CN106117293B - A kind of method that new anti-bacterial agent sambacide is produced by solid fermentation Fusariumsp Fusarium sp. - Google Patents
A kind of method that new anti-bacterial agent sambacide is produced by solid fermentation Fusariumsp Fusarium sp. Download PDFInfo
- Publication number
- CN106117293B CN106117293B CN201610421717.2A CN201610421717A CN106117293B CN 106117293 B CN106117293 B CN 106117293B CN 201610421717 A CN201610421717 A CN 201610421717A CN 106117293 B CN106117293 B CN 106117293B
- Authority
- CN
- China
- Prior art keywords
- sambacide
- fusarium
- new
- fusariumsp
- solid state
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Expired - Fee Related
Links
- 241001149959 Fusarium sp. Species 0.000 title claims abstract description 32
- 238000000034 method Methods 0.000 title claims abstract description 18
- 239000003242 anti bacterial agent Substances 0.000 title claims abstract description 10
- 239000007787 solid Substances 0.000 title claims abstract description 8
- 238000000855 fermentation Methods 0.000 title claims description 24
- 230000004151 fermentation Effects 0.000 title claims description 24
- 244000061456 Solanum tuberosum Species 0.000 claims abstract description 26
- 235000002595 Solanum tuberosum Nutrition 0.000 claims abstract description 26
- 150000001875 compounds Chemical class 0.000 claims abstract description 22
- 239000001963 growth medium Substances 0.000 claims abstract description 14
- 238000004128 high performance liquid chromatography Methods 0.000 claims abstract description 12
- 238000010563 solid-state fermentation Methods 0.000 claims abstract description 10
- VYPSYNLAJGMNEJ-UHFFFAOYSA-N Silicium dioxide Chemical compound O=[Si]=O VYPSYNLAJGMNEJ-UHFFFAOYSA-N 0.000 claims abstract description 9
- 239000000741 silica gel Substances 0.000 claims abstract description 8
- 229910002027 silica gel Inorganic materials 0.000 claims abstract description 8
- 239000002904 solvent Substances 0.000 claims abstract description 4
- 230000002421 anti-septic effect Effects 0.000 claims abstract 6
- OKKJLVBELUTLKV-UHFFFAOYSA-N Methanol Chemical compound OC OKKJLVBELUTLKV-UHFFFAOYSA-N 0.000 claims description 46
- WEVYAHXRMPXWCK-UHFFFAOYSA-N Acetonitrile Chemical compound CC#N WEVYAHXRMPXWCK-UHFFFAOYSA-N 0.000 claims description 18
- HEDRZPFGACZZDS-UHFFFAOYSA-N Chloroform Chemical compound ClC(Cl)Cl HEDRZPFGACZZDS-UHFFFAOYSA-N 0.000 claims description 16
- 244000005700 microbiome Species 0.000 claims description 15
- 239000000287 crude extract Substances 0.000 claims description 12
- 238000011218 seed culture Methods 0.000 claims description 12
- 238000000926 separation method Methods 0.000 claims description 12
- 230000001954 sterilising effect Effects 0.000 claims description 12
- 238000004659 sterilization and disinfection Methods 0.000 claims description 12
- 229960001866 silicon dioxide Drugs 0.000 claims description 7
- 238000005084 2D-nuclear magnetic resonance Methods 0.000 claims description 6
- 230000004913 activation Effects 0.000 claims description 6
- 238000001514 detection method Methods 0.000 claims description 6
- 238000010828 elution Methods 0.000 claims description 6
- 239000000499 gel Substances 0.000 claims description 6
- 238000002114 high-resolution electrospray ionisation mass spectrometry Methods 0.000 claims description 6
- 239000007788 liquid Substances 0.000 claims description 6
- 238000000238 one-dimensional nuclear magnetic resonance spectroscopy Methods 0.000 claims description 6
- 238000004806 packaging method and process Methods 0.000 claims description 6
- 238000002203 pretreatment Methods 0.000 claims description 6
- 238000012545 processing Methods 0.000 claims description 6
- 235000011962 puddings Nutrition 0.000 claims description 6
- 238000002137 ultrasound extraction Methods 0.000 claims description 4
- 238000003556 assay Methods 0.000 claims description 2
- 238000000605 extraction Methods 0.000 claims description 2
- 239000000203 mixture Substances 0.000 claims description 2
- 239000003480 eluent Substances 0.000 claims 1
- 238000004519 manufacturing process Methods 0.000 abstract description 5
- 230000000844 anti-bacterial effect Effects 0.000 abstract description 4
- 241000588724 Escherichia coli Species 0.000 abstract description 3
- 239000004599 antimicrobial Substances 0.000 abstract description 3
- 238000006243 chemical reaction Methods 0.000 abstract description 3
- 241000191967 Staphylococcus aureus Species 0.000 abstract description 2
- 229910052799 carbon Inorganic materials 0.000 abstract description 2
- 238000001914 filtration Methods 0.000 abstract description 2
