CN112391432A - Fermentation medium and fermentation method for producing pingyangmycin - Google Patents
Fermentation medium and fermentation method for producing pingyangmycin Download PDFInfo
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- 238000000855 fermentation Methods 0.000 title claims abstract description 102
- 230000004151 fermentation Effects 0.000 title claims abstract description 102
- 108700004675 bleomycetin Proteins 0.000 title claims abstract description 24
- QYOAUOAXCQAEMW-UTXKDXHTSA-N bleomycin A5 Chemical compound N([C@H](C(=O)N[C@H](C)[C@@H](O)[C@H](C)C(=O)N[C@@H]([C@H](O)C)C(=O)NCCC=1SC=C(N=1)C=1SC=C(N=1)C(=O)NCCCNCCCCN)[C@@H](O[C@H]1[C@H]([C@@H](O)[C@H](O)[C@H](CO)O1)O[C@@H]1[C@H]([C@@H](OC(N)=O)[C@H](O)[C@@H](CO)O1)O)C=1N=CNC=1)C(=O)C1=NC([C@H](CC(N)=O)NC[C@H](N)C(N)=O)=NC(N)=C1C QYOAUOAXCQAEMW-UTXKDXHTSA-N 0.000 title claims abstract description 24
- 238000004519 manufacturing process Methods 0.000 title abstract description 11
- 239000000843 powder Substances 0.000 claims abstract description 57
- 240000008042 Zea mays Species 0.000 claims abstract description 49
- 235000005824 Zea mays ssp. parviglumis Nutrition 0.000 claims abstract description 49
- 235000002017 Zea mays subsp mays Nutrition 0.000 claims abstract description 49
- 235000005822 corn Nutrition 0.000 claims abstract description 49
- IJGRMHOSHXDMSA-UHFFFAOYSA-N Atomic nitrogen Chemical compound N#N IJGRMHOSHXDMSA-UHFFFAOYSA-N 0.000 claims abstract description 42
- 244000068988 Glycine max Species 0.000 claims abstract description 28
- 235000010469 Glycine max Nutrition 0.000 claims abstract description 28
- 229910052757 nitrogen Inorganic materials 0.000 claims abstract description 21
- OKTJSMMVPCPJKN-UHFFFAOYSA-N Carbon Chemical compound [C] OKTJSMMVPCPJKN-UHFFFAOYSA-N 0.000 claims abstract description 17
- 229910052799 carbon Inorganic materials 0.000 claims abstract description 17
- 229910017053 inorganic salt Inorganic materials 0.000 claims abstract description 11
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 claims abstract description 3
- VWDWKYIASSYTQR-UHFFFAOYSA-N sodium nitrate Chemical compound [Na+].[O-][N+]([O-])=O VWDWKYIASSYTQR-UHFFFAOYSA-N 0.000 claims description 28
- 229920002472 Starch Polymers 0.000 claims description 27
- 239000008107 starch Substances 0.000 claims description 27
- 235000019698 starch Nutrition 0.000 claims description 27
- 240000004808 Saccharomyces cerevisiae Species 0.000 claims description 20
- 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 18
- 239000008103 glucose Substances 0.000 claims description 18
- 235000017060 Arachis glabrata Nutrition 0.000 claims description 17
- 244000105624 Arachis hypogaea Species 0.000 claims description 17
- 235000010777 Arachis hypogaea Nutrition 0.000 claims description 17
- 235000018262 Arachis monticola Nutrition 0.000 claims description 17
- 102000004139 alpha-Amylases Human genes 0.000 claims description 17
- 108090000637 alpha-Amylases Proteins 0.000 claims description 17
- 229940024171 alpha-amylase Drugs 0.000 claims description 17
- 235000020232 peanut Nutrition 0.000 claims description 17
- 239000007836 KH2PO4 Substances 0.000 claims description 15
- 229910000402 monopotassium phosphate Inorganic materials 0.000 claims description 15
- 235000012054 meals Nutrition 0.000 claims description 14
- GNSKLFRGEWLPPA-UHFFFAOYSA-M potassium dihydrogen phosphate Chemical compound [K+].OP(O)([O-])=O GNSKLFRGEWLPPA-UHFFFAOYSA-M 0.000 claims description 11
- 235000019764 Soybean Meal Nutrition 0.000 claims description 6
- 241000187747 Streptomyces Species 0.000 claims description 6
- 238000000034 method Methods 0.000 claims description 6
- 239000004455 soybean meal Substances 0.000 claims description 6
- 239000007788 liquid Substances 0.000 claims description 4
- 235000012343 cottonseed oil Nutrition 0.000 claims description 3
- 210000003746 feather Anatomy 0.000 claims description 3
- 102000004169 proteins and genes Human genes 0.000 claims description 3
- 108090000623 proteins and genes Proteins 0.000 claims description 3
- 150000003839 salts Chemical class 0.000 claims description 3
