CN116333948B - Clostridium aerophilum enrichment medium and preparation method thereof - Google Patents
Clostridium aerophilum enrichment medium and preparation method thereof Download PDFInfo
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- CN116333948B CN116333948B CN202310586453.6A CN202310586453A CN116333948B CN 116333948 B CN116333948 B CN 116333948B CN 202310586453 A CN202310586453 A CN 202310586453A CN 116333948 B CN116333948 B CN 116333948B
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- 241000193403 Clostridium Species 0.000 title claims abstract description 43
- 238000002360 preparation method Methods 0.000 title abstract description 13
- 229940024606 amino acid Drugs 0.000 claims abstract description 46
- 150000001413 amino acids Chemical class 0.000 claims abstract description 46
- HEMHJVSKTPXQMS-UHFFFAOYSA-M Sodium hydroxide Chemical compound [OH-].[Na+] HEMHJVSKTPXQMS-UHFFFAOYSA-M 0.000 claims abstract description 45
- 239000001963 growth medium Substances 0.000 claims abstract description 37
- SEOVTRFCIGRIMH-UHFFFAOYSA-N indole-3-acetic acid Chemical compound C1=CC=C2C(CC(=O)O)=CNC2=C1 SEOVTRFCIGRIMH-UHFFFAOYSA-N 0.000 claims abstract description 34
- 229940088594 vitamin Drugs 0.000 claims abstract description 27
- 229930003231 vitamin Natural products 0.000 claims abstract description 27
- 235000013343 vitamin Nutrition 0.000 claims abstract description 27
- 239000011782 vitamin Substances 0.000 claims abstract description 27
- 229910052751 metal Inorganic materials 0.000 claims abstract description 26
- 239000002184 metal Substances 0.000 claims abstract description 26
- 150000003722 vitamin derivatives Chemical class 0.000 claims abstract description 26
- 239000002609 medium Substances 0.000 claims abstract description 25
- 230000001954 sterilising effect Effects 0.000 claims abstract description 22
- 229920002472 Starch Polymers 0.000 claims abstract description 18
- 235000019698 starch Nutrition 0.000 claims abstract description 18
- 239000008107 starch Substances 0.000 claims abstract description 18
- FBPFZTCFMRRESA-FSIIMWSLSA-N D-Glucitol Natural products OC[C@H](O)[C@H](O)[C@@H](O)[C@H](O)CO FBPFZTCFMRRESA-FSIIMWSLSA-N 0.000 claims abstract description 17
- 238000009835 boiling Methods 0.000 claims abstract description 17
- 239000000600 sorbitol Substances 0.000 claims abstract description 17
- 238000002156 mixing Methods 0.000 claims abstract description 16
- 229930091371 Fructose Natural products 0.000 claims abstract description 15
- RFSUNEUAIZKAJO-ARQDHWQXSA-N Fructose Chemical compound OC[C@H]1O[C@](O)(CO)[C@@H](O)[C@@H]1O RFSUNEUAIZKAJO-ARQDHWQXSA-N 0.000 claims abstract description 15
- 239000005715 Fructose Substances 0.000 claims abstract description 15
- UIIMBOGNXHQVGW-DEQYMQKBSA-M Sodium bicarbonate-14C Chemical compound [Na+].O[14C]([O-])=O UIIMBOGNXHQVGW-DEQYMQKBSA-M 0.000 claims abstract description 15
- VLSOAXRVHARBEQ-UHFFFAOYSA-N [4-fluoro-2-(hydroxymethyl)phenyl]methanol Chemical compound OCC1=CC=C(F)C=C1CO VLSOAXRVHARBEQ-UHFFFAOYSA-N 0.000 claims abstract description 15
- 229940041514 candida albicans extract Drugs 0.000 claims abstract description 15
- 239000000843 powder Substances 0.000 claims abstract description 15
- 229910052979 sodium sulfide Inorganic materials 0.000 claims abstract description 15
- GRVFOGOEDUUMBP-UHFFFAOYSA-N sodium sulfide (anhydrous) Chemical compound [Na+].[Na+].[S-2] GRVFOGOEDUUMBP-UHFFFAOYSA-N 0.000 claims abstract description 15
- 239000012138 yeast extract Substances 0.000 claims abstract description 15
- 238000000034 method Methods 0.000 claims abstract description 11
- 235000001014 amino acid Nutrition 0.000 claims description 44
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Chemical compound O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 claims description 39
- 239000008367 deionised water Substances 0.000 claims description 38
- 229910021641 deionized water Inorganic materials 0.000 claims description 38
- 238000003756 stirring Methods 0.000 claims description 35
- 238000007789 sealing Methods 0.000 claims description 21
- CIWBSHSKHKDKBQ-JLAZNSOCSA-N Ascorbic acid Chemical compound OC[C@H](O)[C@H]1OC(=O)C(O)=C1O CIWBSHSKHKDKBQ-JLAZNSOCSA-N 0.000 claims description 20
- 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 claims description 20
- FAPWRFPIFSIZLT-UHFFFAOYSA-M Sodium chloride Chemical compound [Na+].[Cl-] FAPWRFPIFSIZLT-UHFFFAOYSA-M 0.000 claims description 20
- ONDPHDOFVYQSGI-UHFFFAOYSA-N zinc nitrate Chemical compound [Zn+2].[O-][N+]([O-])=O.[O-][N+]([O-])=O ONDPHDOFVYQSGI-UHFFFAOYSA-N 0.000 claims description 20
- 239000007788 liquid Substances 0.000 claims description 18
- 238000004806 packaging method and process Methods 0.000 claims description 18
- 238000007872 degassing Methods 0.000 claims description 16
- KPGXRSRHYNQIFN-UHFFFAOYSA-L 2-oxoglutarate(2-) Chemical compound [O-]C(=O)CCC(=O)C([O-])=O KPGXRSRHYNQIFN-UHFFFAOYSA-L 0.000 claims description 11
- 239000004475 Arginine Substances 0.000 claims description 10
- JMGZEFIQIZZSBH-UHFFFAOYSA-N Bioquercetin Natural products CC1OC(OCC(O)C2OC(OC3=C(Oc4cc(O)cc(O)c4C3=O)c5ccc(O)c(O)c5)C(O)C2O)C(O)C(O)C1O JMGZEFIQIZZSBH-UHFFFAOYSA-N 0.000 claims description 10
- AUNGANRZJHBGPY-UHFFFAOYSA-N D-Lyxoflavin Natural products OCC(O)C(O)C(O)CN1C=2C=C(C)C(C)=CC=2N=C2C1=NC(=O)NC2=O AUNGANRZJHBGPY-UHFFFAOYSA-N 0.000 claims description 10
- ZZZCUOFIHGPKAK-UHFFFAOYSA-N D-erythro-ascorbic acid Natural products OCC1OC(=O)C(O)=C1O ZZZCUOFIHGPKAK-UHFFFAOYSA-N 0.000 claims description 10
- WHUUTDBJXJRKMK-UHFFFAOYSA-N Glutamic acid Natural products OC(=O)C(N)CCC(O)=O WHUUTDBJXJRKMK-UHFFFAOYSA-N 0.000 claims description 10
- QIVBCDIJIAJPQS-VIFPVBQESA-N L-tryptophane Chemical compound C1=CC=C2C(C[C@H](N)C(O)=O)=CNC2=C1 QIVBCDIJIAJPQS-VIFPVBQESA-N 0.000 claims description 10
- 229910021380 Manganese Chloride Inorganic materials 0.000 claims description 10
- GLFNIEUTAYBVOC-UHFFFAOYSA-L Manganese chloride Chemical compound Cl[Mn]Cl GLFNIEUTAYBVOC-UHFFFAOYSA-L 0.000 claims description 10
- MTCFGRXMJLQNBG-UHFFFAOYSA-N Serine Natural products OCC(N)C(O)=O MTCFGRXMJLQNBG-UHFFFAOYSA-N 0.000 claims description 10
- QIVBCDIJIAJPQS-UHFFFAOYSA-N Tryptophan Natural products C1=CC=C2C(CC(N)C(O)=O)=CNC2=C1 QIVBCDIJIAJPQS-UHFFFAOYSA-N 0.000 claims description 10
- 229930003268 Vitamin C Natural products 0.000 claims description 10
- WHMDKBIGKVEYHS-IYEMJOQQSA-L Zinc gluconate Chemical compound [Zn+2].OC[C@@H](O)[C@@H](O)[C@H](O)[C@@H](O)C([O-])=O.OC[C@@H](O)[C@@H](O)[C@H](O)[C@@H](O)C([O-])=O WHMDKBIGKVEYHS-IYEMJOQQSA-L 0.000 claims description 10
- DFPAKSUCGFBDDF-ZQBYOMGUSA-N [14c]-nicotinamide Chemical compound N[14C](=O)C1=CC=CN=C1 DFPAKSUCGFBDDF-ZQBYOMGUSA-N 0.000 claims description 10
- 229940103272 aluminum potassium sulfate Drugs 0.000 claims description 10
- ODKSFYDXXFIFQN-UHFFFAOYSA-N arginine Natural products OC(=O)C(N)CCCNC(N)=N ODKSFYDXXFIFQN-UHFFFAOYSA-N 0.000 claims description 10
