained by introducing an aminoethyl group, a quaternary aminoethyl group, a quaternary ammonium group or the like into a polymer such as a cross-linking type polysaccharide, cellulose, acrylamide resin, or the like. Examples thereof include a spacer introduced between the molecule and the exchange group. Specifically, DEAE-Cellulofine (manufactured by Seikagaku Corporation) having a diethylaminoethyl group introduced therein and QAE-Sephadex (manufactured by Pharmacia) having a quaternary aminoethyl group introduced therein may be used.
【００１９】補体Ｃ３ｃ含有画分を陰イオン交換体と接
触させる方法としては、タンク内で接触させる方法、陰
イオン交換体を充填したカラムに通液する方法、回転型
カラム反応器を用いる方法〔特開平２−１３８２９５号
公報〕など、従来より知られている方法を用いることが
できる。補体Ｃ３ｃ含有画分を陰イオン交換体と接触さ
せる際の温度条件については、特に制限はないが、通常
４℃〜４０℃の範囲で行うことが望ましい。４０℃以上
の温度で接触を行うと微生物の繁殖による汚染が著しく
なる。The complement C3c-containing fraction may be contacted with the anion exchanger by contacting in a tank, passing through a column packed with anion exchanger, or using a rotary column reactor. A conventionally known method such as [JP-A-2-138295] can be used. The temperature condition for bringing the complement C3c-containing fraction into contact with the anion exchanger is not particularly limited, but is usually preferably in the range of 4 ° C to 40 ° C. If the contact is performed at a temperature of 40 ° C. or higher, the contamination due to the growth of microorganisms becomes remarkable.
【００２０】陰イオン交換体に吸着させた補体Ｃ３ｃの
溶出は、次のような手順で行う。まず、イオン強度が
０．３未満でｐＨが２〜１０の溶液で補体Ｃ３ｃを吸着
させた陰イオン交換体を洗浄する。この際、洗浄に用い
る溶液のイオン強度が０．３以上になると補体Ｃ３ｃが
溶出してしまう危険性があり、また、ｐＨが２未満ある
いは１０を越えると補体Ｃ３ｃが変性する危険性が高ま
る。したがって、好ましくは、イオン強度が０．２５以
下でｐＨが４〜８の溶液で陰イオン交換体を洗浄すると
良い。洗浄に用いる溶液としては、水に食塩や塩化カリ
ウムなどの塩を溶解してイオン強度を０．３未満に調整
した溶液、あるいは、酢酸緩衝液、クエン酸緩衝液など
の有機酸緩衝液やグリシン、ジメチルグルタル酸緩衝液
などの緩衝液、さらには、リン酸緩衝液などを挙げるこ
とができる。なお、この洗浄に先立って、水あるいは温
湯で予備洗浄を行っておくと洗浄に使用する溶液の液量
を節約することができるので、コスト面で有利となる。Elution of complement C3c adsorbed on the anion exchanger is carried out by the following procedure. First, the anion exchanger having adsorbed complement C3c is washed with a solution having an ionic strength of less than 0.3 and a pH of 2 to 10. At this time, there is a risk that the complement C3c will be eluted if the ionic strength of the solution used for washing is 0.3 or more, and there is a risk that the complement C3c will be denatured if the pH is less than 2 or more than 10. Increase. Therefore, it is preferable to wash the anion exchanger with a solution having an ionic strength of 0.25 or less and a pH of 4 to 8. As a solution used for washing, a solution in which salt such as sodium chloride or potassium chloride is dissolved in water to adjust the ionic strength to less than 0.3, or an organic acid buffer such as acetate buffer or citrate buffer or glycine is used. , A buffer solution such as dimethyl glutarate buffer solution, and a phosphate buffer solution. It should be noted that pre-washing with water or warm water prior to this washing can save the amount of the solution used for washing, which is advantageous in terms of cost.
【００２１】次に、イオン強度が０．３以上、好ましく
は０．４〜１．０でｐＨが２〜１０、好ましくは４〜８
の溶液で補体Ｃ３ｃを陰イオン交換体から溶出させる。
この際、溶出に用いる溶液のイオン強度が０．３未満で
は補体Ｃ３ｃの溶出量が少なくなり、また、この溶液の
ｐＨが２未満あるいは１０を越えると補体Ｃ３ｃが変性
する危険性が高まる。この溶出に用いることのできる溶
液としては、水に食塩や塩化カリウムなどの塩を溶解し
てイオン強度を０．３以上に調整した溶液、あるいは、
クエン酸緩衝液、酢酸緩衝液、マレイン酸緩衝液などの
有機酸緩衝液、リン酸緩衝液、イミダゾール緩衝液、重
炭酸ナトリウム緩衝液、ホウ酸緩衝液、トリス緩衝液な
どを挙げることができる。Next, the ionic strength is 0.3 or more, preferably 0.4 to 1.0, and the pH is 2 to 10, preferably 4 to 8.
Complement C3c is eluted from the anion exchanger with the solution of.
At this time, if the ionic strength of the solution used for elution is less than 0.3, the elution amount of complement C3c will be small, and if the pH of this solution is less than 2 or more than 10, the risk of denaturation of complement C3c will increase. . As the solution that can be used for this elution, a solution in which salt such as sodium chloride or potassium chloride is dissolved in water to adjust the ionic strength to 0.3 or more, or
Examples thereof include citrate buffer, acetate buffer, maleic acid buffer and other organic acid buffers, phosphate buffers, imidazole buffers, sodium bicarbonate buffers, borate buffers, Tris buffers and the like.
【００２２】このようにして得られた補体Ｃ３ｃ含有画
分については、必要に応じて濃縮、脱塩、乾燥などの処
理を行う。濃縮は、真空濃縮法、限外濾過法、逆浸透圧
濾過法などの処理方法を用いることができる。また、脱
塩は、限外濾過膜、透析膜、電気透析膜、あるいはイオ
ン交換樹脂、ゲル濾過用担体などを用いて行うことがで
きる。さらに、乾燥は、凍結乾燥法や噴霧乾燥法などの
処理方法を用いることができる。なお、この場合の補体
Ｃ３ｃの純度は６０％以上となる。The complement C3c-containing fraction thus obtained is subjected to treatments such as concentration, desalting and drying, if necessary. For the concentration, a treatment method such as a vacuum concentration method, an ultrafiltration method or a reverse osmosis filtration method can be used. Further, desalting can be carried out using an ultrafiltration membrane, a dialysis membrane, an electrodialysis membrane, an ion exchange resin, a gel filtration carrier or the like. Further, for the drying, a processing method such as a freeze drying method or a spray drying method can be used. The purity of complement C3c in this case is 60% or more.
【００２３】このようにして得られた補体Ｃ３ｃ含有画
分を病原菌付着阻止能を有する物質として飲食品、医
薬、化粧品、飼料などに添加することもできるが、常法
に従い、さらに精製して高純度の補体Ｃ３ｃを得ること
もできる。The complement C3c-containing fraction thus obtained can be added to foods, drinks, pharmaceuticals, cosmetics, feeds, etc. as a substance having an ability to inhibit the attachment of pathogenic bacteria, but it is further purified by a conventional method. It is also possible to obtain high-purity complement C3c.
