CN106544379B - The identification and its application of S. ahygroscopicus W-273 polyketone product - Google Patents
The identification and its application of S. ahygroscopicus W-273 polyketone product Download PDFInfo
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Abstract
The invention discloses the identifications and its application of S. ahygroscopicus W-273 polyketone product.The present invention provides the extracts for providing S. ahygroscopicus (Streptomyces ahygroscopicus) W-273CGMCC NO.9604 or its tunning or its tunning to prepare the application in nystatin；The extract of S. ahygroscopicus (Streptomyces ahygroscopicus) W-273CGMCC NO.9604 or its tunning or its tunning has the application in bacteria resistance function product in preparation.The experiment proves that, S. ahygroscopicus W-273 can generate a kind of polyketone product, and this polyketone product has the structure of nystatin ingredient A1, has good inhibiting effect to fungi, can have exploitation at the potential of biological control product in agricultural production.
Description
Technical field
The invention belongs to field of biotechnology, especially belong to microbial secondary metabolite field, and in particular to one kind is not
The identification and its application of streptomyces hygroscopicus W-273 polyketone product.
Background technique
Streptomycete is a kind of gram-positive bacterium for deriving from soil, it can produce a large amount of active secondary metabolism and produces
Object, these activated products are functionally divided into antibacterium, antimycotic, antiviral and antitumor, treating tuberculosis, immunosupress etc.；From
Ucleosides, polypeptide, glycopeptide class, beta-lactam and polyketides etc. are divided into structure, wherein studying more
Polyketides mainly include macrolides, ansha, polyethers, anthracycline, angle anthracycline and Tetracyclines etc..Polyketone
Class compound biological synthesis gene cluster is carried out in the form of carboxylic acid condensation reaction.Such as erythromycin (Streptomyces
) and nystatin (Streptomyces noursei) etc. erytheara.
Nystatin is the discovery that a kind of antifungal as caused by streptomycete Streptomyces noursei earliest, belongs to
In a kind of polyenoid class macrolide, there is broad spectrum activity, bacterium unrestraint is acted on.Currently, being medically mainly used for internal treatment of
It treats fungus infection in digestive tract or is used for surface skin fungal infection outside, to agricultural insect the study found that high concentration nystatin
Processing can significantly inhibit Yeast-like symbionts quantity in brown paddy plant hopper body, and then the nutrition for influencing Yeast-like symbionts synthesizes function.
Currently, about other streptomycete bacterial strains can produce nystatin and its agricultural disease prevention and treatment on application be rarely reported.Cause
This, develops the new nystatin producing strains of one kind and is applied in agricultural disease prevention and treatment, with certain creativeness and newly
Newness, and further enrich the type of biological control product.
Summary of the invention
A purpose of the invention is to provide S. ahygroscopicus (Streptomyces ahygroscopicus) W-
The purposes of the extract of 273CGMCC NO.9604 or its tunning or its tunning.
The present invention provides S. ahygroscopicus (Streptomyces ahygroscopicus) W-273CGMCC
The extract of NO.9604 or its tunning or its tunning is preparing the application in nystatin；
Or, S. ahygroscopicus (Streptomyces ahygroscopicus) W-273CGMCC NO.9604 or its hair
The extract of ferment product or its tunning has the application in bacteria resistance function product in preparation.
Above-mentioned Nysfungin is Nysfungin A1.
Another object of the present invention, which is to provide one kind, has bacteria resistance function product, and active constituent is S. ahygroscopicus
The extraction of (Streptomyces ahygroscopicus) W-273CGMCC NO.9604 or its tunning or its tunning
Object.
Among the above, described antibacterial for inhibition Rhodotorula sp；
Or the extract is methanolic extract.
Another object of the present invention is to provide a kind of method for preparing nystatin.
Method provided by the invention, includes the following steps:
1) ferment S. ahygroscopicus (Streptomyces ahygroscopicus) W-273CGMCC NO.9604, receives
Collect tunning；
2) tunning is extracted with organic solvent, obtains methanolic extract to get nystatin is arrived.
