CN106086104A - A kind of additive for improving slimeball rhzomorph fermentation yield and application thereof - Google Patents
A kind of additive for improving slimeball rhzomorph fermentation yield and application thereof Download PDFInfo
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Abstract
The invention discloses a kind of additive for improving slimeball rhzomorph fermentation yield, described additive is methyl oleate, its final concentration of 15 μ l/ml added in slimeball rhzomorph liquid fermentation medium, and the time of interpolation is that Myxococcus xanthus ferments latter 36 hours.Experiment confirms, additive disclosed by the invention can promote that Myxococcus xanthus metabolism produces slimeball rhzomorph, for being not added with the CYE liquid fermentation medium of methyl oleate, after with the addition of methyl oleate, the fermentation yield of slimeball rhzomorph improves 7.75 times, prove that the present invention has had in terms of the fermentation technique of slimeball rhzomorph to promote significantly, and the inventive method reduces the fermentation costs of slimeball rhzomorph, indicate that it has important economic worth and broad prospect of application in fermenting and producing slimeball rhzomorph.
Description
Technical field
The present invention relates to a kind of additive for fermenting and producing, particularly relate to a kind of for improving slimeball rhzomorph fermentation product
The additive of amount and application thereof, belong to antibiotic medicine fermentation preparing technical field.
Background technology
20th century, the most noticeable medical science achieved the discovery of antibiotic beyond doubt.Nineteen twenty-eight it is found that in the world
A kind of antibiotics penicillin, open antibiotic research golden age.People from this grasped anti-microbial pathogen strong
Weapon so that the most fatal antibacterial infects and can be controlled and treat, and therefore the life of countless people saved.But
It is the excessive use recently as antibiotic, occurs in that the drug-resistant bacteria of phenotypic variation and genovariation, even occur in that
Some almost can resist the super drug-resistant bacteria of most antibiotic medicine.In in the past few decades, scientists one
Directly it is devoted to the research and development of new antibiotic, tries hard to race with drug-resistant bacteria.But, a nearest research showed, in the past
30 years in the research and development of new antibiotic the most delayed.Since two thousand five only have 3 class new antibiotic medicines to go through to list
(Bassetti et al.,2013).From finding new compound to they being designed as antibiotic medicine and being approved clinical practice
Need a very long process.During this period, the most delayed public health issue of research and development speed of new antibiotic by precarious can
Danger.Therefore seek to have the new antibiotic of wider antimicrobial spectrum to become when the extremely urgent task of the next item down.
Slime bacteria (Myxobacteria) is to have the bacterial groups of complicated many cells behavior characteristics it is considered to be high
Prokaryote.Further, the secondary metabolite kind of slime bacteria is many, structure is new, mechanism of action is unique, be after actinomycetes again
One microorganism new monoid in medicine source important, that have huge applications potentiality (Reichenbach, 2001；Gerth,et al.,
2003), there is in new drug development the advantage of uniqueness.
Slimeball rhzomorph (Myxovirescin) is a class 28 of report in Myxococcus xanthus (Myxococcus xanthus)
Membered macrolide lactam analog compound (formula 1).Its action target spot is two type signal peptidases of lspA gene code, belongs to two types
Signal peptidase inhibitor, can suppress the intracellular lipoprotein precursor containing LspA shearing site processing (Xiao et al.,
2012)。
Many documents show, slimeball rhzomorph is a class quick fungicide, and to most gram negative bacterias and minority leather
Lan Shi positive bacteria be respectively provided with good bactericidal activity (Gerth et al., 1982；Onishi et al.,1984；Content
et al.,2003).As the broad ectrum antibiotic that a class formation is more novel, slimeball rhzomorph has potential broad prospect of application.
But slimeball rhzomorph yield in Myxococcus xanthus (Simunovic et al., 2006) on the low side at present, one
Determine in degree, to limit its follow-up research and application.Accordingly, it would be desirable to explore the improvement in terms of the fermentation technique of slimeball rhzomorph, promote
Enter the raising of slimeball rhzomorph fermentation yield.Retrieval finds about the patent of this aspect or document, viscous especially with respect to being used for improving
The additive of coccus element fermentation yield is so far without report.
