CN112795607A - Method for improving adenosine fermentation yield - Google Patents
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- OIRDTQYFTABQOQ-KQYNXXCUSA-N adenosine Chemical compound C1=NC=2C(N)=NC=NC=2N1[C@@H]1O[C@H](CO)[C@@H](O)[C@H]1O OIRDTQYFTABQOQ-KQYNXXCUSA-N 0.000 title claims abstract description 90
- 238000000855 fermentation Methods 0.000 title claims abstract description 63
- 230000004151 fermentation Effects 0.000 title claims abstract description 63
- 239000002126 C01EB10 - Adenosine Substances 0.000 title claims abstract description 45
- 229960005305 adenosine Drugs 0.000 title claims abstract description 45
- 238000000034 method Methods 0.000 title claims abstract description 26
- 239000000843 powder Substances 0.000 claims abstract description 33
- 229940041514 candida albicans extract Drugs 0.000 claims abstract description 15
- 239000012138 yeast extract Substances 0.000 claims abstract description 15
- AGPKZVBTJJNPAG-WHFBIAKZSA-N L-isoleucine Chemical compound CC[C@H](C)[C@H](N)C(O)=O AGPKZVBTJJNPAG-WHFBIAKZSA-N 0.000 claims abstract description 14
- 229960000310 isoleucine Drugs 0.000 claims abstract description 14
- AGPKZVBTJJNPAG-UHFFFAOYSA-N isoleucine Natural products CCC(C)C(N)C(O)=O AGPKZVBTJJNPAG-UHFFFAOYSA-N 0.000 claims abstract description 14
- 244000063299 Bacillus subtilis Species 0.000 claims abstract description 9
- 235000014469 Bacillus subtilis Nutrition 0.000 claims abstract description 9
- LRFVTYWOQMYALW-UHFFFAOYSA-N 9H-xanthine Chemical compound O=C1NC(=O)NC2=C1NC=N2 LRFVTYWOQMYALW-UHFFFAOYSA-N 0.000 claims description 36
- WQZGKKKJIJFFOK-GASJEMHNSA-N Glucose Natural products OC[C@H]1OC(O)[C@H](O)[C@@H](O)[C@@H]1O WQZGKKKJIJFFOK-GASJEMHNSA-N 0.000 claims description 29
- 239000008103 glucose Substances 0.000 claims description 29
- 239000001963 growth medium Substances 0.000 claims description 27
- CSNNHWWHGAXBCP-UHFFFAOYSA-L Magnesium sulfate Chemical compound [Mg+2].[O-][S+2]([O-])([O-])[O-] CSNNHWWHGAXBCP-UHFFFAOYSA-L 0.000 claims description 18
- 229940075420 xanthine Drugs 0.000 claims description 18
- 238000012258 culturing Methods 0.000 claims description 17
- 238000011218 seed culture Methods 0.000 claims description 15
- 240000004808 Saccharomyces cerevisiae Species 0.000 claims description 12
- 240000008042 Zea mays Species 0.000 claims description 12
- 235000005824 Zea mays ssp. parviglumis Nutrition 0.000 claims description 12
- 235000002017 Zea mays subsp mays Nutrition 0.000 claims description 12
- 235000005822 corn Nutrition 0.000 claims description 12
- FDGQSTZJBFJUBT-UHFFFAOYSA-N hypoxanthine Chemical compound O=C1NC=NC2=C1NC=N2 FDGQSTZJBFJUBT-UHFFFAOYSA-N 0.000 claims description 12
- 229910052943 magnesium sulfate Inorganic materials 0.000 claims description 9
- 239000002609 medium Substances 0.000 claims description 8
- 239000007788 liquid Substances 0.000 claims description 7
- 239000007787 solid Substances 0.000 claims description 7
- 229920001817 Agar Polymers 0.000 claims description 6
- UGQMRVRMYYASKQ-UHFFFAOYSA-N Hypoxanthine nucleoside Natural products OC1C(O)C(CO)OC1N1C(NC=NC2=O)=C2N=C1 UGQMRVRMYYASKQ-UHFFFAOYSA-N 0.000 claims description 6
- 239000007836 KH2PO4 Substances 0.000 claims description 6