In the above method, the organic solvent is methanol, n-butanol, ethyl alcohol, acetone, ethyl acetate or chloroform；
Or, further including following steps after obtaining methanol extract liquid in step 2): the methanol described in liquid chromatography purification mentions
Liquid is taken, retention time and the consistent product of nystatin standard items is collected, obtains nystatin；
The condition of the liquid chromatography purification are as follows:
Mobile phase is acetonitrile and the isometric mixed solution of water；
Sample introduction speed: 0.5mLmin-1Or 1.0mLmin-1。
Sample volume: 10 μ L；
Column temperature: 25 DEG C；
Detection wavelength: 305nm.
In the above method, the tunning described in methanol extraction includes the following steps:
1) precipitating in the tunning is collected；
2) methanol is added into the precipitating to mix, reaction, the supernatant in collecting reaction product obtains methanol and mentions
Take object.
In the above method, the additional amount of the methanol is the amount for being 1:1 by the tunning and the methanol volume ratio
It is added；
Or the time of the reaction is 30min-60min.
In the above method, the culture medium that the fermentation uses, every L is by 3.0% soy meal of mass percentage, quality percentage
0.8% corn flour of content, mass percentage 1.0%-4.0% oatmeal, mass percentage 0.01%-0.2%NaCl, matter
Measure percentage composition 0.01%-0.2%MgSO4, mass percentage 0.01%-0.2%%K2HPO4, mass percentage
0.01%-0.3%FeSO4, mass percentage 0.01%-1.0%CaCO3, mass percentage 0.01%-0.5% (NH4)2SO4It is formed with water；
Or, the culture medium that the fermentation uses, every L is by 3.0% soy meal of mass percentage, mass percentage
0.8% corn flour, 2.0% oatmeal of mass percentage, mass percentage 0.02%NaCl, mass percentage 0.02%
MgSO4, mass percentage 0.02%K2HPO4, mass percentage 0.02%FeSO4, mass percentage 0.5%CaCO3、
0.25% (NH of mass percentage4)2SO4It is formed with water；
Or, the condition of the fermentation is 28 DEG C, 200rmin-1、96h。
Above-mentioned Nysfungin is Nysfungin A1.
Another object of the present invention, which is to provide, a kind of prepares S. ahygroscopicus (Streptomyces
Ahygroscopicus) the method for the methanolic extract of W-273CGMCC NO.9604 tunning.
Method provided by the invention includes the steps that above-mentioned 1) -2): obtain methanolic extract.
The methanolic extract of above-mentioned method preparation is also the scope of protection of the invention；
Or it is also the scope of protection of the invention that the methanolic extract, which has the application in bacteria resistance function product in preparation,.
S. ahygroscopicus W-273 (Streptomyces ahygroscopicus) was preserved on August 26th, 2014
China Microbiological preservation administration committee common micro-organisms center, address: Yard 1, BeiChen xi Road, Chaoyang District, Beijing City 3, preservation
Number are as follows: CGMCC No.9604, classification naming: S. ahygroscopicus Wuyi mutation (Streptomyces
ahygroscopicus var.wuyiensis)。
The experiment proves that S. ahygroscopicus W-273 can generate a kind of polyketone product, and this polyketone product
Structure with nystatin ingredient A1, to fungi have good inhibiting effect, can have in agricultural production exploitation at
The potential of biological control product.
Detailed description of the invention
Fig. 1 is Wuyiencin biological synthesis gene cluster.
Fig. 2 is the synthesis of polyketide backbone.
Fig. 3 is the chemical structural drawing of nystatin ingredient A1.
Fig. 4 is the Antibacterial Activity of polyketone product.
Fig. 5 is the liquid chromatogram of W-273 fermentation N,N-dimethylformamide extract liquor.
Fig. 6 is nystatin sterling Antibacterial Activity.
Fig. 7 is the Antibacterial Activity of methanol extraction liquid.
Fig. 8 is the liquid chromatogram of W-273 fermented methanol extract liquor.
Fig. 9 is the liquid chromatogram of W-273 fermented methanol extract liquor after optimizing liquid-phase condition.
Figure 10 is the liquid chromatogram of W-273 fermented methanol extract liquor after optimizing liquid-phase condition.
Figure 11 is the mass spectrogram of W-273 fermented methanol extract liquor.