- 235000013312 flour Nutrition 0.000 claims 6
- 241001147844 Streptomyces verticillus Species 0.000 claims 2
- 239000004382 Amylase Substances 0.000 claims 1
- 102000013142 Amylases Human genes 0.000 claims 1
- 108010065511 Amylases Proteins 0.000 claims 1
- 235000019418 amylase Nutrition 0.000 claims 1
- JZCCFEFSEZPSOG-UHFFFAOYSA-L copper(II) sulfate pentahydrate Chemical compound O.O.O.O.O.[Cu+2].[O-]S([O-])(=O)=O JZCCFEFSEZPSOG-UHFFFAOYSA-L 0.000 claims 1
- 238000012262 fermentative production Methods 0.000 claims 1
- 235000019796 monopotassium phosphate Nutrition 0.000 claims 1
- 235000010344 sodium nitrate Nutrition 0.000 claims 1
- RZLVQBNCHSJZPX-UHFFFAOYSA-L zinc sulfate heptahydrate Chemical compound O.O.O.O.O.O.O.[Zn+2].[O-]S([O-])(=O)=O RZLVQBNCHSJZPX-UHFFFAOYSA-L 0.000 claims 1
- 239000001963 growth medium Substances 0.000 abstract description 34
- 239000002609 medium Substances 0.000 abstract description 26
- NWONKYPBYAMBJT-UHFFFAOYSA-L zinc sulfate Chemical compound [Zn+2].[O-]S([O-])(=O)=O NWONKYPBYAMBJT-UHFFFAOYSA-L 0.000 description 15
- 229910000368 zinc sulfate Inorganic materials 0.000 description 15
- 239000011686 zinc sulphate Substances 0.000 description 15
- 229910052927 chalcanthite Inorganic materials 0.000 description 12
- 239000007791 liquid phase Substances 0.000 description 11
- 238000002474 experimental method Methods 0.000 description 7
- OKKJLVBELUTLKV-UHFFFAOYSA-N Methanol Chemical compound OC OKKJLVBELUTLKV-UHFFFAOYSA-N 0.000 description 6
- 238000001514 detection method Methods 0.000 description 4
- ATHGHQPFGPMSJY-UHFFFAOYSA-N spermidine Chemical compound NCCCCNCCCN ATHGHQPFGPMSJY-UHFFFAOYSA-N 0.000 description 4
- QTBSBXVTEAMEQO-UHFFFAOYSA-N Acetic acid Chemical compound CC(O)=O QTBSBXVTEAMEQO-UHFFFAOYSA-N 0.000 description 3
- 244000294611 Punica granatum Species 0.000 description 3
- 235000014360 Punica granatum Nutrition 0.000 description 3
- 238000011081 inoculation Methods 0.000 description 3
- 239000012071 phase Substances 0.000 description 3
- 238000011218 seed culture Methods 0.000 description 3
- 239000006228 supernatant Substances 0.000 description 3
- QGZKDVFQNNGYKY-UHFFFAOYSA-N Ammonia Chemical compound N QGZKDVFQNNGYKY-UHFFFAOYSA-N 0.000 description 2
- 240000000716 Durio zibethinus Species 0.000 description 2
- 235000006025 Durio zibethinus Nutrition 0.000 description 2
- FAPWRFPIFSIZLT-UHFFFAOYSA-M Sodium chloride Chemical compound [Na+].[Cl-] FAPWRFPIFSIZLT-UHFFFAOYSA-M 0.000 description 2
- 239000003153 chemical reaction reagent Substances 0.000 description 2
- 229910000366 copper(II) sulfate Inorganic materials 0.000 description 2
- 230000000694 effects Effects 0.000 description 2
- 244000005700 microbiome Species 0.000 description 2
- 239000000203 mixture Substances 0.000 description 2
- 125000001477 organic nitrogen group Chemical group 0.000 description 2
- FGIUAXJPYTZDNR-UHFFFAOYSA-N potassium nitrate Chemical compound [K+].[O-][N+]([O-])=O FGIUAXJPYTZDNR-UHFFFAOYSA-N 0.000 description 2
- 239000002243 precursor Substances 0.000 description 2
- 229940063673 spermidine Drugs 0.000 description 2
- 230000001954 sterilising effect Effects 0.000 description 2
- 238000004659 sterilization and disinfection Methods 0.000 description 2
- 238000004809 thin layer chromatography Methods 0.000 description 2
- OWEGMIWEEQEYGQ-UHFFFAOYSA-N 100676-05-9 Natural products OC1C(O)C(O)C(CO)OC1OCC1C(O)C(O)C(O)C(OC2C(OC(O)C(O)C2O)CO)O1 OWEGMIWEEQEYGQ-UHFFFAOYSA-N 0.000 description 1
- WRROLJSGZKCWGR-UHFFFAOYSA-N 3-(4-aminobutylamino)propylazanium;chloride Chemical compound Cl.NCCCCNCCCN WRROLJSGZKCWGR-UHFFFAOYSA-N 0.000 description 1
- WSGYTJNNHPZFKR-UHFFFAOYSA-N 3-hydroxypropanenitrile Chemical group OCCC#N WSGYTJNNHPZFKR-UHFFFAOYSA-N 0.000 description 1
- 229920001817 Agar Polymers 0.000 description 1
- 208000019901 Anxiety disease Diseases 0.000 description 1
- 241000894006 Bacteria Species 0.000 description 1
- 108010006654 Bleomycin Proteins 0.000 description 1
- 241000931705 Cicada Species 0.000 description 1
- 239000003109 Disodium ethylene diamine tetraacetate Substances 0.000 description 1