【００２４】なお、精製された補体Ｃ３ｃについて、ア
ミノ酸配列を分析したところ、Ｎ末端側アミノ酸１５残
基は、Ａｓｎ−Ｐｒｏ−Ｍｅｔ−Ｔｙｒ−Ｓｅｒ−Ｍｅ
ｔ−Ｉｌｅ−Ｔｈｒ−Ｐｒｏ−Ａｓｎ−Ｉｌｅ−Ｌｅｕ
−Ａｒｇ−Ｌｅｕ−Ｇｌｕ−であり、ヒト補体Ｃ３のＮ
末端側アミノ酸１３残基と一致した。また、分子量もヒ
ト補体Ｃ３ｃの１４０ｋＤａと近似の値を示し、市販の
ウシ補体Ｃ３ｃ抗体を用いたウェスタンブロッティング
により、強い発色が認められた。次に本発明を実施例を
挙げて具体的に説明する。When the amino acid sequence of the purified complement C3c was analyzed, 15 amino acid residues on the N-terminal side were found to have Asn-Pro-Met-Tyr-Ser-Me.
t-Ile-Thr-Pro-Asn-Ile-Leu
-Arg-Leu-Glu- and N of human complement C3.
It coincided with 13 residues of the terminal amino acid. Further, the molecular weight also showed a value close to 140 kDa of human complement C3c, and strong color development was recognized by Western blotting using a commercially available bovine complement C3c antibody. Next, the present invention will be specifically described with reference to examples.
【００２５】[0025]
【実施例１】スルホン化キトパール（富士紡績（株）
製）１．５Ｌを充填した回転型カラム反応器（東京理化
器械（株）製）に３００ｋｇの生脱脂乳を２００Ｌ／ｈ
の流速で通液した。通液後、回転型カラム反応器を水で
十分洗浄した後、０．１Ｍ塩化カリウムを含む０．０１
Ｍクエン酸−クエン酸ナトリウム緩衝液（ｐＨ５．０）
で洗浄した。補体Ｃ３ｃの溶出は０．８Ｍ塩化カリウム
を含むクエン酸−クエン酸ナトリウム緩衝液（ｐＨ５．
０）で行った。そして、分画分子量５０，０００の限外
濾過膜を用いて濃縮、脱塩した。この段階で補体Ｃ３ｃ
の純度を電気泳動によって求めたところ、４１％であっ
た。[Example 1] Sulfonated chitopearl (Fuji Spinning Co., Ltd.)
200 L / h of 300 kg of raw skim milk in a rotary column reactor (manufactured by Tokyo Rika Kikai Co., Ltd.) filled with 1.5 L
The liquid was passed at a flow rate of. After passing the solution, the rotary column reactor was thoroughly washed with water, and then 0.01 M containing 0.1 M potassium chloride was added.
M citric acid-sodium citrate buffer (pH 5.0)
Washed with. The elution of complement C3c was carried out using a citric acid-sodium citrate buffer solution (pH 5.
0). Then, it was concentrated and desalted using an ultrafiltration membrane having a cut-off molecular weight of 50,000. Complement C3c at this stage
The purity of the product was 41% as determined by electrophoresis.
【００２６】[0026]
【実施例２】実施例１で得られた補体Ｃ３ｃを含む溶出
画分５００ｍｌを水酸化ナトリウムでｐＨ７．０に調整
し、０．０１Ｍリン酸緩衝液（ｐＨ７．０）で平衡化し
たＤＥＡＥ−セルロファインＡＭ（生化学工業（株）
製）を充填したカラムに通液した。通液後、カラムを
０．１５Ｍ塩化カリウムを含む０．０１Ｍリン酸緩衝液
（ｐＨ７．０）で洗浄した。補体Ｃ３ｃの溶出は、０．
５Ｍ塩化カリウムを含む０．０１Ｍリン酸緩衝液（ｐＨ
７．０）で行った。そして、セファデックスＧ−２５
（ファルマシア社製）を用いて脱塩し、凍結乾燥を行っ
て１ｇの粉末を得た。こうして得られた補体Ｃ３ｃの純
度を電気泳動によって求めたところ、７８％であった。Example 2 500 ml of the eluted fraction containing complement C3c obtained in Example 1 was adjusted to pH 7.0 with sodium hydroxide and equilibrated with 0.01 M phosphate buffer (pH 7.0). -Cellulofine AM (Seikagaku Corporation)
The product was passed through a column filled with After passing the solution, the column was washed with a 0.01 M phosphate buffer solution (pH 7.0) containing 0.15 M potassium chloride. The elution of complement C3c was 0.
0.01M phosphate buffer containing 5M potassium chloride (pH
7.0). And Sephadex G-25
(Manufactured by Pharmacia), desalted and freeze-dried to obtain 1 g of powder. The purity of the complement C3c thus obtained was 78% as determined by electrophoresis.
【００２７】[0027]
【実施例３】実施例２で得られた補体Ｃ３ｃ粉末を５０
ｍＭリン酸緩衝液（ｐＨ７．０）２５ｍｌに溶解し、あ
らかじめ５０ｍＭリン酸緩衝液（ｐＨ７．０）で平衡化
したプロテインＧアフィニティーカラム（ファルマシア
社製、直径１．５ｃｍ×１１．５ｃｍ、容量２０ｍｌ）
に通液し、２ｍｌ／分の流速で５０ｍＭリン酸緩衝液
（ｐＨ７．０）を通液して非吸着画分を回収した。この
非吸着画分をさらに５０ｍＭリン酸緩衝液（ｐＨ７．
０）で平衡化したプロテインＧアフィニティーカラム
（ファルマシア社製、直径１．５ｃｍ×１１．５ｃｍ、
容量２０ｍｌ）に通液し、２ｍｌ／分の流速で５０ｍＭ
リン酸緩衝液（ｐＨ７．０）を通液して非吸着画分を回
収した。この二度のアフィニティークロマトグラフィー
によって、免疫グロブリンＧが完全に除去された。得ら
れた非吸着画分４８ｍｌを分画分子量１０，０００の限
外濾過膜（アドバンテック社製）で１５ｍｌとなるまで
濃縮し、０．５Ｍ塩化ナトリウムを含む１０ｍＭイミダ
ゾール−塩酸緩衝液（ｐＨ６．５）で平衡化したハイロ
ード１６／６０スーパーデックス７５（ファルマシア社
製、直径１．６ｃｍ×６０ｃｍ）に５ｍｌずつ通液し、
０．５Ｍ塩化ナトリウムを含む１０ｍＭイミダゾール−
塩酸緩衝液（ｐＨ６．５）を０．４ｍｌ／分の流速で通
液してメインピークを分取し、さらにハイロード１６／
６０スーパーデックス７５（ファルマシア社製、直径
１．６ｃｍ×６０ｃｍ）で処理することによって補体Ｃ
３ｃを得た。得られた補体Ｃ３ｃを脱イオン水に対して
透析した後、凍結乾燥を行い、重量を測定したところ
０．６ｇであった。また、この補体Ｃ３ｃの純度を電気
泳動によって求めたところ、９８％であった。Example 3 The complement C3c powder obtained in Example 2 was added to 50 parts.