Specific embodiment
Experimental method used in following embodiments is conventional method unless otherwise specified.
The materials, reagents and the like used in the following examples is commercially available unless otherwise specified.
The prediction of embodiment 1, S. ahygroscopicus W-273 production polyketone product
One, the excavation of the polyketone biological synthesis gene cluster of S. ahygroscopicus W-273
S. ahygroscopicus W-273 (Streptomyces ahygroscopicus) was preserved on August 26th, 2014
China Microbiological preservation administration committee common micro-organisms center, address: Yard 1, BeiChen xi Road, Chaoyang District, Beijing City 3, preservation
Number are as follows: CGMCC No.9604, classification naming: S. ahygroscopicus Wuyi mutation (Streptomyces
ahygroscopicus var.wuyiensis)。
Using S. ahygroscopicus W-273 (Streptomyces ahygroscopicus var.wuyiensis
Strain W-273) (deposit number: CGMCC NO.9604) as by genome-wide screening and notch being sequenced filling up, finally
12834bp continuous polyketone gene cluster sequence relevant to Wuyiencin biosynthesis is obtained, and utilizes a kind of streptomycete of patent
Primer pair disclosed in middle polyketone biological synthesis gene cluster and its amplimer and kit, is expanded, and is finally obtained complete
Gene cluster sequence.Open reading frame is carried out to polyketone biological synthesis gene cluster sequence obtained using RAST software to divide
Analysis.
Bioinformatic analysis shows that the biological synthesis gene cluster (Fig. 1) includes 22 open reading frame altogether, wherein
WysI, wysJ, wysK, wysA, wysB, wysC6 genes are responsible for the synthesis of the long chain backbone of polyketone；wysR,wysRII,
6 gene coding transcript regulators of wysRIII, wysPI, wysPII, wysPIII；2 gene coding ABC of wysG, wysH turn
Transport albumen；Totally 3 genes are responsible for the synthesis and attachment of glycosyl by wysDI, wysDII, wysDIII；wysL,wysM,wysN,wysE,
Totally 5 genes encode other rear albumen for modifying albumen and unknown function to wysF.
Table 1 is the functional analysis of Wuyiencin biological synthesis gene cluster
WysF gene is located at gene cluster nucleotide sequences 228-1 base, length 228bp, encodes 75 amino acid, compiles
Code heat shock protein.
WysG gene is located at gene cluster nucleotide sequences 2216-396 base, and length is respectively 18211bp, coding 606
A amino acid, coded product belong to the abc transport protein family of ATP dependence.
WysH gene is located at gene cluster nucleotide sequences 3948-2194 base, mrna length 1755bp, encodes 584
Amino acid, coded product belong to the abc transport protein family of ATP dependence.
WysDIII gene is located at gene cluster nucleotide sequences 3964-4083 base, length 1089bp, encodes 362
Amino acid, coded product and GDP- mannose -4,6- dehydratase are extremely similar, thus it is speculated that may be responsible for rising for amino trehalose synthesis
Stage beginning.
WysI gene is located at gene cluster nucleotide sequences 4196-32707 base, length 28512bp, encodes 9503
Amino acid, include 6 complete modules, wysI may be responsible for Wuyiencin polyketone skeleton formed in 9-14 step extend.
WysJ gene is located at gene cluster nucleotide sequences 32866-49158 base, length 16293bp, coding 5430
A amino acid, include 3 complete modules, wysJ may be responsible for Wuyiencin polyketone skeleton formed in 15-17 step extend.
WysK gene is located at gene cluster nucleotide sequences 50124-56294 base, length 6171bp, encodes 2056
Amino acid, includes 1 complete module, and wysK may be responsible for Wuyiencin polyketone skeleton and form middle step 18 extension.
WysL gene is located at gene cluster nucleotide sequences 56653-57837 base, length 1185bp, encodes 394
Amino acid, coding albumen belong to Cytochrome P450 monooxygenase, and there are a CypX superfamily, thus it is speculated that may be responsible for gathering
Rear modification in the hydroxylating of ketone compound part and responsible Wuyiencin biosynthesis.
WysM gene is located at gene cluster nucleotide sequences 58113-57922 base, length 192bp, encodes 63 ammonia
Base acid, encoding ferredoxin.
