CN110777184B - 一种发酵核黄素的发酵培养基及其使用方法 - Google Patents
一种发酵核黄素的发酵培养基及其使用方法 Download PDFInfo
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- CN110777184B CN110777184B CN201911184728.3A CN201911184728A CN110777184B CN 110777184 B CN110777184 B CN 110777184B CN 201911184728 A CN201911184728 A CN 201911184728A CN 110777184 B CN110777184 B CN 110777184B
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- riboflavin
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- glucose
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- 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 title claims abstract description 106
- 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 title claims abstract description 52
- 229960002477 riboflavin Drugs 0.000 title claims abstract description 52
- 235000019192 riboflavin Nutrition 0.000 title claims abstract description 52
- 239000002151 riboflavin Substances 0.000 title claims abstract description 52
- 238000000855 fermentation Methods 0.000 title claims abstract description 49
- 230000004151 fermentation Effects 0.000 title claims abstract description 49
- 238000000034 method Methods 0.000 title claims abstract description 20
- 239000001963 growth medium Substances 0.000 claims abstract description 42
- ULGZDMOVFRHVEP-RWJQBGPGSA-N Erythromycin Chemical compound O([C@@H]1[C@@H](C)C(=O)O[C@@H]([C@@]([C@H](O)[C@@H](C)C(=O)[C@H](C)C[C@@](C)(O)[C@H](O[C@H]2[C@@H]([C@H](C[C@@H](C)O2)N(C)C)O)[C@H]1C)(C)O)CC)[C@H]1C[C@@](C)(OC)[C@@H](O)[C@H](C)O1 ULGZDMOVFRHVEP-RWJQBGPGSA-N 0.000 claims abstract description 18
- 239000002609 medium Substances 0.000 claims abstract description 18
- WQZGKKKJIJFFOK-GASJEMHNSA-N Glucose Natural products OC[C@H]1OC(O)[C@H](O)[C@@H](O)[C@@H]1O WQZGKKKJIJFFOK-GASJEMHNSA-N 0.000 claims abstract description 17
- 239000008103 glucose Substances 0.000 claims abstract description 17
- WRUGWIBCXHJTDG-UHFFFAOYSA-L magnesium sulfate heptahydrate Chemical compound O.O.O.O.O.O.O.[Mg+2].[O-]S([O-])(=O)=O WRUGWIBCXHJTDG-UHFFFAOYSA-L 0.000 claims abstract description 16
- 229940061634 magnesium sulfate heptahydrate Drugs 0.000 claims abstract description 16
- 240000004808 Saccharomyces cerevisiae Species 0.000 claims abstract description 12
- 239000000843 powder Substances 0.000 claims abstract description 11
- LWIHDJKSTIGBAC-UHFFFAOYSA-K potassium phosphate Substances [K+].[K+].[K+].[O-]P([O-])([O-])=O LWIHDJKSTIGBAC-UHFFFAOYSA-K 0.000 claims abstract 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 abstract description 9
- BFNBIHQBYMNNAN-UHFFFAOYSA-N ammonium sulfate Chemical compound N.N.OS(O)(=O)=O BFNBIHQBYMNNAN-UHFFFAOYSA-N 0.000 claims abstract description 9