- 239000001888 Peptone Substances 0.000 claims description 6
- 108010080698 Peptones Proteins 0.000 claims description 6
- 239000008272 agar Substances 0.000 claims description 6
- 210000003056 antler Anatomy 0.000 claims description 6
- QVGXLLKOCUKJST-UHFFFAOYSA-N atomic oxygen Chemical compound [O] QVGXLLKOCUKJST-UHFFFAOYSA-N 0.000 claims description 6
- 150000004676 glycans Chemical class 0.000 claims description 6
- HNDVDQJCIGZPNO-UHFFFAOYSA-N histidine Natural products OC(=O)C(N)CC1=CN=CN1 HNDVDQJCIGZPNO-UHFFFAOYSA-N 0.000 claims description 6
- 229910000357 manganese(II) sulfate Inorganic materials 0.000 claims description 6
- 229910000402 monopotassium phosphate Inorganic materials 0.000 claims description 6
- LPUQAYUQRXPFSQ-DFWYDOINSA-M monosodium L-glutamate Chemical compound [Na+].[O-]C(=O)[C@@H](N)CCC(O)=O LPUQAYUQRXPFSQ-DFWYDOINSA-M 0.000 claims description 6
- 235000013923 monosodium glutamate Nutrition 0.000 claims description 6
- 239000004223 monosodium glutamate Substances 0.000 claims description 6
- 229910052760 oxygen Inorganic materials 0.000 claims description 6
- 239000001301 oxygen Substances 0.000 claims description 6
- 235000019319 peptone Nutrition 0.000 claims description 6
- 229920001282 polysaccharide Polymers 0.000 claims description 6
- 239000005017 polysaccharide Substances 0.000 claims description 6
- 108090000623 proteins and genes Proteins 0.000 claims description 6
- 102000004169 proteins and genes Human genes 0.000 claims description 6
- 238000007790 scraping Methods 0.000 claims description 6
- 229910000396 dipotassium phosphate Inorganic materials 0.000 claims description 4
- 230000004913 activation Effects 0.000 claims description 2
- 229910052603 melanterite Inorganic materials 0.000 claims description 2
- 239000000203 mixture Substances 0.000 claims description 2
- 229910052564 epsomite Inorganic materials 0.000 claims 1
- 238000004519 manufacturing process Methods 0.000 abstract description 7
- 239000000463 material Substances 0.000 abstract description 3
- 230000001580 bacterial effect Effects 0.000 abstract 1
- 238000011109 contamination Methods 0.000 abstract 1
- 238000009776 industrial production Methods 0.000 abstract 1
- 239000013589 supplement Substances 0.000 abstract 1
- 230000000052 comparative effect Effects 0.000 description 4
- BAUYGSIQEAFULO-UHFFFAOYSA-L iron(2+) sulfate (anhydrous) Chemical compound [Fe+2].[O-]S([O-])(=O)=O BAUYGSIQEAFULO-UHFFFAOYSA-L 0.000 description 4
- 229910000359 iron(II) sulfate Inorganic materials 0.000 description 4
- 239000000126 substance Substances 0.000 description 4
- WEVYAHXRMPXWCK-UHFFFAOYSA-N Acetonitrile Chemical compound CC#N WEVYAHXRMPXWCK-UHFFFAOYSA-N 0.000 description 3
- 230000000694 effects Effects 0.000 description 3
- 238000011081 inoculation Methods 0.000 description 3
- 238000012544 monitoring process Methods 0.000 description 3
- 239000000523 sample Substances 0.000 description 3
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 description 3
- XTWYTFMLZFPYCI-KQYNXXCUSA-N 5'-adenylphosphoric acid Chemical compound C1=NC=2C(N)=NC=NC=2N1[C@@H]1O[C@H](COP(O)(=O)OP(O)(O)=O)[C@@H](O)[C@H]1O XTWYTFMLZFPYCI-KQYNXXCUSA-N 0.000 description 2