WysN gene is located at gene cluster nucleotide sequences 59314-58163 base, length 1152bp, encodes 383
Amino acid, coding albumen belong to Cytochrome P450 monooxygenase.
WysDII gene is located at gene cluster nucleotide sequences 60414-59356 base, length 1059bp, coding 352
A amino acid encodes aminopherase, and wysDII albumen contains AAT-I superfamily, is a kind of to tremble phosphoric acid dependent on pyrrole
The enzyme of aldehyde (PLP).
WysDI gene is located at gene cluster nucleotide sequences 61824-60433 base, length 1392bp, encodes 463
Amino acid, encoding glycosyltransferases contain GTB topological structure in protein structure domain, and the subdomain that catalysis can be made to react is protected
It is fixed to keep steady.
WysA gene is located at gene cluster nucleotide sequences 66181-66311 base, length 4131bp, encodes 1376
Amino acid contains KS, DH, AT and ACP structural domain.
WysB gene is located at gene cluster nucleotide sequences 66357-75935 base, length 9579bp, encodes 3192
Amino acid.Coding module 1 and module 2.
WysC gene is located at gene cluster nucleotide sequences 75955-109194 base, length 33240bp, coding
11079 amino acid include 6 complete modules, are maximum polyketide synthases in bacterium.
WysE gene is located at gene cluster nucleotide sequences 109498-10247 base, length 750bp, encodes 249
Amino acid, all there is a thioesterase domain in the albumen of coding, main function is under hydrolyzing polyketone long-chain from PKS
Come.
WysPI gene is located at gene cluster nucleotide sequences 110700-113555 base, length 2856bp, coding 951
A amino acid, the product of coding belong to luxR family protein and contain PAS-like structural domain.
WysPII gene is located at gene cluster nucleotide sequences 113657-116437 base, length 2781bp, coding
926 amino acid, the product of coding belong to luxR family protein and contain PAS-like structural domain.
At wysPIII gene nucleotide sequences 116427-119210 base, being located at gene cluster length is 2784bp, is compiled
927 amino acid of code, the product of coding belong to luxR family protein and contain PAS-like structural domain.
WysR gene is located at gene cluster nucleotide sequences 119737-120318 base, length 582bp, encodes 193
Amino acid, the product of coding belong to luxR family protein and contain PAS-like structural domain.
WysRII gene is located at gene cluster nucleotide sequences 121018-121779 base, length 762bp, coding 253
The albumen WysRII of a amino acid, coding belongs to DeoR family.
WysRIII gene is located at gene cluster nucleotide sequences 122807-121784 base, length 1023bp, coding
340 amino acid encode AsnC family transcript regutation protein.
According to the feature of polyketide synthase related gene, it can speculate that the bacterium can produce polyketide, and this can be deduced
The structure of polyketide.Using acetyl coenzyme A and malonyl coenzyme A as carbochain extension unit, KS structural domain is responsible for catalysis two
The condensation of two carbon units and new C-C key are formed.KR structural domain is responsible for the reduction of carbonyl in two carbon units, contains KR structural domain
Module generally will form two hydroxyl carbon units.AT is acyl group transfer organization domain, is responsible for the load of two carbon units and to KS
The transfer of structural domain.ACP is acyl carrier protein, is responsible for the anchoring of carbochain in extending.DH structural domain is responsible for being catalyzed KR structural domain
The dehydration of two carbon units of the hydroxyl of generation, product are double bond containing two carbon units.There are also seldom a part of structures to contain
There is ER, be responsible for the hydrogenation reaction of double bond containing two carbon unit of catalysis, generates carbon-carbon single bond.The synthesis of poly ketone chain is exactly at these
The completion of two carbon units is constantly loaded under the catalytic action of module.Speculate that product may be polyketide, bone according to gene cluster
Frame structure is illustrated in fig. 2 shown below.The skeleton structure and nystatin main effect component A1 have certain homology, thus it is speculated that the polyketide
There may be similar structure to nystatin, be illustrated in fig. 3 shown below.
