CN112795607B - 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 91
- 238000000855 fermentation Methods 0.000 title claims abstract description 64
- 230000004151 fermentation Effects 0.000 title claims abstract description 64
- 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 27
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- 229940041514 candida albicans extract Drugs 0.000 claims abstract description 15
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- 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 10
- 235000014469 Bacillus subtilis Nutrition 0.000 claims abstract description 10
- 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 30
- 239000008103 glucose Substances 0.000 claims description 30
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- 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
- 239000001888 Peptone Substances 0.000 claims description 6
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- 210000003056 antler Anatomy 0.000 claims description 6
- QVGXLLKOCUKJST-UHFFFAOYSA-N atomic oxygen Chemical compound [O] QVGXLLKOCUKJST-UHFFFAOYSA-N 0.000 claims description 6
- 239000000284 extract Substances 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
- 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
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- 230000004913 activation Effects 0.000 claims description 4
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- 239000000126 substance Substances 0.000 description 4
- WEVYAHXRMPXWCK-UHFFFAOYSA-N Acetonitrile Chemical compound CC#N WEVYAHXRMPXWCK-UHFFFAOYSA-N 0.000 description 3
- 108090000790 Enzymes Proteins 0.000 description 3
- 102000004190 Enzymes Human genes 0.000 description 3
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- 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
- 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
- 241001052560 Thallis Species 0.000 description 2
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- 229910021641 deionized water Inorganic materials 0.000 description 2
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- OKTJSMMVPCPJKN-UHFFFAOYSA-N Carbon Chemical compound [C] OKTJSMMVPCPJKN-UHFFFAOYSA-N 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
- 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
- OIRDTQYFTABQOQ-UHFFFAOYSA-N ara-adenosine Natural products Nc1ncnc2n(cnc12)C1OC(CO)C(O)C1O OIRDTQYFTABQOQ-UHFFFAOYSA-N 0.000 description 1
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- 239000002207 metabolite Substances 0.000 description 1
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- 210000005036 nerve Anatomy 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
- 230000008569 process Effects 0.000 description 1
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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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- General Chemical & Material Sciences (AREA)
- Chemical Kinetics & Catalysis (AREA)
- Biotechnology (AREA)
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- Bioinformatics & Cheminformatics (AREA)
- General Engineering & Computer Science (AREA)
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Abstract
The invention relates to a method for improving the fermentation yield of adenosine, and belongs to the technical field of fermentation engineering. The method takes the bacillus subtilis as a production strain, and the fermentation medium continuously increases the yield of adenosine in the later period of fermentation by adding a certain amount of yeast extract powder and isoleucine, so that the yield of the adenosine is increased to 60.7g/L, and compared with the method which is not used, the yield is increased by 43.5%. The method can not only improve the yield of the adenosine, but also simplify the fermentation process without material supplementation, reduce the probability of bacteria 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 the fermentation yield of adenosine.
Background
Adenosine is also called adenine nucleoside, and the pure product is white crystal powder, odorless and bitter. Adenosine formula C 10 H 13 N 5 O 4 The molecular weight is 267.24. Adenosine belongs to an important nucleotide derivative, and is a product obtained by dephosphorylation of adenine nucleotides. As an endogenous nucleoside distributed in human cells, the nucleoside can directly enter cardiac muscle to generate adenosine through phosphorylation, and participate in cardiac muscle energy metabolism. Adenosine plays an important role in biochemistry, including energy transfer in the form of Adenosine Triphosphate (ATP) or Adenosine Diphosphate (ADP), or signaling in cyclic adenosine monophosphate (cAMP), etc. In addition, adenosine is also an inhibitory nerve transmitter and plays an important role in neurotransmission.
Along with the continuous expansion of the action range of the adenosine, the market demand of the adenosine is continuously increased, the production method of the adenosine mainly comprises a chemical synthesis method, an enzyme method and a fermentation method, and the production of the adenosine in China at present mainly comprises the chemical method and the enzyme method, so that the cost is high, the pollution is serious, and the popularization and the application are severely limited. The fermentation method for producing the adenosine has the advantages of mild reaction conditions, low cost, green, clean, environmental protection and the like. Therefore, under the condition of low carbon and environmental protection, the fermentation method is used for producing the adenosine, the fermentation process of the adenosine is optimized, and the yield of the adenosine is further improved, so that the method has important significance.
Disclosure of Invention
In view of the above problems, the present invention aims to provide a method for improving the yield of adenosine fermentation, which is to add yeast extract and isoleucine into a culture medium to provide enough nutrients for the growth and fermentation of thalli, prolong the stationary growth period of the thalli, facilitate the efficient synthesis of adenosine in the later stage and achieve the purpose of improving the yield of adenosine fermentation.