- ZGTMUACCHSMWAC-UHFFFAOYSA-L EDTA disodium salt (anhydrous) Chemical compound [Na+].[Na+].OC(=O)CN(CC([O-])=O)CCN(CC(O)=O)CC([O-])=O ZGTMUACCHSMWAC-UHFFFAOYSA-L 0.000 description 1
- 229920002774 Maltodextrin Polymers 0.000 description 1
- 239000005913 Maltodextrin Substances 0.000 description 1
- GUBGYTABKSRVRQ-PICCSMPSSA-N Maltose Natural products O[C@@H]1[C@@H](O)[C@H](O)[C@@H](CO)O[C@@H]1O[C@@H]1[C@@H](CO)OC(O)[C@H](O)[C@H]1O GUBGYTABKSRVRQ-PICCSMPSSA-N 0.000 description 1
- 241000382353 Pupa Species 0.000 description 1
- 244000061458 Solanum melongena Species 0.000 description 1
- 235000002597 Solanum melongena Nutrition 0.000 description 1
- 241001052560 Thallis Species 0.000 description 1
- 244000273928 Zingiber officinale Species 0.000 description 1
- 235000006886 Zingiber officinale Nutrition 0.000 description 1
- CFOBMZGYYIVJKZ-UHFFFAOYSA-N [Na].CCCCCC Chemical compound [Na].CCCCCC CFOBMZGYYIVJKZ-UHFFFAOYSA-N 0.000 description 1
- 230000001464 adherent effect Effects 0.000 description 1
- 239000008272 agar Substances 0.000 description 1
- 229910021529 ammonia Inorganic materials 0.000 description 1
- 230000036506 anxiety Effects 0.000 description 1
- 230000001580 bacterial effect Effects 0.000 description 1
- 230000015572 biosynthetic process Effects 0.000 description 1
- 229960001561 bleomycin Drugs 0.000 description 1
- OYVAGSVQBOHSSS-UAPAGMARSA-O bleomycin A2 Chemical compound N([C@H](C(=O)N[C@H](C)[C@@H](O)[C@H](C)C(=O)N[C@@H]([C@H](O)C)C(=O)NCCC=1SC=C(N=1)C=1SC=C(N=1)C(=O)NCCC[S+](C)C)[C@@H](O[C@H]1[C@H]([C@@H](O)[C@H](O)[C@H](CO)O1)O[C@@H]1[C@H]([C@@H](OC(N)=O)[C@H](O)[C@@H](CO)O1)O)C=1N=CNC=1)C(=O)C1=NC([C@H](CC(N)=O)NC[C@H](N)C(N)=O)=NC(N)=C1C OYVAGSVQBOHSSS-UAPAGMARSA-O 0.000 description 1
- 238000007796 conventional method Methods 0.000 description 1
- ARUVKPQLZAKDPS-UHFFFAOYSA-L copper(II) sulfate Chemical compound [Cu+2].[O-][S+2]([O-])([O-])[O-] ARUVKPQLZAKDPS-UHFFFAOYSA-L 0.000 description 1
- 238000012136 culture method Methods 0.000 description 1
- 230000007547 defect Effects 0.000 description 1
- ZPWVASYFFYYZEW-UHFFFAOYSA-L dipotassium hydrogen phosphate Chemical compound [K+].[K+].OP([O-])([O-])=O ZPWVASYFFYYZEW-UHFFFAOYSA-L 0.000 description 1
- 229910000396 dipotassium phosphate Inorganic materials 0.000 description 1
- 235000019301 disodium ethylene diamine tetraacetate Nutrition 0.000 description 1
- 238000010828 elution Methods 0.000 description 1
- 229910052564 epsomite Inorganic materials 0.000 description 1
- 238000009472 formulation Methods 0.000 description 1
- 235000008397 ginger Nutrition 0.000 description 1
- 238000004128 high performance liquid chromatography Methods 0.000 description 1
- 239000012535 impurity Substances 0.000 description 1
- 238000009776 industrial production Methods 0.000 description 1
- 229940035034 maltodextrin Drugs 0.000 description 1
- 239000000463 material Substances 0.000 description 1
- 229910052603 melanterite Inorganic materials 0.000 description 1
- 230000002906 microbiologic effect Effects 0.000 description 1
- 238000012986 modification Methods 0.000 description 1
- 230000004048 modification Effects 0.000 description 1
- 239000000049 pigment Substances 0.000 description 1
- 230000001376 precipitating effect Effects 0.000 description 1
- 238000004321 preservation Methods 0.000 description 1
- 238000011160 research Methods 0.000 description 1
- 238000006748 scratching Methods 0.000 description 1
- 230000002393 scratching effect Effects 0.000 description 1
- 230000024053 secondary metabolic process Effects 0.000 description 1
- 239000011780 sodium chloride Substances 0.000 description 1
- UNFWWIHTNXNPBV-WXKVUWSESA-N spectinomycin Chemical compound O([C@@H]1[C@@H](NC)[C@@H](O)[C@H]([C@@H]([C@H]1O1)O)NC)[C@]2(O)[C@H]1O[C@H](C)CC2=O UNFWWIHTNXNPBV-WXKVUWSESA-N 0.000 description 1
- 229960000268 spectinomycin Drugs 0.000 description 1
- 239000007858 starting material Substances 0.000 description 1
- 238000011146 sterile filtration Methods 0.000 description 1
- 238000006467 substitution reaction Methods 0.000 description 1
- BDHFUVZGWQCTTF-UHFFFAOYSA-M sulfonate Chemical compound [O-]S(=O)=O BDHFUVZGWQCTTF-UHFFFAOYSA-M 0.000 description 1