List of references:
Bassetti,M.,Merelli,M.,Temperoni,C.,&Astilean,A.(2013).New
antibiotics for bad bugs:where are we？.Annals of clinical microbiology and
antimicrobials,12(1),1.
Content,S.,Dutton,C.J.,&Roberts,L.(2003).Myxovirescin analogues via
macrocyclic ring–closing metathesis.Bioorganic&medicinal chemistry letters,13
(3),321-325.
Gerth,K.,Irschik,H.,Reichenbach,H.,&Trowitzsch,W.(1982).The
myxovirescins,a family of antibiotics from Myxococcus virescens
(Myxobacterales).The Journal of antibiotics,35(11),1454-1459.
Gerth,K.,Pradella,S.,Perlova,O.,Beyer,S.,&Müller,R.(2003)
.Myxobacteria:proficient producers of novel natural products with various
biological activities—past and future biotechnological aspects with the
focus on the genus Sorangium.Journal of biotechnology,106(2),233-253.
Onishi,N.,Izaki,K.,&Takahashi,H.(1984).A macrocyclic antibiotic M-
230B produced by Myxococcus xanthus.Isolation and characterization.The
Journal of antibiotics,37(1),13-19.
Reichenbach,H.(2001).Myxobacteria,producers of novel bioactive
substances.Journal of Industrial Microbiology and Biotechnology,27(3),149-
156.
Simunovic,V.,Zapp,J.,Rachid,S.,Krug,D.,Meiser,P.,&Müller,R.(2006)
.Myxovirescin A Biosynthesis is Directed by Hybrid Polyketide Synthases/
Nonribosomal Peptide Synthetase,3-Hydroxy-3-Methylglutaryl-CoA Synthases,and
trans-Acting Acyltransferases.ChemBioChem,7(8),1206-1220.
Xiao,Y.,Gerth,K.,Müller,R.,&Wall,D.(2012).Myxobacterium-produced
antibiotic TA(myxovirescin)inhibits type II signal peptidase.Antimicrobial
agents and chemotherapy,56(4),2014-2021.
Summary of the invention
For the deficiencies in the prior art, the problem of the invention solves the problems that is to provide a kind of for improving slimeball rhzomorph fermentation yield
Additive and application.By the way of adding additive in Myxococcus xanthus fermentation medium, improve slimeball rhzomorph send out
The component of ferment culture medium, thus realize being effectively improved the fermentation yield of slimeball rhzomorph.
Additive for improving slimeball rhzomorph fermentation yield of the present invention, it is characterised in that: described additive is oil
Acid methyl ester, its final concentration of 15 ± 1 μ l/ml added in slimeball rhzomorph liquid fermentation medium.
Wherein: the final concentration of 15 μ l/ that described additive methyl oleate preferably adds in CYE liquid fermentation medium
Ml, the time of interpolation is that Myxococcus xanthus (Myxococcus xanthus) ferments latter 36 hours.
The additive for improving slimeball rhzomorph fermentation yield of the present invention application in fermenting and producing slimeball rhzomorph.
Wherein, the method for described fermenting and producing slimeball rhzomorph is: existed by Myxococcus xanthus (Myxococcus xanthus)
It is transferred in CYE liquid fermentation medium after cultivating 4 ± 1 days with pH 7.6 ± 0.1,30 ± 1 DEG C on CYE solid medium,
Ferment 36 hours, then to CYE liquid under the conditions of pH 7.6 ± 0.1, cultivation temperature 30 ± 1 DEG C, cultivation rotating speed 200 ± 10rpm
Fermentation medium adds the methyl oleate of final concentration of 15 μ l/ml, continues fermentation culture 7 ± 1 days, it is thus achieved that containing slimeball rhzomorph
Fermentation liquid；
Wherein, above-mentioned CYE solid medium composition and content are: casein peptone 10g, yeast extract 5g, MgSO4·
7H2O 1g, 100mM MOPS solution 100ml, agar powder 15g, distilled water 1000ml；Above-mentioned CYE liquid fermentation medium forms
And content is: casein peptone 10g, yeast extract 5g, MgSO4·7H2O 1g, 100mM MOPS solution 100ml, distilled water
1000ml。
In above-mentioned application: described Myxococcus xanthus (Myxococcus xanthus) preferably Myxococcus xanthus
(Myxococcus xanthus) DZE9 bacterial strain.