Protein G affinity column (Pharmacia, diameter 1.5 cm x 11.5 cm, volume 20 ml) dissolved in 25 ml of mM phosphate buffer (pH 7.0) and equilibrated with 50 mM phosphate buffer (pH 7.0) in advance. )
Then, 50 mM phosphate buffer (pH 7.0) was passed at a flow rate of 2 ml / min to collect the non-adsorbed fraction. This non-adsorbed fraction was further added to 50 mM phosphate buffer (pH 7.
0) equilibrated with protein G affinity column (Pharmacia, diameter 1.5 cm × 11.5 cm,
Volume of 20 ml) and 50 mM at a flow rate of 2 ml / min.
A non-adsorbed fraction was recovered by passing a phosphate buffer (pH 7.0). Immunoglobulin G was completely removed by this double affinity chromatography. The resulting non-adsorbed fraction (48 ml) was concentrated to 15 ml with an ultrafiltration membrane (Advantech) having a molecular weight cutoff of 10,000, and 10 mM imidazole-hydrochloric acid buffer solution (pH 6.5) containing 0.5 M sodium chloride. 5 ml each was passed through High Road 16/60 Superdex 75 (Pharmacia, diameter 1.6 cm x 60 cm) equilibrated with),
10 mM imidazole containing 0.5 M sodium chloride
A hydrochloric acid buffer solution (pH 6.5) is passed at a flow rate of 0.4 ml / min to separate the main peak, and then the high load 16 /
Complement C by treatment with 60 Superdex 75 (Pharmacia, diameter 1.6 cm x 60 cm)
3c was obtained. The obtained complement C3c was dialyzed against deionized water, freeze-dried, and the weight was measured to be 0.6 g. The purity of this complement C3c was 98% as determined by electrophoresis.
【００２８】[0028]
【実施例４】０．０１Ｍイミダゾール−塩酸緩衝液（ｐ
Ｈ６．５）で平衡化したＳ−セファロース ファースト
フロー（ファルマシア社製）１０ｍｌを充填したカラム
（直径２ｍｍ×１６ｃｍ）にミルパップ（安積濾紙
（株）製）で濾過したチーズホエー１ｌを３ｍｌ／分の
流速で通液した。通液後、０．１Ｍ塩化ナトリウムを含
む０．０１Ｍイミダゾール−塩酸緩衝液（ｐＨ６．５）
で洗浄し、０．３５Ｍ塩化ナトリウムを含む０．０１Ｍ
イミダゾール−塩酸緩衝液（ｐＨ６．５）で溶出した。
得られた２０ｍｌの溶出画分について、４℃で１５時
間、０．０１Ｍイミダゾール−塩酸緩衝液（ｐＨ６．
５）に対する透析を行った。そして、この透析物を、
０．０１Ｍイミダゾール−塩酸緩衝液（ｐＨ５．５）で
平衡化したＱ−セファロース ファーストフロー（ファ
ルマシア社製）３ｍｌを充填したカラム（直径０．８ｃ
ｍ×６ｃｍ）に２ｍｌ／分の流速で通液した。通液後、
０．１Ｍ塩化ナトリウムを含む０．０１Ｍイミダゾール
−塩酸緩衝液（ｐＨ５．５）で洗浄し、０．４Ｍ塩化ナ
トリウムを含む０．０１Ｍイミダゾール−塩酸緩衝液
（ｐＨ５．５）で溶出した。この溶出画分を分画分子量
１０，０００の限外濾過膜で脱塩し、凍結乾燥を行っ
た。得られた補体Ｃ３ｃの純度を電気泳動によって求め
たところ、８２％であった。また、回収量は２ｍｇであ
った。Example 4 0.01M imidazole-hydrochloric acid buffer solution (p
H-6.5) equilibrated S-Sepharose Fast Flow (Pharmacia) 10 ml packed column (diameter 2 mm x 16 cm) with mill pap (Azumi Filter Paper Co., Ltd.) cheese whey 1 l 3 ml / min. The liquid was passed at a flow rate. After passing the solution, 0.01 M imidazole-hydrochloric acid buffer solution (pH 6.5) containing 0.1 M sodium chloride
Washed with 0.01M containing 0.35M sodium chloride
Elution was carried out with an imidazole-hydrochloric acid buffer solution (pH 6.5).
About 20 ml of the obtained elution fraction, 0.01 M imidazole-hydrochloric acid buffer solution (pH 6.
Dialysis against 5) was performed. And this dialysate,
Column packed with 3 ml of Q-Sepharose Fast Flow (Pharmacia) equilibrated with 0.01 M imidazole-hydrochloric acid buffer (pH 5.5) (diameter 0.8 c
m × 6 cm) at a flow rate of 2 ml / min. After passing the liquid
It was washed with 0.01 M imidazole-hydrochloric acid buffer solution (pH 5.5) containing 0.1 M sodium chloride, and eluted with 0.01 M imidazole-hydrochloric acid buffer solution (pH 5.5) containing 0.4 M sodium chloride. The eluted fraction was desalted with an ultrafiltration membrane having a molecular weight cut off of 10,000 and freeze-dried. The purity of the obtained complement C3c was 82% as determined by electrophoresis. The recovered amount was 2 mg.
【００２９】[0029]
【発明の効果】本発明の方法によると、乳質原料から病
原菌付着阻止能を有する補体Ｃ３ｃを工業的規模で大量
かつ安価に製造できる。したがって、飲食品、医薬、化
粧品、飼料などに病原菌付着阻止能を付与するための素
材として、補体Ｃ３ｃを安定的に供給することができ
る。以下に、本発明の方法によって得られた補体Ｃ３ｃ
の病原菌付着阻止効果と飲食品、医薬、化粧品、飼料へ
の応用例について示す。EFFECT OF THE INVENTION According to the method of the present invention, complement C3c having the ability to inhibit the attachment of pathogenic bacteria can be produced on a large scale and at low cost from a milk material on an industrial scale. Therefore, the complement C3c can be stably supplied as a material for imparting the pathogenic bacterium adhesion-inhibiting ability to foods, drinks, medicines, cosmetics, feeds and the like. Below, complement C3c obtained by the method of the present invention
The following is a description of the effect of inhibiting the attachment of pathogenic bacteria and application examples to food and drink, pharmaceuticals, cosmetics, and feed.
