CN117265035A - Method for improving titer of monensin fermentation liquor and content of monensin A - Google Patents
Method for improving titer of monensin fermentation liquor and content of monensin A Download PDFInfo
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- CN117265035A CN117265035A CN202311207921.0A CN202311207921A CN117265035A CN 117265035 A CN117265035 A CN 117265035A CN 202311207921 A CN202311207921 A CN 202311207921A CN 117265035 A CN117265035 A CN 117265035A
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- 238000000855 fermentation Methods 0.000 title claims abstract description 188
- 230000004151 fermentation Effects 0.000 title claims abstract description 188
- GAOZTHIDHYLHMS-UHFFFAOYSA-N Monensin A Natural products O1C(CC)(C2C(CC(O2)C2C(CC(C)C(O)(CO)O2)C)C)CCC1C(O1)(C)CCC21CC(O)C(C)C(C(C)C(OC)C(C)C(O)=O)O2 GAOZTHIDHYLHMS-UHFFFAOYSA-N 0.000 title claims abstract description 70
- GAOZTHIDHYLHMS-KEOBGNEYSA-N monensin A Chemical compound C([C@@](O1)(C)[C@H]2CC[C@@](O2)(CC)[C@H]2[C@H](C[C@@H](O2)[C@@H]2[C@H](C[C@@H](C)[C@](O)(CO)O2)C)C)C[C@@]21C[C@H](O)[C@@H](C)[C@@H]([C@@H](C)[C@@H](OC)[C@H](C)C(O)=O)O2 GAOZTHIDHYLHMS-KEOBGNEYSA-N 0.000 title claims abstract description 70
- 229930191564 Monensin Natural products 0.000 title claims abstract description 55
- 229960005358 monensin Drugs 0.000 title claims abstract description 55
- 238000000034 method Methods 0.000 title claims abstract description 32
- 239000004183 Monensin A Substances 0.000 title claims abstract description 15
- 235000019376 monensin A Nutrition 0.000 title claims abstract description 15
- 235000014113 dietary fatty acids Nutrition 0.000 claims abstract description 64
- 239000000194 fatty acid Substances 0.000 claims abstract description 64
- 229930195729 fatty acid Natural products 0.000 claims abstract description 64
- 150000004665 fatty acids Chemical class 0.000 claims abstract description 62
- IPCSVZSSVZVIGE-UHFFFAOYSA-N hexadecanoic acid Chemical compound CCCCCCCCCCCCCCCC(O)=O IPCSVZSSVZVIGE-UHFFFAOYSA-N 0.000 claims abstract description 52
- WRIDQFICGBMAFQ-UHFFFAOYSA-N (E)-8-Octadecenoic acid Natural products CCCCCCCCCC=CCCCCCCC(O)=O WRIDQFICGBMAFQ-UHFFFAOYSA-N 0.000 claims abstract description 31
- LQJBNNIYVWPHFW-UHFFFAOYSA-N 20:1omega9c fatty acid Natural products CCCCCCCCCCC=CCCCCCCCC(O)=O LQJBNNIYVWPHFW-UHFFFAOYSA-N 0.000 claims abstract description 31
- QSBYPNXLFMSGKH-UHFFFAOYSA-N 9-Heptadecensaeure Natural products CCCCCCCC=CCCCCCCCC(O)=O QSBYPNXLFMSGKH-UHFFFAOYSA-N 0.000 claims abstract description 31
- 239000005642 Oleic acid Substances 0.000 claims abstract description 31
- ZQPPMHVWECSIRJ-UHFFFAOYSA-N Oleic acid Natural products CCCCCCCCC=CCCCCCCCC(O)=O ZQPPMHVWECSIRJ-UHFFFAOYSA-N 0.000 claims abstract description 31
- QXJSBBXBKPUZAA-UHFFFAOYSA-N isooleic acid Natural products CCCCCCCC=CCCCCCCCCC(O)=O QXJSBBXBKPUZAA-UHFFFAOYSA-N 0.000 claims abstract description 31
- ZQPPMHVWECSIRJ-KTKRTIGZSA-N oleic acid Chemical compound CCCCCCCC\C=C/CCCCCCCC(O)=O ZQPPMHVWECSIRJ-KTKRTIGZSA-N 0.000 claims abstract description 31
- 235000021355 Stearic acid Nutrition 0.000 claims abstract description 30
- QIQXTHQIDYTFRH-UHFFFAOYSA-N octadecanoic acid Chemical compound CCCCCCCCCCCCCCCCCC(O)=O QIQXTHQIDYTFRH-UHFFFAOYSA-N 0.000 claims abstract description 30
- OQCDKBAXFALNLD-UHFFFAOYSA-N octadecanoic acid Natural products CCCCCCCC(C)CCCCCCCCC(O)=O OQCDKBAXFALNLD-UHFFFAOYSA-N 0.000 claims abstract description 30
- 239000008117 stearic acid Substances 0.000 claims abstract description 30
- 235000021314 Palmitic acid Nutrition 0.000 claims abstract description 26
- WQEPLUUGTLDZJY-UHFFFAOYSA-N n-Pentadecanoic acid Natural products CCCCCCCCCCCCCCC(O)=O WQEPLUUGTLDZJY-UHFFFAOYSA-N 0.000 claims abstract description 26
- 241000187747 Streptomyces Species 0.000 claims abstract description 13
- OYHQOLUKZRVURQ-NTGFUMLPSA-N (9Z,12Z)-9,10,12,13-tetratritiooctadeca-9,12-dienoic acid Chemical compound C(CCCCCCC\C(=C(/C\C(=C(/CCCCC)\[3H])\[3H])\[3H])\[3H])(=O)O OYHQOLUKZRVURQ-NTGFUMLPSA-N 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 description 20
- 229910000019 calcium carbonate Inorganic materials 0.000 claims description 10
- 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 8
- 239000008103 glucose Substances 0.000 claims description 8
- ZPWVASYFFYYZEW-UHFFFAOYSA-L dipotassium hydrogen phosphate Chemical compound [K+].[K+].OP([O-])([O-])=O ZPWVASYFFYYZEW-UHFFFAOYSA-L 0.000 claims description 7
- SURQXAFEQWPFPV-UHFFFAOYSA-L iron(2+) sulfate heptahydrate Chemical compound O.O.O.O.O.O.O.[Fe+2].[O-]S([O-])(=O)=O SURQXAFEQWPFPV-UHFFFAOYSA-L 0.000 claims description 7
- DIZPMCHEQGEION-UHFFFAOYSA-H aluminium sulfate (anhydrous) Chemical compound [Al+3].[Al+3].[O-]S([O-])(=O)=O.[O-]S([O-])(=O)=O.[O-]S([O-])(=O)=O DIZPMCHEQGEION-UHFFFAOYSA-H 0.000 claims description 6
- PMZURENOXWZQFD-UHFFFAOYSA-L Sodium Sulfate Chemical compound [Na+].[Na+].[O-]S([O-])(=O)=O PMZURENOXWZQFD-UHFFFAOYSA-L 0.000 claims description 4
- 238000003756 stirring Methods 0.000 claims description 4
- 238000009776 industrial production Methods 0.000 claims description 3
- 238000005273 aeration Methods 0.000 claims 1
- 235000013312 flour Nutrition 0.000 claims 1
- 239000007788 liquid Substances 0.000 abstract description 14
- 238000004519 manufacturing process Methods 0.000 abstract description 6
- 230000008901 benefit Effects 0.000 abstract description 3
- 239000002609 medium Substances 0.000 description 92
- 239000001963 growth medium Substances 0.000 description 24
- 238000002474 experimental method Methods 0.000 description 21
- OYHQOLUKZRVURQ-HZJYTTRNSA-N Linoleic acid Chemical compound CCCCC\C=C/C\C=C/CCCCCCCC(O)=O OYHQOLUKZRVURQ-HZJYTTRNSA-N 0.000 description 20