- 238000001641 gel filtration chromatography Methods 0.000 abstract description 2
- 238000012827 research and development Methods 0.000 abstract description 2
- 238000010898 silica gel chromatography Methods 0.000 abstract description 2
- 241000223218 Fusarium Species 0.000 description 15
- UHPMCKVQTMMPCG-UHFFFAOYSA-N 5,8-dihydroxy-2-methoxy-6-methyl-7-(2-oxopropyl)naphthalene-1,4-dione Chemical compound CC1=C(CC(C)=O)C(O)=C2C(=O)C(OC)=CC(=O)C2=C1O UHPMCKVQTMMPCG-UHFFFAOYSA-N 0.000 description 13
- 239000003795 chemical substances by application Substances 0.000 description 5
- 238000009940 knitting Methods 0.000 description 5
- 150000002825 nitriles Chemical class 0.000 description 5
- 241000894006 Bacteria Species 0.000 description 4
- 229930186096 integracide Natural products 0.000 description 4
- 239000002689 soil Substances 0.000 description 4
- 241000233866 Fungi Species 0.000 description 2
- 241001465754 Metazoa Species 0.000 description 2
- -1 and on taxology Chemical compound 0.000 description 2
- 230000000694 effects Effects 0.000 description 2
- 235000013305 food Nutrition 0.000 description 2
- 239000000463 material Substances 0.000 description 2
- 238000004321 preservation Methods 0.000 description 2
- 239000000126 substance Substances 0.000 description 2
- 238000002604 ultrasonography Methods 0.000 description 2
- 235000013311 vegetables Nutrition 0.000 description 2
- 238000001644 13C nuclear magnetic resonance spectroscopy Methods 0.000 description 1
- 238000005160 1H NMR spectroscopy Methods 0.000 description 1
- FFRBMBIXVSCUFS-UHFFFAOYSA-N 2,4-dinitro-1-naphthol Chemical compound C1=CC=C2C(O)=C([N+]([O-])=O)C=C([N+]([O-])=O)C2=C1 FFRBMBIXVSCUFS-UHFFFAOYSA-N 0.000 description 1
- 108010059892 Cellulase Proteins 0.000 description 1
- 241001478240 Coccus Species 0.000 description 1
- 229920000742 Cotton Polymers 0.000 description 1
- 241000196324 Embryophyta Species 0.000 description 1
- 229930191978 Gibberellin Natural products 0.000 description 1
- 241000713772 Human immunodeficiency virus 1 Species 0.000 description 1
- 108700020129 Human immunodeficiency virus 1 p31 integrase Proteins 0.000 description 1
- 102100034343 Integrase Human genes 0.000 description 1
- 108010061833 Integrases Proteins 0.000 description 1
- 102000004882 Lipase Human genes 0.000 description 1
- 108090001060 Lipase Proteins 0.000 description 1
- 239000004367 Lipase Substances 0.000 description 1
- 206010054949 Metaplasia Diseases 0.000 description 1
- 240000008790 Musa x paradisiaca Species 0.000 description 1
- 235000018290 Musa x paradisiaca Nutrition 0.000 description 1
- 240000007594 Oryza sativa Species 0.000 description 1
- 235000007164 Oryza sativa Nutrition 0.000 description 1
- 235000021307 Triticum Nutrition 0.000 description 1
- 244000098338 Triticum aestivum Species 0.000 description 1
- 240000006677 Vicia faba Species 0.000 description 1
- 235000010749 Vicia faba Nutrition 0.000 description 1
- 235000002098 Vicia faba var. major Nutrition 0.000 description 1
- 235000009754 Vitis X bourquina Nutrition 0.000 description 1
- 235000012333 Vitis X labruscana Nutrition 0.000 description 1
- 240000006365 Vitis vinifera Species 0.000 description 1
- 235000014787 Vitis vinifera Nutrition 0.000 description 1
- 240000008042 Zea mays Species 0.000 description 1
- 235000005824 Zea mays ssp. parviglumis Nutrition 0.000 description 1
- 235000002017 Zea mays subsp mays Nutrition 0.000 description 1
- 241001314279 Zoopagales Species 0.000 description 1
- 238000004458 analytical method Methods 0.000 description 1
- 230000000843 anti-fungal effect Effects 0.000 description 1
- 229940106157 cellulase Drugs 0.000 description 1
- 235000013339 cereals Nutrition 0.000 description 1
- 230000008859 change Effects 0.000 description 1
- 238000004587 chromatography analysis Methods 0.000 description 1
- 235000005822 corn Nutrition 0.000 description 1
- 230000001472 cytotoxic effect Effects 0.000 description 1
- 238000003113 dilution method Methods 0.000 description 1
- 201000010099 disease Diseases 0.000 description 1
- 208000037265 diseases, disorders, signs and symptoms Diseases 0.000 description 1
- 238000005516 engineering process Methods 0.000 description 1
- 239000002828 fuel tank Substances 0.000 description 1
- IXORZMNAPKEEDV-UHFFFAOYSA-N gibberellic acid GA3 Natural products OC(=O)C1C2(C3)CC(=C)C3(O)CCC2C2(C=CC3O)C1C3(C)C(=O)O2 IXORZMNAPKEEDV-UHFFFAOYSA-N 0.000 description 1
- 239000003448 gibberellin Substances 0.000 description 1
- 230000006872 improvement Effects 0.000 description 1
- 239000003112 inhibitor Substances 0.000 description 1
