CN111733202A - Mixed fermentation method of tylosin - Google Patents
Mixed fermentation method of tylosin Download PDFInfo
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- CN111733202A CN111733202A CN202010612978.9A CN202010612978A CN111733202A CN 111733202 A CN111733202 A CN 111733202A CN 202010612978 A CN202010612978 A CN 202010612978A CN 111733202 A CN111733202 A CN 111733202A
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- 238000000855 fermentation Methods 0.000 title claims abstract description 96
- 230000004151 fermentation Effects 0.000 title claims abstract description 96
- WBPYTXDJUQJLPQ-VMXQISHHSA-N tylosin Chemical compound O([C@@H]1[C@@H](C)O[C@H]([C@@H]([C@H]1N(C)C)O)O[C@@H]1[C@@H](C)[C@H](O)CC(=O)O[C@@H]([C@H](/C=C(\C)/C=C/C(=O)[C@H](C)C[C@@H]1CC=O)CO[C@H]1[C@@H]([C@H](OC)[C@H](O)[C@@H](C)O1)OC)CC)[C@H]1C[C@@](C)(O)[C@@H](O)[C@H](C)O1 WBPYTXDJUQJLPQ-VMXQISHHSA-N 0.000 title claims abstract description 53
- 229930194936 Tylosin Natural products 0.000 title claims abstract description 45
- 239000004182 Tylosin Substances 0.000 title claims abstract description 45
- 229960004059 tylosin Drugs 0.000 title claims abstract description 45
- 235000019375 tylosin Nutrition 0.000 title claims abstract description 45
- 238000000034 method Methods 0.000 title claims abstract description 28
- 241000187438 Streptomyces fradiae Species 0.000 claims abstract description 15
- 230000008569 process Effects 0.000 claims abstract description 6
- VTYYLEPIZMXCLO-UHFFFAOYSA-L Calcium carbonate Chemical compound [Ca+2].[O-]C([O-])=O VTYYLEPIZMXCLO-UHFFFAOYSA-L 0.000 claims description 32
- 239000007788 liquid Substances 0.000 claims description 29
- 239000001963 growth medium Substances 0.000 claims description 23
- 240000008042 Zea mays Species 0.000 claims description 16
- 235000005824 Zea mays ssp. parviglumis Nutrition 0.000 claims description 16
- 235000002017 Zea mays subsp mays Nutrition 0.000 claims description 16
- 229910000019 calcium carbonate Inorganic materials 0.000 claims description 16
- 235000005822 corn Nutrition 0.000 claims description 16
- 235000012424 soybean oil Nutrition 0.000 claims description 14
- 239000003549 soybean oil Substances 0.000 claims description 14
- 238000012807 shake-flask culturing Methods 0.000 claims description 10
- 229940041514 candida albicans extract Drugs 0.000 claims description 8
- 239000012138 yeast extract Substances 0.000 claims description 8
- 241000894006 Bacteria Species 0.000 claims description 7
- 238000012258 culturing Methods 0.000 claims description 7
- 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 6
- 239000008103 glucose Substances 0.000 claims description 6
- 241000726221 Gemma Species 0.000 claims description 3
- 239000002054 inoculum Substances 0.000 claims description 3
- 239000000203 mixture Substances 0.000 claims 1
- 230000002503 metabolic effect Effects 0.000 abstract description 4
- 230000000694 effects Effects 0.000 abstract description 3
- 102000004190 Enzymes Human genes 0.000 abstract description 2
- 108090000790 Enzymes Proteins 0.000 abstract description 2
- 230000015572 biosynthetic process Effects 0.000 abstract description 2
- 238000003786 synthesis reaction Methods 0.000 abstract description 2
- 238000001914 filtration Methods 0.000 abstract 1
- 239000000843 powder Substances 0.000 description 16
- 238000011218 seed culture Methods 0.000 description 15
- WCUXLLCKKVVCTQ-UHFFFAOYSA-M Potassium chloride Chemical compound [Cl-].[K+] WCUXLLCKKVVCTQ-UHFFFAOYSA-M 0.000 description 12
- FAPWRFPIFSIZLT-UHFFFAOYSA-M Sodium chloride Chemical compound [Na+].[Cl-] FAPWRFPIFSIZLT-UHFFFAOYSA-M 0.000 description 12
- 230000001502 supplementing effect Effects 0.000 description 12