- 235000020778 linoleic acid Nutrition 0.000 description 20
- OYHQOLUKZRVURQ-IXWMQOLASA-N linoleic acid Natural products CCCCC\C=C/C\C=C\CCCCCCCC(O)=O OYHQOLUKZRVURQ-IXWMQOLASA-N 0.000 description 20
- 239000000843 powder Substances 0.000 description 17
- 238000012360 testing method Methods 0.000 description 16
- 238000011218 seed culture Methods 0.000 description 15
- 235000010469 Glycine max Nutrition 0.000 description 14
- 238000007792 addition Methods 0.000 description 14
- 244000068988 Glycine max Species 0.000 description 13
- HEMHJVSKTPXQMS-UHFFFAOYSA-M Sodium hydroxide Chemical compound [OH-].[Na+] HEMHJVSKTPXQMS-UHFFFAOYSA-M 0.000 description 12
- 239000003549 soybean oil Substances 0.000 description 12
- 235000012424 soybean oil Nutrition 0.000 description 12
- 235000010633 broth Nutrition 0.000 description 11
- QVGXLLKOCUKJST-UHFFFAOYSA-N atomic oxygen Chemical compound [O] QVGXLLKOCUKJST-UHFFFAOYSA-N 0.000 description 8
- 229910052760 oxygen Inorganic materials 0.000 description 8
- 239000001301 oxygen Substances 0.000 description 8
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 description 6
- 230000009194 climbing Effects 0.000 description 5
- 238000013461 design Methods 0.000 description 5
- 230000000694 effects Effects 0.000 description 5
- 239000002054 inoculum Substances 0.000 description 5
- 235000021313 oleic acid Nutrition 0.000 description 5
- 238000005457 optimization Methods 0.000 description 5
- 230000004044 response Effects 0.000 description 5
- 229920001353 Dextrin Polymers 0.000 description 4
- 239000004375 Dextrin Substances 0.000 description 4
- 240000004808 Saccharomyces cerevisiae Species 0.000 description 4
- 230000004913 activation Effects 0.000 description 4
- 230000000052 comparative effect Effects 0.000 description 4
- 238000012258 culturing Methods 0.000 description 4
- 235000019425 dextrin Nutrition 0.000 description 4
- 238000002360 preparation method Methods 0.000 description 4
- 235000015112 vegetable and seed oil Nutrition 0.000 description 4
- 239000008158 vegetable oil Substances 0.000 description 4
- 238000009423 ventilation Methods 0.000 description 4
- VHUUQVKOLVNVRT-UHFFFAOYSA-N Ammonium hydroxide Chemical compound [NH4+].[OH-] VHUUQVKOLVNVRT-UHFFFAOYSA-N 0.000 description 3
- 241001465754 Metazoa Species 0.000 description 3
- 235000011114 ammonium hydroxide Nutrition 0.000 description 3
- 238000000540 analysis of variance Methods 0.000 description 3
- 230000002596 correlated effect Effects 0.000 description 3
- 239000000203 mixture Substances 0.000 description 3
- OKTJSMMVPCPJKN-UHFFFAOYSA-N Carbon Chemical compound [C] OKTJSMMVPCPJKN-UHFFFAOYSA-N 0.000 description 2
- 241000723347 Cinnamomum Species 0.000 description 2
- 235000019483 Peanut oil Nutrition 0.000 description 2
- XUIMIQQOPSSXEZ-UHFFFAOYSA-N Silicon Chemical compound [Si] XUIMIQQOPSSXEZ-UHFFFAOYSA-N 0.000 description 2
- FAPWRFPIFSIZLT-UHFFFAOYSA-M Sodium chloride Chemical compound [Na+].[Cl-] FAPWRFPIFSIZLT-UHFFFAOYSA-M 0.000 description 2
- 229920002472 Starch Polymers 0.000 description 2
- 241000187434 Streptomyces cinnamonensis Species 0.000 description 2
- WQZGKKKJIJFFOK-DVKNGEFBSA-N alpha-D-glucose Chemical compound OC[C@H]1O[C@H](O)[C@H](O)[C@@H](O)[C@@H]1O WQZGKKKJIJFFOK-DVKNGEFBSA-N 0.000 description 2
- 239000010495 camellia oil Substances 0.000 description 2
- 229910052799 carbon Inorganic materials 0.000 description 2
- 210000004027 cell Anatomy 0.000 description 2
- 210000000170 cell membrane Anatomy 0.000 description 2
- 230000008859 change Effects 0.000 description 2
- -1 compound fatty acid Chemical class 0.000 description 2
- 235000005687 corn oil Nutrition 0.000 description 2
- 239000002285 corn oil Substances 0.000 description 2
- 230000003247 decreasing effect Effects 0.000 description 2
- 239000013530 defoamer Substances 0.000 description 2
- 239000003651 drinking water Substances 0.000 description 2
- 235000020188 drinking water Nutrition 0.000 description 2
- 125000005313 fatty acid group Chemical group 0.000 description 2
- 238000011081 inoculation Methods 0.000 description 2
- JVTAAEKCZFNVCJ-UHFFFAOYSA-N lactic acid Chemical compound CC(O)C(O)=O JVTAAEKCZFNVCJ-UHFFFAOYSA-N 0.000 description 2
- 239000000944 linseed oil Substances 0.000 description 2
- 235000021388 linseed oil Nutrition 0.000 description 2
- 239000000463 material Substances 0.000 description 2
- 230000004060 metabolic process Effects 0.000 description 2
- 239000004006 olive oil Substances 0.000 description 2
- 235000008390 olive oil Nutrition 0.000 description 2
- 239000000312 peanut oil Substances 0.000 description 2
- FGIUAXJPYTZDNR-UHFFFAOYSA-N potassium nitrate Chemical compound [K+].[O-][N+]([O-])=O FGIUAXJPYTZDNR-UHFFFAOYSA-N 0.000 description 2
- 230000008569 process Effects 0.000 description 2
- 230000009467 reduction Effects 0.000 description 2
- 238000000611 regression analysis Methods 0.000 description 2
- 230000001105 regulatory effect Effects 0.000 description 2
- 229910052710 silicon Inorganic materials 0.000 description 2
- 239000010703 silicon Substances 0.000 description 2
- 229910052938 sodium sulfate Inorganic materials 0.000 description 2
- 235000011152 sodium sulphate Nutrition 0.000 description 2
- 241000894007 species Species 0.000 description 2
- 239000008107 starch Substances 0.000 description 2
- 235000019698 starch Nutrition 0.000 description 2
- 239000008223 sterile water Substances 0.000 description 2
- 230000001502 supplementing effect Effects 0.000 description 2
- 229920001817 Agar Polymers 0.000 description 1
- 239000002028 Biomass Substances 0.000 description 1