- ZKHQWZAMYRWXGA-KQYNXXCUSA-J ATP(4-) Chemical compound C1=NC=2C(N)=NC=NC=2N1[C@@H]1O[C@H](COP([O-])(=O)OP([O-])(=O)OP([O-])([O-])=O)[C@@H](O)[C@H]1O ZKHQWZAMYRWXGA-KQYNXXCUSA-J 0.000 description 2
- XTWYTFMLZFPYCI-UHFFFAOYSA-N Adenosine diphosphate Natural products C1=NC=2C(N)=NC=NC=2N1C1OC(COP(O)(=O)OP(O)(O)=O)C(O)C1O XTWYTFMLZFPYCI-UHFFFAOYSA-N 0.000 description 2
- ZKHQWZAMYRWXGA-UHFFFAOYSA-N Adenosine triphosphate Natural products C1=NC=2C(N)=NC=NC=2N1C1OC(COP(O)(=O)OP(O)(=O)OP(O)(O)=O)C(O)C1O ZKHQWZAMYRWXGA-UHFFFAOYSA-N 0.000 description 2
- IJGRMHOSHXDMSA-UHFFFAOYSA-N Atomic nitrogen Chemical compound N#N IJGRMHOSHXDMSA-UHFFFAOYSA-N 0.000 description 2
- 241000894006 Bacteria Species 0.000 description 2
- IVOMOUWHDPKRLL-KQYNXXCUSA-N Cyclic adenosine monophosphate Chemical compound C([C@H]1O2)OP(O)(=O)O[C@H]1[C@@H](O)[C@@H]2N1C(N=CN=C2N)=C2N=C1 IVOMOUWHDPKRLL-KQYNXXCUSA-N 0.000 description 2
- 229910052799 carbon Inorganic materials 0.000 description 2
- 239000008367 deionised water Substances 0.000 description 2
- 229910021641 deionized water Inorganic materials 0.000 description 2
- 230000007613 environmental effect Effects 0.000 description 2
- 238000006911 enzymatic reaction Methods 0.000 description 2
- 239000002054 inoculum Substances 0.000 description 2
- 230000000813 microbial effect Effects 0.000 description 2
- 210000004165 myocardium Anatomy 0.000 description 2
- 239000002777 nucleoside Substances 0.000 description 2
- 150000003833 nucleoside derivatives Chemical class 0.000 description 2
- 230000008569 process Effects 0.000 description 2
- 238000003786 synthesis reaction Methods 0.000 description 2
- OKTJSMMVPCPJKN-UHFFFAOYSA-N Carbon Chemical compound [C] OKTJSMMVPCPJKN-UHFFFAOYSA-N 0.000 description 1
- UDMBCSSLTHHNCD-UHFFFAOYSA-N Coenzym Q(11) Natural products C1=NC=2C(N)=NC=NC=2N1C1OC(COP(O)(O)=O)C(O)C1O UDMBCSSLTHHNCD-UHFFFAOYSA-N 0.000 description 1
- 108090000790 Enzymes Proteins 0.000 description 1
- 102000004190 Enzymes Human genes 0.000 description 1
- 241001052560 Thallis Species 0.000 description 1
- 238000009825 accumulation Methods 0.000 description 1
- 230000009471 action Effects 0.000 description 1
- 230000006978 adaptation Effects 0.000 description 1
- UDMBCSSLTHHNCD-KQYNXXCUSA-N adenosine 5'-monophosphate Chemical compound C1=NC=2C(N)=NC=NC=2N1[C@@H]1O[C@H](COP(O)(O)=O)[C@@H](O)[C@H]1O UDMBCSSLTHHNCD-KQYNXXCUSA-N 0.000 description 1
- 229950006790 adenosine phosphate Drugs 0.000 description 1
- GFFGJBXGBJISGV-UHFFFAOYSA-N adenyl group Chemical class N1=CN=C2N=CNC2=C1N GFFGJBXGBJISGV-UHFFFAOYSA-N 0.000 description 1
- 229940024606 amino acid Drugs 0.000 description 1
- 150000001413 amino acids Chemical class 0.000 description 1
- 238000004458 analytical method Methods 0.000 description 1
- 230000015572 biosynthetic process Effects 0.000 description 1
- 238000006243 chemical reaction Methods 0.000 description 1
- 238000007865 diluting Methods 0.000 description 1
- 230000037149 energy metabolism Effects 0.000 description 1
- 238000005516 engineering process Methods 0.000 description 1
- 238000001914 filtration Methods 0.000 description 1
- 230000002068 genetic effect Effects 0.000 description 1
- 210000005260 human cell Anatomy 0.000 description 1
- 230000002401 inhibitory effect Effects 0.000 description 1
