CN110004095A - A kind of preparation method and application of bacillus pumilus antibacterial substance - Google Patents
A kind of preparation method and application of bacillus pumilus antibacterial substance Download PDFInfo
- Publication number
- CN110004095A CN110004095A CN201910373888.6A CN201910373888A CN110004095A CN 110004095 A CN110004095 A CN 110004095A CN 201910373888 A CN201910373888 A CN 201910373888A CN 110004095 A CN110004095 A CN 110004095A
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- China
- Prior art keywords
- compound
- ethyl acetate
- bacillus pumilus
- follows
- antibacterial
- Prior art date
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- 230000000844 anti-bacterial effect Effects 0.000 title claims abstract description 56
- 241000194103 Bacillus pumilus Species 0.000 title claims abstract description 53
- 239000000126 substance Substances 0.000 title claims abstract description 23
- 238000002360 preparation method Methods 0.000 title claims abstract description 18
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- -1 fatty alcohols compounds Chemical class 0.000 claims abstract description 6
- XEKOWRVHYACXOJ-UHFFFAOYSA-N Ethyl acetate Chemical compound CCOC(C)=O XEKOWRVHYACXOJ-UHFFFAOYSA-N 0.000 claims description 158
- OKKJLVBELUTLKV-UHFFFAOYSA-N Methanol Chemical compound OC OKKJLVBELUTLKV-UHFFFAOYSA-N 0.000 claims description 72
- RTZKZFJDLAIYFH-UHFFFAOYSA-N Diethyl ether Chemical compound CCOCC RTZKZFJDLAIYFH-UHFFFAOYSA-N 0.000 claims description 64
- 150000001875 compounds Chemical class 0.000 claims description 33
- HTSGKJQDMSTCGS-UHFFFAOYSA-N 1,4-bis(4-chlorophenyl)-2-(4-methylphenyl)sulfonylbutane-1,4-dione Chemical compound C1=CC(C)=CC=C1S(=O)(=O)C(C(=O)C=1C=CC(Cl)=CC=1)CC(=O)C1=CC=C(Cl)C=C1 HTSGKJQDMSTCGS-UHFFFAOYSA-N 0.000 claims description 26
- NLFBCYMMUAKCPC-KQQUZDAGSA-N ethyl (e)-3-[3-amino-2-cyano-1-[(e)-3-ethoxy-3-oxoprop-1-enyl]sulfanyl-3-oxoprop-1-enyl]sulfanylprop-2-enoate Chemical compound CCOC(=O)\C=C\SC(=C(C#N)C(N)=O)S\C=C\C(=O)OCC NLFBCYMMUAKCPC-KQQUZDAGSA-N 0.000 claims description 26
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- 239000007836 KH2PO4 Substances 0.000 claims description 3
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- 229940041514 candida albicans extract Drugs 0.000 claims description 3
- ZPWVASYFFYYZEW-UHFFFAOYSA-L dipotassium hydrogen phosphate Chemical compound [K+].[K+].OP([O-])([O-])=O ZPWVASYFFYYZEW-UHFFFAOYSA-L 0.000 claims description 3
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- QTBSBXVTEAMEQO-UHFFFAOYSA-N Acetic acid Chemical compound CC(O)=O QTBSBXVTEAMEQO-UHFFFAOYSA-N 0.000 claims 3
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Classifications
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- A01N31/00—Biocides, pest repellants or attractants, or plant growth regulators containing organic oxygen or sulfur compounds
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- A01N35/00—Biocides, pest repellants or attractants, or plant growth regulators containing organic compounds containing a carbon atom having two bonds to hetero atoms with at the most one bond to halogen, e.g. aldehyde radical
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Abstract
The present invention relates to a kind of preparation method and applications of bacillus pumilus antibacterial substance.One bacillus pumilus (Bacillus pumilus) BP, the bacterial strain is stored in China Committee for Culture Collection of Microorganisms's common micro-organisms center on March 21st, 2019, address: Yard 1, BeiChen xi Road, Chaoyang District, Beijing City 3, Institute of Microorganism, Academia Sinica, deposit number CGMCC NO.17424；The invention further relates to prepare antibacterial substance and relevant application using the bacterial strain.The present invention extracts three kinds of fatty alcohols compounds with antibacterial activity from bacillus pumilus (Bacillus pumilus) for the first time, and above-mentioned three kinds of antibacterial substances are not found in existing bacillus pumilus.
