CN105087726B - A method of preparing beauvericin - Google Patents
A method of preparing beauvericin Download PDFInfo
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- CN105087726B CN105087726B CN201410211035.XA CN201410211035A CN105087726B CN 105087726 B CN105087726 B CN 105087726B CN 201410211035 A CN201410211035 A CN 201410211035A CN 105087726 B CN105087726 B CN 105087726B
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- round
- beauvericin
- solid fermentation
- maize pulp
- fusarium
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Abstract
The invention discloses a kind of methods preparing beauvericin.This prepares the method for beauvericin, is included in solid fermentation layer in the round equipped with maize pulp culture medium and goes out fusarium (Fusarium proliferatum) CGMCC3.1777, collects the step of solid fermentation product obtains beauvericin；The maize pulp culture medium is the solid medium that water content is 50% (mass percentage) made of water and maize pulp；The solid fermentation will be carried out in the round is placed in 24 DEG C 28 DEG C, relative humidity is 50% 100% environment.The beauvericin yield of this method reaches 3405mg/g solid fermentation products.
Description
Technical field
The present invention relates to a kind of methods preparing beauvericin in biotechnology.
Background technology
Beauvericin is a kind of six-membered cyclic contracting ester peptide compounds, by 3 methylphenylalanines and (D)-Alpha-hydroxy isoamyl
Sour residue, which is alternately connected, to be formed, molecular formula C45H57N3O9, molecular weight 786.Beauvericin is the secondary metabolite of fungi,
Hamil was extracted from the mycelium of beauveria bassiana in 1969 and is obtained for the first time.Up to now, from Beauveria
Isolated beauvericin in bassiana and several other fungies.Beauvericin is to larvae, artemia, blowfly
There is lethal effect with coleopteran pest etc..Currently, beauvericin is mainly used as insecticide and bacteria remover etc..
In recent years, more and more research shows that beauvericin also has many bioactivity other than desinsection.2000
Year, Nilanonta reports the treating tuberculosis of its homologue and the activity of plasmodium.2004, Fukuda was from Beauveria
Isolated beauvericin and its 3 homologues in sp.FKI-1366, and carried out anti-Candida with Miconazole cooperation
The active measurement of albicans, the results showed that by coordinating beauvericin to use Miconazole, common white vacation silk can not only be inhibited
Yeast, and can effectively inhibit the candida albicans for having drug resistance to Miconazole.2004, Jow etc. had found beauvericin energy
Enough induce the apoptosis of human leukemia cell.2005, the mechanism that Lin etc. induces human lung carcinoma cell apoptosis to beauvericin carried out
Research.However, beauvericin is more toxic mammal, the health of animals or humans can be influenced, mouse and the mankind can be caused
The significant cell death of tumor cell line, and cholesterol acyltransferase is lived in potent and special inhibition mouse liver microbody
Property.2003, Dombrink-Kurtzman also found that beauvericin can induce withering for turkey peripheral blood medium size lymphocyte
It dies, to influence its immune system, the toxicity of beauvericin significantly limits its application.
The ketoconazole of beauvericin and low dosage generates interaction effect, not only substantially increases antimicrobial efficiency (especially to resistance to
Medicine bacterium), while also increasing the antimicrobial spectrum of antimicrobial agent and reducing toxic side effect, and be antifungal pathogen
(fungicidal) non-inhibited (fungistasis).
Based on the above advantage, beauvericin has the potential ability as drug.It is used in order to prepare beauvericin
Later research needs the yield level for improving beauvericin.
Invention content
Technical problem to be solved by the invention is to provide a kind of method preparing beauvericin, the beauvericins of this method
Yield is high.
The method provided by the present invention for preparing beauvericin is included in the round equipped with maize pulp culture medium solid
Body fermentation layer goes out fusarium (Fusarium proliferatum) CGMCC3.1777, collects solid fermentation product and obtains beauvericin
The step of；The maize pulp culture medium is that the solid that the water content made of water and maize pulp is 50% (mass percentage) is trained
Support base；By the round be placed in 24 DEG C -28 DEG C, relative humidity be 50%-100% environment in carry out the solid hair
Ferment.
In the above method, the maize pulp is that corn kernel is crushed to 1mm3The corn kernel crushed material that particle obtains.
In the above method, the solid fermentation can carry out 20 days.