- 238000012360 testing method Methods 0.000 description 1
- 210000003934 vacuole Anatomy 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
- C12P21/00—Preparation of peptides or proteins
- C12P21/005—Glycopeptides, glycoproteins
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- 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
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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
- C12P21/00—Preparation of peptides or proteins
- C12P21/02—Preparation of peptides or proteins having a known sequence of two or more amino acids, e.g. glutathione
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- General Engineering & Computer Science (AREA)
- General Health & Medical Sciences (AREA)
- Chemical Kinetics & Catalysis (AREA)
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- General Chemical & Material Sciences (AREA)
- Proteomics, Peptides & Aminoacids (AREA)
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- Tropical Medicine & Parasitology (AREA)
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- Micro-Organisms Or Cultivation Processes Thereof (AREA)
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Abstract
The invention discloses a fermentation medium and a fermentation method for producing pingyangmycin. The fermentation medium comprises a carbon source, a nitrogen source, inorganic salt and water, wherein the content of the nitrogen source is 4.5-5.9%, and the nitrogen source comprises soybean cake powder and corn steep liquor; the content of the corn steep liquor is more than 0.75 percent and less than 2.2 percent; the percentage is the mass percentage of the fermentation medium. The fermentation culture medium of the invention is used for fermentation of pingyangmycin, which can greatly improve the yield of pingyangmycin (the yield can reach 2.47 times of the yield obtained by using the fermentation culture medium in the prior art), and simultaneously, the fermentation cost is obviously reduced, thereby laying a good foundation for the industrialized production of pingyangmycin.
Description
Technical Field
The invention belongs to the technical field of industrial microorganisms, and particularly relates to a fermentation medium and a fermentation method for producing pingyangmycin.
Background
Influence of organic nitrogen sources on the modification of fermentation components of spectinomycin in Shanghai third pharmaceutical factory (Yu anxiety, longevity, plum blossom)]Microbiological reports, 1980,7(4):14-17.) in 1976 that the addition of corn steep liquor significantly increased the fermentation titer of pingyangmycin, which was then found to be related to the presence of spermidine in the corn steep liquor. The conditions influencing the fermentation of pingyangmycin are studied by a shaking test, and the fermentation conditions are 0.5 percent of glucose, 7 percent of maltose, 0.3 percent of NaCl0.3 percent of NaNO3 0.2％，KH2PO4 0.1％、ZnSO4·7H2O 0.05％、CuSO4The shake flask fermentation was carried out under the conditions of 0.01%, pH6.5, 150ml/750ml triangular flask, and the results indicated that feeding of corn steep liquor during the fermentation (1.5% of corn steep liquor fed at 48 hours from the beginning of the fermentation, and 1.5% fed every 24 hours thereafter) promoted the formation of component A5, making A5 the major component, increasing from 14.8% to 56.8%, regardless of the strain used. In addition, researches show that in experiments of proportioning other organic nitrogen sources in the basic culture medium, other formulas are unchanged, corn steep liquor is supplemented under the conditions of 2.5% of peanut cake powder and 2.5% of cicada pupa powder, and the yield can be improved by 3 times. The pingyangmycin detection method in the document uses thin-layer chromatography, and compared with the existing liquid phase detection method, the thin-layer chromatography method has low accuracy and larger interference, and the referential significance of the pingyangmycin fermentation titer is not large.