In above-mentioned application: after described Myxococcus xanthus (Myxococcus xanthus) is cultivated on CYE solid medium
The bacterium amount being transferred in CYE liquid fermentation medium is preferably 1～2% with volume basis.
In above-mentioned application: described Myxococcus xanthus (Myxococcus xanthus) is in solid training or liquid fermentation and culture
PH is preferably 7.6, uses KOH solution regulation pH.
Concrete, the additive for improving slimeball rhzomorph fermentation yield of the present invention is in fermenting and producing slimeball rhzomorph
Application process be:
(1) it is taken at the Myxococcus xanthus DZE9 bacterial strain bacterium solution 20 μ l preserved under the conditions of-80 DEG C, is added drop-wise to CYE solid plate
On, with in advance the most calcination be cooled to the inoculating loop of room temperature and carry out drawing on CYE solid plate repeatedly on alcohol burner flame
Line operates.
(2) the CYE flat board of line is inverted cultivation 4～5 days under the conditions of 30 DEG C, until there being obvious yellow color colonies to go out
Existing.
(3) with in advance the most calcination be cooled to the inoculation shovel scraping Myxococcus xanthus of room temperature repeatedly on alcohol burner flame
DZE9 bacterial strain, is inoculated in the 300ml conical flask equipped with 50ml CYE fluid medium, and during inoculation, needs inoculation shovel is by waiting
The thalline planted torments uniformly on bottle wall, is broken up by the thalline of inoculation；
(4) at 30 DEG C, shaken cultivation 24h under conditions of 200rpm, to logarithm mid-early stage；
(5) cell concentration is adjusted to 1 × 10 with CYE fluid medium8Cfu/ml, 2% inoculum concentration is by above-mentioned by volume
Myxococcus xanthus DZE9 bacterium solution is transferred in 100ml CYE liquid fermentation medium, at 30 DEG C, and vibration training under conditions of 200rpm
Support and ferment.
(6), after cultivating 36 hours, methyl oleate (final concentration 15 μ l/ml) is added；Simultaneously according to fermentation volume 1% often
Group fermentation medium adds the resin of the most high temperature sterilize.At 30 DEG C, shaken cultivation under conditions of 200rpm, continues to send out
Ferment 7 days.With the CYE culture medium without methyl oleate as a control group.Often group Setup Experiments 4 is parallel.
(7) resin it is collected by filtration in 10ml centrifuge tube with 80 eye mesh screens, rinses resin with sterile deionized water, wash away table
The thalline of face residual.Add 3ml methanol overnight to extract.
(8) lixiviating solution reclaimed filters with the 0.22 organic filter membrane of μm, is transferred in HPLC sample bottle.
(9) in fermentation liquid, the content of slimeball rhzomorph uses HPLC to detect, and chromatographic column is XTerra MS C18column
(5 μm,2.1 × 100mm, Waters, US), testing conditions is as follows: sample size 20 μ l；Flow rate of mobile phase: 0.3mL/min；
Mobile phase A is containing 0.1% first aqueous acid, and Mobile phase B is the acetonitrile solution containing 0.1% formic acid, uses multistage gradient elution
Method, i.e. 0-2min Mobile phase B concentration is 5%；2-10min Mobile phase B concentration is risen to 50% by 5%；10-27min flows
Dynamic phase B concentration is risen to 95% by 50%；The concentration of 27-40min Mobile phase B maintains 95%, and detection wavelength is 239nm.
(10) calculate the yield of slimeball rhzomorph according to the relation curve of absworption peak area with slimeball rhzomorph, experimental result is made even
Average also calculates error.
Data show, in the case of without methyl oleate, in fermentation liquid the content of slimeball rhzomorph be 10.3 ±
3.7mg/L.After with the addition of methyl oleate in CYE fluid medium, in fermentation liquid the content of slimeball rhzomorph be 90.2 ±
18.1mg/L.Improve 7.75 times.These data show: compared with the most additivated liquid fermentation medium, use containing this
The liquid fermentation medium inventing described additive can significantly improve the fermentation yield of slimeball rhzomorph.