- 239000002609 medium Substances 0.000 description 11
- 238000011081 inoculation Methods 0.000 description 10
- 244000046052 Phaseolus vulgaris Species 0.000 description 8
- 235000010627 Phaseolus vulgaris Nutrition 0.000 description 8
- MNNHAPBLZZVQHP-UHFFFAOYSA-N diammonium hydrogen phosphate Chemical compound [NH4+].[NH4+].OP([O-])([O-])=O MNNHAPBLZZVQHP-UHFFFAOYSA-N 0.000 description 8
- 229910000388 diammonium phosphate Inorganic materials 0.000 description 8
- 235000019838 diammonium phosphate Nutrition 0.000 description 8
- 238000001816 cooling Methods 0.000 description 7
- 230000001954 sterilising effect Effects 0.000 description 7
- 235000016068 Berberis vulgaris Nutrition 0.000 description 6
- 241000335053 Beta vulgaris Species 0.000 description 6
- 235000019733 Fish meal Nutrition 0.000 description 6
- VEXZGXHMUGYJMC-UHFFFAOYSA-N Hydrochloric acid Chemical compound Cl VEXZGXHMUGYJMC-UHFFFAOYSA-N 0.000 description 6
- 230000001580 bacterial effect Effects 0.000 description 6
- GVPFVAHMJGGAJG-UHFFFAOYSA-L cobalt dichloride Chemical compound [Cl-].[Cl-].[Co+2] GVPFVAHMJGGAJG-UHFFFAOYSA-L 0.000 description 6
- 239000004467 fishmeal Substances 0.000 description 6
- 235000013312 flour Nutrition 0.000 description 6
- LGQLOGILCSXPEA-UHFFFAOYSA-L nickel sulfate Chemical compound [Ni+2].[O-]S([O-])(=O)=O LGQLOGILCSXPEA-UHFFFAOYSA-L 0.000 description 6
- 229910000363 nickel(II) sulfate Inorganic materials 0.000 description 6
- 239000001103 potassium chloride Substances 0.000 description 6
- 235000011164 potassium chloride Nutrition 0.000 description 6
- 239000011780 sodium chloride Substances 0.000 description 6
- 230000000052 comparative effect Effects 0.000 description 5
- BFNBIHQBYMNNAN-UHFFFAOYSA-N ammonium sulfate Chemical compound N.N.OS(O)(=O)=O BFNBIHQBYMNNAN-UHFFFAOYSA-N 0.000 description 4
- 229910052921 ammonium sulfate Inorganic materials 0.000 description 4
- 235000011130 ammonium sulphate Nutrition 0.000 description 4
- 239000003921 oil Substances 0.000 description 4
- 235000019198 oils Nutrition 0.000 description 4
- 238000003756 stirring Methods 0.000 description 4
- 238000009423 ventilation Methods 0.000 description 4
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 description 4
- 241001052560 Thallis Species 0.000 description 3
- 238000004519 manufacturing process Methods 0.000 description 3
- 238000002360 preparation method Methods 0.000 description 3
- 235000017060 Arachis glabrata Nutrition 0.000 description 2
- 244000105624 Arachis hypogaea Species 0.000 description 2
- 235000010777 Arachis hypogaea Nutrition 0.000 description 2
- 235000018262 Arachis monticola Nutrition 0.000 description 2
- 108010068370 Glutens Proteins 0.000 description 2
- 230000000844 anti-bacterial effect Effects 0.000 description 2
- 230000000845 anti-microbial effect Effects 0.000 description 2
- QVGXLLKOCUKJST-UHFFFAOYSA-N atomic oxygen Chemical compound [O] QVGXLLKOCUKJST-UHFFFAOYSA-N 0.000 description 2
- 235000012343 cottonseed oil Nutrition 0.000 description 2
- 238000001514 detection method Methods 0.000 description 2
- 238000005516 engineering process Methods 0.000 description 2
- 235000021312 gluten Nutrition 0.000 description 2
- 230000006872 improvement Effects 0.000 description 2
- 239000001301 oxygen Substances 0.000 description 2
- 229910052760 oxygen Inorganic materials 0.000 description 2
- 235000020232 peanut Nutrition 0.000 description 2
- 239000000725 suspension Substances 0.000 description 2
- 241000193830 Bacillus <bacterium> Species 0.000 description 1
- 241000204031 Mycoplasma Species 0.000 description 1
- 229930182555 Penicillin Natural products 0.000 description 1
