CN113528402A - Culture medium and fermentation method of streptomyces microflavus - Google Patents
Culture medium and fermentation method of streptomyces microflavus Download PDFInfo
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- CN113528402A CN113528402A CN202110962280.4A CN202110962280A CN113528402A CN 113528402 A CN113528402 A CN 113528402A CN 202110962280 A CN202110962280 A CN 202110962280A CN 113528402 A CN113528402 A CN 113528402A
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- 238000000855 fermentation Methods 0.000 title claims abstract description 41
- 230000004151 fermentation Effects 0.000 title claims abstract description 41
- 241000187395 Streptomyces microflavus Species 0.000 title claims abstract description 36
- 239000001963 growth medium Substances 0.000 title claims abstract description 32
- 238000000034 method Methods 0.000 title claims abstract description 13
- CSNNHWWHGAXBCP-UHFFFAOYSA-L Magnesium sulfate Chemical compound [Mg+2].[O-][S+2]([O-])([O-])[O-] CSNNHWWHGAXBCP-UHFFFAOYSA-L 0.000 claims abstract description 17
- 239000000843 powder Substances 0.000 claims abstract description 15
- FAPWRFPIFSIZLT-UHFFFAOYSA-M sodium chloride Inorganic materials [Na+].[Cl-] FAPWRFPIFSIZLT-UHFFFAOYSA-M 0.000 claims abstract description 13
- 240000004808 Saccharomyces cerevisiae Species 0.000 claims abstract description 11
- 238000011218 seed culture Methods 0.000 claims abstract description 11
- 229920002472 Starch Polymers 0.000 claims abstract description 10
- 229910000396 dipotassium phosphate Inorganic materials 0.000 claims abstract description 10
- 229910052943 magnesium sulfate Inorganic materials 0.000 claims abstract description 10
- 239000008107 starch Substances 0.000 claims abstract description 10
- 235000019698 starch Nutrition 0.000 claims abstract description 10
- VTYYLEPIZMXCLO-UHFFFAOYSA-L Calcium carbonate Chemical compound [Ca+2].[O-]C([O-])=O VTYYLEPIZMXCLO-UHFFFAOYSA-L 0.000 claims abstract description 9
- ZPWVASYFFYYZEW-UHFFFAOYSA-L dipotassium hydrogen phosphate Chemical compound [K+].[K+].OP([O-])([O-])=O ZPWVASYFFYYZEW-UHFFFAOYSA-L 0.000 claims abstract description 8
- FGIUAXJPYTZDNR-UHFFFAOYSA-N potassium nitrate Inorganic materials [K+].[O-][N+]([O-])=O FGIUAXJPYTZDNR-UHFFFAOYSA-N 0.000 claims abstract description 7
- 239000011780 sodium chloride Substances 0.000 claims abstract description 7
- 239000002609 medium Substances 0.000 claims abstract description 6
- 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 5
- 244000046052 Phaseolus vulgaris Species 0.000 claims abstract description 5
- 235000010627 Phaseolus vulgaris Nutrition 0.000 claims abstract description 5
- 229930006000 Sucrose Natural products 0.000 claims abstract description 5
- CZMRCDWAGMRECN-UGDNZRGBSA-N Sucrose Chemical compound O[C@H]1[C@H](O)[C@@H](CO)O[C@@]1(CO)O[C@@H]1[C@H](O)[C@@H](O)[C@H](O)[C@@H](CO)O1 CZMRCDWAGMRECN-UGDNZRGBSA-N 0.000 claims abstract description 5
- 239000002518 antifoaming agent Substances 0.000 claims abstract description 5
- 229910000019 calcium carbonate Inorganic materials 0.000 claims abstract description 5
- 239000008103 glucose Substances 0.000 claims abstract description 5
- 229910000359 iron(II) sulfate Inorganic materials 0.000 claims abstract description 5
- 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 claims abstract 2
- 239000007788 liquid Substances 0.000 claims description 14