In precursor experiments, it was found by Gaoqian (Fujii, A et al [ J ] Antibot.27: 73,1979) et al that the addition of spermidine at the beginning of the fermentation resulted in 100% A5 of the bleomycin-producing strain, followed by sterile filtration of spermidine hydrochloride and addition of 0.36mg/ml to the fermentation medium before inoculation, resulting in a 466% increase in A5 over the control. And the main component is A5. However, the precursor used in the method is expensive and has high production cost, so that the production requirement cannot be met.
Disclosure of Invention
The invention aims to overcome the defects of high production cost, difficult expanded production and the like of fermentation of pingyangmycin in the prior art and provide a fermentation medium and a fermentation method for producing pingyangmycin.
The invention adopts the following technical scheme to solve the technical problems:
the invention provides a fermentation medium for producing pingyangmycin, which comprises a carbon source, a nitrogen source, inorganic salt and water, wherein the content of the nitrogen source is 4.5-5.9%, and the nitrogen source comprises soybean cake powder and corn steep liquor; the content of the corn steep liquor is more than 0.75 percent and less than 2.2 percent; the percentage is the mass percentage of the fermentation medium.
Preferably, the content of the corn steep liquor is 1.2-2.0%, such as 1.4%, 1.6% or 1.8%; the percentage is the mass percentage of the fermentation medium.
Wherein, the content of the soybean cake powder is preferably 1.1 to 3.5%, more preferably 1.7 to 2.9%, further more preferably 1.9 to 2.7%, such as 2.1%, 2.3% or 2.5%; the percentage is the mass percentage of the fermentation medium.
The soybean cake powder is preferably cold-pressed soybean cake powder.
The nitrogen source as described above preferably further comprises one or more selected from the group consisting of cottonseed meal, peanut meal, albumen powder, feather meal, bran, medium temperature soybean meal, yeast powder and hot-pressed soybean meal, preferably peanut meal and yeast powder.
The content of the peanut cake is preferably 0.6-2.4%, preferably 0.8-1.6%, such as 1.0%, 1.2% or 1.4%, and the percentage is the mass percentage of the fermentation medium.
Preferably, the total content of the peanut meal cake and the cold-pressed soybean cake powder is 3.5 percent, and the percentage is the mass percentage of the fermentation medium.
In the present invention, the content of the carbon source may be conventional in the art; preferably, the content of the carbon source is 3.8-9.8%, and the percentage is the mass percentage of the fermentation medium.
The carbon source preferably comprises glucose and starch, and the content of the glucose is 0.3-0.8%, preferably 0.5%; the content of the starch is 3.5-9%, preferably 6%, and the percentage is the mass percentage of the fermentation medium; the starch is preferably alpha-amylase liquefied starch or alpha-amylase gelatinized starch.
The inorganic salt described in the present invention preferably includes NaNO3、KH2PO4、ZnSO4·7H2O and CuSO4·5H2O; wherein:
the NaNO3The content of (C) is preferably 0.2%, and the KH is2PO4The content of (b) is preferably 0.1%, and the ZnSO4·7H2The content of O is preferably 0.05%, and the CuSO is4·5H2The content of O is preferably 0.01 percent, and the percentage is the mass percentage of the fermentation medium.
In a preferred embodiment of the invention, the fermentation medium comprises 2.2% of corn steep liquor, 2.1% of cold-pressed soybean cake powder, 0.2% of yeast powder, 1.4% of peanut cake powder, 0.5% of glucose, 6% of starch (alpha-amylase liquefaction), NaNO30.2％、KH2PO4 0.1％、ZnSO4·7H20.05% of O and CuSO4·5H20.01 percent of O, wherein the percentage is the mass percentage of the fermentation medium.
The invention provides an application of the fermentation medium in fermentation production of pingyangmycin.
The invention provides a fermentation method of pingyangmycin, which comprises the following steps:
(1) inoculating seed solution of Streptomyces pingyangensis (Streptomyces pingyangensis n.sp) into the fermentation culture medium, and performing liquid fermentation culture to obtain fermentation broth;
(2) separating the pingyangmycin from the fermentation liquor.
The Streptomyces verticillatus Pingyang variety in the step (1) is preferably the Streptomyces verticillatus Pingyang variety with the number of CPCC 200554.
On the basis of the common knowledge in the field, the above preferred conditions can be combined randomly to obtain the preferred embodiments of the invention.
The reagents and starting materials used in the present invention are commercially available.