- JGSARLDLIJGVTE-MBNYWOFBSA-N Penicillin G Chemical compound N([C@H]1[C@H]2SC([C@@H](N2C1=O)C(O)=O)(C)C)C(=O)CC1=CC=CC=C1 JGSARLDLIJGVTE-MBNYWOFBSA-N 0.000 description 1
- 241000700605 Viruses Species 0.000 description 1
- 230000008859 change Effects 0.000 description 1
- 238000004134 energy conservation Methods 0.000 description 1
- 238000005265 energy consumption Methods 0.000 description 1
- 238000009655 industrial fermentation Methods 0.000 description 1
- 208000015181 infectious disease Diseases 0.000 description 1
- 231100000053 low toxicity Toxicity 0.000 description 1
- 239000003120 macrolide antibiotic agent Substances 0.000 description 1
- 229940049954 penicillin Drugs 0.000 description 1
- 230000009467 reduction Effects 0.000 description 1
- 238000001228 spectrum 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
- C12P39/00—Processes involving microorganisms of different genera in the same process, simultaneously
-
- 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
- C12P19/44—Preparation of O-glycosides, e.g. glucosides
- C12P19/60—Preparation of O-glycosides, e.g. glucosides having an oxygen of the saccharide radical directly bound to a non-saccharide heterocyclic ring or a condensed ring system containing a non-saccharide heterocyclic ring, e.g. coumermycin, novobiocin
- C12P19/62—Preparation of O-glycosides, e.g. glucosides having an oxygen of the saccharide radical directly bound to a non-saccharide heterocyclic ring or a condensed ring system containing a non-saccharide heterocyclic ring, e.g. coumermycin, novobiocin the hetero ring having eight or more ring members and only oxygen as ring hetero atoms, e.g. erythromycin, spiramycin, nystatin
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- Organic Chemistry (AREA)
- Chemical & Material Sciences (AREA)
- Engineering & Computer Science (AREA)
- Zoology (AREA)
- Life Sciences & Earth Sciences (AREA)
- Wood Science & Technology (AREA)
- Chemical Kinetics & Catalysis (AREA)
- Microbiology (AREA)
- General Chemical & Material Sciences (AREA)
- Biotechnology (AREA)
- Health & Medical Sciences (AREA)
- Biochemistry (AREA)
- Bioinformatics & Cheminformatics (AREA)
- General Engineering & Computer Science (AREA)
- General Health & Medical Sciences (AREA)
- Genetics & Genomics (AREA)
- Preparation Of Compounds By Using Micro-Organisms (AREA)
- Micro-Organisms Or Cultivation Processes Thereof (AREA)
Abstract
The invention relates to a tylosin mixed culture fermentation method, which is characterized in that streptomyces fradiae is used as a fermentation strain, and a cultured spore-state diplococcus is inoculated in the fermentation process for producing tylosin for continuous fermentation. The method utilizes the mixed fermentation of the diplococcus and the streptomyces fradiae to produce the tylosin, wherein the diplococcus and the streptomyces fradiae are mixed and cultured in a fermentation system, and share a metabolic enzyme system and a metabolic intermediate thereof, so that the method has obvious promotion effect on the synthesis of the tylosin, thereby effectively improving the fermentation level and improving the component content of the fermentation liquor, particularly the content of the tylosin A. By the method, the fermentation period of the tylosin can be controlled within 160-170 hours, the fermentation unit is improved by more than 10% compared with the original process, the titer of the tylosin A in the fermentation liquor is improved by more than 10%, and the tylosin A has no influence on the filtration of the fermentation liquor.
Description
Technical Field
The invention relates to biological and new medical technology, in particular to a tylosin mixed culture fermentation method.