Embodiment 2, S. ahygroscopicus W-273 produce nystatin
(1) S. ahygroscopicus W-273 produces the first instance of nystatin
1, polyketone gene cluster tie substance ferments:
(1) seed culture medium FD-1 (1L): 8.0g glucose, 4.5g (NH4)2SO4, 2.0g KH2PO4, 1.0gMg2SO4·7H2O, 21.0g Mops (3-N morphine-propane sulfonic acid), 3ml TMS-1, remaining is water.TMS-1 (1L): 5.0g Fe
SO4·7H2O, 390mg CuSO4·5H2O, 440mgZnSO4·7H2O, 150mg Mn SO4H2O, 10mg NaMoO4·2H2O,
20mgCoCl2·2H2O and 50ml volumn concentration 37%HCl aqueous solution, remaining is water.
(2) fermentation medium (1L): 25.0g glucose, 8.3g (NH4)2SO4, 1.5g KH2PO4, 1.0g Mg2SO4·
7H2O, 21.0g Mops (3-N morphine-propane sulfonic acid), 3ml TMS-1.
By the spore inoculating of streptomycete W-273 to seed culture medium, 28 DEG C, 200rmin-1, 48h, obtain seed liquor.
Seed liquor is inoculated into fermentation medium, 28 DEG C, 200rmin in the ratio of volumn concentration 2.5%-1、
72h obtains fermentation liquid.
Extracting substances: above-mentioned fermentation liquid is extracted with n,N-Dimethylformamide with volume ratio for the ratio of 1:1, room
Temperature extraction 10min, 5000rmin-1It is centrifuged 10min and removes mycelium, obtain n,N-Dimethylformamide extract liquor, be stored in-
20℃。
2, polyketone gene cluster tie substance bacteriostatic test:
(1) it prepares rhodotorula bacterium solution: the rhodothece rubra (Rhodotorula Rubra) of 5d will be cultivated on PDA plate, used
Swab stick, which scrapes to be put into sterile water, breaks up mixing for fungus block with sterilized bead, obtains rhodotorula bacterium solution.
(2) rhodotorula bacterium solution is inoculated into the ratio of volumn concentration 1% and melts the PDA culture for being cooled to about 40 DEG C
In base, inverted plate is shaken up.
(3) Oxford cup of sterilizing is placed on above-mentioned plate, 2 Oxford cups contain 200 μ LN, N- in 2 Oxford cups respectively
The N,N-dimethylformamide extract liquor that dimethylformamide and 200 μ L above-mentioned 1 are obtained.
As a result as shown in figure 4, A is above-mentioned 1 obtained n,N-Dimethylformamide extract liquor；B is N, N- dimethyl formyl
Amine；N,N-Dimethylformamide itself can inhibit the growth of rhodotorula, and fungistatic effect is fine, when with N, N- dimethyl methyl
When amide carrys out extractive fermentation liquid, the bacteriostasis of reagent itself can cover the fungistatic effect of the substance extracted, therefore from antibacterial
From the point of view of test, n,N-Dimethylformamide is not suitable for making the extractant in this experiment.
3、The test of polyketone gene cluster tie substance high performance liquid chromatography:
(1) preparation of product is marked: by nystatin standard items (German Dr.Ehrensorfer company CAS No.1400-61-
9, nystatin A1) it is dissolved in the solution that chromatographic grade n,N-Dimethylformamide is configured to concentration as 4mg/mL, successively it is diluted to
2mg/mL, 1mg/mL, and crossing aperture is 0.22 μm of filter membrane processing.
(2) processing of sample: the N,N-dimethylformamide extract liquor filter membrane that above-mentioned 1 is obtained (aperture is 0.22 μm)
Processing.
(3) liquid-phase condition: mobile phase: [methanol: water: dimethylformamide (550+300+150, that is, 11:6:3)], sample introduction speed
Degree: 1.0mLmin-1；Sample volume: 10 μ L；Column temperature: 25 DEG C；Detection wavelength: 305nm.
Chromatographic column be water generation Science and Technology Ltd. (Waters), Symmetry, C18,5.0 μm, (4.6mm × 250mm)
Product article No.: WAT05427.