The positive progress effects of the invention are as follows:
the fermentation culture medium of the invention is used for fermentation of pingyangmycin, which can greatly improve the yield of pingyangmycin, the titer can be improved from the original 13 mu g/ml to 32.11 mu g/ml (namely, the yield can reach 2.47 times of the yield obtained by using the fermentation culture medium in the prior art), and simultaneously, the fermentation cost is obviously reduced, and a good foundation is laid for the industrial production of pingyangmycin.
Detailed Description
The invention is further illustrated by the following examples, which are not intended to limit the scope of the invention. The experimental methods without specifying specific conditions in the following examples were selected according to the conventional methods and conditions, or according to the commercial instructions.
First, experimental material
1. Instrument for measuring the position of a moving object
2. Reagent
3. Bacterial strain
The invention adopts the strain Streptomyces pingyangensis n.sp purchased from China pharmaceutical microorganism strain preservation management center (CPCC) and the strain number is CPCC 200554.
4. Culture medium
A first culture medium: slant medium (g/L): soluble starch (20g), KNO3(1g)，K2HPO4(0.5g)，MgSO4·7H2O(0.5g)，NaCl(0.5g)，FeSO4·7H2O (0.01g), corn steep liquor (0.5g), agar 20g, and pH of 7.4-7.6
And (2) culture medium II: seed fermentation culture medium: 3% of corn steep liquor, 2% of soybean cake powder, 0.2% of pomegranate flower yeast powder, 1% of glucose, 1% of maltodextrin and KH2PO4 0.1％，ZnSO4·7H2O 0.05％；CuSO4·5H2O 0.01％；pH 6.5。
And (3) culture medium III: basic fermentation medium: 0.75% of corn steep liquor, 3.5% of cold-pressed soybean cake powder, 0.2% of durian yeast powder, 0.5% of glucose, 6% of starch (alpha-amylase liquefaction), and NaNO3 0.2％；KH2PO40.1％,ZnSO4·7H2O 0.05％；CuSO4·5H2O 0.01％；pH 6.5。
Second, the method
1. Culture method
The invention comprises the following processes:
and (3) sterilization conditions: 121 ℃ for 20min
Under the aseptic condition, a freeze-dried tube in which the altemaria verticillata sun-exposed variety is preserved is inoculated into an eggplant bottle filled with a slant culture medium by using a sterilized inoculating needle for culture, the surface light white thalli can be seen on the next day of slant culture, the color is deepened on the third day or the fourth day, a small amount of white spores are seen to be generated, the culture is continued until a large amount of white spores are seen to be generated on the surface of the culture medium, and the culture medium body is changed into ginger yellow due to the generation of the altemaria verticillata sun-exposed variety pigment. The slant can be used for passage or inoculation.
Preparing a seed culture medium according to a seed culture medium formula, slightly scratching a slant culture medium with spores by using a sterilization inoculating shovel under an aseptic condition, digging a slant culture medium which is as thin as possible and is full of spores and about 0.8cm multiplied by 1.5cm, inoculating the slant culture medium into the killed seed culture medium, shaking at 29 ℃ for 24-28 hours at 240rpm, changing the color of a culture in a shaking bottle from dark brown to yellow turbid, shaking, having a flowing sense, having high viscosity and uniformly sliding down adherent walls. The hyphae are clustered and spread to the periphery, are long and free of foreign bacteria, and have the pH value of 6.9-7.0.
Inoculating the cultured shake flask seeds into a sterilized fermentation medium, wherein the inoculation amount is 3ml/35ml, the shaking table is 29 ℃, the rotation speed is 240rpm, and the days are 8-9. The fermentation shake flask becomes thick and deepens color in about 24 hours, a layer of white spore layer appears on the upper wall of the shake flask in 3-4 days, the viscosity of the shake flask is not reduced in 8-9 days, microscopic examination shows that hyphae are broken more, dense vacuoles are generated in the hyphae, the hyphae are thin and weak, and the condition that the hyphae enter the later stage of secondary metabolism and the pH value is 7.0-8.0 is shown.
In the invention, the conditions except the nitrogen source in the three formulas of the culture medium are not changed, and multiple shake flask fermentation experiments are carried out by increasing the types of the nitrogen source and adjusting the proportion and the total amount of the corresponding nitrogen source, so that the fermentation culture medium formula with higher yield is finally obtained.
2. Detection method
Centrifuging the fermentation liquor at 12000rpm, taking the supernatant, adding methanol for precipitating protein impurities according to the ratio of the supernatant to the methanol being 1:3, centrifuging at 12000rpm, taking the supernatant, and measuring the fermentation unit by using a high performance liquid chromatography.