【００３０】[0030]
【試験例１】実施例３で得られた補体Ｃ３ｃ及びこの補
体Ｃ３ｃをプロナーゼ処理して得られた補体Ｃ３ｃ加水
分解物の病原性大腸菌に対する付着阻止効果について、
ヒト小腸細胞ＪＴＣ−１７を用いて調べた。東京医科大
学より分譲を受けたヒト小腸細胞ＪＴＣ−１７を、５％
牛胎児血清を含むダルベッコ変法イーグル培地（５％Ｆ
ＣＳ／ＤＭＥＭ）にて２５ｃｍ2 の底面積をもつ培養フ
ラスコに３７℃、ＣＯ2 濃度５％で培養した。細胞がフ
ラスコの底面一面に増殖したら、培養を止め、常法通り
トリプシン（２００Ｕ／ｍｌ）含有リン酸緩衝生理食塩
水（ＰＢＳ）１ｍｌにて細胞を剥がし取り、１，０００
ｒｐｍで５分間遠心分離することで細胞を集めた。上清
を吸い出し、新たに５％ＦＣＳ／ＤＭＥＭ８ｍｌに細胞
を懸濁して培養した。この細胞懸濁液各１ｍｌを１０ｍ
ｌポリスチレンチューブ（ファルコン社製、２０９５）
に入れ３７℃、３日間培養した。TEST EXAMPLE 1 Regarding the effect of inhibiting complement C3c obtained in Example 3 and the complement C3c hydrolyzate obtained by treating this complement C3c with pronase against pathogenic Escherichia coli,
It was examined using human small intestinal cells JTC-17. 5% of human small intestinal cell JTC-17, which was purchased from Tokyo Medical University
Dulbecco's modified Eagle medium containing fetal bovine serum (5% F
(CS / DMEM) was cultured in a culture flask having a bottom area of 25 cm 2 at 37 ° C. and a CO 2 concentration of 5%. When the cells have grown on the entire bottom surface of the flask, the culture is stopped, and the cells are peeled off with 1 ml of trypsin (200 U / ml) -containing phosphate buffered saline (PBS) as usual, and the cells are removed.
The cells were collected by centrifugation at rpm for 5 minutes. The supernatant was sucked out, and the cells were newly suspended in 8 ml of 5% FCS / DMEM and cultured. 10 ml of each 1 ml of this cell suspension
l Polystyrene tube (Falcon, 2095)
The cells were placed in a plate and cultured at 37 ° C. for 3 days.
【００３１】一方、都立衛生研究所より分譲を受けた病
原性大腸菌Ｈ１０４９７株、Ｐｂ１７６株をそれぞれ１
白金耳取り、普通ブイヨン培地４ｍｌに３７℃で一夜培
養した。培養後、３，０００ｒｐｍ、１０分間、遠心分
離することにより集菌し、これをｐＨ８に調整したＰＢ
Ｓで３回洗浄した。その後、病原性大腸菌をｐＨ８に調
整したＰＢＳ１ｍｌに懸濁し、フルオレッセインイソチ
オシアネート（ＦＩＴＣ）（シグマ社製）１ｍｇを加え
てよく溶解し、４℃で３時間ゆっくり攪拌しながら反応
させ、病原性大腸菌をＦＩＴＣにてラベルした。ラベル
を終えた病原性大腸菌は、３，０００ｒｐｍ、１０分間
の遠心分離にて集菌し、ＰＢＳにて３回洗浄した後、３
ｍｌのＰＢＳに懸濁した。このようにして調製したＦＩ
ＴＣラベルの病原性大腸菌懸濁液４００μｌに、本発明
に係る各試料のＰＢＳ溶液２００μｌを混合し、３７℃
で１時間インキュベートした。On the other hand, 1 each of pathogenic Escherichia coli H10497 strain and Pb176 strain received from Tokyo Metropolitan Institute of Health
Platinum loops were taken and cultured in 4 ml of normal broth medium at 37 ° C. overnight. After culturing, the cells were collected by centrifugation at 3,000 rpm for 10 minutes, and PB adjusted to pH 8
Wash with S three times. Then, the pathogenic Escherichia coli was suspended in 1 ml of PBS adjusted to pH 8, 1 mg of fluorescein isothiocyanate (FITC) (manufactured by Sigma) was added and well dissolved. E. coli was labeled with FITC. Labeled pathogenic E. coli was collected by centrifugation at 3,000 rpm for 10 minutes, washed 3 times with PBS, and then 3
Suspended in ml PBS. FI thus prepared
400 μl of TC-labeled pathogenic Escherichia coli suspension was mixed with 200 μl of PBS solution of each sample according to the present invention, and the mixture was incubated at 37 ° C.
And incubated for 1 hour.
【００３２】そして、３日間の培養を終えたＪＴＣ１７
細胞を１，０００ｒｐｍで５分間遠心分離し、上清の培
地を除き、さらにＰＢＳで３回洗浄し、これに上述の病
原性大腸菌と試料を混合してインキュベートした溶液各
６００μｌを加え、３７℃で３０分間インキュベートし
た。さらに、７５０ｒｐｍ、５分間遠心分離し、細胞の
みを沈殿させ、細胞に付着せず、上清に残った病原性大
腸菌を吸い出した。さらに、ＰＢＳにて細胞を２回洗浄
した。ＰＢＳで洗浄した細胞に２００Ｕトリプシン／Ｐ
ＢＳ９００μｌを加え、激しく攪拌した。これに０．１
％ＳＤＳ１００μｌ、１ｍｌの脱イオン交換水を加えて
激しく攪拌し、３０分間静置することにより細胞を溶解
した。この溶液を蛍光分光光度計（日立フルオレッセン
ススペクトロメーターＦ−３０００）にて励起波長４９
０ｎｍ、蛍光波長５２０ｎｍで相対蛍光強度を測定し、
蛍光ラベルした病原性大腸菌のＪＴＣ−１７細胞への付
着を評価した。Then, JTC17 which had been cultured for 3 days
The cells were centrifuged at 1,000 rpm for 5 minutes, the supernatant medium was removed, the cells were further washed 3 times with PBS, and 600 µl of a solution prepared by mixing the above-mentioned pathogenic Escherichia coli and the sample and incubating them was added to each well, and the temperature was 37 ° C. And incubated for 30 minutes. Further, the cells were centrifuged at 750 rpm for 5 minutes to precipitate only the cells, and the pathogenic Escherichia coli that did not adhere to the cells and remained in the supernatant was sucked out. Furthermore, the cells were washed twice with PBS. 200U trypsin / P was added to the cells washed with PBS.
900 μl of BS was added and vigorously stirred. 0.1 for this
% SDS (100 μl) and 1 ml of deionized exchanged water were added, and the mixture was vigorously stirred and left to stand for 30 minutes to lyse the cells. Excitation wavelength of this solution was 49 with a fluorescence spectrophotometer (Hitachi Fluorescence Spectrometer F-3000).
Relative fluorescence intensity is measured at 0 nm and fluorescence wavelength of 520 nm,
Adhesion of fluorescently labeled pathogenic E. coli to JTC-17 cells was evaluated.
【００３３】なお、試料無添加（ブランク）時の相対蛍
光強度を付着率１００％、蛍光ラベルした病原性大腸菌
のポリスチレンチューブへの非特異的な吸着（ポリスチ
レンチューブ内に検体と同じ濃度の蛍光ラベルした病原
性大腸菌を入れ、検体と同条件で測定した相対蛍光強
度）を付着率０％とし、各試料を添加した際の付着阻止
率を算出し、表１に示した。補体Ｃ３ｃを含むいずれの
試料も、病原性大腸菌のヒト小腸細胞（ＪＴＣ−１７）
への付着は阻止され、０．１％でも効果が期待される。The relative fluorescence intensity when no sample was added (blank) was 100%, and the non-specific adsorption of the fluorescently labeled pathogenic Escherichia coli to the polystyrene tube (fluorescent label having the same concentration as the sample in the polystyrene tube) was used. Adhesion rate of each sample was calculated by setting the relative fluorescence intensity (measured under the same conditions as the sample) of 0% to 100%, and the adhesion inhibition rate when each sample was added is shown in Table 1. All samples containing complement C3c are human intestinal cells of pathogenic E. coli (JTC-17)
Adhesion to is prevented, and even 0.1% is expected to be effective.