- 229910052921 ammonium sulfate Inorganic materials 0.000 claims abstract description 9
- 235000011130 ammonium sulphate Nutrition 0.000 claims abstract description 9
- 229960003276 erythromycin Drugs 0.000 claims abstract description 9
- 235000013379 molasses Nutrition 0.000 claims abstract description 9
- 229910000402 monopotassium phosphate Inorganic materials 0.000 claims abstract description 9
- 235000019796 monopotassium phosphate Nutrition 0.000 claims abstract description 9
- 239000011670 zinc gluconate Substances 0.000 claims abstract description 9
- 235000011478 zinc gluconate Nutrition 0.000 claims abstract description 9
- 229960000306 zinc gluconate Drugs 0.000 claims abstract description 9
- SQUHHTBVTRBESD-UHFFFAOYSA-N Hexa-Ac-myo-Inositol Natural products CC(=O)OC1C(OC(C)=O)C(OC(C)=O)C(OC(C)=O)C(OC(C)=O)C1OC(C)=O SQUHHTBVTRBESD-UHFFFAOYSA-N 0.000 claims abstract description 8
- BCKXLBQYZLBQEK-KVVVOXFISA-M Sodium oleate Chemical compound [Na+].CCCCCCCC\C=C/CCCCCCCC([O-])=O BCKXLBQYZLBQEK-KVVVOXFISA-M 0.000 claims abstract description 8
- CDAISMWEOUEBRE-GPIVLXJGSA-N inositol Chemical compound O[C@H]1[C@H](O)[C@@H](O)[C@H](O)[C@H](O)[C@@H]1O CDAISMWEOUEBRE-GPIVLXJGSA-N 0.000 claims abstract description 8
- 229960000367 inositol Drugs 0.000 claims abstract description 8
- CDAISMWEOUEBRE-UHFFFAOYSA-N scyllo-inosotol Natural products OC1C(O)C(O)C(O)C(O)C1O CDAISMWEOUEBRE-UHFFFAOYSA-N 0.000 claims abstract description 8
- 229960005091 chloramphenicol Drugs 0.000 claims abstract description 7
- WIIZWVCIJKGZOK-RKDXNWHRSA-N chloramphenicol Chemical compound ClC(Cl)C(=O)N[C@H](CO)[C@H](O)C1=CC=C([N+]([O-])=O)C=C1 WIIZWVCIJKGZOK-RKDXNWHRSA-N 0.000 claims abstract description 7
- 229920000136 polysorbate Polymers 0.000 claims abstract description 7
- GNSKLFRGEWLPPA-UHFFFAOYSA-M potassium dihydrogen phosphate Chemical compound [K+].OP(O)([O-])=O GNSKLFRGEWLPPA-UHFFFAOYSA-M 0.000 claims abstract description 7
- YBJHBAHKTGYVGT-ZKWXMUAHSA-N (+)-Biotin Chemical compound N1C(=O)N[C@@H]2[C@H](CCCCC(=O)O)SC[C@@H]21 YBJHBAHKTGYVGT-ZKWXMUAHSA-N 0.000 claims description 14
- 239000004475 Arginine Substances 0.000 claims description 7
- QNAYBMKLOCPYGJ-REOHCLBHSA-N L-alanine Chemical compound C[C@H](N)C(O)=O QNAYBMKLOCPYGJ-REOHCLBHSA-N 0.000 claims description 7
- 235000004279 alanine Nutrition 0.000 claims description 7
- ODKSFYDXXFIFQN-UHFFFAOYSA-N arginine Natural products OC(=O)C(N)CCCNC(N)=N ODKSFYDXXFIFQN-UHFFFAOYSA-N 0.000 claims description 7
- 229960002685 biotin Drugs 0.000 claims description 7
- 235000020958 biotin Nutrition 0.000 claims description 7
- 239000011616 biotin Substances 0.000 claims description 7