Description
Technical field
The present invention relates to a kind of preparation method and applications of bacillus pumilus antibacterial substance, belong to microbial technique
And applied technical field.
Background technique
Currently, a large amount of uses of livestock and poultry breeding industry antibiotic, lead to drug resistance generation and residue of veterinary drug, have seriously affected poultry
Product quality, excretion residue also lead to soil and water pollution.Ecological environment is destroyed, human health is directly endangered.Environment is dirty
Dye causes the deterioration of environment for human survival, and food-safety problem is caused to become increasingly conspicuous.
Antibacterial substance is the research hotspot of recent domestic, can be widely applied to biological feedstuff, biological and ecological methods to prevent plant disease, pests, and erosion, medicine,
Antiseptics for natural food, cosmetics etc..Wherein bacillus can produce a variety of antibacterial materials, and Antibacterial Mechanism is unique, right
Gram-negative bacteria, gram positive bacteria, mould and saccharomycete have inhibiting effect, have broad-spectrum antiseptic, efficiently, safely, be not easy to produce
Raw drug resistance, it is degradable the features such as, since the report bacillus subtilis such as Johnson can produce antibacterial substance, both at home and abroad
Scholar successively isolated a variety of antibacterial substances, including polypeptide, lipopeptid class and Antagonistic protein class etc. from different bacillus.
Bacillus pumilus is one kind of bacillus, is widely present in nature, microscopically observation nutrition is thin
Born of the same parents be it is rod-shaped, Gram-reaction is positive, can move.Colonial morphology is divided into two kinds of translucent and opaque shape.Short and small gemma bar
Bacterium can the very strong cellulase of secretion activity, lipase, zytase and pectin lyase etc., be conducive to macromolecular complex of degrading
Matter.The Antagonisms substance such as antibiotic and antibacterial protein can also be generated, scope of restraining fungi is wide, has inhibition to make a variety of pathogens
With.
Currently, it is domestic about the relevant technologies of production bacillus pumilus class product, such as: Chinese patent literature
CN103563991A (application number 201210271396.4) discloses rice blast biocontrol bacteria Brevibacillus brevis fungicide
Dosage form of water dispersible granule and preparation method.The water dispersible granules of the solid include Brevibacillus brevis TW bacterial strain original powder (2 ×
1012Cfu/g) and auxiliary agent, auxiliary agent include carrier, dispersing agent, wetting agent, disintegrating agent, bright protective agent and binder；Its processing method
It is to mix effective component and auxiliary agent probe's certain proportion, through air-flow crushing, granulation, dry acquisition.
Chinese patent literature CN105494441A (application number 200710021509.1) discloses a kind of containing gemma sprouting
Wettable powder of bacillus pumilus of agent and preparation method thereof, the raw material of the wettable powder is by following composition by weight group
At: 40-70 parts of bacillus pumilus fermentation liquid；Filler: 20-30 parts of bentonite；Wetting agent: 1-4 parts of polyethylene glycol；Dispersing agent:
0.2-1 parts of lauryl sodium sulfate；Gemma germinant: 0.1-0.5 parts of l-Alanine, 2,0.1-0.5 parts of dipicolimic acid 2 calcium；
Uv-protector: 0.5-2 parts of carboxymethyl cellulose, 0.2-1 parts of powder-beta-dextrin；Drying protectant: 1-4 parts of maltose, sodium glutamate
0.2-1 parts.
Chinese patent literature CN103205383A (application number 201310128271.0) discloses a kind of bacillus pumilus
(Bacillus pumilus)E14CGMCC No.6682.The invention also discloses the cultural method of bacillus pumilus E14,
Step: by bacillus pumilus E14 strain inoculated into 2216E fluid nutrient medium, 28 DEG C, 150rpm culture 20h to get bacterium
Liquid.The bacterium solution that bacillus pumilus E14 of the present invention or the method culture obtain has inhibition Vibrio harveyi
The bacteriostatic application of (Vibrio harveyi) can be used for the preparation of shrimp crab cultivation drug, or for adding as antibacterial medicines
It is added in shrimp crab breeding feed.