In order to achieve the above purpose, the invention adopts the following specific scheme:
a method for increasing the fermentation yield of adenosine comprising the steps of:
step one, slant culture: inoculating bacillus subtilis serving as an initial strain into a slant solid culture medium for activation, and culturing for 15-20 hours at 28-32 ℃;
step two, seed culture: scraping strains on the inclined plane solid culture by using an inoculating loop, inoculating the strains to a seed culture medium, and culturing at 200-500 rpm at 30-36 ℃ for 8-10 hours to obtain fermentation seed liquid;
step three, fermentation culture: transferring the fermentation seed liquid obtained in the second step into a fermentation tank filled with a fermentation medium according to the inoculation amount of 12-15%, maintaining the dissolved oxygen at 20-30% at 33-36 ℃, and using NH 3 ·H 2 O is used for regulating the pH value to be 6.6-7.0, and the culture time is 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 extract, 3-5 parts of monosodium glutamate and K 2 HPO 4, 1-3, 0.1-0.2 isoleucine, 0.1-0.2 xanthine, 0.1-0.2 hypoxanthine, mgSO 4 ·7H 2 O1~5，MnSO 4 0.03 to 0.06 and FeSO 4 ·7H 2 O 0.03~0.06。
Further, the slant solid culture medium in the step one 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 pH7.
Further, the seed culture medium in the second step comprises the following components in g/L: 15-20 parts of glucose and 15-25 m parts of corn steep liquorL/L, 5-10 of protein refined powder, 5-10 of yeast powder and KH 2 PO 4 0.5~1.5，MgSO 4 ·7H 2 O0.2-0.8, xanthine 0.1-0.15, histidine 0.01-0.05, pH 6.5-7.0.
The beneficial effects are that:
the invention optimizes the fermentation culture medium by taking bacillus subtilis XGL as a fermentation strain, adds yeast extract powder and isoleucine on the basis of a 50L fermentation tank test, prolongs the production time of metabolites and improves the yield of adenosine.
(1) Compared with the traditional chemical method and enzyme method, the method has the advantages of simple production equipment, little green pollution, low cost and suitability for mass production, and accords with the current low-carbon environment.
(2) The microbial strain used in the invention has stable genetic markers, is not easy to lose, transfers more than ten generations, and basically keeps stable yield.
(3) The invention adds yeast extract powder and amino acid 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 yeast extract powder and isoleucine, the yield is improved by 43.5%.
Detailed Description
A method for improving the fermentation yield of adenosine by adding yeast extract powder and isoleucine, wherein the method adopts a fed-batch fermentation mode to produce the adenosine, and a fermentation strain adopts bacillus subtilis (Bacillus subtilis) XGL (the strain is published in China journal of biological engineering in 12 months 31 of 2011, and the article is "the influence of over-expression purA gene on the accumulation of the adenosine", which is now preserved in the university of Tianjin's institute of biological engineering, strain collection).
The specific operation of the method of the invention comprises the following steps:
step 1, slant culture: inoculating bacillus subtilis XGL serving as an initial strain to an activation inclined plane, and culturing at 28-32 ℃ for 15-20 hours;
step 2, seed culture: scraping the inclined plane strain by an inoculating loop, inoculating the inclined plane strain to a seed culture medium, and culturing at 200-500 rpm for 8-10 hours at the temperature of 30-36 ℃ to obtain fermentation seed liquid;
and 3, fermenting and culturing: according to 1Transferring 2-15% of inoculum size into a fermentation tank filled with a fermentation medium, maintaining dissolved oxygen at 20-30% at 33-36 ℃ by using NH 3 ·H 2 And the pH value is regulated to be 6.6-7.0 by O, and the culture time is 40-50 h.
Wherein, the inclined plane 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 pH 7;
the seed culture medium comprises the following components in g/L: 15-20 parts of glucose, 15-25 mL/L of corn steep liquor, 5-10 parts of protein refined powder, 5-10 parts of yeast powder and KH 2 PO 4 0.5~1.5，MgSO 4 ·7H 2 0.2-0.8 of O, 0.1-0.15 of xanthine, 0.01-0.05 of histidine and 6.5-7.0 of pH;
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 extract, 3-5 parts of monosodium glutamate and K 2 HPO 4, 1-3, 0.1-0.2 isoleucine, 0.1-0.2 xanthine, 0.1-0.2 hypoxanthine, mgSO 4 ·7H 2 O1~5，MnSO 4 0.03~0.06，FeSO 4 ·7H 2 O0.03~0.06。
The invention is described below with reference to specific examples. Those skilled in the art will appreciate that the specific ratios of materials, process conditions, and results thereof described in these specific examples are intended to be illustrative of the present invention only and are not intended to limit the scope of the present invention in any way.