The invention discloses a kind of additive methyl oleate for improving slimeball rhzomorph fermentation yield, it is at slimeball rhzomorph
The final concentration added in liquid fermentation medium is preferably 15 μ l/ml, and the time of adding is preferably fermentation 36 hours.It is experimentally confirmed that
Additive disclosed by the invention can promote that Myxococcus xanthus metabolism produces slimeball rhzomorph, compared to being not added with methyl oleate
For CYE liquid fermentation medium, after with the addition of methyl oleate, the fermentation yield of slimeball rhzomorph improves 7.75 times, it was demonstrated that this
Invent to have had in terms of the fermentation technique of slimeball rhzomorph and promote significantly, and the inventive method reduces being fermented into of slimeball rhzomorph
This, indicate that it has important economic valency in the broad ectrum antibiotic slimeball rhzomorph that fermenting and producing is more novel as a class formation
Value and broad prospect of application.
Accompanying drawing explanation
Fig. 1. use the slimeball rhzomorph in HPLC detection Myxococcus xanthus fermentation liquid.
Fig. 2. slimeball rhzomorph ultraviolet characteristic absorption peak figure.
Fig. 3. myxobacter quality spectrogram.
Detailed description of the invention
Below in conjunction with disclosed in embodiment for improving the additive of slimeball rhzomorph fermentation yield, further describe its with
In the application improved in slimeball rhzomorph fermentation yield.Described content is explanation of the invention rather than restriction.
Embodiment 1:
(1) the bacterium solution 20 μ l of extracting yellow myxobacter DZE9 bacterial strain, (casein peptone 10g, yeast extracts to be added drop-wise to CYE solid
Thing 5g, MgSO4·7H2O 1g, 100mM MOPS (3-(N-morpholine) propane sulfonic acid) solution 100ml, agar powder 15g, distilled water
1000ml, pH 7.6) on flat board, rule on CYE solid plate with aseptic inoculating loop.
(2) the CYE solid plate being connected to Myxococcus xanthus DZE9 bacterial strain is inverted under the conditions of 30 DEG C cultivation 4～5 days, directly
Occur to there being obvious yellow color colonies.
(3) with aseptic inoculation shovel scraping Myxococcus xanthus DZE9 bacterial strain, it is inoculated into equipped with 50ml CYE fluid medium
((casein peptone 10g, yeast extract 5g, MgSO4·7H2O 1g, 100mM MOPS (3-(N-morpholine) propane sulfonic acid) solution
100ml, agar powder 15g, distilled water 1000ml, pH 7.6)) conical flask in.
(4) at 30 DEG C, shaken cultivation 24h under conditions of 200rpm, to logarithm mid-early stage.
(5) cell concentration is adjusted to 1 × 10 with CYE fluid medium8Cfu/ml, 2% inoculum concentration is by above-mentioned by volume
Myxococcus xanthus DZE9 bacterium solution is transferred in 100ml CYE liquid fermentation medium, at 30 DEG C, and vibration training under conditions of 200rpm
Support and ferment.
(6) cultivating after 36 hours, (final concentration divides to add the methyl oleate of variable concentrations respectively in liquid fermentation medium
It is not 0,5,10,15,20 μ l/ml)；Simultaneously according to fermentation volume 1% often organize fermentation medium adds the highest
The resin of temperature sterilizing.At 30 DEG C, shaken cultivation under conditions of 200rpm, continues fermentation 7 days.CYE without methyl oleate trains
Foster base is as a control group.Often group Setup Experiments 4 is parallel.
(7) resin it is collected by filtration in 10ml centrifuge tube with 80 eye mesh screens, rinses resin with sterile deionized water, wash away table
The thalline of face residual.Add 3ml methanol overnight to extract.
(8) lixiviating solution reclaimed filters with the 0.22 organic filter membrane of μm, is transferred in HPLC sample bottle.
(9) in fermentation liquid, the content of slimeball rhzomorph uses HPLC to detect, and chromatographic column is XTerra MS C18column
(5 μm,2.1 × 100mm, Waters, US), testing conditions is as follows: sample size 20 μ l；Flow rate of mobile phase: 0.3mL/min；
Mobile phase A is containing 0.1% first aqueous acid, and Mobile phase B is the acetonitrile solution containing 0.1% formic acid, uses multistage gradient elution
Method, i.e. 0-2min Mobile phase B concentration is 5%；2-10min Mobile phase B concentration is risen to 50% by 5%；10-27min flows
Dynamic phase B concentration is risen to 95% by 50%；The concentration of 27-40min Mobile phase B maintains 95%, and detection wavelength is 239nm.