- KGBXLFKZBHKPEV-UHFFFAOYSA-N boric acid Chemical compound OB(O)O KGBXLFKZBHKPEV-UHFFFAOYSA-N 0.000 claims description 10
- 239000004327 boric acid Substances 0.000 claims description 10
- 229940044175 cobalt sulfate Drugs 0.000 claims description 10
- 229910000361 cobalt sulfate Inorganic materials 0.000 claims description 10
- KTVIXTQDYHMGHF-UHFFFAOYSA-L cobalt(2+) sulfate Chemical compound [Co+2].[O-]S([O-])(=O)=O KTVIXTQDYHMGHF-UHFFFAOYSA-L 0.000 claims description 10
- BVTBRVFYZUCAKH-UHFFFAOYSA-L disodium selenite Chemical compound [Na+].[Na+].[O-][Se]([O-])=O BVTBRVFYZUCAKH-UHFFFAOYSA-L 0.000 claims description 10
- IVTMALDHFAHOGL-UHFFFAOYSA-N eriodictyol 7-O-rutinoside Natural products OC1C(O)C(O)C(C)OC1OCC1C(O)C(O)C(O)C(OC=2C=C3C(C(C(O)=C(O3)C=3C=C(O)C(O)=CC=3)=O)=C(O)C=2)O1 IVTMALDHFAHOGL-UHFFFAOYSA-N 0.000 claims description 10
- 235000003891 ferrous sulphate Nutrition 0.000 claims description 10
- 239000011790 ferrous sulphate Substances 0.000 claims description 10
- 235000013922 glutamic acid Nutrition 0.000 claims description 10
- 239000004220 glutamic acid Substances 0.000 claims description 10
- BAUYGSIQEAFULO-UHFFFAOYSA-L iron(2+) sulfate (anhydrous) Chemical compound [Fe+2].[O-]S([O-])(=O)=O BAUYGSIQEAFULO-UHFFFAOYSA-L 0.000 claims description 10
- 229910000359 iron(II) sulfate Inorganic materials 0.000 claims description 10
- AGBQKNBQESQNJD-UHFFFAOYSA-M lipoate Chemical compound [O-]C(=O)CCCCC1CCSS1 AGBQKNBQESQNJD-UHFFFAOYSA-M 0.000 claims description 10
- 235000019136 lipoic acid Nutrition 0.000 claims description 10
- 235000002867 manganese chloride Nutrition 0.000 claims description 10
- 239000011565 manganese chloride Substances 0.000 claims description 10
- 229940099607 manganese chloride Drugs 0.000 claims description 10
- GRLPQNLYRHEGIJ-UHFFFAOYSA-J potassium aluminium sulfate Chemical compound [Al+3].[K+].[O-]S([O-])(=O)=O.[O-]S([O-])(=O)=O GRLPQNLYRHEGIJ-UHFFFAOYSA-J 0.000 claims description 10
- FDRQPMVGJOQVTL-UHFFFAOYSA-N quercetin rutinoside Natural products OC1C(O)C(O)C(CO)OC1OCC1C(O)C(O)C(O)C(OC=2C(C3=C(O)C=C(O)C=C3OC=2C=2C=C(O)C(O)=CC=2)=O)O1 FDRQPMVGJOQVTL-UHFFFAOYSA-N 0.000 claims description 10
- 229960002477 riboflavin Drugs 0.000 claims description 10
- 235000019192 riboflavin Nutrition 0.000 claims description 10
- 239000002151 riboflavin Substances 0.000 claims description 10
- IKGXIBQEEMLURG-BKUODXTLSA-N rutin Chemical compound O[C@H]1[C@H](O)[C@@H](O)[C@H](C)O[C@@H]1OC[C@H]1[C@H](O)[C@@H](O)[C@H](O)[C@@H](OC=2C(C3=C(O)C=C(O)C=C3OC=2C=2C=C(O)C(O)=CC=2)=O)O1 IKGXIBQEEMLURG-BKUODXTLSA-N 0.000 claims description 10
- ALABRVAAKCSLSC-UHFFFAOYSA-N rutin Natural products CC1OC(OCC2OC(O)C(O)C(O)C2O)C(O)C(O)C1OC3=C(Oc4cc(O)cc(O)c4C3=O)c5ccc(O)c(O)c5 ALABRVAAKCSLSC-UHFFFAOYSA-N 0.000 claims description 10
- 235000005493 rutin Nutrition 0.000 claims description 10
- 229960004555 rutoside Drugs 0.000 claims description 10
- 239000011780 sodium chloride Substances 0.000 claims description 10
- 235000015921 sodium selenite Nutrition 0.000 claims description 10
- 239000011781 sodium selenite Substances 0.000 claims description 10
- 229960001471 sodium selenite Drugs 0.000 claims description 10
- XMVONEAAOPAGAO-UHFFFAOYSA-N sodium tungstate Chemical compound [Na+].[Na+].[O-][W]([O-])(=O)=O XMVONEAAOPAGAO-UHFFFAOYSA-N 0.000 claims description 10
- 229960002663 thioctic acid Drugs 0.000 claims description 10
- VZZHAYFWMLLWGG-UHFFFAOYSA-K triazanium;bismuth;2-hydroxypropane-1,2,3-tricarboxylate Chemical compound [NH4+].[NH4+].[NH4+].[Bi+3].[O-]C(=O)CC(O)(CC([O-])=O)C([O-])=O.[O-]C(=O)CC(O)(CC([O-])=O)C([O-])=O VZZHAYFWMLLWGG-UHFFFAOYSA-K 0.000 claims description 10
- 235000019154 vitamin C Nutrition 0.000 claims description 10
- 239000011718 vitamin C Substances 0.000 claims description 10
- 235000011478 zinc gluconate Nutrition 0.000 claims description 10
- 239000011670 zinc gluconate Substances 0.000 claims description 10
- 229960000306 zinc gluconate Drugs 0.000 claims description 10
- 238000001914 filtration Methods 0.000 claims description 8
- 238000004659 sterilization and disinfection Methods 0.000 claims description 7
- 238000005303 weighing Methods 0.000 claims description 7
- 241000193468 Clostridium perfringens Species 0.000 abstract description 89
- 230000001580 bacterial effect Effects 0.000 abstract description 26
- 230000012010 growth Effects 0.000 abstract description 23
- KPGXRSRHYNQIFN-UHFFFAOYSA-N 2-oxoglutaric acid Chemical compound OC(=O)CCC(=O)C(O)=O KPGXRSRHYNQIFN-UHFFFAOYSA-N 0.000 abstract description 12
- 230000009286 beneficial effect Effects 0.000 abstract description 11
- 239000000126 substance Substances 0.000 abstract description 10
- 238000011160 research Methods 0.000 abstract description 8
- 230000004913 activation Effects 0.000 abstract description 7
- HWXBTNAVRSUOJR-UHFFFAOYSA-N alpha-hydroxyglutaric acid Natural products OC(=O)C(O)CCC(O)=O HWXBTNAVRSUOJR-UHFFFAOYSA-N 0.000 abstract description 6
- 229940009533 alpha-ketoglutaric acid Drugs 0.000 abstract description 6
- 244000005700 microbiome Species 0.000 abstract description 6
- 238000009825 accumulation Methods 0.000 abstract description 4
- 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 abstract description 3
- 239000000463 material Substances 0.000 abstract description 2
- 230000008569 process Effects 0.000 abstract description 2
- 230000002195 synergetic effect Effects 0.000 abstract description 2
- 238000009655 industrial fermentation Methods 0.000 abstract 1
- 239000000243 solution Substances 0.000 description 75
- 238000000855 fermentation Methods 0.000 description 19
- 230000004151 fermentation Effects 0.000 description 19
- 238000012258 culturing Methods 0.000 description 15
- IJGRMHOSHXDMSA-UHFFFAOYSA-N Atomic nitrogen Chemical compound N#N IJGRMHOSHXDMSA-UHFFFAOYSA-N 0.000 description 14
- CURLTUGMZLYLDI-UHFFFAOYSA-N Carbon dioxide Chemical compound O=C=O CURLTUGMZLYLDI-UHFFFAOYSA-N 0.000 description 14
- 230000000694 effects Effects 0.000 description 12
- 239000000306 component Substances 0.000 description 8
- 238000004519 manufacturing process Methods 0.000 description 8
- 239000002504 physiological saline solution Substances 0.000 description 8
- 239000006228 supernatant Substances 0.000 description 8
- 238000001994 activation Methods 0.000 description 7
- 239000001569 carbon dioxide Substances 0.000 description 7
- 229910002092 carbon dioxide Inorganic materials 0.000 description 7
- 229910052757 nitrogen Inorganic materials 0.000 description 7
- 238000004090 dissolution Methods 0.000 description 5
- LFQSCWFLJHTTHZ-UHFFFAOYSA-N Ethanol Chemical compound CCO LFQSCWFLJHTTHZ-UHFFFAOYSA-N 0.000 description 4
- 230000003698 anagen phase Effects 0.000 description 4
- 239000007789 gas Substances 0.000 description 4
- 235000015097 nutrients Nutrition 0.000 description 4
- 235000016709 nutrition Nutrition 0.000 description 4