- XUJGQWFDGVVLSN-OPKOWMNOSA-N CC[C@]1(CC[C@@H](O1)[C@]1(C)CC[C@]2(C[C@H](O)[C@@H](C)[C@H](O2)C(C)C(CCCC(O)=O)OC)O1)[C@@H]1O[C@H](C[C@@H]1C)[C@H]1O[C@@](O)(CO)[C@H](C)C[C@@H]1C Chemical compound CC[C@]1(CC[C@@H](O1)[C@]1(C)CC[C@]2(C[C@H](O)[C@@H](C)[C@H](O2)C(C)C(CCCC(O)=O)OC)O1)[C@@H]1O[C@H](C[C@@H]1C)[C@H]1O[C@@](O)(CO)[C@H](C)C[C@@H]1C XUJGQWFDGVVLSN-OPKOWMNOSA-N 0.000 description 1
- ZXLUKLZKZXJEFX-WALYMESLSA-N CO[C@H]([C@H](C)[C@H]1O[C@@]2(CC[C@](C)(O2)[C@H]2CC[C@](C)(O2)[C@@H]2O[C@H](C[C@@H]2C)[C@H]2O[C@@](O)(CO)[C@H](C)C[C@@H]2C)C[C@H](O)[C@H]1C)[C@H](C)C(O)=O Chemical compound CO[C@H]([C@H](C)[C@H]1O[C@@]2(CC[C@](C)(O2)[C@H]2CC[C@](C)(O2)[C@@H]2O[C@H](C[C@@H]2C)[C@H]2O[C@@](O)(CO)[C@H](C)C[C@@H]2C)C[C@H](O)[C@H]1C)[C@H](C)C(O)=O ZXLUKLZKZXJEFX-WALYMESLSA-N 0.000 description 1
- 206010059866 Drug resistance Diseases 0.000 description 1
- ZXLUKLZKZXJEFX-UHFFFAOYSA-N Monensin B Natural products C1C(O)C(C)C(C(C)C(OC)C(C)C(O)=O)OC11OC(C)(C2OC(C)(CC2)C2C(CC(O2)C2C(CC(C)C(O)(CO)O2)C)C)CC1 ZXLUKLZKZXJEFX-UHFFFAOYSA-N 0.000 description 1
- CXDSHTJJHWKNRY-UHFFFAOYSA-N Monensin C Natural products CCC(C(OC)C(C)C1OC2(CCC(C)(O2)C3CCC(CC)(O3)C4OC(CC4C)C5OC(O)(CO)C(C)CC5C)CC(O)C1C)C(=O)O CXDSHTJJHWKNRY-UHFFFAOYSA-N 0.000 description 1
- 230000002378 acidificating effect Effects 0.000 description 1
- 230000006978 adaptation Effects 0.000 description 1
- 239000008272 agar Substances 0.000 description 1
- 230000001165 anti-coccidial effect Effects 0.000 description 1
- 230000009286 beneficial effect Effects 0.000 description 1
- 230000003115 biocidal effect Effects 0.000 description 1
- 150000001768 cations Chemical class 0.000 description 1
- 230000010261 cell growth Effects 0.000 description 1
- 239000003153 chemical reaction reagent Substances 0.000 description 1
- 239000003795 chemical substances by application Substances 0.000 description 1
- 238000007796 conventional method Methods 0.000 description 1
- 238000001816 cooling Methods 0.000 description 1
- 230000001419 dependent effect Effects 0.000 description 1
- 235000005911 diet Nutrition 0.000 description 1
- 230000037213 diet Effects 0.000 description 1
- 235000019621 digestibility Nutrition 0.000 description 1
- 239000006185 dispersion Substances 0.000 description 1
- 238000013401 experimental design Methods 0.000 description 1
- 230000002349 favourable effect Effects 0.000 description 1
- 235000013373 food additive Nutrition 0.000 description 1
- 239000002778 food additive Substances 0.000 description 1
- 230000003993 interaction Effects 0.000 description 1
- 239000004310 lactic acid Substances 0.000 description 1
- 235000014655 lactic acid Nutrition 0.000 description 1
- 208000006443 lactic acidosis Diseases 0.000 description 1
- WRUGWIBCXHJTDG-UHFFFAOYSA-L magnesium sulfate heptahydrate Chemical compound O.O.O.O.O.O.O.[Mg+2].[O-]S([O-])(=O)=O WRUGWIBCXHJTDG-UHFFFAOYSA-L 0.000 description 1
- 229940061634 magnesium sulfate heptahydrate Drugs 0.000 description 1
- 230000002503 metabolic effect Effects 0.000 description 1
- 238000012986 modification Methods 0.000 description 1
- 230000004048 modification Effects 0.000 description 1
- 230000003204 osmotic effect Effects 0.000 description 1
- 230000036284 oxygen consumption Effects 0.000 description 1
- 244000052769 pathogen Species 0.000 description 1
- 239000002460 polyether antibiotic agent Substances 0.000 description 1
- 235000010333 potassium nitrate Nutrition 0.000 description 1
- 239000004323 potassium nitrate Substances 0.000 description 1
- 230000000630 rising effect Effects 0.000 description 1
- 238000012216 screening Methods 0.000 description 1
- 229930000044 secondary metabolite Natural products 0.000 description 1
- 238000012807 shake-flask culturing Methods 0.000 description 1
- 238000004088 simulation Methods 0.000 description 1
- 239000011734 sodium Substances 0.000 description 1
- 239000011780 sodium chloride Substances 0.000 description 1
- 238000006467 substitution reaction Methods 0.000 description 1
- 238000005303 weighing Methods 0.000 description 1
Classifications
-
- 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
- C12P17/00—Preparation of heterocyclic carbon compounds with only O, N, S, Se or Te as ring hetero atoms
- C12P17/18—Preparation of heterocyclic carbon compounds with only O, N, S, Se or Te as ring hetero atoms containing at least two hetero rings condensed among themselves or condensed with a common carbocyclic ring system, e.g. rifamycin
- C12P17/181—Heterocyclic compounds containing oxygen atoms as the only ring heteroatoms in the condensed system, e.g. Salinomycin, Septamycin
-
- 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
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- 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
- C12N1/38—Chemical stimulation of growth or activity by addition of chemical compounds which are not essential growth factors; Stimulation of growth by removal of a chemical compound
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12R—INDEXING SCHEME ASSOCIATED WITH SUBCLASSES C12C - C12Q, RELATING TO MICROORGANISMS
- C12R2001/00—Microorganisms ; Processes using microorganisms
- C12R2001/01—Bacteria or Actinomycetales ; using bacteria or Actinomycetales
- C12R2001/465—Streptomyces
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- Chemical & Material Sciences (AREA)
- Engineering & Computer Science (AREA)
- Life Sciences & Earth Sciences (AREA)
- Organic Chemistry (AREA)
- Health & Medical Sciences (AREA)
- Zoology (AREA)
- Wood Science & Technology (AREA)
- Genetics & Genomics (AREA)
- Bioinformatics & Cheminformatics (AREA)
- Biotechnology (AREA)
- General Health & Medical Sciences (AREA)
- Biochemistry (AREA)
- General Engineering & Computer Science (AREA)
- Microbiology (AREA)
- General Chemical & Material Sciences (AREA)
- Medicinal Chemistry (AREA)