- QVGXLLKOCUKJST-UHFFFAOYSA-N atomic oxygen Chemical compound [O] QVGXLLKOCUKJST-UHFFFAOYSA-N 0.000 claims description 9
- 229910052760 oxygen Inorganic materials 0.000 claims description 9
- 239000001301 oxygen Substances 0.000 claims description 9
- 230000001105 regulatory effect Effects 0.000 claims description 9
- 238000012258 culturing Methods 0.000 claims description 6
- 229960004793 sucrose Drugs 0.000 claims description 4
- 238000009423 ventilation Methods 0.000 claims description 4
- 239000005720 sucrose Substances 0.000 abstract description 3
- 239000013530 defoamer Substances 0.000 abstract 1
- 239000003337 fertilizer Substances 0.000 description 12
- 230000001580 bacterial effect Effects 0.000 description 7
- 239000002689 soil Substances 0.000 description 7
- HEMHJVSKTPXQMS-UHFFFAOYSA-M Sodium hydroxide Chemical compound [OH-].[Na+] HEMHJVSKTPXQMS-UHFFFAOYSA-M 0.000 description 6
- 230000000694 effects Effects 0.000 description 5
- 230000000813 microbial effect Effects 0.000 description 5
- IJGRMHOSHXDMSA-UHFFFAOYSA-N Atomic nitrogen Chemical compound N#N IJGRMHOSHXDMSA-UHFFFAOYSA-N 0.000 description 4
- 239000000306 component Substances 0.000 description 4
- 235000020344 instant tea Nutrition 0.000 description 4
- 239000013028 medium composition Substances 0.000 description 4
- 239000000203 mixture Substances 0.000 description 4
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 description 3
- 241000894006 Bacteria Species 0.000 description 2
- OAICVXFJPJFONN-UHFFFAOYSA-N Phosphorus Chemical compound [P] OAICVXFJPJFONN-UHFFFAOYSA-N 0.000 description 2
- YPHMISFOHDHNIV-FSZOTQKASA-N cycloheximide Chemical compound C1[C@@H](C)C[C@H](C)C(=O)[C@@H]1[C@H](O)CC1CC(=O)NC(=O)C1 YPHMISFOHDHNIV-FSZOTQKASA-N 0.000 description 2
- 201000010099 disease Diseases 0.000 description 2
- 208000037265 diseases, disorders, signs and symptoms Diseases 0.000 description 2
- 230000035558 fertility Effects 0.000 description 2
- BAUYGSIQEAFULO-UHFFFAOYSA-L iron(2+) sulfate (anhydrous) Chemical compound [Fe+2].[O-]S([O-])(=O)=O BAUYGSIQEAFULO-UHFFFAOYSA-L 0.000 description 2
- 229910052757 nitrogen Inorganic materials 0.000 description 2
- 229910052698 phosphorus Inorganic materials 0.000 description 2
- 239000011574 phosphorus Substances 0.000 description 2
- 230000001954 sterilising effect Effects 0.000 description 2
- 238000005406 washing Methods 0.000 description 2
- 238000005303 weighing Methods 0.000 description 2
- ACTOXUHEUCPTEW-BWHGAVFKSA-N 2-[(4r,5s,6s,7r,9r,10r,11e,13e,16r)-6-[(2s,3r,4r,5s,6r)-5-[(2s,4r,5s,6s)-4,5-dihydroxy-4,6-dimethyloxan-2-yl]oxy-4-(dimethylamino)-3-hydroxy-6-methyloxan-2-yl]oxy-10-[(2s,5s,6r)-5-(dimethylamino)-6-methyloxan-2-yl]oxy-4-hydroxy-5-methoxy-9,16-dimethyl-2-o Chemical compound O([C@H]1/C=C/C=C/C[C@@H](C)OC(=O)C[C@@H](O)[C@@H]([C@H]([C@@H](CC=O)C[C@H]1C)O[C@H]1[C@@H]([C@H]([C@H](O[C@@H]2O[C@@H](C)[C@H](O)[C@](C)(O)C2)[C@@H](C)O1)N(C)C)O)OC)[C@@H]1CC[C@H](N(C)C)[C@@H](C)O1 ACTOXUHEUCPTEW-BWHGAVFKSA-N 0.000 description 1
- 229930192334 Auxin Natural products 0.000 description 1
- 108010092160 Dactinomycin Proteins 0.000 description 1
- 241000196324 Embryophyta Species 0.000 description 1
- 241000233866 Fungi Species 0.000 description 1
- 229930191978 Gibberellin Natural products 0.000 description 1
- 241000238631 Hexapoda Species 0.000 description 1
- ZLMJMSJWJFRBEC-UHFFFAOYSA-N Potassium Chemical compound [K] ZLMJMSJWJFRBEC-UHFFFAOYSA-N 0.000 description 1