- 230000035764 nutrition Effects 0.000 description 4
- 239000000047 product Substances 0.000 description 4
- 230000035755 proliferation Effects 0.000 description 4
- 108090000623 proteins and genes Proteins 0.000 description 4
- 241000894006 Bacteria Species 0.000 description 3
- OKTJSMMVPCPJKN-UHFFFAOYSA-N Carbon Chemical compound [C] OKTJSMMVPCPJKN-UHFFFAOYSA-N 0.000 description 3
- 239000002253 acid Substances 0.000 description 3
- 229910052799 carbon Inorganic materials 0.000 description 3
- 239000003638 chemical reducing agent Substances 0.000 description 3
- 230000000813 microbial effect Effects 0.000 description 3
- 235000018102 proteins Nutrition 0.000 description 3
- 102000004169 proteins and genes Human genes 0.000 description 3
- 238000000746 purification Methods 0.000 description 3
- 102000004190 Enzymes Human genes 0.000 description 2
- 108090000790 Enzymes Proteins 0.000 description 2
- 230000015572 biosynthetic process Effects 0.000 description 2
- 238000005119 centrifugation Methods 0.000 description 2
- 238000006243 chemical reaction Methods 0.000 description 2
- 230000000052 comparative effect Effects 0.000 description 2
- 230000002349 favourable effect Effects 0.000 description 2
- 238000011049 filling Methods 0.000 description 2
- 230000004083 survival effect Effects 0.000 description 2
- 108091003079 Bovine Serum Albumin Proteins 0.000 description 1
- UGFAIRIUMAVXCW-UHFFFAOYSA-N Carbon monoxide Chemical compound [O+]#[C-] UGFAIRIUMAVXCW-UHFFFAOYSA-N 0.000 description 1
- 241001656809 Clostridium autoethanogenum Species 0.000 description 1
- 241000282414 Homo sapiens Species 0.000 description 1
- HCHKCACWOHOZIP-UHFFFAOYSA-N Zinc Chemical compound [Zn] HCHKCACWOHOZIP-UHFFFAOYSA-N 0.000 description 1
- 230000009471 action Effects 0.000 description 1
- 230000003213 activating effect Effects 0.000 description 1
- 238000010170 biological method Methods 0.000 description 1
- 229940098773 bovine serum albumin Drugs 0.000 description 1
- 229910002091 carbon monoxide Inorganic materials 0.000 description 1
- 239000003153 chemical reaction reagent Substances 0.000 description 1
- 239000005515 coenzyme Substances 0.000 description 1
- 230000001276 controlling effect Effects 0.000 description 1
- 230000007423 decrease Effects 0.000 description 1
- 230000007547 defect Effects 0.000 description 1
- 230000003111 delayed effect Effects 0.000 description 1
- 238000013461 design Methods 0.000 description 1
- 238000001514 detection method Methods 0.000 description 1
- 238000011161 development Methods 0.000 description 1
- 230000018109 developmental process Effects 0.000 description 1
- 238000007865 diluting Methods 0.000 description 1
- 238000002474 experimental method Methods 0.000 description 1
- 239000000446 fuel Substances 0.000 description 1
- 238000010353 genetic engineering Methods 0.000 description 1
- 239000012533 medium component Substances 0.000 description 1
- 238000012269 metabolic engineering Methods 0.000 description 1
- 230000004060 metabolic process Effects 0.000 description 1
- 229910021645 metal ion Inorganic materials 0.000 description 1
- 230000004048 modification Effects 0.000 description 1
- 238000012986 modification Methods 0.000 description 1
- -1 molybdate ions Chemical class 0.000 description 1
- 230000003647 oxidation Effects 0.000 description 1
- 238000007254 oxidation reaction Methods 0.000 description 1
- 238000001556 precipitation Methods 0.000 description 1
- 239000002994 raw material Substances 0.000 description 1
- 230000001105 regulatory effect Effects 0.000 description 1
- 238000003786 synthesis reaction Methods 0.000 description 1
- 238000012546 transfer Methods 0.000 description 1
- 235000015099 wheat brans Nutrition 0.000 description 1
- 229910052725 zinc Inorganic materials 0.000 description 1
- 239000011701 zinc Substances 0.000 description 1
Classifications
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N1/00—Microorganisms, e.g. protozoa; Compositions thereof; Processes of propagating, maintaining or preserving microorganisms or compositions thereof; Processes of preparing or isolating a composition containing a microorganism; Culture media therefor
- C12N1/20—Bacteria; Culture media therefor
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12R—INDEXING SCHEME ASSOCIATED WITH SUBCLASSES C12C - C12Q, RELATING TO MICROORGANISMS
- C12R2001/00—Microorganisms ; Processes using microorganisms
- C12R2001/01—Bacteria or Actinomycetales ; using bacteria or Actinomycetales
- C12R2001/145—Clostridium
-
- Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
- Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
- Y02E—REDUCTION OF GREENHOUSE GAS [GHG] EMISSIONS, RELATED TO ENERGY GENERATION, TRANSMISSION OR DISTRIBUTION
- Y02E50/00—Technologies for the production of fuel of non-fossil origin
- Y02E50/10—Biofuels, e.g. bio-diesel
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- Life Sciences & Earth Sciences (AREA)
- Chemical & Material Sciences (AREA)
- Engineering & Computer Science (AREA)
- Health & Medical Sciences (AREA)
- Genetics & Genomics (AREA)
- Biotechnology (AREA)
- Organic Chemistry (AREA)
- Zoology (AREA)
- Bioinformatics & Cheminformatics (AREA)
- Wood Science & Technology (AREA)
- Medicinal Chemistry (AREA)
- Microbiology (AREA)
- Biomedical Technology (AREA)
- Virology (AREA)
- Biochemistry (AREA)
- General Engineering & Computer Science (AREA)
- General Health & Medical Sciences (AREA)
- Tropical Medicine & Parasitology (AREA)
- Micro-Organisms Or Cultivation Processes Thereof (AREA)
- Preparation Of Compounds By Using Micro-Organisms (AREA)
Abstract
The invention relates to a clostridium aerovorum enrichment medium and a preparation method thereof, belonging to the field of microorganisms. According to the invention, through optimizing the culture medium, clostridium perfringens can be rapidly recuperated and proliferated, and the growth rate of clostridium perfringens is improved. The components of the culture medium comprise sodium bicarbonate, yeast extract powder, soluble starch, sorbitol, fructose, alpha-ketoglutaric acid, 3-indoleacetic acid, L-cysteine hydrochloride, sodium hydroxide, sodium sulfide, metal solution, vitamin solution and amino acid solution. The preparation method comprises boiling the above materials under anaerobic condition, sterilizing, and mixing. In the formula, the soluble starch can reduce the accumulation of harmful substances in the bacterial reproduction process, the synergistic effect of sorbitol, alpha-ketoglutaric acid and 3-indoleacetic acid can delay the decay of bacterial, and various amino acids can greatly improve the growth rate of clostridium perfringens in the same culture time, so that the bacterial can be rapidly and threely proliferated to reach higher quantity in a short time, and the method is beneficial to the industrial fermentation activation culture and research of clostridium perfringens.