Above-mentioned configured mark product and treated sample are subjected to high performance liquid chromatography detection according to above-mentioned condition,
As a result as shown in figure 5, A be standard items 1mg/mL, peak area: 57323, retention time: 7.066min；B is W-273
Ferment n,N-Dimethylformamide extract liquor, main matter A peak area: 2756.4, retention time: 2.433min；Main matter B
Peak area: 2983.1, retention time: 3.006min.
Above-mentioned extract liquor is compared by liquid chromatogram and is found, the main component of extracting substance under same liquid-phase condition
Retention time and the retention time of standard items are inconsistent, and two main peaks occur, and there are two analysis reason possibility: first is that extraction
Substance out is not target product；Second is that liquid-phase condition is improper.
Unstable and mycelia the amount slow growth based on thalli growth in fermentation medium in (one) extracts in bacteriostatic test
Agent n,N-Dimethylformamide has inhibiting effect to the growth of rhodotorula, and liquid phase result extracting substance retention time and standard items are not
The problems such as consistent, uses new culture medium prescription, extraction conditions instead again.
(2) S. ahygroscopicus W-273 produces fermentation and the optimization of polyketone product
1, polyketone gene cluster tie substance fermentation
(1) seed fermentation culture medium (1L): 2.0% glucose of mass percentage, 0.6% albumen of mass percentage
Peptone, 0.6% yeast powder of mass percentage, mass percentage 1%NaCl, remaining is water, PH 7.2.
(2) fermentation medium (1L): 3.0% soy meal of mass percentage, 0.8% corn flour of mass percentage, matter
Measure 2.0% oatmeal of percentage composition, mass percentage 0.02%NaCl, mass percentage 0.02%MgSO4, quality percentage
Content 0.02%K2HPO4, mass percentage 0.02%FeSO4, mass percentage 0.5%CaCO3, mass percentage
0.25% (NH4)2SO4, remaining is water, PH 7.2.
By the spore inoculating of streptomycete W-273 to seed culture medium, 28 DEG C, 200rmin-1, for 24 hours, obtain seed liquor.
Seed liquor is inoculated into fermentation medium, 28 DEG C, 200rmin in the ratio of volumn concentration 3%-1, 96h,
Obtain fermentation liquid.
Extracting substances:
By above-mentioned fermentation liquid 6000rmin-1, it is centrifuged 10min, collects primary deposition；
The sterile water isometric with fermentation liquid is added into primary deposition to be washed, 30min, 6000rmin are vibrated-1
It is centrifuged 10min, discards supernatant liquid, collects secondary precipitation；
Methanol is added by the amount that fermentation liquid and methanol volume ratio are 1:1 into secondary precipitation, vibrates 30min-60min,
6000r·min-1It is centrifuged 10min, collects supernatant, that is, methanol extraction liquid.
2, polyketone gene cluster tie substance bacteriostatic test:
(1) it prepares rhodotorula bacterium solution: (the rhodothece rubra Rhodotorula Rubra) that cultivates 5d on PDA plate is used
Swab stick, which scrapes to be put into sterile water, breaks up mixing for fungus block with sterilized bead, obtains rhodotorula bacterium solution.
(2) rhodotorula bacterium solution is inoculated into the ratio of volumn concentration 1% and melts the PDA culture for being cooled to about 40 DEG C
In base, inverted plate is shaken up.
(3) 2 Oxford cups of sterilizing are placed on above-mentioned plate, contain 200 μ L methanol and 200 μ L respectively in 2 Oxford cups
Above-mentioned 1 obtained methanol extraction liquid or concentration are 1mg/mL nystatin solution, and solvent is methanol.Nystatin A1 mark product are purchased from
German Dr.Ehrensorfer company CAS No.1400-61-9.
The result of nystatin solution and methanol is as shown in fig. 6, A is methanol；B: nystatin mark product methanol solution；Methanol
The result of liquor and methanol is extracted as shown in fig. 7, A is methanol；B: methanol extraction liquid；As can be seen that working as nystatin concentration
When for 1mg/mL, the inhibition zone to rhodotorula is 35mm；The inhibition zone of methanol extraction liquid is 25.4mm, and extract liquor is antibacterial
Enclose transparent, sharpness of border；Show that the fermentation, extraction product of streptomycete W-273 can inhibit rhodotorula.