Background
Tylosin (Tylosin), also known as Tylosin, is a macrolide antibiotic with broad antimicrobial spectrum, strongest antimicrobial activity against mycoplasma, low toxicity and broad spectrum, and can be used for treating infections caused by penicillin-resistant bacteria, mainly against G+For most part G-Spirochetes and large viruses also play a role. Tylosin is mainly composed of four components of tylosin A, B, C, D. A, B, C has antibacterial activity except tylosin D component, wherein the antibacterial activity of A component is the highest. Among tylosin currently produced, the a component is the main component of tylosin.
In the prior art, streptomyces fradiae is generally used for pure fermentation in fermentation production of tylosin, for example, the streptomyces fradiae in patent CN111139197A and application thereof in tylosin fermentation are adopted, thalli are enabled to produce tylosin by means of seed culture, fermentation process control and the like, the method has the advantages of low tylosin fermentation unit, high difficulty in improving fermentation level and small fermentation process change space. At present, the improvement of the fermentation level is an urgent problem to be solved by fermentation enterprises, and the further improvement of the industrial fermentation level, energy conservation and consumption reduction are urgent needs for improving the international competitiveness of the product at present.
Disclosure of Invention
The invention aims to provide a tylosin mixed strain fermentation method which can effectively improve the content of a tylosin A component in tylosin fermentation and improve the fermentation level.
The technical scheme adopted for realizing the aim of the invention is as follows:
a tylosin mixed culture fermentation method is characterized in that: in the fermentation culture process of producing tylosin by taking streptomyces fradiae as a fermentation strain, the cultured gemma diplococcus is inoculated and then fermentation is continued.
The diplococcus is inoculated in fermentation culture for 90-100 hours.
The inoculation amount of the diplococcus is 0.05-0.1%.
The preparation method of the gemma diplococcus comprises the following steps: inoculating the diplococcus liquid into a sterilized shake flask culture medium, culturing for 10-14 hours at the temperature of 29-33 ℃ in a shaking table at 170-220 revolutions, and performing microscopic examination on the bacteria to form spores;
the shake flask culture medium consists of: 2.0-3.0% of glucose, and corn steep liquor: 0.1-0.2% of yeast extract, 0.2-0.3% of calcium carbonate and a proper amount of soybean oil.
The control of the fermentation process is based on the prior disclosed tylosin fermentation production process, such as the tylosin production process published in the journal of industrial technology.
The method utilizes the mixed fermentation of the diplococcus and the streptomyces fradiae to produce the tylosin, wherein the diplococcus and the streptomyces fradiae are mixed and cultured in a fermentation system, and share a metabolic enzyme system and a metabolic intermediate thereof, so that the method has obvious promotion effect on the synthesis of the tylosin, thereby effectively improving the fermentation level and improving the component content of the fermentation liquor, particularly the content of the tylosin A. By the method, the fermentation period of the tylosin can be controlled at 160-170 hours, the fermentation unit is improved by more than 10% compared with the original process, the titer of the tylosin A in the fermentation broth is improved by more than 10%, and the fermentation broth is not influenced.
Detailed Description
The invention is illustrated below by way of examples, which are to be understood as being illustrative and not limiting. The scope and core content of the invention are to be determined by the claims.
The following examples diplococcal medium consists of: 2.0-3.0% of glucose, and corn steep liquor: 0.1-0.2% of yeast extract, 0.2-0.3% of calcium carbonate and a proper amount of soybean oil.
Example 1
1. Preparation of diplococcus bacterial liquid
Culture medium of dicoccus: 2.0 percent of glucose, 0.1 percent of corn steep liquor, 0.2 percent of yeast extract, 0.15 percent of calcium carbonate and a proper amount of soybean oil. Sterilizing at 119-122 ℃, preserving heat for 25 minutes, and cooling for seed waiting.
And (3) selecting a ring of the preserved diplococcus liquid, inoculating the diplococcus liquid into a sterilized shake flask culture medium, placing the shake flask culture medium in a shaking table for 180 revolutions, culturing the diplococcus liquid at the temperature of 30 ℃ for 10 hours, checking the mycelia into sporulous shapes by using a microscope, and placing the diplococcus liquid in a refrigerator at the temperature of 4 ℃ for later use.