- Tropical Medicine & Parasitology (AREA)
- Virology (AREA)
- Biomedical Technology (AREA)
- Chemical Kinetics & Catalysis (AREA)
- Preparation Of Compounds By Using Micro-Organisms (AREA)
Abstract
The invention provides a method for improving the titer of a monensin fermentation liquid and the content of monensin A, belonging to the technical field of biological fermentation. The invention uses the streptomyces cinnamomi to ferment and produce the monensin, and adds the combined fatty acid into the basic fermentation medium; the combined fatty acid consists of 1-2 parts of oleic acid, 2-3 parts of linoleic acid, 0.2-0.4 part of stearic acid and 0.4-0.6 part of palmitic acid according to parts by mass. According to the invention, the content of the component A of the monensin and the titer of the monensin fermentation liquid are improved by adding the combined fatty acid into the basic fermentation medium, the highest production level of the monensin can reach 26707 mug/mL, the highest content of the component A of the monensin can reach 98.06%, the yield and the quality are improved, and the method has obvious economic benefit.
Description
Technical Field
The invention belongs to the technical field of biological fermentation, and particularly relates to a method for improving the titer of a monensin fermentation liquid and the content of monensin A.
Background
Monensin is a polyether antibiotic produced by fermentation of Streptomyces cinnamomi (Streptomyces cinnamonensis) and has been found to have broad-spectrum anticoccidial activity. The monensin mainly comprises four acidic components, wherein monensin A and monensin B occupy higher proportion, monensin C and monensin D occupy little proportion, and monensin A is the main active component. Monensin can bind Na + 、K + And (3) equivalent monovalent cations, which are transferred from the environment into the cell membrane, so that osmotic pressure at two sides of the cell membrane is changed, and the cell is finally dead. The monensin can improve the digestibility of dry matter in diet, reduce the production of lactic acid, prevent lactic acidosis in animals, and can be used as food additive and weighting agent for animals. And because of the advantages of difficult drug resistance of pathogenic organisms, the preparation method is still active in the antibiotic industry of animal husbandry, and has great market demand. But at present due to technical barriersThe fermentation yield of monensin is low, the production cost is always high, and the content of monensin A is not high. Therefore, developing a novel fermentation production process, improving the titer of the monensin fermentation liquor and the duty ratio of monensin A, and reducing the production cost of monensin is one of the technical problems to be solved in the field.
Disclosure of Invention
Therefore, the invention aims to provide a method for improving the titer of a monensin fermentation liquor and the content of monensin A, wherein the production level of monensin can reach 26707 mug/mL, and the content of monensin A component can reach 98.06%.
In order to achieve the above object, the present invention provides the following technical solutions:
the invention provides a method for improving the titer of a monensin fermentation broth and the content of monensin A, wherein the monensin is produced by fermenting Streptomyces cinnamomi, and combined fatty acid is added into a basic fermentation medium; the combined fatty acid consists of 1-2 parts of oleic acid, 2-3 parts of linoleic acid, 0.2-0.4 part of stearic acid and 0.4-0.6 part of palmitic acid according to parts by mass.
Preferably, the addition amount of the combined fatty acid is 4-5% of the mass of the basic fermentation medium.
Preferably, the basic fermentation medium comprises 3 to 4 percent of soybean cake powder, 3 to 4 percent of oral glucose, 0.2 to 0.25 percent of anhydrous sodium sulfate, 0.007 to 0.008 percent of dipotassium hydrogen phosphate, 0.005 to 0.015 percent of ferrous sulfate heptahydrate, 0.06 to 0.07 percent of aluminum sulfate, 0.25 percent of calcium carbonate and 7.1 to 7.3 percent of pH.
Preferably, the fermentation temperature is 32-35 ℃.
Preferably, the fermentation stirring rotating speed is 300-500 r/min.
Preferably, the fermentation ventilation is 5-7L/min.
Preferably, the fermentation time is 10 to 15d.
Preferably, the fermentation pH is 6.6 to 7.5.
Preferably, the fermentation mode is fed-batch fermentation.
The invention also provides application of the method in industrial production of monensin.
The invention has the beneficial effects that:
the method is simple and easy to implement, and can obviously improve the titer of the monensin A component and the fermentation liquor, so that the titer of the monensin reaches 15034 mug/mL after 12d fermentation of a shake flask system, and the titer is improved by 23.65 percent compared with a culture medium without adding combined fatty acid; the titer of monensin reaches 26707 mug/mL after fermentation in a fermentation tank for 12d, and is improved by 11.53 percent compared with a culture medium without adding combined fatty acid, the A component ratio is improved from 93.37 percent to 98.06 percent, and the method has obvious economic benefit.
Drawings
Fig. 1: the effect of different oleic acid, linoleic acid, stearic acid and palmitic acid addition amounts on fermentation.
Fig. 2: optimizing metabolism curve of Streptomyces cinnamomi on 10L tank, A: titer, thallus concentration and pH; b: dissolved oxygen, rotational speed and air flow.
Fig. 3: and (3) a monensin A component ratio chart before and after optimization, wherein: a control group; and B experimental group.