Chinese patent literature CN102965299A (application number 201210290374.2) discloses a bacillus pumilus
The zymotechnique of LD-b1 and its in prevention and treatment Cucumber Target Leaf Spot, gray mold of cucumber, cucumber timberrot, canker of apple fruit, eggplant are withered
Disease, the red viral disease of wheat, the application on Cabbage Leaf Spot and graw mold of tomato.Curly hair is refined etc. to have investigated bacillus pumilus fermentation liquid
Influence to Tomato Seeds Germination and growth of seedling, the results showed that bacillus pumilus fermentation liquid is to Tomato Seeds Germination, radical bud
The plant height and plant weight of growth and seedling have apparent facilitation, wherein it is the most significant with the effect of 50 times of dilutions, and
The effect of foliage-spray is substantially better than pouring root and seed soaking in seedling method of application.
Chinese patent literature CN105494441A (application number 201510951106.4) discloses a kind of containing gemma sprouting
Wettable powder of bacillus pumilus of agent and preparation method thereof, the raw material of the wettable powder is by following composition by weight group
At: 40-70 parts of bacillus pumilus fermentation liquid；Filler: 20-30 parts of bentonite；Wetting agent: 1-4 parts of polyethylene glycol；Dispersing agent:
0.2-1 parts of lauryl sodium sulfate；Gemma germinant: 0.1-0.5 parts of l-Alanine, 2,0.1-0.5 parts of dipicolimic acid 2 calcium；
Uv-protector: 0.5-2 parts of carboxymethyl cellulose, 0.2-1 parts of powder-beta-dextrin；Drying protectant: 1-4 parts of maltose, sodium glutamate
0.2-1 parts.
The focus of above-mentioned technology is placed on controlling plant diseases more and promotes plant growth direction, not to short and small gemma bar
The relevant report that the active material of bacterium is further studied.
Summary of the invention
In view of the deficiencies of the prior art, the present invention provides a kind of preparation methods of bacillus pumilus antibacterial substance
And application.
Technical solution of the present invention is as follows:
One bacillus pumilus (Bacillus pumilus) BP, during which has been stored on March 21st, 2019
State's Microbiological Culture Collection administration committee common micro-organisms center, address: Yard 1, BeiChen xi Road, Chaoyang District, Beijing City 3, in
Institute of microbiology, the academy of sciences, state, deposit number CGMCC NO.17424.
The cultural method of above-mentioned bacillus pumilus, steps are as follows:
(1) above-mentioned bacillus pumilus is inoculated in seed culture medium, in 35~38 DEG C, 160~200 revs/min of item
It under part, cultivates 14~16 hours, seed liquor is made；
(2) seed liquor made from step (1) is seeded in fermentation medium in the ratio of percent by volume 3~5%,
28~32 DEG C, 160~200 revs/min of 45~50h of culture, fermentation liquid is made；
The fermentation medium, every liter of component are as follows:
Glucose 30.0g, K2HPO4×3H2O 7.0g, KH2PO43.0g, (NH4)2SO41.5g, sodium citrate 3H2O
0.5g, MgSO4·7H2O 0.1g, pH7.0~7.2 add water to be settled to 1L.
Preferred according to the present invention, in the step (1), seed culture medium is LB broth bouillon, and every liter of component is as follows:
10 grams of peptone, 10 grams of sodium chloride, 5 grams of yeast extract, water is settled to 1000 milliliters.