- 238000011031 large-scale manufacturing process Methods 0.000 description 1
- 239000012528 membrane Substances 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
- 239000002858 neurotransmitter agent Substances 0.000 description 1
- 229910052757 nitrogen Inorganic materials 0.000 description 1
- 125000003729 nucleotide group Chemical group 0.000 description 1
- 235000015097 nutrients Nutrition 0.000 description 1
- 230000009965 odorless effect Effects 0.000 description 1
- 125000001477 organic nitrogen group Chemical group 0.000 description 1
- 230000002018 overexpression Effects 0.000 description 1
- 230000026731 phosphorylation Effects 0.000 description 1
- 238000006366 phosphorylation reaction Methods 0.000 description 1
- 238000011020 pilot scale process Methods 0.000 description 1
- 230000002035 prolonged effect Effects 0.000 description 1
- 101150002764 purA gene Proteins 0.000 description 1
- 239000012488 sample solution Substances 0.000 description 1
- 230000011664 signaling Effects 0.000 description 1
- 230000005062 synaptic transmission Effects 0.000 description 1
- 238000005303 weighing Methods 0.000 description 1
Classifications
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- C—CHEMISTRY; METALLURGY
- 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
- C12P19/00—Preparation of compounds containing saccharide radicals
- C12P19/26—Preparation of nitrogen-containing carbohydrates
- C12P19/28—N-glycosides
- C12P19/38—Nucleosides
- C12P19/40—Nucleosides having a condensed ring system containing a six-membered ring having two nitrogen atoms in the same ring, e.g. purine nucleosides
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- Life Sciences & Earth Sciences (AREA)
- Chemical & Material Sciences (AREA)
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- Zoology (AREA)
- Engineering & Computer Science (AREA)
- Wood Science & Technology (AREA)
- Microbiology (AREA)
- General Chemical & Material Sciences (AREA)
- Chemical Kinetics & Catalysis (AREA)
- Biotechnology (AREA)
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- Biochemistry (AREA)
- Bioinformatics & Cheminformatics (AREA)
- General Engineering & Computer Science (AREA)
- General Health & Medical Sciences (AREA)
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Abstract
The invention relates to a method for improving adenosine fermentation yield, and belongs to the technical field of fermentation engineering. According to the method, bacillus subtilis is taken as a production strain, and a certain amount of yeast extract powder and isoleucine are added into a fermentation medium, so that the adenosine yield is continuously increased in the later fermentation period, the adenosine yield is increased to 60.7g/L, and the adenosine yield is increased by 43.5% compared with that in the case of not using the method. The method can improve the yield of adenosine, has simple fermentation process without material supplement, reduces the probability of bacterial contamination, and is very suitable for industrial production.
Description
Technical Field
The invention belongs to the technical field of fermentation engineering, and particularly relates to a method for improving adenosine fermentation yield.