Description
Technical Field
The invention belongs to the technical field of microorganisms, and particularly relates to a clostridium aerovorum enrichment medium and a preparation method thereof.
Background
At present, the greenhouse effect is still one of the major problems faced by human beings, the gas causing the greenhouse effect mainly comprises water vapor and the gas is secondly carbon dioxide, the conversion and utilization of industrial carbon-containing gas are realized through a biological method, and the generation of valuable chemicals while reducing carbon emission is an important research direction.
Clostridium aerovorans is a major group of chemolithotrophic microorganisms, belonging to the gram-positive genus clostridium anaerobicus. The clostridium perfringens can utilize carbon dioxide and carbon monoxide to synthesize various chemicals and fuels, is a main chemolithospermum and has good industrial application prospect. The reported clostridium perfringens mainly comprises clostridium Yankeei, clostridium autoethanogenum, clostridium carboxydotrophicum and the like.
In recent years, researchers at home and abroad make many attempts to optimize and improve the fermentation performance of clostridium perfringens, for example, improve the alcohol and acid production efficiency of clostridium perfringens by optimizing the ratio of carbon-containing gas, improve the alcohol content of clostridium perfringens fermentation products by controlling the concentration of molybdate ions, improve the material conversion efficiency by improving the gas-liquid mass transfer rate of fermentation liquid, and also the influence of zinc element on the fermentation of clostridium perfringens has been studied by scholars. In addition, due to the development of molecular genetic manipulation tools in recent years, metabolic engineering design, modification and synthesis biology research of clostridium aerovorans have also been advanced and discovered to some extent.
However, the research on improving the growth and propagation rate of clostridium perfringens by using fermentation liquor or culture medium is very few at present, the prior clostridium perfringens fermentation process needs to be started and put into a fermentation tank after the prior clostridium perfringens is started, the process is complicated, the clostridium perfringens culture genes on the market lack some necessary components, especially amino acid solution, can not realize the rapid proliferation of clostridium perfringens in the prior clostridium perfringens activation stage, the survival rate is low, and after the clostridium perfringens is put into fermentation for use, the bacterial cells are greatly degenerated due to the factors of harmful substance accumulation, nutrition competition and the like caused by the fact that the bacterial count reaches a certain number, the bacterial count needs to be frequently supplemented to maintain the bacterial count, the prior clostridium perfringens is not suitable for large-scale bacterium increasing culture of clostridium perfringens, and the prior culture medium on the market has little contribution to scientific research of clostridium perfringens.
Disclosure of Invention
Based on the background, the invention provides the clostridium perfringens enrichment medium and the preparation method thereof by optimizing the medium components, so that clostridium perfringens can be greatly proliferated in a short time in an activation period, quickly enter a logarithmic growth phase, and is beneficial to the growth of clostridium perfringens, applicable to the enrichment culture and fermentation of clostridium perfringens and more beneficial to the scientific research of clostridium perfringens.
The technical scheme adopted by the invention is as follows:
an aerothrice increasing culture medium, which is characterized in that: 1-5g of sodium bicarbonate, 2-4g of yeast extract powder, 0.1-2g of soluble starch, 1-2g of sorbitol, 2-3g of fructose, 0.5-0.8g of alpha-ketoglutarate, 0.01-0.03g of 3-indoleacetic acid, 0.1-0.3g of L-cysteine hydrochloride, 0.01-0.05g of sodium hydroxide, 0.1-0.3g of sodium sulfide, 5-8mL of metal solution, 5-8mL of vitamin solution and 5-10mL of amino acid solution;
the amino acid solution comprises 5g of arginine, 2g of serine, 3g of tryptophan and 0.5g of glutamic acid per liter.
Further preferred is the following scheme: the metal solution comprises 5mg of sodium selenite, 12mg of sodium tungstate, 0.01g of zinc gluconate, 2g of bismuth ammonium citrate, 5g of sodium chloride, 0.7g of manganese chloride, 0.15g of cobalt sulfate, 0.05g of ferrous sulfate, 0.3g of zinc nitrate, 0.04g of aluminum potassium sulfate and 0.03g of boric acid.
Further preferred is the following scheme: the vitamin solution comprises 0.2mg of nicotinamide, 7mg of rutin, 3mg of riboflavin, 5mg of vitamin C and 8mg of lipoic acid per liter.
A preparation method of clostridium aerovorum enrichment medium is characterized in that:
(1) Adding 5mg of sodium selenite, 12mg of sodium tungstate, 0.01g of zinc gluconate, 2g of bismuth ammonium citrate, 5g of sodium chloride, 0.7g of manganese chloride, 0.15g of cobalt sulfate, 0.05g of ferrous sulfate, 0.3g of zinc nitrate, 0.04g of aluminum potassium sulfate and 0.03g of boric acid into a container I containing one liter of deionized water, stirring and dissolving to prepare a metal solution;
(2) Adding 0.2mg of nicotinamide, 7mg of rutin, 3mg of riboflavin, 5mg of vitamin C and 8mg of lipoic acid into a second container containing one liter of deionized water, stirring and dissolving to prepare a vitamin solution;
(3) Putting 900mL of deionized water into a container III, weighing 1-5g of sodium bicarbonate, 2-4g of yeast extract powder, 0.1-2g of soluble starch, 0.5-0.8g of alpha-ketoglutarate and 0.01-0.03g of 3-indoleacetic acid, adding into the deionized water, adding 5-8mL of metal solution of the container I, stirring and dissolving 5-8mL of vitamin solution of the container II, boiling, degassing, anaerobically subpackaging and sealing, and sterilizing at 121 ℃ for 15 minutes;
(4) Adding 1-2g of sorbitol, 2-3g of fructose, 0.01-0.05g of sodium hydroxide, 0.1-0.3g of L-cysteine hydrochloride and 0.1-0.3g of sodium sulfide into a container IV, adding 100ml of deionized water, stirring and dissolving, boiling, degassing, sub-packaging and sealing anaerobically, and sterilizing at 121 ℃ for 15 minutes;
(5) Under anaerobic condition, adding 5g of arginine, 2g of serine, 3g of tryptophan, 0.5g of glutamic acid and 1g of bovine serum albumin into a container, adding 1 liter of deionized water, stirring for dissolution, filtering for sterilization, packaging and sealing to prepare an amino acid solution;
(6) And (3) immediately before use, taking 5-10mL of the amino acid solution in the container five, and uniformly mixing with the liquid prepared in the steps (3) - (4) under anaerobic conditions to obtain the amino acid solution.