- 229930027945 nicotinamide-adenine dinucleotide Natural products 0.000 claims description 7
- 244000063299 Bacillus subtilis Species 0.000 claims description 6
- 235000014469 Bacillus subtilis Nutrition 0.000 claims description 6
- 239000007640 basal medium Substances 0.000 claims description 5
- IFGCUJZIWBUILZ-UHFFFAOYSA-N sodium 2-[[2-[[hydroxy-(3,4,5-trihydroxy-6-methyloxan-2-yl)oxyphosphoryl]amino]-4-methylpentanoyl]amino]-3-(1H-indol-3-yl)propanoic acid Chemical compound [Na+].C=1NC2=CC=CC=C2C=1CC(C(O)=O)NC(=O)C(CC(C)C)NP(O)(=O)OC1OC(C)C(O)C(O)C1O IFGCUJZIWBUILZ-UHFFFAOYSA-N 0.000 claims description 4
- -1 45g/L Chemical compound 0.000 claims description 3
- WIIZWVCIJKGZOK-IUCAKERBSA-N 2,2-dichloro-n-[(1s,2s)-1,3-dihydroxy-1-(4-nitrophenyl)propan-2-yl]acetamide Chemical compound ClC(Cl)C(=O)N[C@@H](CO)[C@@H](O)C1=CC=C([N+]([O-])=O)C=C1 WIIZWVCIJKGZOK-IUCAKERBSA-N 0.000 claims description 2
- ACFIXJIJDZMPPO-NNYOXOHSSA-N NADPH Chemical compound C1=CCC(C(=O)N)=CN1[C@H]1[C@H](O)[C@H](O)[C@@H](COP(O)(=O)OP(O)(=O)OC[C@@H]2[C@H]([C@@H](OP(O)(O)=O)[C@@H](O2)N2C3=NC=NC(N)=C3N=C2)O)O1 ACFIXJIJDZMPPO-NNYOXOHSSA-N 0.000 claims 2
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- 238000004519 manufacturing process Methods 0.000 description 6
- XJLXINKUBYWONI-DQQFMEOOSA-N [[(2r,3r,4r,5r)-5-(6-aminopurin-9-yl)-3-hydroxy-4-phosphonooxyoxolan-2-yl]methoxy-hydroxyphosphoryl] [(2s,3r,4s,5s)-5-(3-carbamoylpyridin-1-ium-1-yl)-3,4-dihydroxyoxolan-2-yl]methyl phosphate Chemical compound NC(=O)C1=CC=C[N+]([C@@H]2[C@H]([C@@H](O)[C@H](COP([O-])(=O)OP(O)(=O)OC[C@@H]3[C@H]([C@@H](OP(O)(O)=O)[C@@H](O3)N3C4=NC=NC(N)=C4N=C3)O)O2)O)=C1 XJLXINKUBYWONI-DQQFMEOOSA-N 0.000 description 5
- 235000009697 arginine Nutrition 0.000 description 5
- 238000006243 chemical reaction Methods 0.000 description 5
- IJGRMHOSHXDMSA-UHFFFAOYSA-N Atomic nitrogen Chemical compound N#N IJGRMHOSHXDMSA-UHFFFAOYSA-N 0.000 description 4
- 241000894006 Bacteria Species 0.000 description 4
- PYMYPHUHKUWMLA-LMVFSUKVSA-N aldehydo-D-ribose Chemical compound OC[C@@H](O)[C@@H](O)[C@@H](O)C=O PYMYPHUHKUWMLA-LMVFSUKVSA-N 0.000 description 4
- 230000004907 flux Effects 0.000 description 4
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- 239000000126 substance Substances 0.000 description 4
- QIVBCDIJIAJPQS-VIFPVBQESA-N L-tryptophane Chemical compound C1=CC=C2C(C[C@H](N)C(O)=O)=CNC2=C1 QIVBCDIJIAJPQS-VIFPVBQESA-N 0.000 description 3
- OAICVXFJPJFONN-UHFFFAOYSA-N Phosphorus Chemical compound [P] OAICVXFJPJFONN-UHFFFAOYSA-N 0.000 description 3
- QIVBCDIJIAJPQS-UHFFFAOYSA-N Tryptophan Natural products C1=CC=C2C(CC(N)C(O)=O)=CNC2=C1 QIVBCDIJIAJPQS-UHFFFAOYSA-N 0.000 description 3