Background
Adenosine, also known as adenosine, is a pure white crystalline powder, odorless, bitter in taste. Adenosine formula C10H13N5O4And the molecular weight is 267.24. Adenosine is an important nucleotide derivative and is a dephosphorylated product of adenine nucleotide. As an endogenous nucleoside distributed throughout human cells, the nucleoside can directly enter cardiac muscle to generate adenylic acid through phosphorylation and participate in cardiac muscle energy metabolism. Adenosine plays a biochemical important role, including the transfer of energy in the form of Adenosine Triphosphate (ATP) or Adenosine Diphosphate (ADP), or the signaling of cyclic adenosine monophosphate (cAMP), among others. In addition, adenosine is an inhibitory neurotransmitter, and plays an important role in neurotransmission.
Along with the continuous expansion of the action range of adenosine, the market demand is also continuously increased, the production method of adenosine mainly comprises a chemical synthesis method, an enzymatic method and a fermentation method, and the production of adenosine at home at present mainly comprises the chemical method and the enzymatic method, so that the cost is high, the pollution is serious, and the popularization and the application are severely limited. The method for producing adenosine by fermentation has the advantages of mild reaction conditions, low cost, greenness, cleanness, environmental protection and the like. Therefore, under the condition of low carbon and environmental protection, the adenosine is produced by using a fermentation method, the fermentation process of the adenosine is optimized, the yield of the adenosine is further improved, and the method has important significance.
Disclosure of Invention
In view of the above problems, the present invention aims to provide a method for increasing adenosine fermentation yield, which is characterized in that yeast extract powder and isoleucine are added into a culture medium to provide sufficient nutrients for the growth and fermentation of bacteria, so as to prolong the stable growth period of the bacteria, facilitate the efficient synthesis of adenosine at the later stage, and achieve the purpose of increasing adenosine fermentation yield.
In order to achieve the purpose, the invention adopts the specific scheme that:
a method for improving the fermentation yield of adenosine comprises the following steps:
step one, slant culture: inoculating bacillus subtilis serving as an original strain into a slant solid culture medium for activation, and culturing for 15-20 h at 28-32 ℃;
step two, seed culture: scraping strains on the slant solid culture by using an inoculating ring, inoculating the strains to a seed culture medium, and culturing at 30-36 ℃ and 200-500 rpm for 8-10 h to obtain a fermented seed solution;
step three, fermentation culture: transferring the fermentation seed liquid obtained in the step two into a fermentation tank filled with a fermentation culture medium according to the inoculation amount of 12-15%, maintaining the dissolved oxygen at 20-30% at 33-36 ℃, and using NH3·H2Regulating the pH value to 6.6-7.0 by O, and culturing for 40-50 h;
the liquid fermentation medium comprises the following components in g/L: 80-120 parts of glucose, 30-50 mL/L of corn steep liquor, 3-5 parts of yeast extract powder, 5-10 parts of polysaccharide antler essence, 3-5 parts of monosodium glutamate and K2HPO4,1 to 3, isoleucine 0.1 to 0.2, xanthine 0.1 to 0.2, hypoxanthine 0.1 to 0.2, MgSO4·7H2O1~5，MnSO40.03 to 0.06 and FeSO4·7H2O 0.03~0.06。
Further, the slant solid culture medium in the first step comprises the following components in g/L: 1-3 parts of glucose, 5-10 parts of peptone, 2-5 parts of yeast powder, 0.1-0.15 part of xanthine and 20-30 parts of agar, and the pH value is 7.0-7.2.
Further, the seed culture medium of the second step comprises the following components in g/L: 15-20% of glucose, 15-25 mL/L of corn steep liquor, 5-10% of protein fine powder, 5-10% of yeast powder and KH2PO40.5~1.5，MgSO4·7H20.2 to 0.8O, 0.1 to 0.15 xanthine and 0.01 to 0.05 histidine, pH6.5 to 7.0.
Has the advantages that:
according to the invention, bacillus subtilis XGL is used as a fermentation strain, a fermentation medium is optimized, yeast extract powder and isoleucine are added on the basis of a 50L fermentation tank pilot scale, the production time of metabolites is prolonged, and the yield of adenosine is improved.
(1) Compared with the traditional chemical method and enzyme method, the method has the advantages of simple production equipment, little pollution, accordance with the current low-carbon environment, low cost and suitability for large-scale production.