- 235000019421 lipase Nutrition 0.000 description 1
- 244000144972 livestock Species 0.000 description 1
- 230000014759 maintenance of location Effects 0.000 description 1
- 230000004060 metabolic process Effects 0.000 description 1
- 239000002207 metabolite Substances 0.000 description 1
- 230000015689 metaplastic ossification Effects 0.000 description 1
- 239000000401 methanolic extract Substances 0.000 description 1
- 229930014626 natural product Natural products 0.000 description 1
- NCYVXEGFNDZQCU-UHFFFAOYSA-N nikethamide Chemical compound CCN(CC)C(=O)C1=CC=CN=C1 NCYVXEGFNDZQCU-UHFFFAOYSA-N 0.000 description 1
- 230000001473 noxious effect Effects 0.000 description 1
- 231100000208 phytotoxic Toxicity 0.000 description 1
- 230000000885 phytotoxic effect Effects 0.000 description 1
- 239000002243 precursor Substances 0.000 description 1
- 238000002360 preparation method Methods 0.000 description 1
- 239000011347 resin Substances 0.000 description 1
- 229920005989 resin Polymers 0.000 description 1
- 235000009566 rice Nutrition 0.000 description 1
- 239000004576 sand Substances 0.000 description 1
- 229930000044 secondary metabolite Natural products 0.000 description 1
- 230000001568 sexual effect Effects 0.000 description 1
- 238000003860 storage Methods 0.000 description 1
- 238000012360 testing method Methods 0.000 description 1
- 238000004809 thin layer chromatography Methods 0.000 description 1
- 239000003053 toxin Substances 0.000 description 1
- 231100000765 toxin Toxicity 0.000 description 1
Classifications
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07J—STEROIDS
- C07J9/00—Normal steroids containing carbon, hydrogen, halogen or oxygen substituted in position 17 beta by a chain of more than two carbon atoms, e.g. cholane, cholestane, coprostane
-
- 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
- C12N1/14—Fungi; Culture media therefor
- C12N1/145—Fungal isolates
-
- 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
- C12P33/00—Preparation of steroids
-
- 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
- C12R2001/645—Fungi ; Processes using fungi
- C12R2001/77—Fusarium
-
- Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
- Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
- Y02A—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE
- Y02A50/00—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE in human health protection, e.g. against extreme weather
- Y02A50/30—Against vector-borne diseases, e.g. mosquito-borne, fly-borne, tick-borne or waterborne diseases whose impact is exacerbated by climate change
Landscapes
- Chemical & Material Sciences (AREA)
- Organic Chemistry (AREA)
- Life Sciences & Earth Sciences (AREA)
- Health & Medical Sciences (AREA)
- Engineering & Computer Science (AREA)
- Zoology (AREA)
- Wood Science & Technology (AREA)
- Genetics & Genomics (AREA)
- Biotechnology (AREA)
- General Health & Medical Sciences (AREA)
- Bioinformatics & Cheminformatics (AREA)
- General Engineering & Computer Science (AREA)
- Biochemistry (AREA)
- Microbiology (AREA)
- Medicinal Chemistry (AREA)
- Mycology (AREA)
- Tropical Medicine & Parasitology (AREA)
- Biomedical Technology (AREA)
- Virology (AREA)
- Botany (AREA)
- Chemical Kinetics & Catalysis (AREA)
- General Chemical & Material Sciences (AREA)
- Preparation Of Compounds By Using Micro-Organisms (AREA)
- Micro-Organisms Or Cultivation Processes Thereof (AREA)
Abstract
The present invention discloses a kind of method that new anti-bacterial agent sambacide is produced by solid state fermentation Fusariumsp Fusarium sp.B10.1 (CGMCC No.11819).I.e.：Potato culture medium ferments through Fusarium sp.B10.1 (CGMCC No.11819), is extracted by suitable solvent supersonic, filtering, after concentration, through isolated one new compound sambacide of silica gel and gel filtration chromatography.With reference to TLC TLCs and high performance liquid chromatography, substantial amounts of sambacide generation can detect.Sambacide has significant antibacterial action to staphylococcus aureus and Escherichia coli, is a new antiseptic.It this method provide the production method of a new antimicrobial compound.This method is a kind of efficient; with novelty; reaction condition is gentle; green non-pollution; easily extension produces; the method that operation equipment simply largely produces antimicrobial compound sambacide, the demand of modern environment protection and low-carbon economy is not only met, and solid foundation has been established for the further research and development of later stage industrial volume production.