The invention has the beneficial effects that:
(1) The clostridium perfringens enrichment medium can be used for activating clostridium perfringens, improving the activation efficiency and survival rate, enabling clostridium perfringens to rapidly enter a logarithmic growth phase, and making a foundation for subsequent fermentation and scientific research, and can be used for large-scale fermentation culture of clostridium perfringens;
(2) The traditional fermentation medium needs to be added with raw materials such as wheat bran and the like which are not purified, and the purification difficulty of later-stage strains or fermentation products is increased, so that the method can be used for fermentation purification with higher requirements, can directly input strains for use without a strain activation step, and saves fermentation time;
(3) The clostridium perfringens enrichment medium optimizes the nutrition components of the medium, particularly adds various amino acids, can provide a large amount of nutrition for clostridium perfringens in a frozen state and with weak other vital activities, simultaneously reduces the accumulation of harmful substances in the activation process, ensures that clostridium perfringens rapidly grows and splits to reach the logarithmic growth phase, obtains higher number of bacteria in a short time, improves the efficiency of strain in the early fermentation period, and is beneficial to the activation of the strain and popularization and application of clostridium perfringens fermentation;
(4) According to the invention, components such as soluble starch, sorbitol, alpha-ketoglutaric acid and 3-indoleacetic acid are added, wherein the soluble starch reduces the accumulation of harmful substances in the microbial fermentation process, and other components can regulate and control the vital activities of microorganisms through synergistic effect, so that strains are kept in an active state, the natural decay of microorganisms is delayed, the stability of the number of living bacteria is maintained to a certain extent, and the feeding frequency is reduced;
(5) According to the preparation method of the culture medium, the reducing agent is added under the anaerobic condition of 100% nitrogen, so that the oxidation of the culture medium is avoided, the growth of clostridium perfringens is not facilitated, the working procedures such as boiling and degassing are added, the preparation environment of the culture medium is strictly controlled, the anaerobic characteristic of the culture medium is ensured, and the growth of clostridium perfringens is ensured.
Drawings
In order to more clearly illustrate the embodiments of the invention or the technical solutions in the prior art, the drawings that are required in the embodiments or the description of the prior art will be briefly described, it being obvious that the drawings in the following description are only some embodiments of the invention, and that other drawings may be obtained according to these drawings without inventive effort for a person skilled in the art.
FIG. 1 is a graph showing the growth of Clostridium aerovorans over time in example 1.
FIG. 2 is a graph showing the growth effect of Clostridium aerovorans cultured for 2 days in example 1.
FIG. 3 is a comparative graph of the turbidity of clostridium perfringens of examples 1-4 and the control group.
FIG. 4 is a graph showing the growth effect of Clostridium aerovorans cultured for 2 days in example 2.
FIG. 5 is a graph showing the growth effect of Clostridium aerovorans cultured for 2 days in example 3.
FIG. 6 is a graph showing the growth effect of Clostridium aerovorans cultured for 2 days in example 4.
FIG. 7 is a graph showing the growth effect of Clostridium aerovorans cultured for 2 days in the control group.
Description of the embodiments
In order to make the objects, technical solutions and advantages of the present invention more apparent, the technical solutions of the present invention will be described in detail below. It will be apparent that the embodiments described are only some, but not all, embodiments of the invention. All other embodiments, based on the examples herein, which are within the scope of the invention as defined by the claims, will be within the scope of the invention as defined by the claims.
The strain source in the examples below was ATCC 55383, and the reagents or apparatus were not identified to the manufacturer and were all conventional products commercially available through regular channels.
A clostridium aerovorum enrichment medium and a preparation method thereof are as follows:
the formula of the culture medium is as follows:
the culture medium per liter contains 1-5g of sodium bicarbonate, 2-4g of yeast extract powder, 0.1-2g of soluble starch, 1-2g of sorbitol, 2-3g of fructose, 0.5-0.8g of alpha-ketoglutarate, 0.01-0.03g of 3-indoleacetic acid, 0.1-0.3g of L-cysteine hydrochloride, 0.01-0.05g of sodium hydroxide, 0.1-0.3g of sodium sulfide, 5-8mL of metal solution, 5-8mL of vitamin solution and 5-10mL of amino acid solution.
Wherein:
the vitamin solution comprises 0.2mg of nicotinamide, 7mg of rutin, 3mg of riboflavin, 5mg of vitamin C and 8mg of lipoic acid per liter;
the vitamin solution in the proportion participates in the formation of various prosthetic groups and coenzyme in clostridium perfringens through the combined action of various vitamins, maintains and regulates the normal metabolism of microbial cells.
The amino acid solution comprises 5g of arginine, 2g of serine, 3g of tryptophan and 0.5g of glutamic acid per liter.
Amino acids are important components of proteins during the growth of microbial cells, and proteins determine the function of the individual structures of the microorganism. The formula screens out four amino acids through multiple experiments, and through proper proportion, clostridium perfringens can rapidly utilize amino acids to synthesize important proteins required by life in an activation period, so that clostridium perfringens can rapidly proliferate and enter a logarithmic growth phase.
The metal solution comprises 5mg of sodium selenite, 12mg of sodium tungstate, 0.01g of zinc gluconate, 2g of bismuth ammonium citrate, 5g of sodium chloride, 0.7g of manganese chloride, 0.15g of cobalt sulfate, 0.05g of ferrous sulfate, 0.3g of zinc nitrate, 0.04g of aluminum potassium sulfate and 0.03g of boric acid.
The enzyme activity of clostridium perfringens needs to be regulated and controlled by a plurality of metal ions, the enzyme activity also affects the alcohol production and acid production rate of clostridium perfringens, and the metal solution with the proportion can maintain the alcohol production and acid production of clostridium perfringens, and is favorable for the growth and propagation of clostridium perfringens under certain conditions.
The formula is optimized, so that not only can various microelements required by clostridium perfringens growth be met, but also 4 amino acids favorable for clostridium perfringens growth can be screened out from 20 amino acids through combination, the nutrition is more comprehensive, the growth division rate of clostridium perfringens can be improved, and the clostridium perfringens can rapidly enter the logarithmic phase.
Preparation of the culture medium:
(1) Adding 5mg of sodium selenite, 12mg of sodium tungstate, 0.01g of zinc gluconate, 2g of bismuth ammonium citrate, 5g of sodium chloride, 0.7g of manganese chloride, 0.15g of cobalt sulfate, 0.05g of ferrous sulfate, 0.3g of zinc nitrate, 0.04g of aluminum potassium sulfate and 0.03g of boric acid into a container I containing one liter of deionized water, stirring and dissolving to prepare a metal solution;
(2) Adding 0.2mg of nicotinamide, 7mg of rutin, 3mg of riboflavin, 5mg of vitamin C and 8mg of lipoic acid into a second container containing one liter of deionized water, stirring and dissolving to prepare a vitamin solution;
(3) Putting 900mL of deionized water into a container III, weighing 1-5g of sodium bicarbonate, 2-4g of yeast extract powder, 0.1-2g of soluble starch, 0.5-0.8g of alpha-ketoglutarate and 0.01-0.03g of 3-indoleacetic acid, adding into the deionized water, adding 5-8mL of metal solution of the container I, stirring and dissolving 5-8mL of vitamin solution of the container II, boiling, degassing, anaerobically subpackaging and sealing, and sterilizing at 121 ℃ for 15 minutes;
(4) Adding 1-2g of sorbitol, 2-3g of fructose, 0.01-0.05g of sodium hydroxide, 0.1-0.3g of L-cysteine hydrochloride and 0.1-0.3g of sodium sulfide into a container IV, adding 100ml of deionized water, stirring and dissolving, boiling, degassing, sub-packaging and sealing anaerobically, and sterilizing at 121 ℃ for 15 minutes;
(5) Adding 5g of arginine, 2g of serine, 3g of tryptophan and 0.5g of glutamic acid into a container under anaerobic condition, adding 1 liter of deionized water, stirring for dissolving, filtering for sterilization, packaging and sealing to prepare an amino acid solution;
(6) And (3) immediately before use, taking 5-10mL of the amino acid solution in the container five, and uniformly mixing with the liquid prepared in the steps (3) - (4) under anaerobic conditions to obtain the amino acid solution.