From the antibacterial substance of bacillus pumilus, structural formula difference is as follows:
Compound I, title are as follows: 6,7- (Alpha-hydroxy) diethyldodecanes, English name: 6,7- (α-hydroxy)-
Diethyl-dodecane:
Compound II, title are as follows: 4,5- dibutyl -1,2- (Alpha-Methyl)-cyclohexanedimethanol, English name: 4,5-
Dibutyl-1,2- (α-methyl)-cyclohexanedimethanol:
Compound III, title are as follows: 4,5- dibutyl -1,2- (Alpha-Methyl)-hexamethylene Ketene dimethyl, English name: 4,5-
Dibutyl-1,2- (α-methyl)-cyclohexanedimethanol:
The preparation method of the above-mentioned antibacterial substance from bacillus pumilus, steps are as follows:
(i) above-mentioned fermentation liquid is separated by solid-liquid separation, takes supernatant, it is concentrated, concentrate is made；
(ii) concentrate made from step (ii) is extracted through ethyl acetate, extraction phase is taken, through silica gel column chromatography after concentration
Separation, respectively use petroleum ether, ethyl acetate, methanol different solutions difference gradient elution, collect have antibacterial activity petroleum ether/
Ethyl acetate=50:50 eluent is concentrated, dry, and concentrated dry thing is made；
(iii) antibacterial substance point is made through high performance liquid chromatography separation in concentrated dry thing made from step (ii)
It Wei not above compound I, compound II, compound III.
It is preferred according to the present invention, in the step (i), be separated by solid-liquid separation under conditions of 3500~4500 revs/min from
The heart 25~35 minutes.
It is preferred according to the present invention, in the step (i), it is concentrated to be concentrated into the 8 of original solution concentration using rotary evaporation
~12 times.
Preferred according to the present invention, in the step (ii), the volume ratio of concentrate and ethyl acetate is 1:1；Preferably,
Extraction times are 2~4 times.
Preferred according to the present invention, in the step (ii), silicagel column is the silicagel column of 200~300 mesh.
It is preferred according to the present invention, in the step (ii), petroleum ether, ethyl acetate, methanol different solutions difference gradient
It is specific as follows, be volume ratio:
Petroleum ether, petrol ether/ethyl acetate=75:25, petrol ether/ethyl acetate=50:50；Petrol ether/ethyl acetate
=25:75；Petrol ether/ethyl acetate=0:100；Ethyl acetate/methanol=75:25；Ethyl acetate/methanol=50:50；Acetic acid
Ethyl ester/methanol=25:75, ethyl acetate/methanol=0:100.
Preferred according to the present invention, in the step (ii), dry is to blow drying mode using nitrogen.
Preferred according to the present invention, in the step (iii), high performance liquid chromatography separation condition is as follows:
Column is prepared using YMC-Pack SIL column, 250mm × 10mm；The ethyl acetate and 40% that mobile phase is 60% is just
Butanol, wavelength 260nm, 5 μ L of sample volume, flow velocity 2.0mL/min, column temperature is 25 DEG C, pressure 40bar.
The above-mentioned compound I with antibacterial activity, compound II, compound III inhibit in pathogenic bacteria drug in preparation
Using.
The above-mentioned compound I with antibacterial activity, compound II, compound III are preparing the application in food preservative.
The above-mentioned compound I with antibacterial activity, compound II, compound III are preparing the application in antibacterial cosmetic.
The above-mentioned compound I with antibacterial activity, compound II, compound III are in the application for preparing antibiotic health care product.
The above-mentioned compound I with antibacterial activity, compound II, compound III are preparing the application in pesticide.
The above-mentioned compound I with antibacterial activity, compound II, compound III are preparing the application in antibacterial chewing gum.
Beneficial effect
1, present invention firstly discovers that a bacillus pumilus (Bacillus pumilus) BP, the bacterial strain can be generated
Three kinds of fatty alcohols compounds with antibacterial activity, and above-mentioned three kinds of antibacterial activities are not found in existing bacillus pumilus
Substance；
2, present invention firstly discloses the compound that three kinds of enol classes have antibacterial activity, such compound has very strong
Inhibit the effect of pathogenic bacteria, and prepare that with short production cycle, product is stable, content is high using microbial fermentation, which can be wide
It is general to be applied in pesticide, medicine, health care product and Cosmetic Manufacture.