Description
Technical field
Produced the present invention relates to one kind by solid state fermentation Fusariumsp Fusarium sp.B10.1 (CGMCC No.11819)
New anti-bacterial agent sambacide method；Specifically it is found that a new anti-bacterial agent sambacide.Microorganism belonging to genus metabolism production
Thing separates analysis field,
The microorganism that the present invention uses：Fusariumsp Fusarium sp.B10.1 (preserving numbers：CGMCC No.11819) preservation
In China Committee for Culture Collection of Microorganisms's common micro-organisms center, depositary institution address：Chaoyang District, Beijing City North Star west
The institute 3 of road 1；The preservation time：On January 15th, 2016.
Background technology：
Fusarium (Fusarium) is also known as Fusarium, and on taxology, Fusarium asexual period is belonged to originally in partly knowing
Bacterium subphylum, knurl seat Zoopagales.Sexual period is Ascomycotina, and Perfect stage is often Gibberella (Gibberella).
Each kind of Fusarium is widely distributed in soil and organism, and is once neutralized from the permafrost of the arctic
It is separated in the sand of the Sahara desert.Their well-growns in the anthropogenic soil of temperate zone and torrid areas, are often quilts
One of fungi that phytopathologist is separated to.Sickle-like bacteria can also cause the disease of human and animal, substantial amounts of stock
Rot, usually produce noxious material, pollute the food of people and the feed of livestock.As other many soil fungis, it
Be adapted to and survived in soil, one of its function is that can have quickly in morphology and physiologically for new environment
Change and the ability adapted to.They can be lived among the deep layer subsoil of broad range, can also be from the food of many storages
Separated with chemical substance, or even in the fuel tank of aircraft, also once with resin bud branch it is mould along be separated.
Fusarium it is widely distributed with nature in, sickle-like bacteria can produce plant stimulin (gibberellin), can make crops
Volume increase；Some kinds can produce cellulase, lipase, pectase etc.；Also some kinds can produce toxin, pollution grain, vegetables and feeding
Material, people and animals, which eat by mistake, to be poisoned；Sickle-like bacteria can also infect diversified economy crop, cause rice, wheat, corn, broad bean, vegetables etc.
Head blight, the droop of cotton, banana blight etc..
Integracides is one kind 4,4- dimethyl lumistane compounds, and being being capable of very effective selection HIV-1
The inhibitor of integrase, there is different kind organism activity to include antibacterial antifungal activity, and cytotoxic activity.HIV-1 integrases press down
The structure activity study of preparation shows, integracides related natural products 3- sulfuric acid esters have stronger than precursor compound
Work.
Triterpene compound (such as：Integracides the Fusarium from Vesonder and Burmeister) has been reported
Active metabolite separates Fusarium, finds potential phytotoxic secondary metabolite.Therefore, from Fusarium
Spp. the related compound for finding integracides has significant meaning.
The content of the invention
The object of the invention is intended to produce new anti-bacterial agent by Fusariumsp Fusarium sp.B10.1 solid fermentations
Sambacide method.The present invention provides a kind of high conversion rate, and equipment requirement is low, simple and easy to operate, green non-pollution, is adapted to
The method that industrialization produces sambacide.