2. Inoculating the diplococcus liquid into tylosin fermentation liquid for mixed culture
Taking Streptomyces fradiae ACCC40012 as a fermentation strain, and fermenting according to a conventional fermentation method:
primary seed culture medium: 6g/L of peanut cake powder, 4g/L of yeast extract powder, 5g/L of corn steep liquor, 12g/L of soybean oil and 2g/L of calcium carbonate.
Secondary seed culture medium: 12g/L of gluten powder, 10g/L of cottonseed cake powder, 10g/L of bean cake powder, 8g/L of corn steep liquor, 15g/L of soybean oil, 0.4g/L of diammonium hydrogen phosphate and 3g/L of calcium carbonate.
Fermentation medium: 0.6% of bean cake powder, 0.5% of fish meal, 1.8% of corn flour, 0.5% of beet hydrochloride, 0.01% of diammonium hydrogen phosphate, 0.08% of potassium chloride, 0.1% of sodium chloride, 0.0004% of cobalt chloride, 0.0004% of nickel sulfate and 0.2% of calcium carbonate.
First-order seed culture: the method comprises the steps of sterilizing a primary seed culture medium, cooling, maintaining the pressure by using sterile air, inoculating a cultured Streptomyces fradiae mother bottle fermentation liquor into a primary seed tank for culture under the protection of flame, controlling the inoculation amount to be 10% of the volume of the primary seed culture medium, and transferring the primary seeds until the concentration of thalli is 20%, the pH value is 6-8 and no other bacteria are polluted.
Secondary seed culture: sterilizing the secondary seed culture medium, cooling, maintaining the pressure with sterile air, transferring the primary seed liquid into a secondary seed tank for culture, wherein the inoculation amount is 10 percent, and the strain is transferred when the thallus concentration is 30 percent, the fermentation period is 34 hours and no other bacteria pollution exists.
Fermentation culture: inoculating the secondary seed liquid into a sterilized fermentation medium according to the inoculation amount of 20% for culture, controlling the culture temperature to be 31-34 ℃, and the ventilation volume to be 1: 1.1-1.4 v/v/min, stirring and fermenting, supplementing ammonium sulfate according to pH in the fermentation process, supplementing soybean oil according to residual oil content, supplementing water according to bacterial concentration, inoculating cultured diplococcus spongiensis after fermenting for 100 hours, wherein the inoculation amount is 0.05%, and putting the diplococcus spongiensis in a tank after growing for 165 hours.
Detection shows that the bacillus is inoculated to have an obvious promotion effect on tylosin fermentation, the tylosin fermentation titer is 13500 mug/ml, the tylosin fermentation titer is improved by more than 10% compared with a control, and the tylosin A component is improved by more than 4%.
Example 2
1. Preparation of diplococcus bacterial liquid
Culture medium of dicoccus: 2.5 percent of glucose, 1.5 percent of corn steep liquor, 0.25 percent of yeast extract, 0.2 percent of calcium carbonate and a proper amount of soybean oil. Sterilizing at 119-122 ℃, preserving heat for 20-25 minutes, and cooling for seed waiting.
And (3) selecting a ring of the preserved diplococcus liquid, inoculating the diplococcus liquid into a sterilized shake flask culture medium, placing the shake flask culture medium in a shaking table for 180 revolutions, culturing the diplococcus liquid at the temperature of 30 ℃ for 10 hours, checking the mycelia into sporulous shapes by using a microscope, and placing the diplococcus liquid in a refrigerator at the temperature of 4 ℃ for later use.
2. Inoculating the diplococcus liquid into tylosin fermentation liquid for mixed culture
Fermentation medium: 0.7 percent of bean cake powder, 0.6 percent of fish meal, 2.0 percent of corn flour, 0.5 percent of beet hydrochloride, 0.01 percent of diammonium hydrogen phosphate, 0.08 percent of potassium chloride, 0.1 percent of sodium chloride, 0.0004 percent of cobalt chloride, 0.0004 percent of nickel sulfate and 0.2 percent of calcium carbonate.