- QVGXLLKOCUKJST-UHFFFAOYSA-N atomic oxygen Chemical compound [O] QVGXLLKOCUKJST-UHFFFAOYSA-N 0.000 description 3
- 239000002054 inoculum Substances 0.000 description 3
- 235000016709 nutrition Nutrition 0.000 description 3
- 230000035764 nutrition Effects 0.000 description 3
- 229910052760 oxygen Inorganic materials 0.000 description 3
- 239000001301 oxygen Substances 0.000 description 3
- 229910052698 phosphorus Inorganic materials 0.000 description 3
- 239000011574 phosphorus Substances 0.000 description 3
- 239000002243 precursor Substances 0.000 description 3
- 238000002360 preparation method Methods 0.000 description 3
- 239000002994 raw material Substances 0.000 description 3
- 238000011218 seed culture Methods 0.000 description 3
- 230000001954 sterilising effect Effects 0.000 description 3
- 102000004190 Enzymes Human genes 0.000 description 2
- 108090000790 Enzymes Proteins 0.000 description 2
- CSNNHWWHGAXBCP-UHFFFAOYSA-L Magnesium sulfate Chemical compound [Mg+2].[O-][S+2]([O-])([O-])[O-] CSNNHWWHGAXBCP-UHFFFAOYSA-L 0.000 description 2
- 101100379086 Mus musculus Ankrd2 gene Proteins 0.000 description 2
- DFPAKSUCGFBDDF-UHFFFAOYSA-N Nicotinamide Chemical compound NC(=O)C1=CC=CN=C1 DFPAKSUCGFBDDF-UHFFFAOYSA-N 0.000 description 2
- GZCGUPFRVQAUEE-SLPGGIOYSA-N aldehydo-D-glucose Chemical compound OC[C@@H](O)[C@@H](O)[C@H](O)[C@@H](O)C=O GZCGUPFRVQAUEE-SLPGGIOYSA-N 0.000 description 2
- 235000001014 amino acid Nutrition 0.000 description 2
- 150000001413 amino acids Chemical class 0.000 description 2
- PYMYPHUHKUWMLA-UHFFFAOYSA-N arabinose Natural products OCC(O)C(O)C(O)C=O PYMYPHUHKUWMLA-UHFFFAOYSA-N 0.000 description 2
- 230000008901 benefit Effects 0.000 description 2
- SRBFZHDQGSBBOR-UHFFFAOYSA-N beta-D-Pyranose-Lyxose Natural products OC1COC(O)C(O)C1O SRBFZHDQGSBBOR-UHFFFAOYSA-N 0.000 description 2
- UYEUUXMDVNYCAM-UHFFFAOYSA-N dioxotetrahydropteridine Natural products N1=CC=NC2=NC(O)=NC(O)=C21 UYEUUXMDVNYCAM-UHFFFAOYSA-N 0.000 description 2
- 238000006911 enzymatic reaction 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
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- 229910052757 nitrogen Inorganic materials 0.000 description 2
- 235000015097 nutrients Nutrition 0.000 description 2
- 230000037361 pathway Effects 0.000 description 2
- PJNZPQUBCPKICU-UHFFFAOYSA-N phosphoric acid;potassium Chemical compound [K].OP(O)(O)=O PJNZPQUBCPKICU-UHFFFAOYSA-N 0.000 description 2
- 235000010482 polyoxyethylene sorbitan monooleate Nutrition 0.000 description 2
- 229920000053 polysorbate 80 Polymers 0.000 description 2
- PUZPDOWCWNUUKD-UHFFFAOYSA-M sodium fluoride Chemical compound [F-].[Na+] PUZPDOWCWNUUKD-UHFFFAOYSA-M 0.000 description 2