(10) calculate the yield of slimeball rhzomorph according to the relation curve of absworption peak area with slimeball rhzomorph, experimental result is made even
Average also calculates error.Experimental result is as shown in table 1.
The impact on slimeball rhzomorph fermentation yield of the table 1 variable concentrations methyl oleate.
| Methyl oleate final concentration (μ l/ml) | Slimeball rhzomorph fermentation yield (mg/L) |
| 0 | 10.3±3.7 |
| 5 | 51.9±9.8 |
| 10 | 70.7±12.5 |
| 15 | 90.2±18.1 |
| 20 | 78.9±11.6 |
Data from table 1 we can see that: in the case of without methyl oleate, slimeball rhzomorph in fermentation liquid
Content is 10.3 ± 3.7mg/L.After with the addition of methyl oleate in CYE fluid medium, the fermentation yield of slimeball rhzomorph obtains
Arrive raising in various degree.Slimeball rhzomorph fermentation yield when the final concentration of methyl oleate reaches 15 μ l/ml, in fermentation liquid
Reach the highest, i.e. 90.2 ± 18.1mg/L, improve 7.75 times.But, when the final concentration of methyl oleate is further increased to 20 μ
During l/ml, the fermentation yield of slimeball rhzomorph has declined, and have dropped 12.5% during compared to final concentration of 15 μ l/ml, but still
Ratio is without high in the case of methyl oleate.
Embodiment 2:
(1) the bacterium solution 20 μ l of extracting yellow myxobacter DZE9 bacterial strain, (casein peptone 10g, yeast extracts to be added drop-wise to CYE solid
Thing 5g, MgSO4·7H2O 1g, 100mM MOPS (3-(N-morpholine) propane sulfonic acid) solution 100ml, agar powder 15g, distilled water
1000ml, pH 7.6) on flat board, rule on CYE solid plate with aseptic inoculating loop.
(2) the CYE solid plate being connected to Myxococcus xanthus DZE9 bacterial strain is inverted under the conditions of 30 DEG C cultivation 4～5 days, directly
Occur to there being obvious yellow color colonies.
(3) with aseptic inoculation shovel scraping Myxococcus xanthus DZE9 bacterial strain, it is inoculated into equipped with 50ml CYE fluid medium
((casein peptone 10g, yeast extract 5g, MgSO4·7H2O 1g, 100mM MOPS (3-(N-morpholine) propane sulfonic acid) solution
100ml, agar powder 15g, distilled water 1000ml, pH 7.6)) conical flask in.
(4) at 30 DEG C, shaken cultivation 24h under conditions of 200rpm, to logarithm mid-early stage.
(5) cell concentration is adjusted to 1 × 10 with CYE fluid medium8Cfu/ml, 2% inoculum concentration is by above-mentioned by volume
Myxococcus xanthus DZE9 bacterium solution is transferred in 100ml CYE liquid fermentation medium respectively, calculates in often group by fermentation volume 1%
Fermentation medium adds the resin of the most high temperature sterilize.
(6) add in liquid fermentation medium when of choosing fermentation 0 hour, 24 hours, 36 hours and 48 hours respectively
Add the methyl oleate of final concentration of 15 μ l/ml, at 30 DEG C, shaken cultivation continuous fermentation 7 days under conditions of 200rpm, often organize experiment
Set up 4 parallel.
(7) resin it is collected by filtration in 10ml centrifuge tube with 80 eye mesh screens, rinses resin with sterile deionized water, wash away table
The thalline of face residual.Add 3ml methanol overnight to extract.
(8) lixiviating solution reclaimed filters with the 0.22 organic filter membrane of μm, is transferred in HPLC sample bottle.