Tylosin fermentation was carried out according to the procedure of example 1, and the cultured suspension of dicoccus was inoculated at an inoculum size of 0.08% for 90 hours of growth. And (3) putting the fermentation system into a tank after 165 hours of growth, wherein the tank putting titer is 13800 microgram/ml, which is improved by 13.5% compared with a control, and the A component of the fermentation liquid is 88% and is improved by more than 4%.
Example 3
1. Preparing a diplococcus liquid:
culture medium of dicoccus: 3.0 percent of glucose, 0.2 percent of corn steep liquor, 0.3 percent of yeast extract, 0.25 percent of calcium carbonate and a proper amount of soybean oil. Sterilizing at 119-122 ℃, preserving heat for 25 minutes, and cooling for seed waiting.
And (3) selecting a ring of the preserved diplococcus liquid, inoculating the diplococcus liquid into a sterilized shake flask culture medium, placing the shake flask culture medium in a shaking table for 180 revolutions, culturing the diplococcus liquid at the temperature of 30 ℃ for 10 hours, checking the mycelia into sporulous shapes by using a microscope, and placing the diplococcus liquid in a refrigerator at the temperature of 4 ℃ for later use.
2. Inoculating the diplococcus liquid into tylosin fermentation liquid for mixed culture
Fermentation medium: 0.8% of bean cake powder, 0.7% of fish meal, 2.2% of corn flour, 0.5% of beet hydrochloride, 0.01% of diammonium hydrogen phosphate, 0.08% of potassium chloride, 0.1% of sodium chloride, 0.0004% of cobalt chloride, 0.0004% of nickel sulfate and 0.2% of calcium carbonate.
Tylosin fermentation was carried out according to the procedure of example 1, and the cultured suspension of dicoccus was inoculated at an inoculum size of 0.08% for 100 h of growth. And (3) putting the fermentation system into a tank after 165 hours of growth, wherein the tank putting titer is 14100 mug/ml, which is improved by 14.3% compared with a control, and the A component of the fermentation liquid is up to 88% and is improved by more than 4%.
Comparative example 1
Taking Streptomyces fradiae ACCC40012 as a fermentation strain, and fermenting according to a conventional fermentation method:
primary seed culture medium: 6g/L of peanut cake powder, 4g/L of yeast extract powder, 5g/L of corn steep liquor, 12g/L of soybean oil and 2g/L of calcium carbonate.
Secondary seed culture medium: 12g/L of gluten powder, 10g/L of cottonseed cake powder, 10g/L of bean cake powder, 8g/L of corn steep liquor, 15g/L of soybean oil, 0.4g/L of diammonium hydrogen phosphate and 3g/L of calcium carbonate.
Fermentation medium: 0.6% of bean cake powder, 0.5% of fish meal, 1.8% of corn flour, 0.5% of beet hydrochloride, 0.01% of diammonium hydrogen phosphate, 0.08% of potassium chloride, 0.1% of sodium chloride, 0.0004% of cobalt chloride, 0.0004% of nickel sulfate and 0.2% of calcium carbonate.
First-order seed culture: the method comprises the steps of sterilizing a primary seed culture medium, cooling, maintaining the pressure by using sterile air, inoculating a cultured Streptomyces fradiae mother bottle fermentation liquor into a primary seed tank for culture under the protection of flame, controlling the inoculation amount to be 10% of the volume of the primary seed culture medium, and transferring the primary seeds until the concentration of thalli is 20%, the pH value is 6-8 and no other bacteria are polluted.
Secondary seed culture: sterilizing the secondary seed culture medium, cooling, maintaining the pressure with sterile air, transferring the primary seed liquid into a secondary seed tank for culture, wherein the inoculation amount is 10 percent, and the strain is transferred when the concentration of the strain is 30 percent, the period is 34 hours and no other bacteria are polluted.
Fermentation culture: inoculating the secondary seed liquid into a sterilized fermentation medium according to the inoculation amount of 20% for culture, controlling the culture temperature to be 31-34 ℃, and the ventilation volume to be 1: 1.1-1.4 v/v/min, stirring and fermenting, supplementing ammonium sulfate according to pH in the fermentation process, supplementing soybean oil according to residual oil content, supplementing water according to bacterial concentration, growing for 165 hours, and putting into a tank.
Through detection, the tank placing titer is 11800 mug/ml, and the component A in the fermentation liquor is 83%.