The purpose of the present invention is realized by technical scheme in detail below：One kind passes through Fusariumsp Fusarium
Sp.B10.1 fermentations produce new anti-bacterial agent sambacide, it is characterised in that are sent out by Fusariumsp Fusarium sp.B10.1 solids
It is sambacide that ferment, which produces a noval chemical compound, and its chemical constitution is：
The method that the present invention produces new anti-bacterial agent sambacide by Fusariumsp Fusarium sp.B10.1 fermentations, it is special
Sign is to carry out solid fermentation as follows：
(1) will intend being activated first with microorganism Fusariumsp Fusarium sp.B10.1, i.e., by Fusarium
After sp.B10.1 is inoculated into the PDA slant mediums by 121 DEG C of high-temperature sterilization processing, 3-7d is cultivated in constant incubator
Afterwards, it is placed in 4 DEG C of refrigerators；
(2) strain of activation is connected in PDB seed culture mediums, 3-7d is cultivated in constant-temperature table；
(3) after taking clean potato to be diced, appropriate small pudding is taken to be placed in tissue culture flasks；By the group equipped with potato
The 30min that sterilizes is placed in 121 DEG C of high-temperature sterilization boxes after knitting blake bottle capping packaging, cools down tissue culture flasks after taking-up；
(4) seed culture medium of step (2) is inoculated on the potato by step (3) pre-treatment in an aseptic environment,
It is placed in incubator after cultivating 5-40d and takes out after capping；
(5) through microorganism Fusariumsp Fusarium sp.B10.1 fermentation after potato culture medium by methanol ultrasonic extraction,
Filter and be concentrated to give crude extract；The structural formula for the sambacide that separation crude extract obtains is as follows：
(6) detect, then separated by silicagel column, separation process uses gradient elution, washes through TLC TLCs
De- agent is chloroform and carbinol mixture, wherein chloroform：Methanol presses 20:1—5:1 mixes, in the compound being then isolated to
Gel column purifies, and identifies to obtain new compound structure by 1D/2D NMR and HRESIMS；
(7) using following HPLC chromatogram condition measure sambacide yield.
The fermentation process is solid state fermentation.
The fermentation time is 5-40d.
The fermentation temperature is 20-30 DEG C.
The Extraction solvent is methanol.
Assay is carried out by following HPLC conditions：
(1) chromatographic column：ODS performance liquid chromatographic columns；
(2) gradient elution program：0-10min, 30-60% acetonitriles；10-20min, 60% acetonitrile；20-25,60-30% second
Nitrile；
(3) Detection wavelength：247nm；
(4) flow velocity：1.5mL/min；
(5) column temperature：25℃.
Potato culture medium of the present invention ferments through sickle mycete Fusarium sp.B10.1, is carried by suitable solvent supersonic
Take, filter, after concentration, through isolated one new compound sambacide of silica gel and gel filtration chromatography.With reference to TLC thin layers
Chromatography and high performance liquid chromatography, it can detect substantial amounts of sambacide generation.Sambacide is to golden yellow grape
Coccus and Escherichia coli have significant antibacterial action, are a new anti-bacterial agents.
The inventive method is a kind of efficient, has innovative, and reaction condition is gentle, green non-pollution, easily expands
Big metaplasia production, the method that operation equipment simply largely produces antimicrobial compound sambacide, not only meet modern environment
Protection and the demand of low-carbon economy, and established solid foundation for the further research and development of later stage industrial volume production.
Brief description of the drawings
Fig. 1 is noval chemical compound sambacide in the present invention1H-NMR；
Fig. 2 is noval chemical compound sambacide in the present invention13C-NMR；
Fig. 3 is the HPLC chromatogram of noval chemical compound sambacide in the present invention；
Fig. 4 is the HPLC chromatogram of Fusarium sp.B10.1 methanol extracts in the present invention.
Embodiment
The present invention is expanded on further with reference to embodiment, but the present invention is not limited to the specific embodiment, this area skill
Art personnel are it should be appreciated that present invention encompasses all possible alternative in right, improvement project and wait
Efficacious prescriptions case.
Concrete technical scheme of the present invention is：
(1) it will intend being activated first with microorganism Fusarium sp.B10.1, i.e., meet Fusarium sp.B10.1
After kind arrives the PDA slant mediums by 121 DEG C of high-temperature sterilizations processing, after cultivating 3-7d in constant incubator, 4 DEG C of ice are placed in
In case；
(2) strain of activation is connected in PDB seed culture mediums, 3-7d is cultivated in constant-temperature table；
(3) after taking clean potato to be diced, appropriate small pudding is taken to be placed in tissue culture flasks；By the group equipped with potato
The 30min that sterilizes is placed in 121 DEG C of high-temperature sterilization boxes after knitting blake bottle capping packaging, cools down tissue culture flasks after taking-up；
(4) seed culture medium of step (2) is inoculated on the potato by step (3) pre-treatment in an aseptic environment,
It is placed in incubator after cultivating 5-40d and takes out after capping.
(5) through microorganism Fusarium sp.B10.1 fermentation after potato culture medium by methanol ultrasonic extraction, filtering and
It is concentrated to give crude extract；The structural formula for the sambacide that separation crude extract obtains is as follows respectively：
(6) detect, then separated by silicagel column, separation process uses gradient elution, washes through TLC TLCs
De- agent is chloroform：Methanol (20:1—5:1) in the compound, being then isolated to gel column purify, by 1D/2D NMR with
And HRESIMS identifies to obtain new compound structure.