The analytical methods used for the adenosine content of the fermentation products in the following examples were as follows:
the adenosine product in the fermentation broth was measured using an instrument Agilent1100 High Performance Liquid Chromatograph (HPLC). The column was a KromasilC18 column (250 mm x 416mm i.d.,5 μm), column temperature 30 ℃, detector was an ultraviolet detector (259 nm), mobile phase was water: acetonitrile=90: 10; the flow rate is 1.0ml/min; the sample injection amount was 20. Mu.L. Accurately weighing a standard substance of adenosine, and preparing a standard substance solution with the mass concentration of 1g/L by deionized water; and diluting the pretreated fermentation liquor sample to be detected to a proper concentration by deionized water, filtering by a 0.22 mu m microporous filter membrane, taking the diluted fermentation liquor sample as a fermentation sample solution to be detected, and calculating the adenosine yield according to the peak area of the fermentation sample.
Example 1:
the slant culture medium formula is calculated in g/L: glucose 2, peptone 5, yeast powder 5, xanthine 0.1, agar 20, ph7.0;
seed medium formulation in g/L: glucose 20, corn steep liquor 20mL/L, refined protein powder 10, yeast powder 5 and KH 2 PO 4 1，MgSO 4 ·7H 2 O0.5, xanthine 0.15, histidine 0.05, pH7.0;
fermentation medium is calculated in g/L: glucose 80, corn steep liquor 40mL/L, polysaccharide antler extract 6, yeast extract 5, monosodium glutamate 4, K 2 HPO 4 3, xanthine 0.15, hypoxanthine 0.2, mgSO 4 ·7H 2 O1，MnSO 4 0.05，FeSO 4 ·7H 2 O0.05。
Inoculating bacillus subtilis XGL as an initial strain to an activated inclined plane, culturing for 18 hours at 30 ℃, scraping the inclined plane strain by an inoculating loop, inoculating to a seed culture medium, culturing for 9 hours at 36 ℃ and 200rpm, transferring to a 50L fermentation tank filled with a fermentation culture medium according to 15% (v/v) inoculum size, maintaining dissolved oxygen at 20-30% at 36 ℃, and culturing with NH 3 ·H 2 And regulating the pH value to 6.4 by O, monitoring the residual glucose content in the fermentation liquid on line, and feeding glucose until the residual glucose content in the fermentation liquid is 10g/L when the residual glucose content is reduced to 10g/L, fermenting for 40h, wherein the adenosine yield reaches 54.3g/L.
Example 2:
the slant culture medium formula is calculated in g/L: glucose 2, peptone 5, yeast powder 5, xanthine 0.1, agar 20, ph7.0;
seed medium formulation in g/L: glucose 20, corn steep liquor 20mL/L, refined protein powder 10, yeast powder 5 and KH 2 PO 4 1，MgSO 4 ·7H 2 O0.5, xanthine 0.15, histidine 0.05, pH7.0;
fermentation medium is calculated in g/L: glucose 80, corn steep liquor 40mL/L, polysaccharide antler extract 6, monosodium glutamate 4, K 2 HPO 4 3, 0.1 to 0.2 of isoleucine, 0.15 of xanthine, 0.2 of hypoxanthine and MgSO 4 ·7H 2 O1，MnSO 4 0.05，FeSO 4 ·7H 2 O0.05。
Inoculating bacillus subtilis XGL serving as an initial strain to an activation inclined plane, culturing for 18 hours at 30 ℃, scraping the inclined plane strain by an inoculating loop, inoculating to a seed culture medium, and culturing for 9 hours at 36 ℃ and 200 rpm; transferring to a 50L fermentation tank containing fermentation medium according to 15% (v/v) inoculum size, maintaining dissolved oxygen at 36 deg.C at 20-30%, and using NH 3 ·H 2 And regulating the pH value to 6.4 by O, monitoring the residual glucose content in the fermentation liquid on line, and feeding glucose until the residual glucose content in the fermentation liquid is 10g/L when the residual glucose content is reduced to 10g/L, fermenting for 40h, wherein the adenosine yield reaches 50.7g/L.