Detailed Description
The invention provides a method for improving the titer of a monensin fermentation broth and the content of monensin A, wherein the monensin is produced by fermenting Streptomyces cinnamomi, and combined fatty acid is added into a basic fermentation medium; the combined fatty acid consists of 1-2 parts of oleic acid, 2-3 parts of linoleic acid, 0.2-0.4 part of stearic acid and 0.4-0.6 part of palmitic acid according to parts by mass. Preferably, the combined fatty acid of the invention consists of 1.2 to 1.4 parts of oleic acid, 2.7 to 2.8 parts of linoleic acid, 0.3 to 0.35 part of stearic acid and 0.45 to 0.5 part of palmitic acid.
The addition amount of the combined fatty acid is 4-5% of the mass of the basic fermentation medium, preferably 4.2-4.971%, and more preferably 4.5-4.6%.
In the invention, the streptomyces cinnamomi is activated firstly, then is subjected to expansion culture in a seed culture medium, and then the cultured seed liquid is transferred into a fermentation bottle/fermentation tank for fermentation culture, so as to obtain the monensin fermentation liquid.
The activation medium is preferably a high-grade first slant medium. In the present invention, the activation culture temperature is preferably 32 to 35 ℃, more preferably 33 ℃; the activation culture time is preferably 9 to 12 days, more preferably 10 days.
The invention picks 1cm from the activation culture medium 2 Culturing mature spores, inoculating the spores into a primary seed culture medium for shake flask culture, wherein the seed culture medium (mass fraction) is preferably as follows: 1.5 to 2.5 percent of dextrin, 1 to 2 percent of soybean cake powder, 0.5 to 1 percent of glucose, 0.2 to 0.3 percent of yeast powder and 0.1 percent of calcium carbonate. The seed culture medium is prepared by drinking water. As an alternative implementation mode, the invention firstly uses a small amount of water to dissolve and mix the soybean cake powder, then boils the soybean cake powder in a microwave oven for 3 times, mixes the soybean cake powder with dextrin, glucose and yeast powder uniformly, adjusts the pH to about 6.8 by using 6M sodium hydroxide, and then adds calcium carbonate to 0.1% to obtain a seed culture medium. In the present invention, the culture temperature of the seed culture medium is preferably 32 to 35 ℃, more preferably 33 ℃; the shaking condition is preferably 180r/min, and the culture time is preferably 20-30 h, more preferably 22-24 h.
The invention inoculates the cultured seed liquid into the fermentation medium (combined fatty acid + basal fermentation medium) at an inoculum size of 9-12% (V/V), preferably 10%. The basic fermentation culture medium comprises 3-4% of soybean cake powder, 3-4% of oral glucose, 0.2-0.25% of anhydrous sodium sulfate, 0.007-0.008% of dipotassium hydrogen phosphate, 0.005-0.015% of ferrous sulfate heptahydrate, 0.06-0.07% of aluminum sulfate, 0.25% of calcium carbonate and 7.1-7.3% of pH7. The fermentation medium is prepared by drinking water. As an alternative implementation mode, the invention firstly uses a small amount of water to dissolve and mix the soybean cake powder uniformly, then boils the soybean cake powder in a microwave oven for 3 times, then mixes the soybean cake powder with oral glucose, anhydrous sodium sulfate, dipotassium hydrogen phosphate, ferrous sulfate heptahydrate and aluminum sulfate uniformly, adjusts the pH to about 7.2 by using 6M sodium hydroxide, then adds 0.25% of calcium carbonate to obtain a basic fermentation medium, and then adds 4-5% of combined fatty acid into the basic fermentation medium to obtain the fermentation medium.
In the present invention, the fermentation temperature is preferably 32 to 35 ℃, more preferably 33 ℃; the fermentation stirring rotating speed is preferably 300-500 r/min, more preferably 350-400 r/min; the fermentation time is preferably 10 to 15 days, more preferably 12 to 13 days; the fermentation ventilation is preferably 5 to 7L/min, more preferably 6L/min. The invention controls the dissolved oxygen in the fermentation process to be not less than 30% by using the air flow and the rotating speed. The fermentation pH of the invention is 6.6-7.5, and as an alternative implementation mode, the invention uses ammonia water to control the pH not lower than 6.6.
When fermentation is performed using a fermenter, the present invention preferably performs a fed-batch fermentation of the fermenter. As an alternative embodiment, the present invention starts feeding at 144h of fermentation culture, 2% (volume fraction) of feeding medium, which is the combined fatty acid according to the present invention. The fermentation culture time of the invention is 12d.
The invention also provides application of the method in the industrial production of monensin, and as an optional implementation mode, the volume of the fermentation tank is 10L, and the volume of the fermentation medium is 6L.
The technical solutions provided by the present invention are described in detail below with reference to examples, but they should not be construed as limiting the scope of the present invention.
In the following examples, conventional methods are used unless otherwise specified.
Materials, reagents and the like used in the examples described below are commercially available unless otherwise specified.
In a specific embodiment of the invention, the slant medium, the seed medium and the fermentation medium are:
high first slant medium (mass fraction): 2% of soluble starch, 0.1% of potassium nitrate, 0.05% of dipotassium hydrogen phosphate, 0.05% of magnesium sulfate heptahydrate, 0.001% of ferrous sulfate heptahydrate, 0.05% of sodium chloride and 3.0% of agar. The preparation method comprises the following steps: dissolving soluble starch completely, weighing other components, dissolving sequentially, fixing volume, cooling, and adjusting pH to 7.5.
Seed culture medium (mass fraction): dextrin 2%, soybean cake powder 1.5%, glucose 0.8%, yeast powder 0.2% and calcium carbonate 0.1%. The preparation method comprises the following steps: the soybean cake powder is firstly dissolved and uniformly mixed by a small amount of water, then boiled for 3 times in a microwave oven, then dextrin, glucose and yeast powder are weighed, dissolved in sequence, and after constant volume and temperature reduction, the pH is regulated to about 6.8 by 6M sodium hydroxide, and then 0.1 percent of calcium carbonate is added.
Basic fermentation medium (mass fraction): 3.5% of soybean cake powder, 3.5% of oral glucose, 0.2% of anhydrous sodium sulfate, 0.008% of dipotassium hydrogen phosphate, 0.005% of ferrous sulfate heptahydrate, 0.06% of aluminum sulfate and 0.25% of calcium carbonate. The preparation method comprises the following steps: the soybean cake powder is firstly dissolved and uniformly mixed with a small amount of water, then boiled in a microwave oven for 3 times, then oral glucose, anhydrous sodium sulfate, dipotassium hydrogen phosphate, ferrous sulfate heptahydrate and aluminum sulfate are weighed, and are dissolved in sequence, after constant volume and temperature reduction, the pH is regulated to about 7.2 by using 6M sodium hydroxide, and 0.25 percent of calcium carbonate is added.