(7) antibacterial activity test is carried out to compound sambacide using two times of gradient dilution methods.As a result show：
Sambacide staphylococcus aureuses and Escherichia coli have significant bacteriostasis (MIC≤16 μ g/mL).
(8) using following HPLC chromatogram condition measure sambacide yield.
A, chromatographic column：ODS performance liquid chromatographic columns；
B, gradient elution program：0-10min, 30-60% acetonitriles；10-20min, 60% acetonitrile；20-25,60-30% second
Nitrile；
C, Detection wavelength：247nm；
D, flow velocity：1.5mL/min；
E, column temperature：25℃.
Embodiment 1
(1) will intend being activated first with microorganism sickle mycete Fusarium sp.B10.1, i.e., by Fusarium
After sp.B10.1 is inoculated into the PDA slant mediums by 121 DEG C of high-temperature sterilization processing, after cultivating 3d in constant incubator,
It is placed in 4 DEG C of refrigerators；
(2) strain of activation is connected in PDB seed culture mediums, 3d is cultivated in constant-temperature table；
(3) after taking clean potato to be diced, appropriate small pudding is taken to be placed in tissue culture flasks；By the group equipped with potato
The 30min that sterilizes is placed in 121 DEG C of high-temperature sterilization boxes after knitting blake bottle capping packaging, cools down tissue culture flasks after taking-up；
(4) seed culture medium of step (2) is inoculated on the potato by step (3) pre-treatment in an aseptic environment,
It is placed in incubator after cultivating 30d and takes out after capping；
(5) through microorganism Fusariumsp Fusarium sp.B10.1 fermentation after potato culture medium by methanol ultrasonic extraction,
Filter and be concentrated to give crude extract；The structural formula for the sambacide that separation crude extract obtains is as follows respectively：
(6) detect, then separated by silicagel column, separation process uses gradient elution, washes through TLC TLCs
De- agent is chloroform：Methanol (20:1—5:1) in the compound, being then isolated to gel column purify, by 1D/2D NMR with
And HRESIMS identifies to obtain compound structure；
(7) following HPLC chromatogram condition measure sambacide yield is used as 17.27 ± 0.52g/Kg
A, chromatographic column：ODS performance liquid chromatographic columns；
B, gradient elution program：0-10min, 30-60% acetonitriles；10-20min, 60% acetonitrile；20-25,60-30% second
Nitrile；
C, Detection wavelength：247nm；
D, flow velocity：1.5mL/min；
E, column temperature：25℃.
Embodiment 2
(1) will intend being activated first with microorganism sickle mycete Fusarium sp.B10.1, i.e., by Fusarium
After sp.B10.1 is inoculated into the PDA slant mediums by 121 DEG C of high-temperature sterilization processing, after cultivating 3d in constant incubator,
It is placed in 4 DEG C of refrigerators；
(2) strain of activation is connected in PDB seed culture mediums, 3d is cultivated in constant-temperature table；
(3) after taking clean potato to be diced, appropriate small pudding is taken to be placed in tissue culture flasks；By the group equipped with potato
The 30min that sterilizes is placed in 121 DEG C of high-temperature sterilization boxes after knitting blake bottle capping packaging, cools down tissue culture flasks after taking-up；
(4) seed culture medium of step (2) is inoculated on the potato by step (3) pre-treatment in an aseptic environment,
It is placed in incubator after cultivating 20d and takes out after capping.
(5) the potato culture medium after microorganism sickle mycete Fusarium sp.B10.1 fermentations carries by methanol ultrasound
Take, filter and be concentrated to give crude extract；The structural formula for the sambacide that separation crude extract obtains is as follows respectively：
(6) detect, then separated by silicagel column, separation process uses gradient elution, washes through TLC TLCs
De- agent is chloroform：Methanol (20:1—5:1) in the compound, being then isolated to gel column purify, by 1D/2D NMR with
And HRESIMS identifies to obtain compound structure.
(7) following HPLC chromatogram condition measure sambacide yield is used as 19.045 ± 0.819g/Kg
A, chromatographic column：ODS performance liquid chromatographic columns；
B, gradient elution program：0-10min, 30-60% acetonitriles；10-20min, 60% acetonitrile；20-25,60-30% second
Nitrile；
C, Detection wavelength：247nm；
D, flow velocity：1.5mL/min；
E, column temperature：25℃.