Detailed description of the invention
Fig. 1 is the Mass Spectrometer Method result figure of compound I；
The hydrogen that Fig. 2 is compound I composes testing result figure；
The carbon that Fig. 3 is compound I composes testing result figure；
Fig. 4 is the Mass Spectrometer Method result figure of compound II；
The hydrogen that Fig. 5 is compound II composes testing result figure；
The carbon that Fig. 6 is compound II composes testing result figure；
Fig. 7 is the Mass Spectrometer Method result figure of compound III；
The hydrogen that Fig. 8 is compound III composes testing result figure；
The carbon that Fig. 9 is compound III composes testing result figure；
Figure 10 is the bacteriostatic activity test result photo of concentrated dry thing made from 3 step of embodiment (ii)；
Figure 11 is the bacteriostatic activity test result photo of three kinds of compounds made from embodiment 3；
Wherein: 1, the inhibition zone of compound I；2, the inhibition zone of compound II；3, the inhibition zone of compound III；
Figure 12 is the antibacterial result curve figure that compound I is directed to different strains；
Figure 13 is the antibacterial result curve figure that compound II is directed to different strains；
Figure 14 is the antibacterial result curve figure that compound III is directed to different strains；
Specific embodiment
To further explain the present invention, below in conjunction with specific embodiment to technical solutions according to the invention into
Row is more detailed to be introduced, but embodiment is merely to illustrate, and does not have any restrictions to the present invention, based on present invention teach that made
What is replaced or transformation, all belongs to the scope of protection of the present invention.
Biomaterial
Bacillus pumilus (Bacillus pumilus) BP, it is micro- which has been stored in China on March 21st, 2019
Biological inoculum preservation administration committee common micro-organisms center (abbreviation CGMCC, address: Yard 1, BeiChen xi Road, Chaoyang District, Beijing City
No. 3, Institute of Microorganism, Academia Sinica), deposit number is CGMCC NO.17424.
Bacillus pumilus BP-5 is purchased from Chinese microorganism strain preservation, number: CGMCC 1.8167.
Embodiment 1
One bacillus pumilus (Bacillus pumilus) BP, during which has been stored on March 21st, 2019
State's Microbiological Culture Collection administration committee common micro-organisms center (abbreviation CGMCC, address: BeiChen West Road, Chaoyang District, BeiJing City 1
Number institute 3, Institute of Microorganism, Academia Sinica), deposit number is CGMCC NO.17424.
The biological property of the bacterial strain: for bacillus, thallus thin rod shape, Gram-positive.It is grown on the inclined-plane LB
For transparence, Antibacterial Constituents can be generated.
Embodiment 2
The cultural method of short (Bacillus pumilus) BP of bacillus pumilus, steps are as follows:
(1) above-mentioned bacillus pumilus is inoculated in seed culture medium, under conditions of 37 DEG C, 180 revs/min, culture
16 hours, seed liquor is made；
The seed culture medium is LB broth bouillon, and every liter of component is as follows:
10 grams of peptone, 10 grams of sodium chloride, 5 grams of yeast extract, water is settled to 1000 milliliters；
(2) seed liquor made from step (1) is seeded in fermentation medium in the ratio of percent by volume 3%, 30
DEG C, fermentation liquid is made in 180 revs/min of culture 48h；
The fermentation medium, every liter of component are as follows:
Glucose 30.0g, K2HPO4×3H2O 7.0g, KH2PO43.0g, (NH4)2SO41.5g, sodium citrate 3H2O
0.5g, MgSO4·7H2O 0.1g, pH7.0~7.2 add water to be settled to 1L.