(2) The microbial strains used in the invention have stable genetic markers and are not easy to lose, and the microbial strains are transferred after more than ten generations, so that the yield is basically kept stable.
(3) According to the invention, the yeast extract powder and the amino acid are added into the fermentation tank bottom material, so that the production strength of adenosine is improved to 60.7g/L, and compared with the method without adding the yeast extract powder and isoleucine, the yield is improved by 43.5%.
Detailed Description
A method for improving adenosine fermentation yield is characterized in that yeast extract powder and isoleucine are added to improve the adenosine fermentation yield, the method adopts a fed-batch mode to ferment and produce adenosine, and a fermentation strain adopts Bacillus subtilis XGL (which is published in China bioengineering journal in 2011, 12 and 31 days, and the article is 'influence of overexpression purA gene on adenosine accumulation', and is now preserved in the strain collection center of institute of bioengineering, university of Tianjin technology).
The specific operation of the method comprises the following processes:
step 1, slant culture: inoculating bacillus subtilis XGL as an original strain to an activated inclined plane, and culturing for 15-20 h at 28-32 ℃;
step 2, seed culture: scraping slant strains by using an inoculating ring, inoculating the slant strains to a seed culture medium, and culturing at 30-36 ℃ and 200-500 rpm for 8-10 h to serve as a fermentation seed solution;
step 3, fermentation culture: transferring the strain into a fermentation tank filled with a fermentation culture medium according to the inoculation amount of 12-15%, maintaining the dissolved oxygen at 20-30% at 33-36 ℃, and using NH3·H2Adjusting the pH value to 6.6-7.0 by O, and culturing for 40-50 h.
Wherein the slant solid culture medium comprises the following components in g/L: 1-3 parts of glucose, 5-10 parts of peptone, 2-5 parts of yeast powder, 0.1-0.15 part of xanthine, 20-30 parts of agar and 7.0-7.2 parts of pHs;
the seed culture medium comprises the following components in g/L: 15-20% of glucose, 15-25 mL/L of corn steep liquor, 5-10% of protein fine powder, 5-10% of yeast powder and KH2PO40.5~1.5，MgSO4·7H20.2 to 0.8O, 0.1 to 0.15 xanthine, 0.01 to 0.05 histidine, pH6.5 to 7.0;
the liquid fermentation medium comprises the following components in g/L: 80-120 parts of glucose, 30-50 mL/L of corn steep liquor, 3-5 parts of yeast extract powder, 5-10 parts of polysaccharide antler essence, 3-5 parts of monosodium glutamate and K2HPO4,1 to 3, isoleucine 0.1 to 0.2, xanthine 0.1 to 0.2, hypoxanthine 0.1 to 0.2, MgSO4·7H2O1~5，MnSO40.03~0.06，FeSO4·7H2O0.03~0.06。
The invention is illustrated below with reference to specific examples. It will be understood by those skilled in the art that the specific material ratios, process conditions and results described in these specific examples are merely illustrative of the invention and do not limit the scope of the invention in any way.
The analysis method adopted for the adenosine content of the fermentation product in each of the following examples is as follows:
the product adenosine in the fermentation broth is measured by an Agilent1100 High Performance Liquid Chromatograph (HPLC). The chromatographic column was a kromasil c18 column (250mm × 416mm i.d., 5 μm), the column temperature was 30 ℃, the detector was an ultraviolet detector (259 nm), the mobile phase was water: acetonitrile = 90: 10; the flow rate is 1.0 ml/min; the amount of the sample was 20. mu.L. Accurately weighing an adenosine standard substance, and preparing a standard substance solution with the mass concentration of 1g/L by using deionized water; diluting the pretreated fermentation liquid sample to be detected to a proper concentration by using deionized water, filtering by using a 0.22 mu m microporous filter membrane to be used as a fermentation sample solution to be detected, and calculating the adenosine yield of the fermentation sample according to the peak area.