Embodiment 3
(1) will intend being activated first with microorganism sickle mycete Fusarium sp.B10.1, i.e., by Fusarium
After sp.B10.1 is inoculated into the PDA slant mediums by 121 DEG C of high-temperature sterilization processing, after cultivating 3d in constant incubator,
It is placed in 4 DEG C of refrigerators；
(2) strain of activation is connected in PDB seed culture mediums, 3d is cultivated in constant-temperature table；
(3) after taking clean potato to be diced, appropriate small pudding is taken to be placed in tissue culture flasks；By the group equipped with potato
The 30min that sterilizes is placed in 121 DEG C of high-temperature sterilization boxes after knitting blake bottle capping packaging, cools down tissue culture flasks after taking-up；
(4) seed culture medium of step (2) is inoculated on the potato by step (3) pre-treatment in an aseptic environment,
It is placed in incubator after cultivating 40d and takes out after capping.
(5) the potato culture medium after microorganism sickle mycete Fusarium sp.B10.1 fermentations carries by methanol ultrasound
Take, filter and be concentrated to give crude extract；The structural formula for the sambacide that separation crude extract obtains is as follows respectively：
(6) detect, then separated by silicagel column, separation process uses gradient elution, washes through TLC TLCs
De- agent is chloroform：Methanol (20:1—5:1) in the compound, being then isolated to gel column purify, by 1D/2D NMR with
And HRESIMS identifies to obtain compound structure.
(7) following HPLC chromatogram condition measure sambacide yield is used as 13.265 ± 13.265g/Kg
A, chromatographic column：ODS performance liquid chromatographic columns；
B, gradient elution program：0-10min, 30-60% acetonitriles；10-20min, 60% acetonitrile；20-25,60-30% second
Nitrile；
C, Detection wavelength：247nm；
D, flow velocity：1.5mL/min；
E, column temperature：25℃.
Claims (6)
1. a kind of fermented by solid state fermentation Fusariumsp Fusarium sp. B10.1 produces new anti-bacterial agent sambacide side
Method, it is characterised in that carry out solid fermentation as follows：
（1）It will intend being activated first with microorganism Fusariumsp Fusarium sp. B10.1, i.e., by Fusarium sp.
After B10.1 is inoculated into the PDA slant mediums by 121 °C of high-temperature sterilization processing, after cultivating 3-7 d in constant incubator,
It is placed in 4 °C of refrigerators；
（2）The strain of activation is connected in PDB seed culture mediums, 3-7 d are cultivated in constant-temperature table；
（3）After taking clean potato to be diced, appropriate small pudding is taken to be placed in tissue culture flasks；Tissue equipped with potato is trained
30 min that sterilize are placed in 121 °C of high-temperature sterilization boxes after supporting bottle capping packaging, cool down tissue culture flasks after taking-up；
（4）By step（2）Seed culture medium be inoculated into an aseptic environment by step（3）On the potato of pre-treatment, capping
After be placed in incubator cultivate 5-40 d after take out；
（5）Potato culture medium after microorganism Fusariumsp Fusarium sp. B10.1 fermentations is by methanol ultrasonic extraction, mistake
Filter and be concentrated to give crude extract；The structural formula for the sambacide that separation crude extract obtains is as follows：
；
（6）Detect, then separated by silicagel column, separation process uses gradient elution, eluant, eluent through TLC TLCs
For chloroform and carbinol mixture, wherein chloroform：Methanol presses 20:1—5:1 mixes, gel in the compound being then isolated to
Post purifies, and identifies to obtain new compound structure by 1D/2D NMR and HRESIMS；
（7）Using following HPLC chromatogram condition measure sambacide yield.
2. new antiseptic is produced by solid state fermentation Fusariumsp Fusarium sp. B10.1 fermentations according to claim 1
Sambacide method, it is characterised in that the fermentation process is solid state fermentation.
3. new antiseptic is produced by solid state fermentation Fusariumsp Fusarium sp. B10.1 fermentations according to claim 1
Sambacide method, it is characterised in that the fermentation time is 5-40 d.
4. new antiseptic is produced by solid state fermentation Fusariumsp Fusarium sp. B10.1 fermentations according to claim 1
Sambacide method, it is characterised in that the fermentation temperature is 20-30 °C.
5. new antiseptic is produced by solid state fermentation Fusariumsp Fusarium sp. B10.1 fermentations according to claim 1
Sambacide method, it is characterised in that the Extraction solvent is methanol.