The method uses boiling and degassing and nitrogen filling to strictly control anaerobic conditions of a preparation culture medium, and prepares a reducing agent, amino acid and basic components separately, and the reducing agent, the amino acid and the basic components are mixed in use, so that anaerobic environment of clostridium perfringens during growth is ensured, and complex precipitation of the amino acid and the basic components is avoided.
Culturing and observing:
the clostridium perfringens is frozen and stored for 30 days at the temperature of minus 20 ℃ in advance and is taken out for standby. Filling an anaerobic incubator with 80% carbon dioxide and 20% nitrogen, inoculating two groups of diluted and equivalent clostridium perfringens into the clostridium perfringens enrichment culture medium under the anaerobic condition, culturing in the incubator at 37 ℃ for 2 days, taking out one group, culturing for 5 days, centrifuging the taken out bacterial liquid, filtering to separate out bacterial bodies, diluting, performing turbidity detection, and comparing with the clostridium perfringens culture medium which is not optimized.
Example 1
1. The embodiment provides a clostridium aerovorum enrichment medium, which comprises the following formula:
each liter of the culture medium contains 3g of sodium bicarbonate, 4g of yeast extract powder, 0.5g of soluble starch, 1g of sorbitol, 3g of fructose, 0.8g of alpha-ketoglutarate, 0.01g of 3-indoleacetic acid, 0.2g of L-cysteine hydrochloride, 0.03g of sodium hydroxide, 0.1g of sodium sulfide, 5mL of metal solution, 8mL of vitamin solution and 10mL of amino acid solution.
The method comprises the following steps:
(1) Adding 5mg of sodium selenite, 12mg of sodium tungstate, 0.01g of zinc gluconate, 2g of bismuth ammonium citrate, 5g of sodium chloride, 0.7g of manganese chloride, 0.15g of cobalt sulfate, 0.05g of ferrous sulfate, 0.3g of zinc nitrate, 0.04g of aluminum potassium sulfate and 0.03g of boric acid into a container I containing one liter of deionized water, stirring and dissolving to prepare a metal solution;
(2) Adding 0.2mg of nicotinamide, 7mg of rutin, 3mg of riboflavin, 5mg of vitamin C and 8mg of lipoic acid into a second container containing one liter of deionized water, stirring and dissolving to prepare a vitamin solution;
(3) Putting 900mL of deionized water into a container III, weighing 3g of sodium bicarbonate, 4g of yeast extract powder, 0.5g of soluble starch, 0.8g of alpha-ketoglutaric acid and 0.01g of 3-indoleacetic acid, adding 5mL of metal solution of the container I, stirring and dissolving 8mL of vitamin solution of the container II, boiling, degassing, anaerobically packaging and sealing, and sterilizing at 121 ℃ for 15 minutes;
(4) Adding 1g of sorbitol, 3g of fructose, 0.03g of sodium hydroxide, 0.2g of L-cysteine hydrochloride and 0.1g of sodium sulfide into a container IV, adding 100ml of deionized water, stirring for dissolution, boiling, degassing, anaerobically packaging, sealing, and sterilizing at 121 ℃ for 15 minutes;
(5) Adding 5g of arginine, 2g of serine, 3g of tryptophan and 0.5g of glutamic acid into a container under anaerobic condition, adding 1 liter of deionized water, stirring for dissolving, filtering for sterilization, packaging and sealing to prepare an amino acid solution;
(6) And (3) immediately before use, mixing 10mL of the amino acid solution in the fifth container with the liquid prepared in the steps (3) - (4) under anaerobic conditions uniformly to obtain the amino acid solution.
2. Culturing:
the anaerobic incubator is filled with 80% carbon dioxide and 20% nitrogen, clostridium aerovorum is inoculated into the clostridium aerovorum enrichment medium under the anaerobic condition, one group is placed into the incubator at 37 ℃ for 2 days, and the other group is cultivated for 5 days.
The graph of clostridium perfringens growth over time in example 1 is shown in figure 1.
The results of the culture of Clostridium aerophilum in the medium prepared in example 1 for 2 days are shown in FIG. 2.
3. Setting a control
Taking non-optimized clostridium perfringens culture medium as a control, inoculating clostridium perfringens liquid into the culture medium under anaerobic condition, and culturing one group at 37 ℃ for 2 days and culturing the other group for 5 days. The above control group was not noted in the same manner as in example 1 of the present invention.
The results of the control group clostridium aerovorans culture for 2 days are shown in fig. 7.
And (3) carrying out low-temperature centrifugation on clostridium aerovorum bacterial liquid after 2 days and 5 days of culture, removing supernatant, adding 10ml of 0.9% sterile physiological saline, uniformly mixing on a vortex mixer, carrying out low-temperature centrifugation again, removing supernatant, adding 10ml of 0.9% sterile physiological saline, uniformly mixing, and then placing into a turbidimeter to detect specific turbidity, wherein the higher the specific turbidity is, the higher the bacterial content is. FIG. 3 is a comparative graph of the turbidity of clostridium perfringens of examples 1-4 and the control group. Compared with a control group, the embodiment is inoculated with an equivalent bacterial count in an equivalent culture medium, and after 2 days of culture, the clostridium perfringens of the embodiment can be rapidly proliferated, so that the clostridium perfringens is beneficial to enrichment culture of clostridium perfringens, after 5 days of culture, the bacterial count is stable and slightly reduced, while in the control group, the clostridium perfringens is slowly proliferated, and after a certain time, nutrient substances in the culture medium cannot meet the growth requirements of clostridium perfringens, so that the clostridium perfringens is not beneficial to enrichment culture of clostridium perfringens.
As can be seen from the comparison of the results of FIG. 3, the present embodiment 1 is a preferred embodiment of the present invention.
Example 2
1. The embodiment provides a clostridium aerovorum enrichment medium, which comprises the following formula:
each liter of the culture medium contains 1g of sodium bicarbonate, 2g of yeast extract powder, 0.7g of soluble starch, 1.5g of sorbitol, 2.5g of fructose, 0.8g of alpha-ketoglutarate, 0.01g of 3-indoleacetic acid, 0.3g of L-cysteine hydrochloride, 0.02g of sodium hydroxide, 0.1g of sodium sulfide, 6mL of metal solution, 6mL of vitamin solution and 5mL of amino acid solution.
The method comprises the following steps:
(1) Adding 5mg of sodium selenite, 12mg of sodium tungstate, 0.01g of zinc gluconate, 2g of bismuth ammonium citrate, 5g of sodium chloride, 0.7g of manganese chloride, 0.15g of cobalt sulfate, 0.05g of ferrous sulfate, 0.3g of zinc nitrate, 0.04g of aluminum potassium sulfate and 0.03g of boric acid into a container I containing one liter of deionized water, stirring and dissolving to prepare a metal solution;
(2) Adding 0.2mg of nicotinamide, 7mg of rutin, 3mg of riboflavin, 5mg of vitamin C and 8mg of lipoic acid into a second container containing one liter of deionized water, stirring and dissolving to prepare a vitamin solution;
(3) Putting 900mL of deionized water into a container III, weighing 1g of sodium bicarbonate, 2g of yeast extract powder, 0.7g of soluble starch, 0.8g of alpha-ketoglutaric acid and 0.01g of 3-indoleacetic acid, adding 6mL of metal solution of the container I, stirring and dissolving 6mL of vitamin solution of the container II, boiling, degassing, anaerobically packaging and sealing, and sterilizing at 121 ℃ for 15 minutes;
(4) Adding 1.5g of sorbitol, 2.5g of fructose, 0.02g of sodium hydroxide, 0.3g of L-cysteine hydrochloride and 0.1g of sodium sulfide into a container IV, adding 100ml of deionized water, stirring for dissolution, boiling, degassing, sub-packaging and sealing anaerobically, and sterilizing at 121 ℃ for 15 minutes;
(5) Adding 5g of arginine, 2g of serine, 3g of tryptophan and 0.5g of glutamic acid into a container under anaerobic condition, adding 1 liter of deionized water, stirring for dissolving, filtering for sterilization, packaging and sealing to prepare an amino acid solution;
(6) And (3) immediately before use, taking 5mL of the amino acid solution in the container five, and uniformly mixing the amino acid solution with the liquid prepared in the steps (3) - (4) under anaerobic conditions to obtain the amino acid solution.