Example 1:
the formula of the slant culture medium is calculated by g/L: glucose 2, peptone 5, yeast powder 5, xanthine 0.1, agar 20, pH 7.0;
the formula of the seed culture medium is calculated in g/L: 20 parts of glucose, 20mL/L of corn steep liquor, 10 parts of protein fine powder, 5 parts of yeast powder and KH2PO41，MgSO4·7H2O0.5, xanthine 0.15, histidine 0.05, pH 7.0;
the fermentation medium is measured by g/L: 80 parts of glucose, 40mL/L of corn steep liquor, 6 parts of polysaccharide antler essence, 5 parts of yeast extract powder, 4 parts of monosodium glutamate and K2HPO43, xanthine 0.15, hypoxanthine 0.2, MgSO4·7H2O1，MnSO40.05，FeSO4·7H2O0.05。
Inoculating Bacillus subtilis XGL as original strain to activated slant, culturing at 30 deg.C for 18 hr, scraping slant strain with inoculating loop, inoculating to seed culture medium, culturing at 36 deg.C and 200rpm for 9 hr, inoculating to 50L fermentation tank containing fermentation culture medium at an inoculum size of 15% (v/v), maintaining dissolved oxygen at 36 deg.C to 30%, and culturing with NH3·H2Adjusting pH, maintaining the pH at 6.4, monitoring the residual glucose amount in the fermentation broth on line, and feeding glucose until the residual glucose amount in the fermentation broth is 10g/L when the residual glucose amount in the fermentation broth is reduced to 10g/L, fermenting for 40h, wherein the adenosine yield reaches 54.3 g/L.
Example 2:
the formula of the slant culture medium is calculated by g/L: glucose 2, peptone 5, yeast powder 5, xanthine 0.1, agar 20, pH 7.0;
the formula of the seed culture medium is calculated in g/L: 20 parts of glucose, 20mL/L of corn steep liquor, 10 parts of protein fine powder, 5 parts of yeast powder and KH2PO41，MgSO4·7H2O0.5, xanthine 0.15, histidine 0.05, pH 7.0;
the fermentation medium is measured by g/L: 80 percent of glucose, 40mL/L of corn steep liquor, 6 percent of polysaccharide antler essence, 4 percent of monosodium glutamate and K2HPO43, isoleucine 0.1-0.2, xanthine 0.15, hypoxanthine 0.2, MgSO 04·7H2O1，MnSO40.05，FeSO4·7H2O0.05。
Inoculating bacillus subtilis XGL as an original strain to an activated inclined plane, culturing at 30 ℃ for 18h, scraping the inclined plane strain by using an inoculating ring, inoculating to a seed culture medium, and culturing at 36 ℃ and 200rpm for 9h; transferring the mixture into a 50L fermentation tank filled with a fermentation medium according to the inoculation amount of 15% (v/v), maintaining the dissolved oxygen at 20-30% at 36 ℃, and using NH3·H2Adjusting pH, maintaining the pH at 6.4, monitoring the residual glucose amount in the fermentation broth on line, and feeding glucose until the residual glucose amount in the fermentation broth is 10g/L when the residual glucose amount in the fermentation broth is reduced to 10g/L, fermenting for 40h, wherein the adenosine yield reaches 50.7 g/L.
Example 3:
the formula of the slant culture medium is calculated by g/L: glucose 2, peptone 5, yeast powder 5, xanthine 0.1, agar 20, pH 7.0;
the formula of the seed culture medium is calculated in g/L: 20 parts of glucose, 20mL/L of corn steep liquor, 10 parts of protein fine powder, 5 parts of yeast powder and KH2PO41，MgSO4·7H2O0.5, xanthine 0.15, histidine 0.05, pH 7.0;
the fermentation medium is measured by g/L: 80 parts of glucose, 40mL/L of corn steep liquor, 6 parts of polysaccharide antler essence, 5 parts of yeast extract powder, 4 parts of monosodium glutamate and K2HPO43, isoleucine 0.1-0.2, xanthine 0.15, hypoxanthine 0.2, MgSO 04·7H2O1，MnSO40.05，FeSO4·7H2O0.05。
Inoculating Bacillus subtilis XGL as original strain to activated slant, culturing at 30 deg.C for 18 hr, scraping slant strain with inoculating loop, inoculating to seed culture medium, culturing at 36 deg.C and 200rpm for 9 hr, inoculating to 50L fermentation tank containing fermentation culture medium at an inoculum size of 15% (v/v), maintaining dissolved oxygen at 36 deg.C to 30%, and culturing with NH3·H2Adjusting pH with O, maintaining pH at 6.4, and online monitoring glucose in fermentation brothWhen the glucose residual quantity is reduced to 10g/L, glucose is fed in until the residual sugar quantity of the fermentation liquor is maintained to be 10g/L, and the adenosine yield reaches 60.7g/L after fermentation for 40 hours.