The fermentation strain is: streptomyces cinnamomum (Streptomyces cinnamonensis), purchased from Nasiy, bulgaria, is Streptomyces cinnamomum MO2206002.
Example 1
1. In this example, the addition of fatty acid species to the fermentation medium was investigated:
(1) The fermentation method (shake flask fermentation) comprises the following steps:
preparing inclined plane spores: streptomyces cinnamomi is inoculated in a first-order inclined plane culture medium of Gao's, and cultured in an incubator at 33 ℃ for 10d.
Seed culture: picking 1cm under aseptic condition 2 Mature spores were inoculated into 500mL triangular flasks containing 100mL of seed medium and cultured at 180r/min on a shaker at 33℃for 24h.
Fermentation culture: transferring the seed cultured in the step 2 into a triangular flask (the capacity of the fermentation flask is 500 mL) filled with 50mL of fermentation medium according to 10% (v/v) inoculum size under aseptic condition, and culturing for 12d at the temperature of shaking table 33 ℃ and under the condition of 200 r/min.
(2) The experimental groupings were designed as:
blank group: the basic fermentation medium is used as a fermentation medium.
Experiment group 1: based on the amount of the basic fermentation medium, 4.2% of soybean oil is directly added into the basic fermentation medium to be used as a fermentation medium.
Experiment group 2: based on the amount of the basic fermentation medium, oleic acid 4.2% is directly added into the basic fermentation medium to be used as a fermentation medium.
Experiment group 3: based on the amount of the basic fermentation medium, 4.2% of linoleic acid is directly added into the basic fermentation medium to be used as a fermentation medium.
Experiment group 4: based on the amount of the basic fermentation medium, 4.2% of stearic acid was directly added to the basic fermentation medium, and the resultant was used as a fermentation medium.
Experimental group 5: based on the amount of the basic fermentation medium, 4.2% palmitic acid was directly added to the basic fermentation medium and used as a fermentation medium.
The titers of monensin in each group of fermentation broths were compared and the results are shown in table 1.
TABLE 1 fermentation titers of different fatty acids
As can be seen from table 1, the addition of single fatty acid increased significantly over the blank, but was lower than the addition of the soy group (containing complex fatty acids). The addition of vegetable oil was not significantly different from the blank in terms of biomass. The addition of the compound fatty acid can obviously improve the titer of the monensin fermentation broth.
2. Single factor experiments were performed on oleic acid, linoleic acid, stearic acid, palmitic acid and response surface experiments were established.
(1) Single factor experiment
The influence of the addition amounts of different oleic acid, linoleic acid, stearic acid and palmitic acid in the fermentation medium on the fermentation is respectively studied, and the titers are compared. The results of the single-factor experiments are shown in FIG. 1.
As can be seen from fig. 1, the titer had a peak in a certain range, before which the monensin yield increased with increasing oleic acid concentration, and after which the monensin yield gradually decreased with increasing fatty acid concentration, with the highest fatty acid concentration being the optimal concentration. The optimal concentration of oleic acid was 1.2%, the optimal concentration of linoleic acid was 2.8%, the optimal concentration of stearic acid was 0.3%, and the optimal concentration of palmitic acid was 0.5%.
(2) Response surface method optimization
Plackett-Burman (PB) test results and analysis:
based on the single factor experiment of fatty acid concentration optimization, a Plackett-Burman experiment with the Design Expert Design 4 factor 2 level is used to find out an influence factor with obvious effect on monensin potency and a monensin potency (mug/mL) as a response value by taking oleic acid (A), linoleic acid (B), palmitic acid (C) and stearic acid (D) as factors, and the experiment is carried out.
The effect of 4 single factors on monensin fermentation titers was analyzed using Plackett-Burman and the results of the analysis of variance are shown in table 2. Regression analysis of the data resulted in a multiple primary regression equation for the response values: y (potency) =10799.25+1110.58a-720.75B-313.92C-942.42D. Among them, oleic acid, linoleic acid and stearic acid are the main effect factors which have significant influence on the fermentation titer result. Model signal to noise ratio Adeq precision= 10.503 is greater than 4 and the model can be used for experimental prediction. Determining the coefficient R 2 0.8499, describing the model linear equation determining coefficient Adj R 2 = 0.7641, the representative equation can explain the 76.41% variation. The reason that 84.99% can be explained by screening, and 3 significant influence factors corrected are used for climbing experiments.
Table 2 PB variables and horizontal values of test design and test results
TABLE 3 regression analysis of Plackett-Burman test results
* : significant differences (P < 0.05); * *: very significant difference (P < 0.01)
Steepest climbing test optimization result
According to PB test results, the steepest climbing experiment is carried out by using three significant influence factors of oleic acid, linoleic acid and stearic acid, wherein the oleic acid coefficient is positive and positively correlated with the yield of monensin, and the linoleic acid and stearic acid are negatively correlated. Experiments were performed with oleic acid 1.1%, linoleic acid 2.9% and stearic acid 0.35% as initial starting points, and with palmitic acid 0.5%, and the experimental design and results are shown in table 4. Test 3 treatment group monensin titers were highest, so the center point was designed using this set of data parameters as the response surface.
Table 4 optimization results of steepest hill climbing test
Box-Behnken (BB) test results and analysis
BB test with 3 factor 3 level is designed according to the steepest climbing experiment result, the design and the level are shown in table 5, the BB experiment design and the result are shown in table 6, and a nonlinear regression equation is obtained by fitting with monensin titer (Y) as a dependent variable and the addition concentrations of oleic acid (A), linoleic acid (B) and stearic acid (C) as independent variables:
Y＝14427+2071.75A+772.13B+816.38C+1074.5AB+1024.5AC+2072.25BC-3705.13A 2 -2834.87B 2 -1729.88C 2 。
table 5 BB test factor level table
Table 6 BB test results and analysis
The analysis of variance of BB test is shown in Table 7, the significance of model p=0.0063 is higher, R 2 For 0.9101, a closer to 1 indicates a better model correlation, with a signal to noise ratio Adeq precision= 15.809 #>4) The model is less interfered by external factors; coefficient of variation=6.98, which indicates that the model has extremely small degree of dispersion and the mismatching term= 0.5343%>0.05 The missing term is not significant. The comprehensive situation shows that the model has high reliability and can be used for simulation prediction of fermentation test results. The influence of square terms and interaction terms in the quadratic polynomial regression equation is obvious, which shows that the influence of each factor on fermentation titer is complex and is not a pure linear relationship.
Table 7 BB test analysis of variance results
* : significant differences (P < 0.05); * *: very significant difference (P < 0.01)
From the experimental results, the optimal proportion of the compound fatty acid is 1.381% of oleic acid, 2.777% of linoleic acid, 0.313% of stearic acid and 0.5% of palmitic acid.