In the above method, the solid fermentation, which is included in, to be accessed the layer in the maize pulp culture medium and goes out fusarium
The round is shaken in the 1st day of (Fusarium proliferatum) CGMCC3.1777 makes training in the round
Object mixing is supported, the layer is accessed in the maize pulp culture medium and goes out fusarium (Fusarium proliferatum)
The 4th day, the 8th day and the 16th day of CGMCC3.1777 carries out following steps：Shaking the round makes the round
Interior culture is broken (to be crushed to 8mm3-25mm3Block, wherein 8mm3-15mm3The volume of block account for the 8mm3-
25mm3The 70% of block total volume is more than 15mm3To 25mm3The volume of block account for the 8mm3-25mm3Block is total
The 30% of volume) and make the culture mixing, the other time equal stationary culture of the solid fermentation.Wherein, in the jade
It is accessed in rice residue culture medium on the day of the layer goes out fusarium (Fusarium proliferatum) CGMCC3.1777 and is denoted as the 0th
It.
In the above method, by the round is placed in 28 DEG C, relative humidity is carries out institute in the environment of 50%-100%
State solid fermentation.
In the above method, by the round be placed in 28 DEG C, relative humidity be 50% environment in carry out the solid
Fermentation.
The above method may also include the step of extraction obtains beauvericin from the solid fermentation product.
In the above method, in the round equipped with maize pulp culture medium, the volume of maize pulp culture medium can be institute
State the 2/5 of round volume.
It is demonstrated experimentally that equipped with the solid culture that the water content made of water and maize pulp is 50% (mass percentage)
Solid fermentation layer goes out fusarium (Fusarium proliferatum) in the round of base (maize pulp culture medium)
CGMCC3.1777, the beauvericin content in solid fermentation product are 3405mg/g solid fermentation products.Wherein, which sends out
Round is being placed in 28 DEG C, is being carried out in the environment that relative humidity is 50% by ferment, and institute is accessed in the maize pulp culture medium
It states layer and goes out shaking the round on the 1st day and making the fermentation for fusarium (Fusariumproliferatum) CGMCC3.1777
Culture mixing in container, in the maize pulp culture medium accessing the layer goes out fusarium (Fusarium
Proliferatum) the 4th day, the 8th day and the 16th day of CGMCC3.1777 carries out following steps：Shake the round
Make the culture in the round is broken (to be crushed to 8mm3-25mm3Block) and make the culture mixing, it is described solid
The other time equal stationary culture of body fermentation.Wherein, it accesses the layer in the maize pulp culture medium and goes out fusarium (Fusarium
Proliferatum it) is denoted as on the day of CGMCC3.1777 the 0th day.
Description of the drawings
Fig. 1 is standard items HPLC-UV detection.
Fig. 2 is the detected sample HPLC-UV detection during experiment numbers are 3.
Specific implementation mode
The present invention is further described in detail With reference to embodiment, the embodiment provided is only for explaining
The bright present invention, the range being not intended to be limiting of the invention.Experimental method in following embodiments is unless otherwise specified
Conventional method.The materials, reagents and the like used in the following examples is commercially available unless otherwise specified.
Layer in following embodiments goes out fusarium (Fusarium proliferatum) CGMCC3.1777 (CGMCC strain mesh
Recording fourth edition (2012), scientific and technical literature publishing house) public can be from Chinese Microbiological Culture Collection management before 2012
(abbreviation CGMCC, address are committee's common micro-organisms center：Yard 1, BeiChen xi Road, Chaoyang District, Beijing City 3) it obtains,
The deposit number of China Committee for Culture Collection of Microorganisms's common micro-organisms center is CGMCC3.1777.
Culture medium as used in the following examples is as follows：
YPD fluid nutrient mediums：10g yeast extracts, 20g peptones, 20g glucose are settled to 1000ml with distilled water, adjust
PH7.0~7.2,115 DEG C of sterilizing 30min.
YPD solid mediums：10g yeast extracts, 20g peptones, 20g glucose, 20g agar are settled to distilled water
1000ml adjusts pH7.0~7.2,115 DEG C of sterilizing 30min.