2. Culturing:
the anaerobic incubator is filled with 80% carbon dioxide and 20% nitrogen, clostridium aerovorum is inoculated into the clostridium aerovorum enrichment medium under the anaerobic condition, one group is placed into the incubator at 37 ℃ for 2 days, and the other group is cultivated for 5 days.
The results of the culture of Clostridium aerophilum in the medium prepared in example 2 for 2 days are shown in FIG. 4.
3. Setting a control
Taking non-optimized clostridium perfringens culture medium as a control, inoculating clostridium perfringens liquid into the culture medium under anaerobic condition, and culturing one group at 37 ℃ for 2 days and culturing the other group for 5 days. The above control group was not noted in the same manner as in example 1 of the present invention.
The results of the control group clostridium aerovorans culture for 2 days are shown in fig. 7.
And (3) centrifuging the clostridium aerophilum bacterial liquid after 2 days and 5 days of culture at low temperature, removing the supernatant, adding 10ml of 0.9% sterile physiological saline, uniformly mixing on a vortex mixer, centrifuging at low temperature again, removing the supernatant, adding 10ml of 0.9% sterile physiological saline, uniformly mixing, and then placing into a turbidimeter to detect specific turbidity. Compared with the control group, the embodiment is inoculated with an equivalent bacterial count in an equivalent culture medium, and the clostridium perfringens of the embodiment can be rapidly proliferated after 2 days of culture, which is beneficial to the enrichment culture of clostridium perfringens, but the bacterial count is obviously less than that of embodiment 1, and the bacterial count is stable and slightly reduced after 5 days of culture. In the control group, the proliferation of clostridium perfringens is slow, and nutrient substances in a culture medium in the later period of culture cannot meet the growth requirement of clostridium perfringens, so that the enrichment culture of clostridium perfringens is not facilitated.
Example 3
1. The embodiment provides a clostridium aerovorum enrichment medium, which comprises the following formula:
each liter of the culture medium contains 2g of sodium bicarbonate, 3g of yeast extract powder, 1g of soluble starch, 1g of sorbitol, 2.5g of fructose, 0.6g of alpha-ketoglutarate, 0.01g of 3-indoleacetic acid, 0.2g of L-cysteine hydrochloride, 0.04g of sodium hydroxide, 0.2g of sodium sulfide, 5mL of metal solution, 7mL of vitamin solution and 7mL of amino acid solution.
The method comprises the following steps:
(1) Adding 5mg of sodium selenite, 12mg of sodium tungstate, 0.01g of zinc gluconate, 2g of bismuth ammonium citrate, 5g of sodium chloride, 0.7g of manganese chloride, 0.15g of cobalt sulfate, 0.05g of ferrous sulfate, 0.3g of zinc nitrate, 0.04g of aluminum potassium sulfate and 0.03g of boric acid into a container I containing one liter of deionized water, stirring and dissolving to prepare a metal solution;
(2) Adding 0.2mg of nicotinamide, 7mg of rutin, 3mg of riboflavin, 5mg of vitamin C and 8mg of lipoic acid into a second container containing one liter of deionized water, stirring and dissolving to prepare a vitamin solution;
(3) Putting 900mL of deionized water into a container III, weighing 2g of sodium bicarbonate, 3g of yeast extract powder, 1g of soluble starch, 0.6g of alpha-ketoglutarate and 0.01g of 3-indoleacetic acid, adding 5mL of metal solution of the container I, stirring and dissolving 7mL of vitamin solution of the container II, boiling, degassing, anaerobically packaging and sealing, and sterilizing at 121 ℃ for 15 minutes;
(4) Adding 1g of sorbitol, 2.5g of fructose, 0.04g of sodium hydroxide, 0.2g of L-cysteine hydrochloride and 0.2g of sodium sulfide into a container IV, adding 100ml of deionized water, stirring for dissolution, boiling, degassing, sub-packaging and sealing anaerobically, and sterilizing at 121 ℃ for 15 minutes;
(5) Adding 5g of arginine, 2g of serine, 3g of tryptophan and 0.5g of glutamic acid into a container under anaerobic condition, adding 1 liter of deionized water, stirring for dissolving, filtering for sterilization, packaging and sealing to prepare an amino acid solution;
(6) And (3) immediately before use, taking 7mL of the amino acid solution in the container five, and uniformly mixing the amino acid solution with the liquid prepared in the steps (3) - (4) under anaerobic conditions to obtain the amino acid solution.
2. Culturing:
the anaerobic incubator is filled with 80% carbon dioxide and 20% nitrogen, clostridium aerovorum is inoculated into the clostridium aerovorum enrichment medium under the anaerobic condition, one group is placed into the incubator at 37 ℃ for 2 days, and the other group is cultivated for 5 days.
The results of the culture of Clostridium aerophilum in the medium prepared in example 3 for 2 days are shown in FIG. 5.
3. Setting a control
Taking non-optimized clostridium perfringens culture medium as a control, inoculating clostridium perfringens liquid into the culture medium under anaerobic condition, and culturing one group at 37 ℃ for 2 days and culturing the other group for 5 days. The above control group was not noted in the same manner as in example 1 of the present invention.
The results of the control group clostridium aerovorans culture for 2 days are shown in fig. 7.
And (3) centrifuging the clostridium aerophilum bacterial liquid after 2 days and 5 days of culture at low temperature, removing the supernatant, adding 10ml of 0.9% sterile physiological saline, uniformly mixing on a vortex mixer, centrifuging at low temperature again, removing the supernatant, adding 10ml of 0.9% sterile physiological saline, uniformly mixing, and then placing into a turbidimeter to detect specific turbidity. Compared with a control group, the embodiment is inoculated with an equivalent bacterial count in an equivalent culture medium, and after 2 days of culture, the clostridium perfringens of the embodiment can be rapidly proliferated, which is beneficial to the enrichment culture of clostridium perfringens, but the bacterial count is obviously less than that of embodiment 1 and more than that of embodiment 2, and after 5 days of culture, the bacterial count is stable and slightly reduced. In the control group, the proliferation of clostridium perfringens is slow, and nutrient substances in a culture medium in the later period of culture cannot meet the growth requirement of clostridium perfringens, so that the enrichment culture of clostridium perfringens is not facilitated.
Example 4
1. The embodiment provides a clostridium aerovorum enrichment medium, which comprises the following formula:
each liter of the culture medium contains 2.5g of sodium bicarbonate, 3.5g of yeast extract powder, 0.5g of soluble starch, 2g of sorbitol, 3g of fructose, 0.6g of alpha-ketoglutarate, 0.02g of 3-indoleacetic acid, 0.25g of L-cysteine hydrochloride, 0.05g of sodium hydroxide, 0.25g of sodium sulfide, 6mL of metal solution, 6mL of vitamin solution and 9mL of amino acid solution.