- 239000004187 Spiramycin Substances 0.000 description 1
- 241000187747 Streptomyces Species 0.000 description 1
- 241001052560 Thallis Species 0.000 description 1
- 241000607479 Yersinia pestis Species 0.000 description 1
- 229930183665 actinomycin Natural products 0.000 description 1
- 239000013543 active substance Substances 0.000 description 1
- 239000002363 auxin Substances 0.000 description 1
- 230000033228 biological regulation Effects 0.000 description 1
- 239000004062 cytokinin Substances 0.000 description 1
- UQHKFADEQIVWID-UHFFFAOYSA-N cytokinin Natural products C1=NC=2C(NCC=C(CO)C)=NC=NC=2N1C1CC(O)C(CO)O1 UQHKFADEQIVWID-UHFFFAOYSA-N 0.000 description 1
- 230000006378 damage Effects 0.000 description 1
- 238000007865 diluting Methods 0.000 description 1
- 239000012153 distilled water Substances 0.000 description 1
- 230000007613 environmental effect Effects 0.000 description 1
- IXORZMNAPKEEDV-UHFFFAOYSA-N gibberellic acid GA3 Natural products OC(=O)C1C2(C3)CC(=C)C3(O)CCC2C2(C=CC3O)C1C3(C)C(=O)O2 IXORZMNAPKEEDV-UHFFFAOYSA-N 0.000 description 1
- 239000003448 gibberellin Substances 0.000 description 1
- 239000003630 growth substance Substances 0.000 description 1
- SEOVTRFCIGRIMH-UHFFFAOYSA-N indole-3-acetic acid Chemical compound C1=CC=C2C(CC(=O)O)=CNC2=C1 SEOVTRFCIGRIMH-UHFFFAOYSA-N 0.000 description 1
- 238000009776 industrial production Methods 0.000 description 1
- 230000005764 inhibitory process Effects 0.000 description 1
- 238000011081 inoculation Methods 0.000 description 1
- 239000002054 inoculum Substances 0.000 description 1
- 238000004519 manufacturing process Methods 0.000 description 1
- 239000002207 metabolite Substances 0.000 description 1
- 238000012986 modification Methods 0.000 description 1
- 230000004048 modification Effects 0.000 description 1
- 238000009335 monocropping Methods 0.000 description 1
- 238000010899 nucleation Methods 0.000 description 1
- 150000007524 organic acids Chemical class 0.000 description 1
- 239000000575 pesticide Substances 0.000 description 1
- 239000011591 potassium Substances 0.000 description 1
- 229910052700 potassium Inorganic materials 0.000 description 1
- 230000000750 progressive effect Effects 0.000 description 1
- 238000011160 research Methods 0.000 description 1
- 238000003900 soil pollution Methods 0.000 description 1
- 229960001294 spiramycin Drugs 0.000 description 1
- 229930191512 spiramycin Natural products 0.000 description 1
- 235000019372 spiramycin Nutrition 0.000 description 1
- 239000008223 sterile water Substances 0.000 description 1
- 230000004936 stimulating effect Effects 0.000 description 1
- 239000000126 substance 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
Abstract
The invention discloses a culture medium and a fermentation method of streptomyces microflavus, belonging to the technical field of biology; comprises a seed culture medium and a fermentation culture medium; the seed culture medium comprises the following components: soluble starch, yeast powder, glucose, K2HPO4，MgSO4，FeSO4NaCl, defoamer, pH7.2-7.4; the fermentation medium comprises the following components: soluble starch, yeast powder, sucrose, bean cake powder, K2HPO4，MgSO4，NaCl，CaCO3，KNO3Defoaming agent, pH7.2-7.6. The culture medium and the fermentation method can effectively improve the growth amount of the streptomyces microflavus.