【表１】 ──────────────────────────────────── 付着阻止率（％） 試 料 ─────────────── Ｈ１０４０７ Ｐｂ１７６ ──────────────────────────────────── ０．１％ｂＣ３ｃ ３４ ４２ １．０％ｂＣ３ｃ ６６ ７８ １．０％ｂＣ３ｃプロナーゼ処理１０分間 ６３ ７７ １．０％ｂＣ３ｃプロナーゼ処理２０分間 ４９ ５８ １．０％ｂＣ３ｃプロナーゼ処理６０分間 ２５ ３１ １．０％ＢＳＡ ３ ４ ──────────────────────────────────── ｂＣ３ｃ：ウシ補体Ｃ３ｃ、ＢＳＡ：ウシ血清アルブミン[Table 1] ──────────────────────────────────── Adhesion inhibition rate (%) Test material ── ───────────── H10407 Pb176 ─────────────────────────────────── -0.1% bC3c 34 42 1.0% bC3c 66 78 1.0% bC3c pronase treatment 10 minutes 63 77 1.0% bC3c pronase treatment 20 minutes 49 58 1.0% bC3c pronase treatment 60 minutes 25 31 1. 0% BSA 34 ──────────────────────────────────── bC3c: Bovine complement C3c, BSA: Bovine serum albumin
【００３４】[0034]
【試験例２】実施例３で得られた補体Ｃ３ｃ及びこの補
体Ｃ３ｃをプロナーゼ処理して得られた補体Ｃ３ｃ加水
分解物のう歯原菌に対する付着阻止効果について、ポリ
スチレンチューブを用いて調べた。３種のストレプトコ
ッカス・ミュータンス（ＡＴＣＣ ２７６０７、ＡＴＣ
Ｃ２５１７５、ＡＴＣＣ ２７３５１）を一白金耳取
り、ブレインハートインフージョン（ＢＨＩ）培地４ｍ
ｌで一晩培養した。その後、３０００ｒｐｍ、１０分間
遠心分離することにより集菌し、上清の培地を吸い出
し、さらにこれをＰＢＳで３回洗浄し、これを６５０ｎ
ｍで吸光度０．８となるようにＰＢＳに懸濁した。この
菌体懸濁液と本発明に係る各試料のＰＢＳ溶液を等量混
合し、１０ｍｌのポリスチレンチューブ（ファルコン社
製、２０９５）に入れ、よく攪拌し、３７℃、３時間イ
ンキュベートした。インキュベート終了後、６５０ｎｍ
で吸光度を測定し、次の式より付着率を求めることによ
って、各試料を添加した際の付着阻止率を算出した。付
着率（％）＝｛（０．４−Ｘ）／０．４｝×１００TEST EXAMPLE 2 The effect of inhibiting complement C3c obtained in Example 3 and the complement C3c hydrolyzate obtained by treating this complement C3c with pronase against dental caries, using a polystyrene tube. Examined. Three Streptococcus mutans (ATCC 27607, ATC
C25175, ATCC 27351), 1 platinum loop, Brain Heart Infusion (BHI) medium 4m
Incubate overnight in 1. Thereafter, the cells were collected by centrifugation at 3000 rpm for 10 minutes, the supernatant medium was sucked out, and this was further washed 3 times with PBS to obtain 650 n.
It was suspended in PBS so that the absorbance was 0.8 at m. Equal amounts of this bacterial cell suspension and the PBS solution of each sample according to the present invention were mixed, placed in a 10 ml polystyrene tube (Falcon, 2095), stirred well, and incubated at 37 ° C. for 3 hours. 650nm after incubation
The absorbance was measured with and the adhesion rate was calculated from the following formula to calculate the adhesion inhibition rate when each sample was added. Adhesion rate (%) = {(0.4-X) /0.4} × 100
【００３５】その結果を表２に示す。なお、試験は全て
２連で行った。補体Ｃ３ｃを含むいずれの試料も、う歯
原菌のポリスチレンチューブへの付着を阻止した。The results are shown in Table 2. All tests were conducted in duplicate. All samples containing complement C3c blocked the attachment of cariogenic bacteria to polystyrene tubes.
【表２】 ──────────────────────────────────── 付着阻止率（％） 試 料 ──────────────────────── ＡＴＣＣ ＡＴＣＣ ＡＴＣＣ ２７６０７ ２５１７５ ２７３５１ ──────────────────────────────────── ０．１％ｂＣ３ｃ １５ ２２ １５ １．０％ｂＣ３ｃ ４６ ４８ ５８ １．０％ｂＣ３ｃ ４１ ４１ ５３ （プロナーゼ処理１０分間） １．０％ｂＣ３ｃ ３４ ３５ ４３ （プロナーゼ処理２０分間） １．０％ｂＣ３ｃ １７ ２１ ２６ （プロナーゼ処理６０分間） １．０％ＢＳＡ ３ ４ ３ ──────────────────────────────────── ｂＣ３ｃ：ウシ補体Ｃ３ｃ、ＢＳＡ：ウシ血清アルブミン[Table 2] ──────────────────────────────────── Adhesion inhibition rate (%) Test material ── ────────────────────── ATCC ATCC ATCC 27607 25175 27351 ─────────────────────── ────────────── 0.1% bC3c 15 22 15 1.0% bC3c 46 48 58 1.0% bC3c 41 41 53 (Pronase treatment 10 minutes) 1.0% bC3c 34 35 43 (Pronase treatment 20 minutes) 1.0% bC3c 17 21 26 (Pronase treatment 60 minutes) 1.0% BSA 3 4 3 ───────────────────── ──────────────── bC3c: Bovine complement C3c, BSA: Bovine serum albumin
【００３６】[0036]
【試験例３】実施例３で得られた補体Ｃ３ｃの病原性大
腸菌腸管付着阻止効果について、無菌マウスを用いて調
べた。無菌マウスＩＣＲを５匹ずつ４群に分け、各群に
以下の試料を毎日０．２ｍｌずつ経口投与した。 Ａ群 ＰＢＳ・０．２ｍｌ Ｂ群 ｂＣ３ｃ・０．１ｍｇ／０．２ｍｌ・ＰＢＳ Ｃ群 ｂＣ３ｃ・０．５ｍｇ／０．２ｍｌ・ＰＢＳ Ｄ群 ＢＳＡ・０．５ｍｇ／０．２ｍｌ・ＰＢＳ なお、各試料は０．２２μｍのフィルターで濾過し、除
菌してから用いた。TEST EXAMPLE 3 The effect of complement C3c obtained in Example 3 on the intestinal adhesion of pathogenic Escherichia coli was examined using sterile mice. Sterile mouse ICR was divided into 4 groups of 5 mice, and 0.2 ml of the following sample was orally administered to each group every day. Group A PBS / 0.2 ml Group B bC3c / 0.1 mg / 0.2 ml / PBS Group C bC3c / 0.5 mg / 0.2 ml / PBS Group D BSA / 0.5 mg / 0.2 ml / PBS Each sample Was filtered through a 0.22 μm filter to remove bacteria and used.