Embodiment 3
From the preparation method of the antibacterial substance of bacillus pumilus (Bacillus pumilus) BP, step is such as
Under:
(i) fermentation liquid for preparing embodiment 2 is centrifuged 35 minutes under conditions of 3500 revs/min, supernatant is taken, using rotation
Turn evaporation, be concentrated into 8 times of original solution concentration, concentrate is made；
(ii) concentrate made from step (ii) is extracted through ethyl acetate, the volume ratio of concentrate and ethyl acetate is 1:
1, take extraction phase, extract 4 times, merge extraction phase, concentration, the then silica gel column chromatography separation through 200 mesh, respectively with petroleum ether,
Ethyl acetate, methanol different solutions difference gradient elution, collecting, there is petrol ether/ethyl acetate=50:50 of antibacterial activity to wash
De- liquid, concentration, nitrogen drying is dry, and concentrated dry thing is made；
The petroleum ether, ethyl acetate, methanol different solutions difference gradient are specific as follows, are volume ratio: petroleum ether, stone
Oily ether/ethyl acetate=75:25, petrol ether/ethyl acetate=50:50；Petrol ether/ethyl acetate=25:75；Petroleum ether/second
Acetoacetic ester=0:100；Ethyl acetate/methanol=75:25；Ethyl acetate/methanol=50:50；Ethyl acetate/methanol=25:75,
Ethyl acetate/methanol=0:100；
Escherichia coli bacteriostatic activity is detected using agar diffusion method mode, as shown in Figure 10；
(iii) antibacterial substance point is made through high performance liquid chromatography separation in concentrated dry thing made from step (ii)
It Wei not above compound I, compound II, compound III；
The high performance liquid chromatography separation condition is as follows:
Column is prepared using YMC-Pack SIL column, 250mm × 10mmI.D, S-5 μm, 12nm, SL 12S-2510WT,
Ser.No.114EA80134；Mobile phase is 60% ethyl acetate (Eta-00060458HPLC) and 40% n-butanol (a-
He-00010273HPLC), wavelength 260nm, 5 μ L of sample volume, flow velocity 2.0mL/min, column temperature is 25 DEG C, pressure 40bar.
Mass spectrum, hydrogen spectrum, carbon spectrum detection, testing result such as Fig. 1-are carried out to compound I, compound II, compound III respectively
Shown in 9；Compound I, title are obtained by the above results are as follows: 6,7- (Alpha-hydroxy) diethyldodecanes, English name: 6,7-
(α-hydroxy)-diethyl-dodecane, structural formula are as follows:
Compound II, title are as follows: 4,5- dibutyl -1,2- (Alpha-Methyl)-cyclohexanedimethanol, English name: 4,5-
Dibutyl-1,2- (a-methyl)-cyclohexanedimethanol, structural formula are as follows:
Compound III, title are as follows: 4,5- dibutyl -1,2- (Alpha-Methyl)-hexamethylene Ketene dimethyl, English name: 4,5-
Dibutyl-1,2- (a-methyl)-cyclohexanedimethanol, structural formula are as follows:
Carry out the bacteriostatic experiment of Escherichia coli respectively to above-mentioned three kinds of compounds, test compound concentrations are respectively 2.6mg/
ML, 3mg/mL, 3.2mg/mL are diluted according to experiment, as a result as shown in figure 11.
Embodiment 4
From the preparation method of the antibacterial substance of bacillus pumilus (Bacillus pumilus), step is such as
Under:
(i) fermentation liquid for preparing embodiment 2 is centrifuged 25 minutes under conditions of 4500 revs/min, supernatant is taken, using rotation
Turn evaporation, be concentrated into 12 times of original solution concentration, concentrate is made；
(ii) concentrate made from step (ii) is extracted through ethyl acetate, the volume ratio of concentrate and ethyl acetate is 1:
1, take extraction phase, extract 2 times, merge extraction phase, concentration, the then silica gel column chromatography separation through 200 mesh, respectively with petroleum ether,
Ethyl acetate, methanol different solutions difference gradient elution, collecting, there is petrol ether/ethyl acetate=50:50 of antibacterial activity to wash
De- liquid, concentration, nitrogen drying is dry, and concentrated dry thing is made；
The petroleum ether, ethyl acetate, methanol different solutions difference gradient are specific as follows, are volume ratio: petroleum ether, stone
Oily ether/ethyl acetate=75:25, petrol ether/ethyl acetate=50:50；Petrol ether/ethyl acetate=25:75；Petroleum ether/second
Acetoacetic ester=0:100；Ethyl acetate/methanol=75:25；Ethyl acetate/methanol=50:50；Ethyl acetate/methanol=25:75,
Ethyl acetate/methanol=0:100；
(iii) antibacterial substance point is made through high performance liquid chromatography separation in concentrated dry thing made from step (ii)
It Wei not above compound I, compound II, compound III；
The high performance liquid chromatography separation condition is as follows:
Column is prepared using YMC-Pack SIL column, 250mm × 10mm；The ethyl acetate and 40% that mobile phase is 60% is just
Butanol, wavelength 260nm, 5 μ L of sample volume, flow velocity 2.0mL/min, column temperature is 25 DEG C, pressure 40bar.