- 239000000243 solution Substances 0.000 description 2
- 102100039375 Ankyrin repeat domain-containing protein 2 Human genes 0.000 description 1
- 235000016068 Berberis vulgaris Nutrition 0.000 description 1
- 241000335053 Beta vulgaris Species 0.000 description 1
- OKTJSMMVPCPJKN-UHFFFAOYSA-N Carbon Chemical compound [C] OKTJSMMVPCPJKN-UHFFFAOYSA-N 0.000 description 1
- 101000961307 Homo sapiens Ankyrin repeat domain-containing protein 2 Proteins 0.000 description 1
- ODKSFYDXXFIFQN-BYPYZUCNSA-P L-argininium(2+) Chemical compound NC(=[NH2+])NCCC[C@H]([NH3+])C(O)=O ODKSFYDXXFIFQN-BYPYZUCNSA-P 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
- 229910019142 PO4 Inorganic materials 0.000 description 1
- 229930003270 Vitamin B Natural products 0.000 description 1
- 240000008042 Zea mays Species 0.000 description 1
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- WQZGKKKJIJFFOK-VFUOTHLCSA-N beta-D-glucose Chemical compound OC[C@H]1O[C@@H](O)[C@H](O)[C@@H](O)[C@@H]1O WQZGKKKJIJFFOK-VFUOTHLCSA-N 0.000 description 1
- 229940041514 candida albicans extract Drugs 0.000 description 1
- 229910052799 carbon Inorganic materials 0.000 description 1
- 230000000052 comparative effect Effects 0.000 description 1
- 150000001875 compounds Chemical class 0.000 description 1
- 235000005822 corn Nutrition 0.000 description 1
- 235000012343 cottonseed oil Nutrition 0.000 description 1
- 238000012258 culturing Methods 0.000 description 1
- ZPWVASYFFYYZEW-UHFFFAOYSA-L dipotassium hydrogen phosphate Chemical compound [K+].[K+].OP([O-])([O-])=O ZPWVASYFFYYZEW-UHFFFAOYSA-L 0.000 description 1
- 229910000396 dipotassium phosphate Inorganic materials 0.000 description 1
- 235000019797 dipotassium phosphate Nutrition 0.000 description 1
- 229940079593 drug Drugs 0.000 description 1
- 239000003814 drug Substances 0.000 description 1
- 230000000694 effects Effects 0.000 description 1
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- 235000021112 essential micronutrients Nutrition 0.000 description 1
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- 229960000304 folic acid Drugs 0.000 description 1
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- 230000034659 glycolysis Effects 0.000 description 1
- 230000036541 health Effects 0.000 description 1
- 238000002347 injection Methods 0.000 description 1
- 239000007924 injection Substances 0.000 description 1
- 229910052943 magnesium sulfate Inorganic materials 0.000 description 1
- 235000019341 magnesium sulphate Nutrition 0.000 description 1
- 239000000463 material Substances 0.000 description 1
- 230000037353 metabolic pathway Effects 0.000 description 1