【００３７】一方、病原性大腸菌Ｐｂ１７６株を普通ブ
イヨン培地で培養後、集菌し、ＰＢＳで３回洗浄した。
各試料投与後、１週間目の各マウスに１０4 ＣＦＵ／
０．２ｍｌの病原性大腸菌懸濁液を経口投与し、さらに
１週間各試料を投与し続けた。その後、マウスを無菌的
に解剖し、腸管をホモゲナイズし、その１ｍｌを寒天ゲ
ル平板に接種し、病原性大腸菌の菌数を数えた。 Ａ群 ３．７×１０8 ＣＦＵ Ｂ群 ５．１×１０7 ＣＦＵ Ｃ群 １．９×１０6 ＣＦＵ Ｄ群 ２．１×１０8 ＣＦＵOn the other hand, the pathogenic Escherichia coli Pb176 strain was cultured in a normal broth medium, then the cells were collected and washed 3 times with PBS.
After administration of each sample, 10 4 CFU /
0.2 ml of pathogenic Escherichia coli suspension was orally administered, and each sample was continuously administered for another week. Then, the mouse was aseptically dissected, the intestinal tract was homogenized, and 1 ml thereof was inoculated on an agar gel plate to count the number of pathogenic Escherichia coli. Group A 3.7 × 10 8 CFU Group B 5.1 × 10 7 CFU Group C 1.9 × 10 6 CFU Group D 2.1 × 10 8 CFU
【００３８】このように、Ｂ群及びＣ群ではＡ群に比べ
て有意に病原性大腸菌の菌数が少なく、顕著な付着阻止
効果が示された。さらに、ＢＳＡを投与したＤ群では効
果のないことから、病原性大腸菌の付着阻止効果は補体
Ｃ３ｃの特異的な作用であることが判る。As described above, the numbers of pathogenic Escherichia coli in the groups B and C were significantly smaller than those in the group A, and a remarkable adhesion-inhibiting effect was shown. Further, since there is no effect in the D group to which BSA was administered, it can be seen that the adhesion-inhibiting effect of pathogenic Escherichia coli is a specific action of complement C3c.
【００３９】[0039]
【試験例４】実施例３で得られた補体Ｃ３ｃの虫歯予防
効果について、ウィスターラットを用いて調べた。３週
令ウィスターラットに、全小麦粉６ｇ、ショ糖５６ｇ、
脱脂粉乳２８ｇ、アルファルファ葉粗粉末３ｇ、肝１
ｇ、ビール酵母４ｇ、食塩２ｇからなるショ糖強化食を
投与して飼育した。ラットを３匹ずつ５群に分け、各群
に以下の試料を飲料水として給水ボトルに入れて自由摂
取させた。 Ａ群 脱イオン水 Ｂ群 ０．１％ｂＣ３ｃ Ｃ群 １％ｂＣ３ｃ Ｄ群 １％ＢＳＡTEST EXAMPLE 4 The dental caries preventive effect of complement C3c obtained in Example 3 was examined using Wistar rats. 3 weeks old Wistar rat, 6 g of whole wheat flour, 56 g of sucrose,
Skim milk powder 28g, alfalfa leaf coarse powder 3g, liver 1
g, brewer's yeast 4 g, and salt 2 g were administered and fed. Rats were divided into 5 groups of 3 animals, and the following samples were placed in a water bottle as drinking water and freely ingested in each group. Group A Deionized water Group B 0.1% bC3c Group C 1% bC3c Group D 1% BSA
【００４０】１週間後、ストレプトコッカス・ミュータ
ンスを口腔内に接種し、さらに２ヶ月間飼育した。その
後、歯を顎ごと摘出し、実体顕微鏡下で虫歯の発生状況
を観察して虫歯の程度を調べた。全く虫歯の発生してい
ないもの０点とし、全部の歯に虫歯が広がっているもの
１０点として、その範囲で各群の得点を合計した。その
結果を表３に示した。Ｂ群、Ｃ群及びＤ群において、有
意に虫歯の発生を防止できることが判った。After one week, Streptococcus mutans was inoculated into the oral cavity, and the animals were further raised for 2 months. After that, the teeth were extracted together with the jaws, and the degree of tooth decay was examined by observing the occurrence of tooth decay under a stereoscopic microscope. The score of each group was totaled within that range, with 0 points showing no caries at all and 10 points having caries spread over all the teeth. The results are shown in Table 3. It was found that the occurrence of caries can be significantly prevented in groups B, C and D.
【表３】 ──────────────────────────────────── 試料 虫歯発生状況得点合計 ──────────────────────────────────── Ａ群 脱イオン水 ４６ Ｂ群 ０．１％ｂＣ３ｃ ３３ Ｃ群 １％ｂＣ３ｃ １６ Ｄ群 １％ＢＳＡ ４３ ──────────────────────────────────── ｂＣ３ｃ：ウシ補体Ｃ３ｃ、ＢＳＡ：ウシ血清アルブミン[Table 3] ──────────────────────────────────── Specimen Cavity occurrence score Total ──── ──────────────────────────────── A group Deionized water 46 B group 0.1% bC3c 33 C group 1% bC3c 16 D group 1% BSA 43 ──────────────────────────────────── bC3c: Bovine complement C3c , BSA: Bovine serum albumin
【００４１】[0041]
【試験例５】実施例３で得られた補体Ｃ３ｃの病原性大
腸菌皮膚付着阻止効果について、無菌マウスを用いて調
べた。無菌マウスＩＣＲの背中の体毛を約２ｃｍ角で剃
り落とし、アルコール綿で消毒した。この無菌マウスを
４匹ずつ５群に分け、体毛を剃った部分を中心に注射針
で傷を付け、各群の無菌マウスに以下の試料を塗布し
た。 Ａ群 脱イオン水 Ｂ群 ０．１％ｂＣ３ｃ Ｃ群 １％ｂＣ３ｃ Ｄ群 １％ｂＣ３ｃトリプシン処理２０分間 Ｅ群 １％ＢＳＡ[Test Example 5] The effect of complement C3c obtained in Example 3 on the skin adhesion of pathogenic Escherichia coli was examined using a sterile mouse. The hair on the back of the aseptic mouse ICR was shaved off at about 2 cm square and disinfected with alcohol cotton. This sterile mouse was divided into 5 groups of 4 mice each, and the wounded body was injured with an injection needle around the shaved part, and the following samples were applied to the sterile mice in each group. Group A Deionized water Group B 0.1% bC3c Group C 1% bC3c Group D 1% bC3c Trypsin treatment 20 minutes Group E 1% BSA
【００４２】試料を塗布した直後に、病原性大腸菌Ｈ１
０４０７株を１０4 ＣＦＵずつ塗布した。１時間後、Ｐ
ＢＳにて傷口を洗い、１週間そのまま無菌的に飼育し
た。その後、皮膚を１ｃｍ角に切り取り、ホモゲナイズ
した。この液１ｍｌを寒天培地を入れたシャーレに接種
し、３７℃、一夜培養後、病原性大腸菌の菌数を測定し
た。 Ａ群 ８．７×１０7 ＣＦＵ Ｂ群 １．１×１０7 ＣＦＵ Ｃ群 ３．６×１０6 ＣＦＵ Ｄ群 ８．９×１０6 ＣＦＵ Ｅ群 ４．１×１０8 ＣＦＵ Ｂ群、Ｃ群及びＤ群は、Ａ群及びＥ群より有意に病原性
大腸菌の付着が少なかった。Immediately after applying the sample, the pathogenic E. coli H1
0407 strain was applied by 10 4 CFU each. 1 hour later, P
The wound was washed with BS and kept aseptically for 1 week. Then, the skin was cut into 1 cm squares and homogenized. 1 ml of this solution was inoculated into a petri dish containing an agar medium and cultured overnight at 37 ° C., after which the number of pathogenic Escherichia coli was measured. Group A 8.7 × 10 7 CFU B group 1.1 × 10 7 CFU C group 3.6 × 10 6 CFU D group 8.9 × 10 6 CFU E group 4.1 × 10 8 CFU B group, C group The groups D and D had significantly less adherence of pathogenic E. coli than the groups A and E.