Example 2
A method for improving the titer of a monensin fermentation broth (shake flask fermentation):
oleic acid 1.381%, linoleic acid 2.777%, stearic acid 0.313% and palmitic acid 0.5% were directly added to the basal fermentation medium based on the amount of the basal fermentation medium, and used as the example fermentation medium.
1. Preparing inclined plane spores: streptomyces cinnamomi is inoculated in a first-order inclined plane culture medium of Gao's, and cultured in an incubator at 33 ℃ for 10d.
2. Seed culture: picking 1cm under aseptic condition 2 Mature spores were inoculated into 500mL triangular flasks containing 100mL of seed medium and cultured at 180r/min on a shaker at 33℃for 24h.
3. Fermentation culture: transferring the seed cultured in the step 2 into a triangular flask (the capacity of the fermentation flask is 500 mL) filled with 50mL of fermentation medium according to 10% (v/v) inoculum size under aseptic condition, and culturing for 12d at the temperature of shaking table 33 ℃ and under the condition of 200 r/min.
Example 3
A method for improving the titer of a monensin fermentation broth (fermentation in a fermenter):
oleic acid 1.381%, linoleic acid 2.777%, stearic acid 0.313% and palmitic acid 0.5% were directly added to the basal fermentation medium based on the amount of the basal fermentation medium, and used as the example fermentation medium.
1. Preparing inclined plane spores: streptomyces cinnamomi is inoculated in a first-order inclined plane culture medium of Gao's, and cultured in an incubator at 33 ℃ for 10d.
2. Seed culture: picking 1cm under aseptic condition 2 The mature spores are inoculated into 500mL triangular shake flasks filled with 100mL of seed culture medium, and cultured for 24 hours at the temperature of 33 ℃ and the speed of 180r/min on a shaking table to obtain primary seed liquid. The activated primary seed liquid is transferred to 3 triangular shake flasks with 200mL seed culture medium at 10% inoculum size, and cultured for 24h at 180r/min on a shaker at 33℃to obtain secondary seed liquid.
3. Fermentation culture: transferring the secondary seed liquid cultured in the step 2 into a fermentation tank (the capacity of the fermentation tank is 10L) filled with 6L of fermentation medium according to the inoculation amount of 10% (V/V) under the aseptic condition, adding 0.2% (V/V) of organic silicon defoamer into the fermentation tank, and culturing for 12d. The initial fermentation condition is that the temperature is 33 ℃, the rotating speed is 300rpm, the ventilation amount is 6L/min, the dissolved oxygen is not lower than 30% in the air flow and rotating speed control process, the pH is not lower than 6.6 by using ammonia water, and the volume is maintained to be 6L by using sterile water.
Example 4
A method for improving the titer of a monensin fermentation broth (fed-batch fermentation in a fermenter):
oleic acid 1.381%, linoleic acid 2.777%, stearic acid 0.313% and palmitic acid 0.5% were directly added to the basal fermentation medium based on the amount of the basal fermentation medium, and used as the example fermentation medium.
1. Preparing inclined plane spores: streptomyces cinnamomi is inoculated in a first-order inclined plane culture medium of Gao's, and cultured in an incubator at 33 ℃ for 10d.
2. Seed culture: picking 1cm under aseptic condition 2 The mature spores are inoculated into 500mL triangular shake flasks filled with 100mL of seed culture medium, and cultured for 24 hours at the temperature of 33 ℃ and the speed of 180r/min on a shaking table to obtain primary seed liquid. The activated primary seed liquid is transferred to 3 triangular shake flasks with 200mL seed culture medium at 10% inoculum size, and cultured for 24h at 180r/min on a shaker at 33℃to obtain secondary seed liquid.
3. Fermentation culture: transferring the secondary seed liquid cultured in the step 2 into a fermentation tank (the capacity of the fermentation tank is 10L) filled with 6L of fermentation medium according to the inoculation amount of 10% (V/V) under the aseptic condition, and adding 0.2% (V/V) of organic silicon defoamer into the fermentation tank. The initial fermentation condition is that the temperature is 33 ℃, the rotating speed is 300rpm, the ventilation amount is 6L/min, the dissolved oxygen is not lower than 30% in the air flow and rotating speed control process, the pH is not lower than 6.6 by using ammonia water, and the volume is maintained to be 6L by using sterile water.
4. And (3) material supplementing: feeding is started when 144h of fermentation culture is carried out, 2% (volume fraction) of the feed is fed, and the feed culture medium is combined fatty acid (oleic acid 1.381: linoleic acid 2.777: stearic acid 0.313: palmitic acid 0.5). The fermentation culture time is 12d.
Comparative example 1
The difference between this comparative example and example 4 is that: the combined fatty acids were replaced with an equal amount of soybean oil.
Example 5
The samples were taken every 24 hours during the fermentation of example 4 and comparative example 1, and the metabolic parameters of monensin in the fermentor were as shown in FIG. 2. In fig. 2, a: titer, thallus concentration and pH; b: dissolved oxygen, rotational speed and air flow.
As is clear from FIG. 2, the cell concentration increased rapidly in the first 72 hours, and the later period became stable gradually, and the combined fatty acid group reached the maximum value in 72 hours, 24 hours earlier than the control group, which was related to the combined fatty acid being more favorable for cell growth. The pH change trend is consistent, the overall trend is that the pH is increased firstly and then decreased, the pH fluctuation is caused by supplementing the carbon source at 144 hours, and the later pH increase is mainly caused by the depletion of the carbon source in the culture medium. The dissolved oxygen is maintained to be more than 30% by adjusting the stirring rotation speed and the air flow, the dissolved oxygen generally shows a trend of descending first and then rising, and compared with the dissolved oxygen of a control group, the whole fermentation process of the combined fatty acid group is lower, so that the experimental group has vigorous metabolism and larger oxygen consumption. The 24h strain starts to produce secondary metabolites, the yield of monensin is positively correlated with the fermentation time in a fermentation period of 0-288h, the potency of the combined fatty acid increases rapidly, the potency is high, and the difference between the two increases with time.
In example 4, the batch feeding titer of the 10L tank reaches 26707 mug/mL, and the final tank discharge cost performance is improved by 11.53% compared with the comparative example 1; the A component ratio is increased from 93.37% to 98.06%, see in particular FIG. 3.
Example 6
This example explores the addition of different fatty acids to the fermentation medium (fermentation process is the same as in example 2), 3 sets of replicates were run per experiment:
experimental group: based on the amount of the basic fermentation medium, oleic acid 1.381%, linoleic acid 2.777%, stearic acid 0.313% and palmitic acid 0.5% were directly added to the basic fermentation medium, and used as a fermentation medium.