Maize pulp culture medium：With water and maize pulp, (maize pulp is that corn kernel is crushed to 1mm3The jade that particle obtains
Rice seed crushed material) solid medium that water content is 50% (mass percentage) is made, 121 DEG C of sterilizing 30min consolidate this
Body culture medium is blended to 8mm3-25mm3Block obtains maize pulp culture medium.8mm3-25mm3In block, 8mm3-15mm3's
The volume of block accounts for the 8mm3-25mm3The 70% of block total volume is more than 15mm3To 25mm3Block volume
Account for the 8mm3-25mm3The 30% of block total volume.
The preparation of embodiment 1, beauvericin
1, the preparation of seed liquor
Take the layer preserved in -80 DEG C of refrigerators go out fusarium (Fusarium proliferatum) CGMCC3.1777 strains (
Frozen in 25% glycerine), after defrosting, dipped a little with oese, the streak inoculation on YPD solid mediums.It is trained in YPD solids
Visible colonies after being cultivated one day at 28 DEG C on base are supported, visible fluffy colony after two days is grown very vigorous after three days.Culture 3
Culture in YPD solid mediums is inoculated into YPD fluid nutrient mediums after it, at 28 DEG C, 140r/min shaken cultivations 2
It, obtains layer and goes out fusarium (Fusarium proliferatum) CGMCC3.1777 seed liquors.
2, solid fermentation
400ml maize pulp culture mediums are packed into the triangular flask that volume is 1000mL, the layer that access 5ml steps 1 obtain goes out
Fusarium (Fusarium proliferatum) CGMCC3.1777 seed liquors, the triangular flask after being inoculated with.Then after being inoculated with
Triangular flask be respectively placed in the environment of temperature and relative humidity that experiment numbers in table 1 are 1-6 and carry out solid fermentation, experiment is compiled
Number for 1-6 solid fermentations time such as table 1, accordingly shaken according to the vibration mode of table 1 in solid fermentation process.Fermentation
After, collect solid fermentation product (all substances in triangular flask).Solid fermentation product is impregnated with 100ml methanol, takes leaching
Liquid is taken, volume is measured, takes 1ml leaching liquids, with the filtering with microporous membrane of 0.45um, take filtrate, which is detected sample,
The detected sample is subjected to HPLC analyses.In HPLC analyses, with beauvericin (Beijing Tai Le fine jades Science and Technology Ltd., product
Catalog number (Cat.No.) MSS1033-1) be standard items according to the retention time of standard items it is qualitative and using calibration curve method (external standard method) carry out
Quantitative analysis beauvericin.HPLC analysis in use Agilent TC-C18 analytical columns, 30 DEG C of column temperature, autosampler sample introduction,
5 μ l of sample size, mobile phase are the liquid being made of first alcohol and water, and the volume ratio of first alcohol and water is 84: 16 in mobile phase, and flow velocity is
1ml/min is detected at wavelength 210nm using UV detectors and is automatically formed separating spectrum.In 6 experiments of table 1, Mei Geshi
It tests in triplicate, repeats the triangular flask after 10 inoculations every time.
The results are shown in Table 1, shows to be consolidated the triangular flask after inoculation in 28 DEG C, the environment that relative humidity is 50%
Body ferments 20 days, and the solid fermentation of the vibration mode of A is used to test the white of (in table 1 experiment numbers for 3) in solid fermentation process
The yield highest of stiff rhzomorph is 3405.3 ± 19.6mg beauvericins/g solid fermentation products.The test sample to be checked that experiment numbers are 3
The chromatographic peak (Fig. 1 and Fig. 2) that product beauvericin occur in 7 minutes or so in the corresponding retention time of beauvericin standard items.