- 230000004060 metabolic process Effects 0.000 description 1
- 239000002207 metabolite Substances 0.000 description 1
- 230000004048 modification Effects 0.000 description 1
- 238000012986 modification Methods 0.000 description 1
- 229960003966 nicotinamide Drugs 0.000 description 1
- 235000005152 nicotinamide Nutrition 0.000 description 1
- 239000011570 nicotinamide Substances 0.000 description 1
- 235000003715 nutritional status Nutrition 0.000 description 1
- 230000008520 organization Effects 0.000 description 1
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- 238000007254 oxidation reaction Methods 0.000 description 1
- 239000010452 phosphate Substances 0.000 description 1
- 125000002467 phosphate group Chemical group [H]OP(=O)(O[H])O[*] 0.000 description 1
- 230000035790 physiological processes and functions Effects 0.000 description 1
- 229910000160 potassium phosphate Inorganic materials 0.000 description 1
- 235000011009 potassium phosphates Nutrition 0.000 description 1
- 230000001376 precipitating effect Effects 0.000 description 1
- 230000002035 prolonged effect Effects 0.000 description 1
- 230000036647 reaction Effects 0.000 description 1
- 238000006722 reduction reaction Methods 0.000 description 1
- 238000011160 research Methods 0.000 description 1
- 150000003839 salts Chemical class 0.000 description 1
- 239000011775 sodium fluoride Substances 0.000 description 1
- 235000013024 sodium fluoride Nutrition 0.000 description 1
- 239000002904 solvent Substances 0.000 description 1
- 241000894007 species Species 0.000 description 1
- 238000004659 sterilization and disinfection Methods 0.000 description 1
- 238000001308 synthesis method Methods 0.000 description 1
- 230000009897 systematic effect Effects 0.000 description 1
- 239000011573 trace mineral Substances 0.000 description 1
- 235000013619 trace mineral Nutrition 0.000 description 1
- 235000019156 vitamin B Nutrition 0.000 description 1
- 239000011720 vitamin B Substances 0.000 description 1
- 239000012138 yeast extract Substances 0.000 description 1
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- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
- C12P25/00—Preparation of compounds containing alloxazine or isoalloxazine nucleus, e.g. riboflavin
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- 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
- C12N1/38—Chemical stimulation of growth or activity by addition of chemical compounds which are not essential growth factors; Stimulation of growth by removal of a chemical compound
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Abstract