Example 3:
the slant culture medium formula is calculated in g/L: glucose 2, peptone 5, yeast powder 5, xanthine 0.1, agar 20, ph7.0;
seed medium formulation in g/L: glucose 20, corn steep liquor 20mL/L, refined protein powder 10, yeast powder 5 and KH 2 PO 4 1，MgSO 4 ·7H 2 O0.5, xanthine 0.15, histidine 0.05, pH7.0;
fermentation medium is calculated in g/L: glucose 80, corn steep liquor 40mL/L, polysaccharide antler extract 6, yeast extract 5, monosodium glutamate 4, K 2 HPO 4 3, 0.1 to 0.2 of isoleucine, 0.15 of xanthine, 0.2 of hypoxanthine and MgSO 4 ·7H 2 O1，MnSO 4 0.05，FeSO 4 ·7H 2 O0.05。
Inoculating bacillus subtilis XGL as an initial strain to an activated inclined plane, culturing for 18 hours at 30 ℃, scraping the inclined plane strain by an inoculating loop, inoculating to a seed culture medium, culturing for 9 hours at 36 ℃ and 200rpm, transferring to a 50L fermentation tank filled with a fermentation culture medium according to 15% (v/v) inoculum size, maintaining dissolved oxygen at 20-30% at 36 ℃, and culturing with NH 3 ·H 2 And regulating the pH value to 6.4 by O, monitoring the residual glucose content in the fermentation liquid on line, and feeding glucose until the residual glucose content in the fermentation liquid is 10g/L when the residual glucose content is reduced to 10g/L, fermenting for 40h, wherein the adenosine yield reaches 60.7g/L.
Comparative example 1:
the fermentation tank was not subjected to fermentation culture by adding yeast extract and isoleucine, and the other conditions were the same as in example 1. The final test shows that the adenosine content is 42.3g/L.
As can be seen from example 1 and comparative example 1, the addition of 5g/L yeast extract in the fermenter can increase the yield of adenosine by 28.4%, which indicates that the addition of the organic nitrogen source yeast extract can provide sufficient nitrogen source for the growth of the cells and prolong the stationary growth period of the cells. As can be seen from example 2 and comparative example 1, the effect of adding isoleucine to the culture medium on the yield of adenosine was also relatively large, the yield of adenosine was increased by 20%, the activity of the cells was improved by isoleucine, and the yield of adenosine in the lower tank was increased. As can be seen from the example 3 and the comparative example 1, the addition of yeast extract powder and isoleucine can obviously improve the yield of adenosine, the yield of adenosine is improved by 43.5%, and the effect is obvious.
It should be noted that the above-mentioned embodiments are to be understood as illustrative, and not limiting, the scope of the invention, which is defined by the appended claims. It will be apparent to those skilled in the art that various modifications and adaptations can be made to the present invention without departing from its spirit or scope.
Claims (3)
1. A method for increasing the fermentation yield of adenosine comprising the steps of:
step one, slant culture: inoculating bacillus subtilis XGL serving as an initial strain into a slant solid culture medium for activation, and culturing at 28-32 ℃ for 15-20 hours;
step two, seed culture: scraping strains on the inclined plane solid culture by using an inoculating loop, inoculating the strains to a seed culture medium, and culturing at 200-500 rpm at 30-36 ℃ for 8-10 hours to obtain fermentation seed liquid;
step three, fermentation culture: transferring the fermentation seed liquid obtained in the second step into a fermentation tank filled with a fermentation medium according to the inoculation amount of 12-15%, maintaining the dissolved oxygen at 20-30% at 33-36 ℃, and using NH 3 ·H 2 O is used for regulating the pH value to be 6.6-7.0, and the culture time is 40-50 h;
a fermentation medium comprising, 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 extract, 3-5 parts of monosodium glutamate and K 2 HPO 4 1-3, 0.1-0.2 isoleucine, 0.1-0.2 xanthine, 0.1-0.2 hypoxanthine, mgSO 4 ·7H 2 O 1~5，MnSO 4 0.03 to 0.06 and FeSO 4 ·7H 2 O 0.03~0.06。
2. The method according to claim 1, characterized in that: 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, 20-30 parts of agar and 7.0-7.2 parts of pH7.
3. The method according to claim 1, characterized in that: the seed culture medium in the second step comprises the following components in g/L: 15-20 parts of glucose, 15-25 mL/L of corn steep liquor, 5-10 parts of protein refined powder, 5-10 parts of yeast powder and KH 2 PO 4 0.5~1.5，MgSO 4 ·7H 2 O0.2-0.8, xanthine 0.1-0.15, histidine 0.01-0.05, pH 6.5-7.0.
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