1) Mobile phase: mobile phase a ═ hexane sodium sulfonate 7.53g/L, disodium EDTA 3.72g/L, acetic acid 0.08mol/L, pH adjusted to 4.3 with pure ammonia, mobile phase B ═ methanol-acetonitrile (70-30)
2) Liquid phase conditions: a chromatographic column: agilent Eclipse Plus C18(4.6 mm. times.250 mm, 5 μm);
the flow rate is 1.0 mL/min; injecting 20 mu L of sample; the detection wavelength is 254 nm; the column temperature is 40 ℃; gradient elution.
Unless otherwise specified, "%" referred to in the following examples is a mass percentage of a specific component based on the total mass of the medium.
The starch (alpha-amylase liquefaction) in the following examples can be replaced by alpha-amylase gelatinized starch, and the effect of the culture medium containing the starch and the starch is basically the same under the condition that other components are kept unchanged.
Example 1
Taking the basic fermentation medium (the third culture medium) as a reference, keeping other nitrogen sources unchanged under the condition that components such as a carbon source, inorganic salts and the like are kept unchanged, exploring the using amount of corn steep liquor, adjusting the using amount of the corn steep liquor from 0.75% to 1%, performing shake flask fermentation for 8 days, and measuring a fermentation unit by adopting a high performance liquid phase.
TABLE 1
| Culture medium III | Example 1 | |
| Corn steep liquor dosage (%) | 0.75 | 1 |
| Relative potency | 1.00 | 1.04 |
The optimized formula comprises: 1% of corn steep liquor, 3.5% of cold-pressed soybean cake powder, 0.2% of yeast powder, 0.5% of glucose, 6% of starch (alpha-amylase liquefaction), and NaNO3 0.2％、KH2PO4 0.1％、ZnSO4·7H20.05% of O and CuSO4·5H2O 0.01％。
The titer obtained by fermentation using the above medium III was 13. mu.g/ml.
Example 2
Taking a basic culture medium formula as a control, keeping other nitrogen sources unchanged under the condition that components such as a carbon source, inorganic salt and the like are kept unchanged, exploring the using amount of corn steep liquor, adjusting the using amount of the corn steep liquor from 1% to 1.2%, performing shake flask fermentation for 8 days, and measuring a fermentation unit by adopting a high performance liquid phase.
TABLE 2
| Corn steep liquor dosage (%) | 1 | 1.2 |
| Relative potency | 1.00 | 1.12 |
The optimized formula comprises: 1.2% of corn steep liquor, 3.5% of cold-pressed soybean cake powder, 0.2% of yeast powder, 0.5% of glucose, 6% of starch (alpha-amylase liquefaction), and NaNO3 0.2％；KH2PO4 0.1％、ZnSO4·7H2O 0.05％；CuSO4·5H2O 0.01％。
Example 3
Taking a basic culture medium formula as a control, keeping other nitrogen sources unchanged under the condition that components such as a carbon source, inorganic salt and the like are kept unchanged, exploring the using amount of corn steep liquor, adjusting the using amount of the corn steep liquor from 1.2% to 1.4%, performing shake flask fermentation for 8 days, and measuring a fermentation unit by adopting a high performance liquid phase.
TABLE 3
| Corn steep liquor dosage (%) | 1.2 | 1.4 |
| Relative potency | 1 | 1.09 |
The optimized formula comprises: 1.4% of corn steep liquor, 3.5% of cold-pressed soybean cake powder, 0.2% of yeast powder, 0.5% of glucose, 6% of starch (alpha-amylase liquefaction), and NaNO3 0.2％、KH2PO4 0.1％、ZnSO4·7H2O 0.05％；CuSO4·5H2O 0.01％。
Example 4
Taking a basic culture medium formula as a control, keeping other nitrogen sources unchanged under the condition that components such as a carbon source, inorganic salt and the like are kept unchanged, exploring the using amount of corn steep liquor, adjusting the using amount of the corn steep liquor from 1.4% to 1.6%, carrying out shake flask fermentation for 8 days, and measuring a fermentation unit by adopting a high performance liquid phase.
TABLE 4
| Corn steep liquor dosage (%) | 1.4 | 1.6 |
| Relative potency | 1 | 1.18 |
The optimized formula comprises: 1.6% of corn steep liquor, 3.5% of cold-pressed soybean cake powder, 0.2% of yeast powder, 0.5% of glucose, 6% of starch (alpha-amylase liquefaction), and NaNO3 0.2％；KH2PO4 0.1％、ZnSO4·7H2O 0.05％；CuSO4·5H2O 0.01％。
Example 5
Taking a basic culture medium formula as a control, keeping other nitrogen sources unchanged under the condition that components such as a carbon source, inorganic salt and the like are kept unchanged, exploring the using amount of corn steep liquor, adjusting the using amount of the corn steep liquor from 1.6% to 1.8%, carrying out shake flask fermentation for 8 days, and measuring a fermentation unit by adopting a high performance liquid phase.