本发明公开了一种发酵核黄素的发酵培养基及其使用方法，涉及微生物技术领域。所述发酵培养基包括基础培养基和流加培养基，其中所述基础培养基的成分和含量为：葡萄糖10‑20g/L、糖蜜30‑60g/L、油酸钠15‑25mL/L、酵母粉5‑8g/L、磷酸二氢钾1.5‑2g/L、七水合硫酸镁0.1‑0.3g/L、葡萄糖酸锌0.3‑0.5g/L、吐温10‑15mL/L、红霉素5‑15mg/L、氯霉素5‑15mg/L，所述流加培养基的成分和含量为：肌醇5‑10g/L、硫酸铵5‑8g/L、葡萄糖30‑50g/L、复合因子500‑900mg/L。本发明解决了现有技术中依赖经验甚于理论，使得核黄素发酵单位偏低的技术问题。
Description
技术领域
本发明属于微生物技术领域，具体涉及一种发酵核黄素的发酵培养基及其使用方法。
背景技术
核黄素(维生素B2，VitaminB2，Riboflavin)分子式为C17H20O6N4，分子量为376.36。化学名称为7,8-二甲基-10-[(2S,3S,4R)-2,3,4,5-四羟基戊基]-3,10-二氢苯并蝶啶-2,4-二酮，系统命名为7,8-二甲基-10-(1’-D-核糖基)异咯嗪[7,8-dimethy-10-(1’-D-ribityl)soalloxazine]；核黄素是机体必需微量营养素之一，具有广泛的生理机能，世界卫生组织(WHO)将其列为评价人体生长发育和营养状况的六大指标之一，核黄素在医药、食品营养强化剂和饲料添加剂工业中广泛应用；《美国药典》规格的核黄素可制成供口服的片剂，供注射的水溶液，或者可以含有烟酰胺或其增溶剂。作为动物饲料的一种添加剂，核黄素通常加入28mg/kg，具体的用量取决于动物的种类和年龄；
核黄素有三种生产方法：(1)生物发酵法：生物发酵法又分为传统的酵母菌发酵法和枯草芽孢杆菌发酵法；(2)化学合成法：以D-葡萄糖为原料，经化学反应合成；(3)化学半合成法：以D-葡萄糖为原料经发酵生成D-核糖，再以D-核糖为原料进行化学合成。
目前在工业上应用的核黄素生产方法主要是酵母菌发酵法和枯草芽孢杆菌发酵法。核黄素发酵工业延续至今已有相当长的历史，和其他微生物发酵产品一样，它的菌体生长，产物形成等所涉及的一系列时刻变化着的生物化学和质量、能量传递过使核黄素发酵表现出相当程度的不确定性。同时又由于反应机理复杂，无合适的模型用以描述过程，使人们在其发酵操作上依赖经验甚于理论，这给核黄素生产水平提高带来了一定的困难。核黄素的获得途径主要是微生物发酵，而微生物的生长、繁殖和产物的合成都需要发酵培养提供必要的营养和能量，所以合适的培养基成分对于核黄素效价的提高尤为重要，但是现有技术中核黄素发酵培养基成分没有针对性，导致核黄素发酵单位偏低等问题。
发明内容
本发明为解决现有技术中依赖经验甚于理论，使得核黄素发酵单位偏低的技术问题，提供了一种发酵核黄素的发酵培养基及其使用方法。
本发明为解决上述技术问题所采用的技术方案是：
一种发酵核黄素的发酵培养基，包括基础培养基和流加培养基，其中所述基础培养基的成分和含量为：葡萄糖10-20g/L、糖蜜30-60g/L、油酸钠15-25mL/L、酵母粉5-8g/L、磷酸二氢钾1.5-2g/L、七水合硫酸镁0.1-0.3g/L、葡萄糖酸锌0.3-0.5g/L、吐温10-15mL/L、红霉素5-15mg/L、氯霉素5-15mg/L，所述流加培养基的成分和含量为：肌醇5-10g/L、硫酸铵5-8g/L、葡萄糖30-50g/L、复合因子500-900mg/L。
优选的，所述复合因子包括NADPH 5-10mg/L、色氨酸100-190mg/L、丙氨酸95-150mg/L、精氨酸100-150mg/L、生物素100-150mg/L、七水合硫酸镁100-250mg/L。
优选的，所述基础培养基的成分和含量为：葡萄糖16g/L、糖蜜54g/L、油酸钠23mL/L、酵母粉8g/L、磷酸二氢钾1.8g/L、七水合硫酸镁0.15g/L、葡萄糖酸锌0.5g/L、吐温8013mL/L、红霉素10mg/L、氯霉素10mg/L。
优选的，所述流加培养基的成分和含量为：肌醇8g/L、硫酸铵7g/L、葡萄糖45g/L、NADPH 8mg/L、色氨酸170mg/L、丙氨酸120mg/L、精氨酸130mg/L、生物素125mg/L、七水合硫酸镁175mg/L。
更进一步的，本发明提供了一种发酵核黄素的发酵培养基的使用方法，首先使用所述基础培养基进行核黄素生产菌株发酵，培养5-10h后开始流加所述流加培养基，流加速度依据培养基中的总糖量确定，保持所述总糖量为在6-8g/L。
采用上述技术方案，本发明取得的技术进步是：本发明提供的一种发酵核黄素的发酵培养基具有以下优势：
①突破了人们长期以来依赖经验大于理论的思想，仔细研究分析了反应机理，采用了基础培养基和流加培养基相结合的方式，提高了核黄素发酵的确定性，更有针对性，明显提高核黄素的发酵效价，能使核黄素发酵48小时平均效价达到50g/L以上，最高能达到58g/L。
②本发明的基础培养基中采用葡萄糖、糖蜜、油酸钠相结合的碳源组成，使得氧化磷酸戊糖途径的代谢通量得到增强，进而增加了前体物5-磷酸核酮糖的供给，进而使得核黄素的产量得到相应的提高。
③本发明的基础培养基中采用酵母粉、磷酸二氢钾相结合的氮、磷源组成保证了菌体生长初期的营养供应，确保了菌体的生长速率，为核黄素的产量的提高奠定了基础。
④本发明的基础培养基中采用七水合硫酸镁和葡萄糖酸锌有两方面的作用，一是微量元素供给，另一个是作为酶促反应中多种酶的激活剂。
⑤本发明的基础培养基中采用吐温80是为了保证培养基的流体状态；采用红霉素、氯霉素是为了保证菌体的遗传特性。
⑥本发明的流加培养基组成主要是发挥以下作用：抑制支路途径，提高核黄素合成支路的通量；增强酶的活性，提高酶促反应效率；增加前体物质及能力供应，促进核黄素的合成。
⑦本发明的流加培养基中采用肌醇、硫酸铵、葡萄糖、复合因子的组合，既可解除营养供应的瓶颈，延长代谢产物产生的时间，提高核黄素的产量；又可以减少糖酵解途径对营养的利用，间接促进核黄素代谢途径中HMP途径的通量，提供核黄素合成所需要的前体物质，促进代谢通量，提高核黄素的产量。