The experiment condition and beauvericin yield of 1,6 experiment of table
In the vibration mode of table 1, A is that access layer goes out fusarium (Fusarium in maize pulp culture medium
Proliferatum) the 1st day vibrations triangular flask of CGMCC3.1777 makes the culture mixing in the triangular flask, in the jade
Accessed in rice residue culture medium the layer go out the 4th day, the 8th day of fusarium (Fusarium proliferatum) CGMCC3.1777 and
It proceeds as follows within 16th day：Vibrations triangular flask makes the culture in triangular flask is broken (to be crushed to 8mm3-25mm3Block,
Wherein 8mm3-15mm3The volume of block account for the 8mm3-25mm3The 70% of block total volume is more than 15mm3To 25mm3
The volume of block account for the 8mm3-25mm3The 30% of block total volume)；Wherein, it is connect in the maize pulp culture medium
Enter on the day of the layer goes out fusarium (Fusarium proliferatum) CGMCC3.1777 and is denoted as the 0th day.The vibration mode of table 1
In, B is that access layer goes out shaking for the 1st day for fusarium (Fusarium proliferatum) CGMCC3.1777 in maize pulp culture medium
Dynamic triangular flask makes the culture mixing in the triangular flask, and accessing the layer in the maize pulp culture medium goes out fusarium
The 4th day of (Fusarium proliferatum) CGMCC3.1777 proceeds as follows：Vibrations triangular flask makes in triangular flask
Culture is broken (to be crushed to 8mm3-25mm3Block, wherein 8mm3-15mm3The volume of block account for the 8mm3-25mm3
The 70% of block total volume is more than 15mm3To 25mm3The volume of block account for the 8mm3-25mm3Block total volume
30%)；Wherein, the layer is accessed in the maize pulp culture medium goes out fusarium (Fusarium proliferatum)
It is denoted as on the day of CGMCC3.1777 the 0th day.
Claims (7)
1. a kind of method preparing beauvericin is included in solid fermentation layer in the round equipped with maize pulp culture medium and goes out sickle
Spore (Fusarium proliferatum) CGMCC3.1777 collects the step of solid fermentation product obtains beauvericin；It is described
Maize pulp culture medium is the solid medium that water content is 50% (mass percentage) made of water and maize pulp；It will be described
The solid fermentation is carried out in the environment that round is placed in 24 DEG C -28 DEG C, relative humidity is 50%-100%.
2. according to the method described in claim 1, it is characterized in that：The solid fermentation carries out 20 days.
3. method according to claim 1 or 2, it is characterised in that：The solid fermentation is included in the maize pulp culture
That accesses that the layer goes out fusarium (Fusarium proliferatum) CGMCC3.1777 in base shakes the round on the 1st day
Make the culture mixing in the round, accessing the layer in the maize pulp culture medium goes out fusarium (Fusarium
Proliferatum) the 4th day, the 8th day and the 16th day of CGMCC3.1777 carries out following steps：Shake the round
So that the culture in the round is crushed and makes the culture mixing.
4. method according to claim 1 or 2, it is characterised in that：By the round be placed in 28 DEG C, relative humidity be
The solid fermentation is carried out in the environment of 50%-100%.
5. according to the method described in claim 4, it is characterized in that：By the round be placed in 28 DEG C, relative humidity be
The solid fermentation is carried out in 50% environment.
6. method according to claim 1 or 2, it is characterised in that：The method includes from the solid fermentation product
The step of extraction obtains beauvericin.
7. method according to claim 1 or 2, it is characterised in that：In the round equipped with maize pulp culture medium,
The volume of maize pulp culture medium is the 2/5 of the round volume.
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| CN201410211035.XA CN105087726B (en) | 2014-05-19 | 2014-05-19 | A method of preparing beauvericin |
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| CN201410211035.XA CN105087726B (en) | 2014-05-19 | 2014-05-19 | A method of preparing beauvericin |
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Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101240249A (en) * | 2008-01-25 | 2008-08-13 | 中国农业大学 | Dioscorea zingiberensis endogenesis fusarium capable of producing beauvericin and antibacterial activity thereof |
| CN101669939A (en) * | 2009-09-22 | 2010-03-17 | 中山大学 | Application of enniatine compound for preparing anti-drug-resistant tubercle bacillus drugs |
-
2014
- 2014-05-19 CN CN201410211035.XA patent/CN105087726B/en not_active Expired - Fee Related
Patent Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101240249A (en) * | 2008-01-25 | 2008-08-13 | 中国农业大学 | Dioscorea zingiberensis endogenesis fusarium capable of producing beauvericin and antibacterial activity thereof |
| CN101669939A (en) * | 2009-09-22 | 2010-03-17 | 中山大学 | Application of enniatine compound for preparing anti-drug-resistant tubercle bacillus drugs |
Non-Patent Citations (2)
| Title |
|---|
| 产白僵菌素镰孢菌发酵培养基的初步研究;霍秦秦等;《中国抗生素杂志》;20121130;第37卷(第11期);第1.1、1.3-1.5、2.2节、图4 * |
| 镰刀菌Fusarium sp.F1制备白僵菌素的初步研究;朱颖雪等;《化学世界》;20071231;第156-157页 * |
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