【００４３】[0043]
【参考例１】次のような配合組成の補体Ｃ３ｃ配合飲料
を製造した。Ａ液・補体Ｃ３ｃ２ｇ及び脱イオン水１ｋ
ｇ、Ｂ液・ショ糖０．９６ｇ、クエン酸１６ｇ、クエン
酸ナトリウム１６ｇ、ビタミンＢ2 ０．０２ｇ、ビタミ
ンＢ０．０４４ｇ、ビタミンＣ０．１１ｇ、葉酸０．０
０８ｇ、香料２ｇ、５倍濃縮オレンジ果汁４０ｇ、水１
ｋｇ。このＡ液とＢ液を別々に６３℃、３０分間保持
し、殺菌を行った後、５〜１０℃に冷却し、混合、貯蔵
した。次いで、この混合液を殺菌済紙容器に無菌的に充
填し、感染防御能を付加した飲料を製造した。[Reference Example 1] A complement C3c blended beverage having the following blending composition was produced. Liquid A / Complement C3c2g and deionized water 1k
g, B liquid / sucrose 0.96 g, citric acid 16 g, sodium citrate 16 g, vitamin B 2 0.02 g, vitamin B 0.044 g, vitamin C 0.11 g, folic acid 0.0
08 g, fragrance 2 g, 5 times concentrated orange juice 40 g, water 1
kg. The solutions A and B were separately held at 63 ° C. for 30 minutes, sterilized, cooled to 5 to 10 ° C., mixed, and stored. Then, this mixed solution was aseptically filled in a sterilized paper container to produce a beverage having an infection protection ability.
【００４４】[0044]
【参考例２】次に、虫歯予防用チューインガムの配合例
を示す。（数値は重量％） ソルビトール ６８ ガム基質 ２３ グリセロール ６ 補体Ｃ３ｃ １ 調味料 １ 着色料 ０．５ 炭酸水素ナトリウム ０．５Reference Example 2 Next, a compounding example of chewing gum for preventing tooth decay will be shown. (The numerical value is% by weight) Sorbitol 68 Gum substrate 23 Glycerol 6 Complement C3c 1 Seasoning 1 Coloring agent 0.5 Sodium hydrogencarbonate 0.5
【００４５】[0045]
【参考例３】次に、虫歯予防用歯磨粉の配合例を示す。
（数値は重量％） 配合例１ 配合例２ リン酸水素カルシウム二水和物 ４５ ４５ グリセリン １０ １０ ソルビトール ２５ ２５ 補体Ｃ３ｃ ２ １ カルボキシメチルセルロースナトリウム ０．５ ０．５ カラギーナン ０．３ ０．３ ビーガム ０．２ ０．２ サッカリンナトリウム ０．２ ０．２ ラウリル硫酸ナトリウム １．２ １．２ ビタミンＥ酢酸塩 ０．１ ０．１ 香料 １．０ １．０ 水 １４．５ １５．５Reference Example 3 Next, a compounding example of toothpaste for preventing tooth decay will be shown.
(Numerical values are% by weight) Formulation Example 1 Formulation Example 2 Calcium hydrogen phosphate dihydrate 45 45 Glycerin 10 10 Sorbitol 25 25 Complement C3c 2 1 Carboxymethylcellulose sodium 0.5 0.5 Carrageenan 0.3 0.3 Veegum 0.2 0.2 Saccharin sodium 0.2 0.2 Sodium lauryl sulfate 1.2 1.2 Vitamin E acetate 0.1 0.1 Flavor 1.0 1.0 Water 14.5 15.5
【００４６】[0046]
【参考例４】次に、ニキビ予防用ローションの配合例を
示す。（数値は重量％） 配合例１ 配合例２ クエン酸素カルシウム二水和物 ０．１ ０．１ スルホ石灰酸亜鉛 ０．２ ０．２ グリセリン ５．０ ５．０ ポリオキシエチレン（２０Ｍ） １．０ １．０ オレインアルコールエーテル ０．５ ０．５ エチルアルコール ２０ ２０ 香料・防腐剤 ２．０ ２．０ 補体Ｃ３ｃ ０．５ １．０ 水 ７０．７ ７０．２ このローションは、１日１〜３回顔面に塗布する。Reference Example 4 Next, a formulation example of a lotion for preventing acne will be shown. (Numerical values are% by weight) Formulation example 1 Formulation example 2 Citric oxygen calcium dihydrate 0.1 0.1 Zinc sulfocalcate 0.2 0.2 Glycerin 5.0 5.0 Polyoxyethylene (20M) 1. 0 1.0 Olein alcohol ether 0.5 0.5 Ethyl alcohol 20 20 Perfume / preservative 2.0 2.0 Complement C3c 0.5 1.0 Water 70.7 70.2 This lotion is 1 day a day Apply to face 3 times.
【００４７】[0047]
【参考例５】次に、動物用飼料の配合例を示す。 脱脂粉乳 ６０．０ｇ ホエー蛋白濃縮物 １４．３ｇ 乳脂肪 １７．２ｇ グルコース ５．０ｇ ビタミン・ミネラル ２．５ｇ 補体Ｃ３ｃ １．０ｇ[Reference Example 5] Next, a compounding example of animal feed will be shown. Nonfat dry milk 60.0 g Whey protein concentrate 14.3 g Milk fat 17.2 g Glucose 5.0 g Vitamin and mineral 2.5 g Complement C3c 1.0 g
【００４８】[0048]
【参考例６】次に、医薬用カプセル剤の配合例を示す。 乳糖 ５．０ｇ 微結晶セルロース ２．５ｇ 補体Ｃ３ｃ ２．５ｇReference Example 6 Next, a formulation example of a pharmaceutical capsule is shown. Lactose 5.0 g Microcrystalline cellulose 2.5 g Complement C3c 2.5 g
【００４９】[0049]
【参考例７】次に、医薬用錠剤の調製例を示す。補体Ｃ
３ｃ１００ｍｇ及び微結晶セルロース１００ｍｇを含む
錠剤を常法に従って調製し、シロップゼラチン沈降性炭
酸カルシウムで糖衣を施した。この錠剤は、１日１〜３
錠を目安に摂取される。Reference Example 7 Next, a preparation example of a pharmaceutical tablet will be shown. Complement C
A tablet containing 100 mg of 3c and 100 mg of microcrystalline cellulose was prepared according to a conventional method, and sugar-coated with syrup gelatin-precipitable calcium carbonate. This tablet is 1 to 3 times a day
Ingested as a guide.