Description
Technical Field
The invention relates to the technical field of biology, in particular to a culture medium and a fermentation method of streptomyces microflavus.
Background
For years, the problems of soil quality rapid reduction, soil hardening, plant diseases and insect pests aggravation, continuous cropping obstacle and the like are frequent and increasingly serious due to the large amount of fertilizer and pesticide, so that the quality of agricultural products is reduced, the cost is increased, and the environmental destruction and ecological imbalance are caused.
The microbial fertilizer is also called biological fertilizer, inoculant or bacterial fertilizer, and is a fertilizer product which takes the life activities of microbes as the core and enables crops to obtain specific fertilizer effects, can promote the growth of agricultural products, can maintain the health of soil and ecology, and is an effective guarantee for treating soil pollution and improving ecological environment. The streptomyces microflavus strain commonly used for the microbial fertilizer is streptomyces actinomycetes, and the metabolite of the streptomyces microflavus strain contains growth regulators such as auxin, gibberellin, cytokinin, organic acid and the like, so that the streptomyces microflavus strain has the effects of converting nitrogen, phosphorus and potassium in soil, improving the soil fertility and reducing the using amount of a chemical fertilizer; the produced active substances such as actinomycin, cycloheximide, spiramycin and the like have inhibition effects on gram-positive and gram-negative bacteria, yeast and filamentous fungi, and are commonly used for preventing diseases and protecting seedlings in agriculture; meanwhile, the fertilizer can be used as bacterial fertilizer, can convert nitrogen and phosphorus elements in soil to improve soil fertility, and has the effect of stimulating crop growth.
At present, related researches on streptomyces microflavus microbial fertilizers are few, and the problem of low thallus growth amount still exists in streptomyces microflavus microbial fermentation, so that how to improve the thallus growth amount of streptomyces microflavus fermentation is a problem to be solved urgently by technical personnel in the field.
Disclosure of Invention
In view of the above, the invention provides a culture medium and a fermentation method of streptomyces microflavus, which can effectively improve the growth amount of streptomyces microflavus.
In order to achieve the purpose, the invention adopts the following technical scheme:
the culture medium of streptomyces microflavus comprises a seed culture medium and a fermentation culture medium;
the seed culture medium comprises the following components:
20g/L of soluble starch, 10g/L of yeast powder, 5g/L of glucose and K2HPO40.2g/L，MgSO40.5g/L，FeSO40.01g/L, NaCl0.5g/L, 0.1mL/L of antifoaming agent, pH7.2-7.4;
the fermentation medium comprises the following components:
20g/L of soluble starch, 10g/L of yeast powder, 5g/L of cane sugar, 15g/L of bean cake powder and K2HPO40.1g/L，MgSO40.5g/L，NaCl6g/L，CaCO30.5g/L，KNO30.5g/L, 0.1mL/L of defoaming agent, and pH 7.2-7.6.
The culture medium of the streptomyces microflavus is applied to fermentation of the streptomyces microflavus, and the growth amount of thalli can be effectively improved.
The fermentation method of streptomyces microflavus comprises the following steps:
(1) inoculating streptomyces microflavus into the seed culture medium, and culturing to obtain a seed solution;
(2) inoculating the seed liquid into the fermentation culture medium, and culturing to obtain fermentation liquid.
Preferably, in the step (1),
the rotation speed of the seed culture process is 80-100r/min, the culture temperature is 28-30 ℃, the dissolved oxygen is controlled to be more than 40%, and the seed is cultured for 46 h.
Preferably, in the step (2),
the rotating speed in the fermentation culture process is 100-200r/min, the culture temperature is 30-32 ℃, the dissolved oxygen is regulated and controlled by more than 40% 30h before fermentation, the rotating speed and ventilation are regulated and controlled at the middle and later stages of fermentation, the dissolved oxygen is regulated and controlled to be not less than 20%, and the culture is carried out for 112 h.