Three kinds of compounds of separation are detected, as a result with embodiment 3.
Comparative example 1
Using existing bacillus pumilus (BP-5), it is purchased from Chinese microorganism strain preservation, is numbered:
CGMCC1.8167。
It is prepared according to the method for embodiment 2-3, is not as a result obtained relevant compound I~III (fatty alcohols chemical combination
Object).
Experimental example
Antibacterial test: the OD by detecting pathogenic bacteria different periods600It measures the pathogenic bacteria growing state, sentences
It is disconnected whether to there is fungistatic effect.Specific steps are as follows:
Pathogenic bacterium solution is configured to 10 before test5Cfu/mL, on superclean bench, according to test dosage, in each EP
100 μ L pathogenic bacteria bacterium solutions are added in pipe, high speed centrifugation 1min, removal supernatant retains thallus, and adds the fresh LB broth cultivation of 900 μ L
Base is supported, marks and corresponds to 100 μ L samples to be tested of addition, mix.Respectively 0 hour and 15 hours, OD is measured600, record data.
Every group of test is repeated 3 times.
Through detecting, compound I is as shown in figure 12 for the antibacterial result of different strains；Compound II is for different strains
Antibacterial result is as shown in figure 13；Compound III is as shown in figure 14 for the antibacterial result of different strains.
Claims (10)
1. a bacillus pumilus (Bacillus pumilus) BP, the bacterial strain are stored in China on March 21st, 2019
Microbiological Culture Collection administration committee common micro-organisms center, address: Yard 1, BeiChen xi Road, Chaoyang District, Beijing City 3, China
Institute of microbiology, the academy of sciences, deposit number CGMCC NO.17424.
2. the cultural method of bacillus pumilus as described in claim 1, which is characterized in that steps are as follows:
(1) above-mentioned bacillus pumilus is inoculated in seed culture medium, under conditions of 35~38 DEG C, 160~200 revs/min,
Seed liquor is made in culture 14~16 hours；
(2) seed liquor made from step (1) is seeded in fermentation medium in the ratio of percent by volume 3~5%, 28~
32 DEG C, 160~200 revs/min of 45~50h of culture, fermentation liquid is made；
The fermentation medium, every liter of component are as follows:
Glucose 30.0g, K2HPO4×3H2O 7.0g, KH2PO43.0g, (NH4)2SO41.5g, sodium citrate 3H2O
0.5g, MgSO4·7H2O 0.1g, pH7.0~7.2 add water to be settled to 1L.
3. cultural method as claimed in claim 2, which is characterized in that in the step (1), seed culture medium is LB broth cultivation
Base is supported, every liter of component is as follows:
10 grams of peptone, 10 grams of sodium chloride, 5 grams of yeast extract, water is settled to 1000 milliliters.
4. from the antibacterial substance of bacillus pumilus (Bacillus pumilus) BP described in claim 1, structure
Formula difference is as follows:
Compound I, title are as follows: 6,7- (Alpha-hydroxy) diethyldodecanes:
Compound II, title are as follows: 4,5- dibutyl -1,2- (Alpha-Methyl)-cyclohexanedimethanol:
Compound III, title are as follows: 4,5- dibutyl -1,2- (Alpha-Methyl)-hexamethylene Ketene dimethyl:
5. from the preparation method of the antibacterial substance of bacillus pumilus described in claim 4, which is characterized in that step
It is as follows:
(i) above-mentioned fermentation liquid is separated by solid-liquid separation, takes supernatant, it is concentrated, concentrate is made；
(ii) concentrate made from step (ii) is extracted through ethyl acetate, takes extraction phase, is separated after concentration through silica gel column chromatography,
Petroleum ether, ethyl acetate, methanol different solutions difference gradient elution are used respectively, collect the petroleum ether/acetic acid with antibacterial activity
Ethyl ester=50:50 eluent is concentrated, dry, and concentrated dry thing is made；
(iii) by concentrated dry thing made from step (ii) through high performance liquid chromatography separation, obtained antibacterial substance is respectively
Above compound I, compound II, compound III.