The method comprises the following steps:
(1) Adding 5mg of sodium selenite, 12mg of sodium tungstate, 0.01g of zinc gluconate, 2g of bismuth ammonium citrate, 5g of sodium chloride, 0.7g of manganese chloride, 0.15g of cobalt sulfate, 0.05g of ferrous sulfate, 0.3g of zinc nitrate, 0.04g of aluminum potassium sulfate and 0.03g of boric acid into a container I containing one liter of deionized water, stirring and dissolving to prepare a metal solution;
(2) Adding 0.2mg of nicotinamide, 7mg of rutin, 3mg of riboflavin, 5mg of vitamin C and 8mg of lipoic acid into a second container containing one liter of deionized water, stirring and dissolving to prepare a vitamin solution;
(3) Putting 900mL of deionized water into a container III, weighing 2.5g of sodium bicarbonate, 3.5g of yeast extract powder, 0.5g of soluble starch, 0.6g of alpha-ketoglutaric acid and 0.02g of 3-indoleacetic acid, adding 6mL of metal solution of the container I, stirring and dissolving 6mL of vitamin solution of the container II, boiling, degassing, sub-packaging and sealing anaerobically, and sterilizing at 121 ℃ for 15 minutes;
(4) Adding 2g of sorbitol, 3g of fructose, 0.05g of sodium hydroxide, 0.25g of L-cysteine hydrochloride and 0.25g of sodium sulfide into a container IV, adding 100ml of deionized water, stirring for dissolution, boiling, degassing, anaerobically packaging, sealing, and sterilizing at 121 ℃ for 15 minutes;
(5) Adding 5g of arginine, 2g of serine, 3g of tryptophan and 0.5g of glutamic acid into a container under anaerobic condition, adding 1 liter of deionized water, stirring for dissolving, filtering for sterilization, packaging and sealing to prepare an amino acid solution;
(6) And (3) immediately before use, taking 9mL of the amino acid solution in the container five, and uniformly mixing the amino acid solution with the liquid prepared in the steps (3) - (4) under anaerobic conditions to obtain the amino acid solution.
2. Culturing:
the anaerobic incubator is filled with 80% carbon dioxide and 20% nitrogen, clostridium aerovorum is inoculated into the clostridium aerovorum enrichment medium under the anaerobic condition, one group is placed into the incubator at 37 ℃ for 2 days, and the other group is cultivated for 5 days.
The results of the culture of Clostridium aerophilum in the medium prepared in example 4 for 2 days are shown in FIG. 6.
3. Setting a control
Taking non-optimized clostridium perfringens culture medium as a control, inoculating clostridium perfringens liquid into the culture medium under anaerobic condition, and culturing one group at 37 ℃ for 2 days and culturing the other group for 5 days. The above control group was not noted in the same manner as in example 1 of the present invention.
The results of the control group clostridium aerovorans culture for 2 days are shown in fig. 7.
And (3) centrifuging the clostridium aerophilum bacterial liquid after 2 days and 5 days of culture at low temperature, removing the supernatant, adding 10ml of 0.9% sterile physiological saline, uniformly mixing on a vortex mixer, centrifuging at low temperature again, removing the supernatant, adding 10ml of 0.9% sterile physiological saline, uniformly mixing, and then placing into a turbidimeter to detect specific turbidity. Compared with the control group, the embodiment is inoculated with the same bacterial count in the same culture medium, and after 2 days of culture, the clostridium perfringens of the embodiment can be rapidly proliferated, which is beneficial to the enrichment culture of clostridium perfringens, but the bacterial count is less than that of embodiment 1 and more than that of embodiments 2 and 3, and after 5 days of culture, the bacterial count is stable and slightly reduced. In the control group, the proliferation of clostridium perfringens is slow, and nutrient substances in a culture medium in the later period of culture cannot meet the growth requirement of clostridium perfringens, so that the enrichment culture of clostridium perfringens is not facilitated.
In summary, the clostridium perfringens enrichment medium of the invention is adopted in examples 1-4, so that clostridium perfringens can be rapidly proliferated in a short time and decline is slow after the bacterial count reaches the highest value along with the increase of the culture days in a certain time; compared with examples 2-4, the clostridium aerovorum enrichment medium prepared according to the preparation method of example 1 has the best enrichment effect, and the culture period is 2 days. The rapid enrichment culture of clostridium perfringens can fill the defect of the existing clostridium perfringens culture medium, does not need to be activated again for frozen strains, can be directly put into production, improves the production efficiency, can be used for fermentation production with higher product purification requirements, and is beneficial to various scientific researches of clostridium perfringens in the future.
It will be apparent that the described embodiments are merely some, but not all embodiments of the invention. All other embodiments, based on the examples herein, which are within the scope of the invention as defined by the claims, will be within the scope of the invention as defined by the claims.
Claims (2)
1. An aerothrice increasing culture medium, which is characterized in that: 1-5g of sodium bicarbonate, 2-4g of yeast extract powder, 0.1-2g of soluble starch, 1-2g of sorbitol, 2-3g of fructose, 0.5-0.8g of alpha-ketoglutarate, 0.01-0.03g of 3-indoleacetic acid, 0.1-0.3g of L-cysteine hydrochloride, 0.01-0.05g of sodium hydroxide, 0.1-0.3g of sodium sulfide, 5-8mL of metal solution, 5-8mL of vitamin solution and 5-10mL of amino acid solution;
the amino acid solution comprises 5g of arginine, 2g of serine, 3g of tryptophan and 0.5g of glutamic acid per liter;
the metal solution comprises 5mg of sodium selenite, 12mg of sodium tungstate, 0.01g of zinc gluconate, 2g of bismuth ammonium citrate, 5g of sodium chloride, 0.7g of manganese chloride, 0.15g of cobalt sulfate, 0.05g of ferrous sulfate, 0.3g of zinc nitrate, 0.04g of aluminum potassium sulfate and 0.03g of boric acid per liter;
the vitamin solution comprises 0.2mg of nicotinamide, 7mg of rutin, 3mg of riboflavin, 5mg of vitamin C and 8mg of lipoic acid per liter.
2. A method for preparing clostridium aerovorum enrichment medium according to claim 1, wherein:
(1) Adding 5mg of sodium selenite, 12mg of sodium tungstate, 0.01g of zinc gluconate, 2g of bismuth ammonium citrate, 5g of sodium chloride, 0.7g of manganese chloride, 0.15g of cobalt sulfate, 0.05g of ferrous sulfate, 0.3g of zinc nitrate, 0.04g of aluminum potassium sulfate and 0.03g of boric acid into a container I containing one liter of deionized water, stirring and dissolving to prepare a metal solution;
(2) Adding 0.2mg of nicotinamide, 7mg of rutin, 3mg of riboflavin, 5mg of vitamin C and 8mg of lipoic acid into a second container containing one liter of deionized water, stirring and dissolving to prepare a vitamin solution;
(3) Putting 900mL of deionized water into a container III, weighing 1-5g of sodium bicarbonate, 2-4g of yeast extract powder, 0.1-2g of soluble starch, 0.5-0.8g of alpha-ketoglutarate and 0.01-0.03g of 3-indoleacetic acid, adding into the deionized water, adding 5-8mL of metal solution of the container I, stirring and dissolving 5-8mL of vitamin solution of the container II, boiling, degassing, anaerobically subpackaging and sealing, and sterilizing at 121 ℃ for 15 minutes;
(4) Adding 1-2g of sorbitol, 2-3g of fructose, 0.01-0.05g of sodium hydroxide, 0.1-0.3g of L-cysteine hydrochloride and 0.1-0.3g of sodium sulfide into a container IV, adding 100ml of deionized water, stirring and dissolving, boiling, degassing, sub-packaging and sealing anaerobically, and sterilizing at 121 ℃ for 15 minutes;
(5) Adding 5g of arginine, 2g of serine, 3g of tryptophan and 0.5g of glutamic acid into a container under anaerobic condition, adding 1 liter of deionized water, stirring for dissolving, filtering for sterilization, packaging and sealing to prepare an amino acid solution;
(6) And (3) immediately before use, taking 5-10mL of the amino acid solution in the container five, and uniformly mixing with the liquid prepared in the steps (3) - (4) under anaerobic conditions to obtain the amino acid solution.
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