According to the technical scheme, the culture medium and the fermentation method of the streptomyces microflavus disclosed by the invention can effectively improve the growth amount of the streptomyces microflavus, and further provide a powerful guarantee for the industrial production of the streptomyces microflavus microbial fertilizer.
Detailed Description
The technical solutions in the embodiments of the present invention are clearly and completely described below, and it is obvious that the described embodiments are only a part of the embodiments of the present invention, and not all embodiments. All other embodiments, which can be derived by a person skilled in the art from the embodiments given herein without making any creative effort, shall fall within the protection scope of the present invention.
Example 1
1. Seed culture
The medium in the seed tank was configured as in tables 1 and 2:
TABLE 115L seed tank-No. 1 Medium composition and content
| Composition (I) | Content (wt.) | Mass to be added when volume of feed liquid is 10L |
| Soluble starch | 22g/L | 220g |
| Yeast powder | 8g/L | 80g |
| Glucose | 5g/L | 50g |
| K2HPO4 | 0.2g/L | 2g |
| MgSO4 | 0.5g/L | 5g |
| FeSO4 | 0.01g/L | 0.1g |
| NaCl | 0.5g/L | 5g |
| Instant tea | 0.1mL/L | 1mL |
TABLE 215L seed tank-No. 2 Medium composition and content
| Composition (I) | Content (wt.) | Mass to be added when volume of feed liquid is 10L |
| Soluble starch | 20g/L | 200g |
| Yeast powder | 10g/L | 100g |
| Glucose | 5g/L | 50g |
| K2HPO4 | 0.2g/L | 2g |
| MgSO4 | 0.5g/L | 5g |
| FeSO4 | 0.01g/L | 0.1g |
| NaCl | 0.5g/L | 5g |
| Instant tea | 0.1mL/L | 1mL |
Weighing corresponding culture medium components according to tables 1 and 2, dissolving in water, diluting to 10L, adjusting pH to 7.2-7.4 with NaOH, and sterilizing at 121 deg.C for 30 min.
Washing the slope of a tomato-shaped bottle of streptomyces microflavus (purchased from China agricultural culture Collection center ACCC41134) with sterile water, inoculating the washing liquid of one strain slope into a 15L seeding tank, culturing at a rotation speed of 80r/min and a temperature of 30 ℃ and dissolved oxygen regulation of more than 40%, and culturing for 46 h.
Microscopic examination shows that mycelium cultured in the No. 2 culture medium is stronger than that cultured in the No. 1 culture medium, and the bacterial content of the streptomyces microflavus in the seed liquid obtained by the No. 2 culture medium culture is 0.51 multiplied by 109The bacterial content of the streptomyces microflavus in the seed liquid obtained by the CFU/mL culture medium No. 1 culture is 0.37 multiplied by 109CFU/mL。
2. Fermentation production
The culture medium of the fermenter was configured as shown in tables 3 and 4:
TABLE 350L fermenter No. 3 Medium composition and content
| Composition (I) | Content (wt.) | Mass to be added when the volume of feed liquid is 35L |
| Soluble starch | 20g/L | 700g |
| Yeast powder | 8g/L | 280g |
| Sucrose | 5g/L | 175g |
| Bean cake powder | 18g/L | 630g |
| K2HPO4 | 0.1g/L | 3.5g |
| MgSO4 | 0.5g/L | 17.5g |
| NaCl | 6g/L | 210g |
| CaCO3 | 0.5g/L | 17.5g |
| KNO3 | 0.5g/L | 17.5g |
| Instant tea | 0.1mL/L | 3.5mL |
TABLE 450L fermenter No. 4 Medium composition and content
| Composition (I) | Content (wt.) | Mass to be added when the volume of feed liquid is 35L |
| Soluble starch | 20g/L | 700g |
| Yeast powder | 10g/L | 350g |
| Sucrose | 5g/L | 175g |
| Bean cake powder | 15g/L | 525g |
| K2HPO4 | 0.1g/L | 3.5g |
| MgSO4 | 0.5g/L | 17.5g |
| NaCl | 6g/L | 210g |
| CaCO3 | 0.5g/L | 17.5g |
| KNO3 | 0.5g/L | 17.5g |
| Instant tea | 0.1mL/L | 3.5mL |
Weighing the components of the culture medium according to the table 3 and the table 4, adding distilled water to dissolve and fix the volume to 35L, adjusting the pH to 7.2-7.6 by NaOH, and sterilizing for 30min at 121 ℃.