6. new antiseptic is produced by solid state fermentation Fusariumsp Fusarium sp. B10.1 fermentations according to claim 1
Sambacide method, it is characterised in that carry out assay by following HPLC conditions：
（1）Chromatographic column：ODS performance liquid chromatographic columns；
（2）Gradient elution program：0-10 min, 30-60% acetonitriles;10-20 min, 60% acetonitrile; 20–25 min,
60-30% acetonitriles；
（3）Detection wavelength：247 nm；
（4）Flow velocity：1.5 mL/min；
（5）Column temperature：25 ℃.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201610421717.2A CN106117293B (en) | 2016-06-15 | 2016-06-15 | A kind of method that new anti-bacterial agent sambacide is produced by solid fermentation Fusariumsp Fusarium sp. |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201610421717.2A CN106117293B (en) | 2016-06-15 | 2016-06-15 | A kind of method that new anti-bacterial agent sambacide is produced by solid fermentation Fusariumsp Fusarium sp. |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| CN106117293A CN106117293A (en) | 2016-11-16 |
| CN106117293B true CN106117293B (en) | 2018-02-16 |
Family
ID=57270386
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN201610421717.2A Expired - Fee Related CN106117293B (en) | 2016-06-15 | 2016-06-15 | A kind of method that new anti-bacterial agent sambacide is produced by solid fermentation Fusariumsp Fusarium sp. |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN106117293B (en) |
Family Cites Families (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO2000036132A1 (en) * | 1998-12-14 | 2000-06-22 | Merck & Co., Inc. | Hiv integrase inhibitors |
| CN101880633B (en) * | 2010-06-25 | 2012-07-25 | 河北省农林科学院植物保护研究所 | Fusarium proliferatum, and bacterium agent and application thereof |
-
2016
- 2016-06-15 CN CN201610421717.2A patent/CN106117293B/en not_active Expired - Fee Related
Also Published As
| Publication number | Publication date |
|---|---|
| CN106117293A (en) | 2016-11-16 |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| CN101961014B (en) | Method for preparing microbial pesticide for preventing and controlling fungal diseases of vegetables | |
| CN104430407B (en) | Tetramycin P is suppressing the application in Rhizoctonia solani and botrytis cinerea | |
| CN103360351B (en) | Isopimarane diterpenoid compounds and application thereof | |
| Zhang et al. | Evaluation of antimicrobial activities of extracts of endophytic fungi from Artemisia annua | |
| CN104031960B (en) | Extracting method and application of extracellular antimicrobial lipopeptides of bacillus amyloliquefaciens | |
| CN104450580A (en) | Preparation method of actinomycin D and application thereof | |
| CN101223879B (en) | Method of preparing microorganisms pesticide for preventing vegetable fungi disease | |
| CN104450835B (en) | A kind of preparation method of compound | |
| CN101961013B (en) | Application of toyocamycin in controlling tomato gray mold | |
| CN106117293B (en) | A kind of method that new anti-bacterial agent sambacide is produced by solid fermentation Fusariumsp Fusarium sp. | |
| CN103864733B (en) | A kind of butenolide class meta-bolites and application thereof | |
| CN114621092A (en) | Phenolic compound in mangrove plant-derived fungi and preparation method thereof | |
| Encheva-Malinova et al. | Antimicrobial activity of Antarctic Streptomycetes against pepper bacterial spot causing agents | |
| CN105803015A (en) | Method for converting dicentrine into (4S,6aR)-4-hydroxyl dicentrine through Clonostachys sp. Fermentation | |
| CN101775357B (en) | Plant pathogenic fungi fusarium moniliforme natural antagonist | |
| CN101468977A (en) | Phomopsis lactone compounds | |
| KR101363163B1 (en) | Naramycin of suppression against bacterial blight of rice caused by bacterial pathogen | |
| CN108220198A (en) | It is a kind of that there is the marine actinomycete for inhibiting fungi activity | |
| CN103222481B (en) | Use of Streptomyces sioyaensis metabolite in prevention and control of Thanatephorus cucumeris | |
| CN103222478B (en) | Use of Streptomyces sioyaensis metabolite in prevention and control of Fusarium oxyspirum niveum | |
| CN103222480B (en) | Use of Streptomyces sioyaensis metabolite in prevention and control of Rhizoctonia solani | |
| CN106978457A (en) | A kind of antibiotic FusaricidinA preparation method | |
| CN108002999B (en) | Naphthalene compound and preparation method and application thereof | |
| CN108002998B (en) | Naphthalene compound and preparation method and application thereof | |
| CN101818172A (en) | Method for separating Wuyi rhzomorph antimicrobial active substance |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| C06 | Publication | ||
| PB01 | Publication | ||
| C10 | Entry into substantive examination | ||
| SE01 | Entry into force of request for substantive examination | ||
| GR01 | Patent grant | ||
| GR01 | Patent grant | ||
| CF01 | Termination of patent right due to non-payment of annual fee | ||
| CF01 | Termination of patent right due to non-payment of annual fee |
Granted publication date: 20180216 |