Comparative example 2
Fermentation medium: 0.7 percent of bean cake powder, 0.6 percent of fish meal, 2.0 percent of corn flour and 0.5 percent of beet hydrochloride; diammonium hydrogen phosphate 0.01%, potassium chloride 0.08%, sodium chloride 0.1%, cobalt chloride 0.0004%, nickel sulfate 0.0004%, and calcium carbonate 0.2%.
Tylosin fermentation is carried out according to the process of comparative example 1, the spores of streptomyces fradiae ACCC40012 are cultured by primary and secondary seeds, the secondary seed solution is inoculated in a sterilized fermentation medium according to the inoculation amount of 20 percent for culture, the culture temperature is 32-34 ℃, and the ventilation volume is 1: 1.1-1.4 v/v/min, stirring, dissolving more than 30% of oxygen in the fermentation process, supplementing ammonium sulfate according to pH, supplementing soybean oil according to oil residue, supplementing water according to bacterial concentration, and culturing for 165 hours to obtain fermentation liquor containing tylosin, wherein the fermentation process is pure fermentation, the tank-placing titer is 12150 mug/ml, and the component A in the fermentation liquor is 84%.
Comparative example 3
Fermentation medium: 0.8% of bean cake powder, 0.7% of fish meal, 2.2% of corn flour, 0.5% of beet hydrochloride, 0.01% of diammonium hydrogen phosphate, 0.08% of potassium chloride, 0.1% of sodium chloride, 0.0004% of cobalt chloride, 0.0004% of nickel sulfate and 0.2% of calcium carbonate.
Tylosin fermentation is carried out according to the process of comparative example 1, the spores of streptomyces fradiae ACCC40012 are cultured by primary and secondary seeds, the secondary seed solution is inoculated in a sterilized fermentation medium according to the inoculation amount of 20 percent for culture, the culture temperature is 32-34 ℃, and the ventilation volume is 1: 1.1-1.4 v/v/min, stirring, dissolving more than 30% of oxygen in the fermentation process, supplementing ammonium sulfate according to pH, supplementing soybean oil according to oil residue, supplementing water according to bacterial concentration, and culturing for 165 hours to obtain fermentation liquor containing tylosin, wherein the fermentation process is pure fermentation, the tank-placing titer is 12340 mug/ml, and the component A in the fermentation liquor is 84%.
Claims (4)
1. A tylosin mixed culture fermentation method is characterized in that: in the fermentation culture process of producing tylosin by taking streptomyces fradiae as a fermentation strain, the cultured gemma diplococcus is inoculated and then fermentation is continued.
2. The fermentation method of the tylosin mixture according to claim 1, wherein: the diplococcus is inoculated in fermentation culture for 90-100 hours.
3. The mixed tylosin fermentation process according to claim 1 or 2, wherein the inoculum size of the diplococcus is 0.05-0.1%.
4. The fermentation method of mixed tylosin according to claim 3, wherein said diplococcus sporogenes is prepared by: inoculating the diplococcus liquid into a sterilized shake flask culture medium, culturing for 10-14 hours at the temperature of 29-33 ℃ in a shaking table at 170-220 revolutions, and performing microscopic examination on the bacteria to form spores;
the shake flask culture medium consists of: 2.0-3.0% of glucose, and corn steep liquor: 0.1-0.2% of yeast extract, 0.2-0.3% of calcium carbonate and a proper amount of soybean oil.
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| CN112779306A (en) * | 2021-01-26 | 2021-05-11 | 浙江普洛生物科技有限公司 | Method for screening optimal proportion of calcium carbonate from fermentation liquor and application thereof |
Citations (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US20020012977A1 (en) * | 2000-05-31 | 2002-01-31 | Pacey Michael Stephen | Enzymatic oxidations |
| CN103074402A (en) * | 2013-02-05 | 2013-05-01 | 宁夏泰瑞制药股份有限公司 | Culture medium for producing tylosin through fermentation of streptomyces fradiae and fermentation method |
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| CN112779306B (en) * | 2021-01-26 | 2022-05-27 | 安徽普洛生物科技有限公司 | Method for screening optimal proportion of calcium carbonate from fermentation liquor and application thereof |
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