───────────────────────────────────────────────────── フロントページの続き (51)Int.Cl.5 識別記号 庁内整理番号 ＦＩ 技術表示箇所 Ａ６１Ｋ 37/54 8314−4Ｃ ─────────────────────────────────────────────────── ─── Continuation of the front page (51) Int.Cl. 5 Identification code Office reference number FI technical display location A61K 37/54 8314-4C
Claims (6)
換体と接触させて補体Ｃ３ｃを陽イオン交換体に吸着さ
せた後、溶出することを特徴とする補体Ｃ３ｃの製造
法。1. A method for producing complement C3c, which comprises contacting a dairy raw material containing complement C3c with a cation exchanger so that the complement C3c is adsorbed on the cation exchanger and then eluting.
を溶出するに際し、イオン強度が０．１５以上でｐＨが
２〜１０の溶液を用いる請求項１記載の補体Ｃ３ｃの製
造法。2. Complement C3c adsorbed on a cation exchanger
The method for producing complement C3c according to claim 1, wherein a solution having an ionic strength of 0.15 or more and a pH of 2 to 10 is used in eluting the.
た後、補体Ｃ３ｃを溶出するに先立って、イオン強度が
０．１５未満でｐＨが２〜１０の溶液を用いて陽イオン
交換体を洗浄する請求項１又は２記載の補体Ｃ３ｃの製
造法。3. After adsorbing complement C3c to a cation exchanger, prior to eluting complement C3c, cation exchange is performed using a solution having an ionic strength of less than 0.15 and a pH of 2 to 10. The method for producing complement C3c according to claim 1 or 2, wherein the body is washed.
で溶出した補体Ｃ３ｃを、さらに陰イオン交換体と接触
させて補体Ｃ３ｃを陰イオン交換体に吸着させた後、溶
出することを特徴とする補体Ｃ３ｃの製造法。4. The complement C3c eluted by the method according to any one of claims 1 to 3 is further contacted with an anion exchanger to adsorb the complement C3c to the anion exchanger and then eluted. A method for producing complement C3c, which is characterized in that
を溶出するに際し、イオン強度が０．３以上でｐＨが２
〜１０の溶液を用いる請求項４記載の補体Ｃ３ｃの製造
法。5. Complement C3c adsorbed on an anion exchanger
The ionic strength is 0.3 or more and the pH is 2
The method for producing complement C3c according to claim 4, wherein the solution of 10 to 10 is used.
た後、補体Ｃ３ｃを溶出するに先立って、イオン強度が
０．３未満でｐＨが２〜１０の溶液を用いて陰イオン交
換体を洗浄する請求項４又は５記載の補体Ｃ３ｃの製造
法。6. Anion exchange using a solution having an ionic strength of less than 0.3 and a pH of 2 to 10 after adsorbing the complement C3c to an anion exchanger and prior to eluting the complement C3c. The method for producing complement C3c according to claim 4 or 5, wherein the body is washed.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| JP5032518A JPH06228200A (en) | 1993-01-29 | 1993-01-29 | Production of complement c3c |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| JP5032518A JPH06228200A (en) | 1993-01-29 | 1993-01-29 | Production of complement c3c |
Publications (1)
| Publication Number | Publication Date |
|---|---|
| JPH06228200A true JPH06228200A (en) | 1994-08-16 |
Family
ID=12361192
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| JP5032518A Pending JPH06228200A (en) | 1993-01-29 | 1993-01-29 | Production of complement c3c |
Country Status (1)
| Country | Link |
|---|---|
| JP (1) | JPH06228200A (en) |
Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| JP2012180368A (en) * | 2012-05-21 | 2012-09-20 | Snow Brand Milk Products Co Ltd | Bone resorption inhibitor |
| WO2015041218A1 (en) | 2013-09-17 | 2015-03-26 | 株式会社カネカ | Novel antibody purification method and antibody obtained therefrom, and novel antibody purification method using cation exchanger and antibody obtained therefrom |
-
1993
- 1993-01-29 JP JP5032518A patent/JPH06228200A/en active Pending
Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| JP2012180368A (en) * | 2012-05-21 | 2012-09-20 | Snow Brand Milk Products Co Ltd | Bone resorption inhibitor |
| WO2015041218A1 (en) | 2013-09-17 | 2015-03-26 | 株式会社カネカ | Novel antibody purification method and antibody obtained therefrom, and novel antibody purification method using cation exchanger and antibody obtained therefrom |
| US10519195B2 (en) | 2013-09-17 | 2019-12-31 | Kaneka Corporation | Antibody purification method, antibody obtained therefrom, novel antibody purification method using cation exchanger, and antibody obtained therefrom |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| CA2063063C (en) | Antimicrobial peptide and an antimicrobial agent | |
| US5656591A (en) | Antimicrobial agents and method for treating products therewith | |
| EP0474506B1 (en) | Antimicrobial peptides | |
| US8603560B2 (en) | Milk protein isolate and process for its preparation | |
| NZ547859A (en) | Treatment method and composition for mastitis | |
| EP0457565A2 (en) | Milk-protein hydrolyzates and compositions for use as hair and skin treating agent | |
| EP0510912A1 (en) | Antimicrobial peptide and antimicrobial agent | |
| Fadaei | Milk Proteins-derived antibacterial peptides as novel functional food ingredients | |
| AU2001270540B2 (en) | Cosmetic composition comprising human serum albumin obtained from transgenic non-human animals | |
| EP0629213B1 (en) | Use of an immunostimulatory agent | |
| JP3746081B2 (en) | Animal skin disease treatment | |
| JP3261174B2 (en) | Antimicrobial agent and method of treating articles using this antimicrobial agent | |
| JPH06228200A (en) | Production of complement c3c | |
| JP4847341B2 (en) | Antibacterial composition | |
| KR20090016730A (en) | Agent for maintaining the hardness of tooth structure | |
| JP2771068B2 (en) | Antimicrobial peptides and antimicrobial agents | |
| JPH05255109A (en) | Antibacterial agent and treating method for article using the same | |
| JP2000159687A (en) | Agent for inhibiting settlement of non-beneficiary intestinal microorganism | |
| JPH05238947A (en) | Bacterial toxin-neutralizing agent | |
| AU659440B2 (en) | Antimicrobial peptide and an antimicrobial agent | |
| CA2102509C (en) | Immunostimulatory agent | |
| JPH11286452A (en) | Agent for protecting infection of pathogenic bacterium and virus | |
| JPH07309774A (en) | Antifungal agent | |
| El-Zahar | Antimicrobial activity of sheep yoghurt prepared by different commercial starter strains | |
| NZ241911A (en) | Immunostimulatory agent comprising a peptide derived from lactoferrin |