Comparative example 1:
the fermentation was carried out in a fermentor without adding yeast extract powder and isoleucine under the same conditions as in example 1. The final adenosine content was determined to be 42.3 g/L.
As can be seen from example 1 and comparative example 1, the addition of 5g/L yeast extract powder in the fermenter can increase the adenosine yield by 28.4%, which indicates that the addition of organic nitrogen source yeast extract powder can provide sufficient nitrogen source for the growth of the thallus and prolong the stable growth period of the thallus. As can be seen from the example 2 and the comparative example 1, the effect of adding isoleucine into the culture medium on the yield of adenosine is large, the yield of adenosine is increased by 20%, and isoleucine can improve the activity of thalli and the yield of adenosine in a tank. As can be seen from example 3 and comparative example 1, the addition of yeast extract and isoleucine at the same time can significantly improve the adenosine yield by 43.5%, and the effect is relatively obvious.
It should be noted that the above-mentioned embodiments illustrate rather than limit the scope of the invention, which is defined by the appended claims. It will be apparent to those skilled in the art that certain insubstantial modifications and adaptations of the present invention can be made without departing from the spirit and scope of the invention.
Claims (3)
1. A method for improving the fermentation yield of adenosine comprises the following steps:
step one, slant culture: inoculating bacillus subtilis serving as an original strain into a slant solid culture medium for activation, and culturing for 15-20 h at 28-32 ℃;
step two, seed culture: scraping strains on the slant solid culture by using an inoculating ring, inoculating the strains to a seed culture medium, and culturing at 30-36 ℃ and 200-500 rpm for 8-10 h to obtain a fermented seed solution;
step three, fermentation culture: inoculating 12-15% of the fermented seed liquid obtained in the step twoTransferring the mixture to a fermentation tank filled with a fermentation culture medium, maintaining the dissolved oxygen at 20-30% at 33-36 ℃, and using NH3·H2Regulating the pH value to 6.6-7.0 by O, and culturing for 40-50 h;
the liquid fermentation medium comprises the following components in g/L: 80-120 parts of glucose, 30-50 mL/L of corn steep liquor, 3-5 parts of yeast extract powder, 5-10 parts of polysaccharide antler essence, 3-5 parts of monosodium glutamate and K2HPO41 to 3, isoleucine 0.1 to 0.2, xanthine 0.1 to 0.2, hypoxanthine 0.1 to 0.2, MgSO4·7H2O 1~5，MnSO40.03 to 0.06 and FeSO4·7H2O 0.03~0.06。
2. The method of claim 1, wherein: the slant solid culture medium in the first step comprises the following components in g/L: 1-3 parts of glucose, 5-10 parts of peptone, 2-5 parts of yeast powder, 0.1-0.15 part of xanthine and 20-30 parts of agar, and the pH value is 7.0-7.2.
3. The method of claim 1, wherein: the seed culture medium of the second step comprises the following components in g/L: 15-20% of glucose, 15-25 mL/L of corn steep liquor, 5-10% of protein fine powder, 5-10% of yeast powder and KH2PO40.5~1.5，MgSO4·7H20.2 to 0.8O, 0.1 to 0.15 xanthine and 0.01 to 0.05 histidine, pH6.5 to 7.0.
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