Control group 1: based on the amount of the basic fermentation medium, 4.971% of soybean oil is directly added to the basic fermentation medium to be used as a fermentation medium.
Control group 2: based on the amount of the basic fermentation medium, 4.971% of peanut oil is directly added into the basic fermentation medium to be used as a fermentation medium.
Control group 3: based on the amount of the basic fermentation medium, 4.971% of corn oil is directly added to the basic fermentation medium to be used as a fermentation medium.
Control group 4: the olive oil 4.971% is directly added to the basic fermentation medium based on the amount of the basic fermentation medium, and is used as a fermentation medium.
Control group 5: based on the amount of the basic fermentation culture medium, the linseed oil 4.971% is directly added into the basic fermentation culture medium to be used as a fermentation culture medium.
Control group 6: based on the amount of the basic fermentation medium, 4.971% of camellia oil is directly added into the basic fermentation medium to be used as a fermentation medium.
The fatty acid composition of soybean oil, peanut oil, corn oil, olive oil, linseed oil and camellia oil are shown in table 8.
TABLE 8 fatty acid Components in different vegetable oils
The titers of monensin in each group of fermentation broths were compared and the results are shown in table 9.
TABLE 9 fermentation titers of different vegetable oils
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As is clear from table 9, the optimal fatty acid combinations of oleic acid 1.381%, linoleic acid 2.777%, stearic acid 0.313% and palmitic acid 0.5% significantly increased the titer of the monensin fermentation broth, and the titers in the monensin fermentation broth were also significantly reduced due to the change in the ratio of each fatty acid in the fatty acid combinations when other vegetable oils were added.
Example 7
In this example, the ratio of addition of different fatty acid combinations in the fermentation medium was investigated (fermentation method is the same as in example 2), and 3 groups of experiments were performed in parallel:
experimental group: based on the amount of the basic fermentation medium, oleic acid 1.381%, linoleic acid 2.777%, stearic acid 0.313% and palmitic acid 0.5% were directly added to the basic fermentation medium, and used as a fermentation medium.
Control group 1: based on the amount of the basic fermentation medium, 4.971% of the combined fatty acid is directly added into the basic fermentation medium to be used as a fermentation medium. Linoleic acid in the combined fatty acids compared to the experimental group: stearic acid: the proportion of palmitic acid is unchanged, and oleic acid is removed.
Control group 2: based on the amount of the basic fermentation medium, 4.971% of the combined fatty acid is directly added into the basic fermentation medium to be used as a fermentation medium. Compared with the experimental group, oleic acid in the combined fatty acids: stearic acid: the proportion of palmitic acid is unchanged, and linoleic acid is removed.
Control group 3: based on the amount of the basic fermentation medium, 4.971% of the combined fatty acid is directly added into the basic fermentation medium to be used as a fermentation medium. Compared with the experimental group, oleic acid in the combined fatty acids: linoleic acid: the ratio of palmitic acid is unchanged, and stearic acid is removed.
Control group 4: based on the amount of the basic fermentation medium, 4.971% of the combined fatty acid is directly added into the basic fermentation medium to be used as a fermentation medium. Compared with the experimental group, oleic acid in the combined fatty acids: linoleic acid: the stearic acid ratio was unchanged and palmitic acid was removed.
The titers of monensin in each group of fermentation broths were compared and the results are shown in table 10.
TABLE 10 fermentation titers of different species of combined fatty acids
As is clear from Table 10, oleic acid 1.381%, linoleic acid 2.777%, stearic acid 0.313% and palmitic acid 0.5% are the optimal fatty acid combinations, and the absence of any fatty acid has an effect on the potency of monensin.
Example 8
In this example, the amount of fatty acid added to the fermentation medium was investigated (fermentation method was the same as in example 2), and 3 groups of experiments were performed in parallel:
according to the optimal fatty acid combination ratio: the experimental tests were carried out by 1.381% oleic acid, 2.777% linoleic acid, 0.313% stearic acid and 0.5% palmitic acid, and the experimental groups 1 to 6 were respectively set according to 4.971% fatty acid added to each group of experimental basic medium, the fatty acid addition was 0% fatty acid combination+100% soybean oil, 20% fatty acid combination+80% soybean oil, 40% fatty acid combination+60% soybean oil, 60% fatty acid combination+40% soybean oil, 80% fatty acid combination+20% soybean oil, 100% fatty acid combination+0% soybean oil, and the results of the 3 parallel experiments are shown in table 11.
TABLE 11 fermentation titers for different combinations of fatty acid additions
As can be seen from table 11, the optimized combined fatty acid was substituted for soybean oil in the fermentation medium, and the titer of monensin was also increased as the substitution ratio of the combined fatty acid was gradually increased, and the titer was highest when reaching 100%. In 3 parallel experiments, the average titer of the monensin reaches 15034 mug/mL, and the monensin is basically consistent with the predicted value of the curved surface model, and the reliability of the model predicted result is high. Compared with the method only added with the soybean oil group (monensin titer 12159 mug/mL), the yield of the combined fatty acid formula is improved by 23.65% only by adding the combined fatty acid.
The foregoing is merely a preferred embodiment of the present invention and it should be noted that modifications and adaptations to those skilled in the art may be made without departing from the principles of the present invention, which are intended to be comprehended within the scope of the present invention.
Claims (10)
1. A method for improving the titer of a monensin fermentation broth and the content of monensin A is characterized in that the monensin is produced by fermenting Streptomyces cinnamomi, and combined fatty acid is added into a basic fermentation medium; the combined fatty acid consists of 1-2 parts of oleic acid, 2-3 parts of linoleic acid, 0.2-0.4 part of stearic acid and 0.4-0.6 part of palmitic acid according to parts by mass.
2. The method according to claim 1, wherein the amount of the combined fatty acids added is 4 to 5% by mass of the basic fermentation medium.
3. The method of claim 1, wherein the basal fermentation medium comprises 3-4% soy cake flour, 3-4% oral glucose, 0.2-0.25% anhydrous sodium sulfate, 0.007-0.008% dipotassium hydrogen phosphate, 0.005-0.015% ferrous sulfate heptahydrate, 0.06-0.07% aluminum sulfate, 0.25% calcium carbonate, and ph 7.1-7.3.
4. The method according to claim 1, wherein the fermentation temperature is 32-35 ℃.
5. The method according to claim 1, wherein the fermentation stirring speed is 300-500 r/min.
6. The method of claim 1, wherein the fermentation aeration is 5 to 7L/min.
7. The method of claim 1, wherein the fermentation time is 10 to 15d.
8. The method according to claim 1, wherein the fermentation pH is 6.6 to 7.5.
9. The method of claim 1, wherein the fermentation is fed-batch fermentation.
10. Use of the method of any one of claims 1 to 9 in the industrial production of monensin.
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