TABLE 5
| Corn steep liquor dosage (%) | 1.6 | 1.8 |
| Relative potency | 1 | 1.12 |
The optimized formula comprises: 1.8% of corn steep liquor, 3.5% of cold-pressed soybean cake powder, 0.2% of yeast powder, 0.5% of glucose, 6% of starch (alpha-amylase liquefaction), and NaNO3 0.2％；KH2PO40.1％、ZnSO4·7H2O 0.05％、CuSO4·5H2O 0.01％。
Example 6
Taking a basic culture medium formula as a control, keeping other nitrogen sources unchanged under the condition that components such as a carbon source, inorganic salt and the like are kept unchanged, exploring the using amount of corn steep liquor, adjusting the using amount of the corn steep liquor from 1.8% to 2.0%, performing shake flask fermentation for 8 days, and measuring a fermentation unit by adopting a high performance liquid phase.
TABLE 6
| Corn steep liquor dosage (%) | 1.8 | 2.0 |
| Relative potency | 1 | 1.17 |
The optimized formula comprises: 2.0% of corn steep liquor, 3.5% of cold-pressed soybean cake powder, 0.2% of yeast powder, 0.5% of glucose, 6% of starch (alpha-amylase liquefaction), and NaNO3 0.2％、KH2PO4 0.1％、ZnSO4·7H2O 0.05％、CuSO4·5H2O 0.01％。
Example 7
Taking a basic culture medium formula as a control, keeping other nitrogen sources unchanged under the condition that components such as a carbon source, inorganic salt and the like are kept unchanged, exploring the using amount of corn steep liquor, adjusting the using amount of the corn steep liquor from 2.0% to 2.2%, performing shake flask fermentation for 8 days, and measuring a fermentation unit by adopting a high performance liquid phase.
TABLE 7
| Corn steep liquor dosage (%) | 2.0 | 2.2 |
| Relative potency | 1 | 1.13 |
The optimized formula comprises: 2.2% of corn steep liquor, 3.5% of cold-pressed soybean cake powder, 0.2% of yeast powder, 0.5% of glucose, 6% of starch (alpha-amylase liquefaction), and NaNO3 0.2％；KH2PO4 0.1％、ZnSO4·7H2O 0.05％；CuSO4·5H2O 0.01％。
Example 8
Taking the optimized culture medium formula as a reference, under the condition that components such as a carbon source, inorganic salts and the like are kept unchanged, under the condition that the contents of corn steep liquor and the pomegranate flower yeast powder are unchanged, the content of cold-pressed soybean cake powder in the basic culture medium is changed from 3.5 percent to 2.5 percent, the rest 1 percent of cold-pressed soybean cake powder is subjected to substitution experiments by adopting cottonseed powder, peanut cake powder, pomegranate flower yeast powder, protein powder, feather meal, bran, medium-temperature soybean cake powder and hot-pressed soybean cake powder, and the mixture is subjected to shake flask fermentation for 8 days and a high-efficiency liquid phase fermentation unit is adopted.
TABLE 8
The optimized formula comprises: 2.2% of corn steep liquor, 2.5% of cold-pressed soybean cake powder, 0.2% of yeast powder, 1% of peanut cake powder, 0.5% of glucose, 6% of starch (alpha-amylase liquefaction), and NaNO3 0.2％、KH2PO40.1％、ZnSO4·7H2O 0.05％；CuSO4·5H2O 0.01％。
Example 9
Taking the optimized culture medium formula as a reference, performing an experiment for adjusting the content ratio of the cold-pressed soybean cake powder to the peanut cake powder under the condition that the total content of the cold-pressed soybean cake powder and the peanut cake powder is 3.5% under the condition that the components such as carbon source, inorganic salt, durian yeast powder and the like are kept unchanged, performing shake-flask fermentation for 8 days, and measuring a fermentation unit by using a high performance liquid phase.
TABLE 9
| Peanut cake powder (%) | 0.6 | 0.8 | 1.0 | 1.2 | 1.4 | 1.6 | 1.8 | 2.0 | 2.2 | 2.4 |
| Relative potency | 0.97 | 0.98 | 1.00 | 1.01 | 1.03 | 0.98 | 0.95 | 0.92 | 0.86 | 0.78 |
According to the results of the above experiments, the optimized formula is as follows: 2.2% of corn steep liquor, 2.1% of cold-pressed soybean cake powder, 0.2% of yeast powder, 1.4% of peanut cake powder, 0.5% of glucose, 6% of starch (alpha-amylase liquefaction), and NaNO3 0.2％、KH2PO40.1％、ZnSO4·7H2O 0.05％、CuSO4·5H2O 0.01％。
The titer obtained after the final fermentation using this optimized formulation was 32.11. mu.g/ml.
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