6. preparation method as claimed in claim 5, which is characterized in that in the step (i), be separated by solid-liquid separation for 3500~
It is centrifuged 25~35 minutes under conditions of 4500 revs/min；
Preferably, it in the step (i), is concentrated to be concentrated into 8~12 times of original solution concentration using rotary evaporation.
7. preparation method as claimed in claim 5, which is characterized in that in the step (ii), concentrate and ethyl acetate
Volume ratio is 1:1；Preferably, extraction times are 2~4 times；
Preferably, in the step (ii), silicagel column is the silicagel column of 200~300 mesh；
Preferably, in the step (ii), it is body that petroleum ether, ethyl acetate, methanol different solutions difference gradient are specific as follows
Product ratio:
Petroleum ether, petrol ether/ethyl acetate=75:25, petrol ether/ethyl acetate=50:50；Petrol ether/ethyl acetate=25:
75；Petrol ether/ethyl acetate=0:100；Ethyl acetate/methanol=75:25；Ethyl acetate/methanol=50:50；Ethyl acetate/
Methanol=25:75, ethyl acetate/methanol=0:100；
Preferably, in the step (ii), dry is to blow drying mode using nitrogen.
8. preparation method as claimed in claim 5, which is characterized in that in the step (iii), high performance liquid chromatography separation item
Part is as follows:
Column is prepared using YMC-Pack SIL column, 250mm × 10mm；Mobile phase is 60% ethyl acetate and 40% positive fourth
Alcohol, wavelength 260nm, 5 μ L of sample volume, flow velocity 2.0mL/min, column temperature is 25 DEG C, pressure 40bar.
9. the compound I, compound II, compound III described in claim 4 with antibacterial activity inhibit pathogenic bacteria medicine in preparation
Application in object；
Preferably, the compound I with antibacterial activity, compound II, compound III are preparing answering in food preservative
With.
10. compound I, compound II, the compound III described in claim 4 with antibacterial activity are preparing antibacterial cosmetic
In application；
Preferably, the compound I with antibacterial activity, compound II, compound III are preparing answering for antibiotic health care product
With；
Preferably, the compound I with antibacterial activity, compound II, compound III are preparing the application in pesticide；
Preferably, the compound I with antibacterial activity, compound II, compound III are preparing answering in antibacterial chewing gum
With.
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| CN110612979A (en) * | 2019-10-12 | 2019-12-27 | 扬州大学 | Disease-resistant growth-promoting composite bacillus water dispersible granule as well as preparation method and application thereof |
| CN111012879A (en) * | 2019-12-16 | 2020-04-17 | 苏州市多爱迷贸易有限公司 | Vaginal mucosa antibacterial gel and preparation method thereof |
| WO2020207048A1 (en) * | 2019-04-10 | 2020-10-15 | 山东省科学院生物研究所 | Preparation method for and application of bacillus pumilus antibacterial active substance |
| CN117305163A (en) * | 2023-09-26 | 2023-12-29 | 中国科学院兰州化学物理研究所 | Bacillus pumilus with plant growth regulating activity, and preparation and application thereof |
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| CN112708646A (en) * | 2020-12-28 | 2021-04-27 | 昆明理工大学 | Preparation method of Pyrophen compound |
| CN113105428B (en) * | 2021-03-10 | 2022-05-17 | 宁波大学 | Xanthone compound and preparation method and application thereof |
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| WO2020207048A1 (en) * | 2019-04-10 | 2020-10-15 | 山东省科学院生物研究所 | Preparation method for and application of bacillus pumilus antibacterial active substance |
| CN110612979A (en) * | 2019-10-12 | 2019-12-27 | 扬州大学 | Disease-resistant growth-promoting composite bacillus water dispersible granule as well as preparation method and application thereof |
| CN111012879A (en) * | 2019-12-16 | 2020-04-17 | 苏州市多爱迷贸易有限公司 | Vaginal mucosa antibacterial gel and preparation method thereof |
| CN117305163A (en) * | 2023-09-26 | 2023-12-29 | 中国科学院兰州化学物理研究所 | Bacillus pumilus with plant growth regulating activity, and preparation and application thereof |
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