Inoculating the seed liquid cultured by the No. 2 culture medium into a sterilized fermentation tank for fermentation, wherein the inoculation amount is 20%, 100-200r/min, the temperature is 31 ℃, the ventilation amount is 0.5-1.0 (V/V.min), the dissolved oxygen is regulated and controlled by more than 40% 30h before fermentation, the rotating speed and ventilation are regulated in the middle and later stages of fermentation, the dissolved oxygen is regulated and controlled to be not less than 20%, and the streptomyces microflavus bacterial liquid is obtained after fermentation culture for 112 h.
Plate count of Streptomyces microflavus in fermentation Medium No. 3The number of bacteria in the liquid is more than 1.3 × 109The bacterial count of the streptomyces microflavus bacterial liquid in the CFU/mL No. 4 fermentation medium is more than 2.0 multiplied by 109CFU/mL。
The embodiments in the present description are described in a progressive manner, each embodiment focuses on differences from other embodiments, and the same and similar parts among the embodiments are referred to each other.
The previous description of the disclosed embodiments is provided to enable any person skilled in the art to make or use the present invention. Various modifications to these embodiments will be readily apparent to those skilled in the art, and the generic principles defined herein may be applied to other embodiments without departing from the spirit or scope of the invention. Thus, the present invention is not intended to be limited to the embodiments shown herein but is to be accorded the widest scope consistent with the principles and novel features disclosed herein.
Claims (5)
1. The culture medium of the streptomyces microflavus is characterized by comprising a seed culture medium and a fermentation culture medium;
the seed culture medium comprises the following components:
20g/L of soluble starch, 10g/L of yeast powder, 5g/L of glucose and K2HPO4 0.2g/L，MgSO40.5g/L，FeSO40.01g/L, NaCl0.5g/L, antifoaming agent 0.1mL/L, pH 7.2-7.4;
the fermentation medium comprises the following components:
20g/L of soluble starch, 10g/L of yeast powder, 5g/L of cane sugar, 15g/L of bean cake powder and K2HPO4 0.1g/L，MgSO4 0.5g/L，NaCl 6g/L，CaCO3 0.5g/L，KNO30.5g/L, 0.1mL/L of defoaming agent and pH 7.2-7.6.
2. Use of a culture medium of Streptomyces microflavus according to claim 1 in a fermentation of Streptomyces microflavus.
3. The fermentation method of streptomyces microflavus is characterized by comprising the following steps:
(1) inoculating streptomyces microflavus into the seed culture medium of claim 1, and culturing to obtain a seed solution;
(2) inoculating the seed solution into the fermentation culture medium of claim 1, and culturing to obtain fermentation liquid.
4. The fermentation process of Streptomyces microflavus according to claim 3, wherein the fermentation broth is obtained by fermenting Streptomyces microflavus,
in the step (1), the step (c),
the rotation speed of the seed culture process is 80-100r/min, the culture temperature is 28-30 ℃, the dissolved oxygen is controlled to be more than 40%, and the seed is cultured for 46 h.
5. The fermentation process of Streptomyces microflavus according to claim 3, wherein the fermentation broth is obtained by fermenting Streptomyces microflavus,
in the step (2),
the rotating speed in the fermentation culture process is 100-200r/min, the culture temperature is 30-32 ℃, the dissolved oxygen is regulated and controlled by more than 40% 30h before fermentation, the rotating speed and ventilation are regulated and controlled at the middle and later stages of fermentation, the dissolved oxygen is regulated and controlled to be not less than 20%, and the culture is carried out for 112 h.
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