⑧本发明的流加培养基中采用NADPH的目的是加速ARPP到ArPP的还原反应，进而为细胞反应提供能量；采用七水合硫酸镁是为了沉淀ArPP上脱去磷酸基团生成ArP时产生的磷以及二氧四氢蝶啶的合成时产生的磷，进而促进反应的快速进行；采用色氨酸、丙氨酸、精氨酸、生物素的组合是为了加快整个氮通路的代谢速度，使其直接使用容易利用的氨基酸，并根据其菌株的特性进行氨基酸的组合和调配，最终确保了核黄素产量的提高。
具体实施方式
为使本发明实施例的目的、技术方案和优点更加清楚，对本发明实施例中的技术方案进行清楚、完整地描述，显然，所描述的实施例是本发明一部分实施例，而不是全部的实施例。
实施例1
配制基础培养基，其的成分和含量为：葡萄糖10g/L、糖蜜60g/L、油酸钠25mL/L、酵母粉8g/L、磷酸二氢钾1.5g/L、七水合硫酸镁0.15g/L、葡萄糖酸锌0.3g/L、吐温80 15mL/L共配制9L，分别装到3个5L的发酵罐中，每罐3L，灭菌备用，并按照比例添加无菌红霉素10mg/L、氯霉素10mg/L；
配制流加培养基，其的成分和含量为：肌醇10g/L、硫酸铵8g/L、葡萄糖50g/L、七水合硫酸镁250mg/L共配制3L，灭菌备用，并按照比例添加无菌NADPH 10mg/L、色氨酸190mg/L、丙氨酸150mg/L、精氨酸150mg/L、生物素150mg/L。
在核黄素发酵培养的过程中，按15％的接种量向本实施例制备的基础培养基中接入产核黄素枯草芽孢杆菌种子培养物，于温度35-38℃下培养，过程中控制溶氧量15～30％，pH6.8(NH3·H2O调节)的条件下，进行发酵，发酵培养5-10h后开始流加所述流加培养基，流加速度依据培养基中的总糖量确定，保持所述总糖量为在6-8g/L，发酵40h后不再进行流加，48h左右结束发酵，检测效价，三个罐的效价分别为52g/L，48g/L，53g/L。
制备方法：首先使用所述基础培养基进行核黄素生产菌株发酵，培养5-10h后开始流加所述流加培养基，流加速度依据培养基中的总糖量确定，保持所述总糖量为在6-8g/L。
实施例2
配制基础培养基，其的成分和含量为：葡萄糖16g/L、糖蜜54g/L、油酸钠23mL/L、酵母粉8g/L、磷酸二氢钾1.8g/L、七水合硫酸镁0.15g/L、葡萄糖酸锌0.5g/L、吐温80 13mL/L，共配制9L，分别装到3个5L的发酵罐中，每罐3L，灭菌备用，并按照比例添加无菌红霉素10mg/L、氯霉素10mg/L；
配制流加培养基，其的成分和含量为：肌醇8g/L、硫酸铵7g/L、葡萄糖45g/L、七水合硫酸镁175mg/L，共配制3L，灭菌备用，并按照比例添加无菌NADPH8mg/L、色氨酸170mg/L、丙氨酸120mg/L、精氨酸130mg/L、生物素125mg/L。
在核黄素发酵培养的过程中，按15％的接种量向本实施例制备的基础培养基中接入产核黄素枯草芽孢杆菌种子培养物，于温度35-38℃下培养，过程中控制溶氧量15～30％，pH6.8(NH3·H2O调节)的条件下，进行发酵，发酵培养5-10h后开始流加所述流加培养基，流加速度依据培养基中的总糖量确定，保持所述总糖量为在6-8g/L，发酵40h后不再进行流加，48h左右结束发酵，检测效价，三个罐的效价分别为56g/L，51g/L，58g/L。
制备方法：首先使用所述基础培养基进行核黄素生产菌株发酵，培养5-10h后开始流加所述流加培养基，流加速度依据培养基中的总糖量确定，保持所述总糖量为在6-8g/L。
对比实施例1
配制基础培养基，其的成分和含量为：葡萄糖25g/L，面包酵母10g/L，酵母浸粉2g/L，棉籽饼粉7g/L，玉米浆20ml/L，甜菜糖蜜20ml/L，磷酸二氢钾0.5g/L，磷酸氢二钾2g/L，硫酸铵3g/L，硫酸镁0.5g/L，氟化钠20mg/L，叶酸2mg/L，共配制9L，分别装到3个5L的发酵罐中，每罐3L，灭菌备用，并按照比例添加无菌红霉素10mg/L、氯霉素10mg/L。
在核黄素发酵培养的过程中，按15％的接种量向本实施例制备的基础培养基中接入产核黄素枯草芽孢杆菌种子培养物，于温度35-38℃下培养，过程中控制溶氧量15～30％，pH6.8(NH3·H2O调节)的条件下，进行发酵，48h左右结束发酵，检测效价，三个罐的效价分别为20g/L，17g/L，16g/L。
应当理解，以上所描述的具体实施例仅用于解释本发明，并不用于限定本发明。由本发明的精神所引伸出的显而易见的变化或变动仍处于本发明的保护范围之中。
Claims (4)
1.一种发酵核黄素的发酵培养基，其特征在于，所述发酵培养基包括基础培养基和流加培养基，其中所述基础培养基的成分和含量为：葡萄糖10-20g/L、糖蜜54-60g/L、油酸钠23-25mL/L、酵母粉8g/L、磷酸二氢钾1.5-2g/L、七水合硫酸镁0.15g/L、葡萄糖酸锌0.3-0.5g/L、吐温13-15mL/L、红霉素10mg/L、氯霉素10mg/L，所述流加培养基的成分和含量为：肌醇8-10g/L、硫酸铵7-8g/L、葡萄糖45-50g/L、复合因子500-900mg/L；
所述复合因子包括NADPH 8-10mg/L、色氨酸170-190mg/L、丙氨酸120-150mg/L、精氨酸130-150mg/L、生物素125-150mg/L、七水合硫酸镁175-250mg/L。
2.根据权利要求1所述的一种发酵核黄素的发酵培养基，其特征在于，所述基础培养基的成分和含量为：葡萄糖16g/L、糖蜜54g/L、油酸钠23mL/L、酵母粉8g/L、磷酸二氢钾1.8g/L、七水合硫酸镁0.15g/L、葡萄糖酸锌0.5g/L、吐温80 13mL/L、红霉素10mg/L、氯霉素10mg/L。
3.根据权利要求1所述的一种发酵核黄素的发酵培养基，其特征在于，所述流加培养基的成分和含量为：肌醇8g/L、硫酸铵7g/L、葡萄糖45g/L、NADPH8mg/L、色氨酸170mg/L、丙氨酸120mg/L、精氨酸130mg/L、生物素125mg/L、七水合硫酸镁175mg/L。
4.根据权利要求1所述的一种发酵核黄素的发酵培养基的使用方法，其特征在于，首先使用所述基础培养基进行核黄素枯草芽孢杆菌生产菌株发酵，培养5-10h后开始流加所述流加培养基，流加速度依据培养基中的总糖量确定，保持所述总糖量为在6-8g/L。
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