(9) in fermentation liquid, the content of slimeball rhzomorph uses HPLC to detect, and chromatographic column is XTerra MS C18column
(5 μm,2.1 × 100mm, Waters, US), testing conditions is as follows: sample size 20 μ l；Flow rate of mobile phase: 0.3mL/min；
Mobile phase A is containing 0.1% first aqueous acid, and Mobile phase B is the acetonitrile solution containing 0.1% formic acid, uses multistage gradient elution
Method, i.e. 0-2min Mobile phase B concentration is 5%；2-10min Mobile phase B concentration is risen to 50% by 5%；10-27min flows
Dynamic phase B concentration is risen to 95% by 50%；The concentration of 27-40min Mobile phase B maintains 95%, and detection wavelength is 239nm.
(10) calculate the yield of slimeball rhzomorph according to the relation curve of absworption peak area with slimeball rhzomorph, experimental result is made even
Average also calculates error.Experimental result is as shown in table 2.
Table 2 slimeball rhzomorph fermentation yield contrasts.
| Methyl oleate adds the time (hour) | Slimeball rhzomorph fermentation yield (mg/L) |
| 0 | 10.3±3.7 |
| 24 | 52.3±9.7 |
| 36 | 60.4±11.9 |
| 48 | 48.7±6.4 |
From table 2 it will be seen that in the different fermentations time, add final concentration of 15 μ l/ml methyl oleates can be different
The raising slimeball rhzomorph of degree.Fermentation time is 36 hours when, especially add methyl oleate, myxobacter in fermentation liquid
The yield of element is the highest, has reached 60.4 ± 11.9mg/L.
Claims (7)
1. the additive being used for improving slimeball rhzomorph fermentation yield, it is characterised in that: described additive is methyl oleate, its
Final concentration of 15 ± 1 μ l/ml added in slimeball rhzomorph liquid fermentation medium.
Additive for improving slimeball rhzomorph fermentation yield the most according to claim 1, it is characterised in that: described interpolation
The final concentration of 15 μ l/ml that agent methyl oleate adds in CYE liquid fermentation medium, the time of interpolation is Myxococcus xanthus
(Myxococcus xanthus) ferments latter 36 hours.
3. for improving the additive of slimeball rhzomorph fermentation yield in fermenting and producing slimeball rhzomorph described in claim 1 or 2
Application.
Application the most according to claim 3, it is characterised in that: the method for described fermenting and producing slimeball rhzomorph is yellow to be glued
Coccus (Myxococcus xanthus) turns after cultivating 4 ± 1 days with pH 7.6 ± 0.1,30 ± 1 DEG C on CYE solid medium
Receive in CYE liquid fermentation medium, in pH 7.6 ± 0.1, cultivation temperature 30 ± 1 DEG C, cultivate rotating speed 200 ± 10rpm condition
Bottom fermentation 36 hours, then adds the methyl oleate of final concentration of 15 μ l/ml in CYE liquid fermentation medium, continues fermentation
Cultivate 7 ± 1 days, it is thus achieved that containing the fermentation liquid of slimeball rhzomorph；
Wherein, described CYE solid medium composition and content are: casein peptone 10g, yeast extract 5g, MgSO4·7H2O1g,
100mM MOPS solution 100ml, agar powder 15g, distilled water 1000ml；Described CYE liquid fermentation medium forms and content is:
Casein peptone 10g, yeast extract 5g, MgSO4·7H2O 1g, 100mM MOPS solution 100ml, distilled water 1000ml.
Application the most according to claim 4, it is characterised in that: described Myxococcus xanthus (Myxococcus xanthus)
Select Myxococcus xanthus (Myxococcus xanthus) DZE9 bacterial strain.
Application the most according to claim 4, it is characterised in that: Myxococcus xanthus (Myxococcus xanthus) is at CYE
The bacterium amount being transferred in CYE liquid fermentation medium after cultivating on solid medium is 1～2% with volume basis.
Application the most according to claim 4, it is characterised in that: described Myxococcus xanthus (Myxococcus xanthus) exists
The pH of solid training or liquid fermentation and culture is 7.6, uses KOH solution regulation pH.
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Cited By (2)
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| CN106755167A (en) * | 2016-11-24 | 2017-05-31 | 山东大学 | A kind of fermentation process for improving macrolide secondary metabolite yield in slime bacteria |
| CN107012240A (en) * | 2017-05-08 | 2017-08-04 | 山东大学 | A kind of method of Rapid identification epothilone gene cluster positive strain |
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