3, polyketone gene cluster tie substance high performance liquid chromatography is tested:
1) high performance liquid chromatography is tested
(1) preparation of product is marked: by nystatin standard items (German Dr.Ehrensorfer company CAS No.1400-61-
9, nystatin A1) it is dissolved in hplc grade methanol preparation 4mg/mL solution, it is successively diluted to 2mg/mL, 1mg/mL, and cross aperture and be
0.22 μm of filter membrane processing.
(2) processing of sample: by methanol extraction liquid filter membrane (diameter is 0.22 μm) processing.
(3) liquid-phase condition: mobile phase: [methanol: water: dimethylformamide (550+300+150, that is, 11:6:3)], sample introduction speed
Degree: 0.5mLmin-1；Sample volume: 10 μ L；Column temperature: 25 DEG C；Detection wavelength: 305nm.
Above-mentioned configured mark product and treated sample are subjected to high performance liquid chromatography detection according to above-mentioned condition,
As a result as shown in figure 8, A is standard items 1mg/mL, peak area: 56548.9 retention times: 10.304min；B is first
Alcohol extract liquor, main matter peak area: 5873.5 retention times: 13.893min；As can be seen that in this liquid phase experiments, discovery
The main matter retention time and standard items of extraction are still inconsistent, but discovery has the appearance time of an extracting substance
(10.398min) and standard items retention time (10.304min) are very close to but peak area very little, dimension attempt other liquid phases
Condition.
2) high performance liquid chromatography experimental condition optimizes
(1) preparation of product is marked: with 1) it is identical.
(2) processing of sample: with 1) it is identical.
(3) liquid-phase condition: mobile phase: (acetonitrile: water=1:1)；Sample introduction speed: 1.0mLmin-1；Sample volume: 10 μ L；Column
Temperature: 25 DEG C；Detection wavelength: 305nm.
Above-mentioned configured mark product and treated sample are subjected to high performance liquid chromatography detection according to above-mentioned condition；
As a result as shown in figure 9, A be standard items 1mg/mL, peak area: 29810, retention time: 2.585min；B is W-273
Fermentation, extraction liquid, peak area: 7087.9 retention times: 2.771min；As can be seen that under new liquid-phase condition, discovery extraction
Main matter retention time and standard items retention time it is almost the same, but retention time is too short, at two minutes or so,
There is the possibility of solvent peak, therefore attempt to reduce sample introduction speed, sees whether the main matter retention time of standard items and extraction can prolong
It is long, to determine whether being solvent peak.
3) high performance liquid chromatography experimental condition double optimization
(1) preparation of product is marked: with 1) it is identical.
(2) processing of sample: with 1) it is identical.
(3) liquid-phase condition: mobile phase: (acetonitrile: water=1:1)；Sample introduction speed: 0.5mLmin-1；Sample volume: 10 μ L；Column
Temperature: 25 DEG C；Detection wavelength: 305nm.
Above-mentioned configured mark product and treated sample are subjected to high performance liquid chromatography detection according to above-mentioned condition；
The results are shown in Figure 10, and A is standard items 1mg/mL, peak area: 61161.6 retention times: 5.145min；B is W-
273 fermentation, extraction liquid, main matter peak area: 6148.8 retention times: 5.172min；As can be seen that reducing sample introduction speed
After find, the main matter retention time and standard items retention time of extraction are still consistent, and retention time extend, illustrate this
Peak is not solvent peak.
In conclusion the method for fermentation condition, extraction in above-mentioned (two) be it is feasible, in liquid phase experiments, by tasting
Try different liquid-phase condition and sample introduction speed, it is believed that (two) liquid-phase condition 3 in) it is feasible, sample introduction speed is 0.5mLmin-1Shi Cui
The main matter taken is consistent with the retention time of standard items, and main peak area is larger, and miscellaneous peak is less.
4) Mass Spectrometer Method
(1) mark product preparation: with 1) in it is identical.
(2) processing of sample: with 1) it is identical.
(3) Mass Spectrometer Method instrument: ultra high efficiency liquid phase --- gas phase flight mass spectrum (Xevo G2-XS QTOF mass spectrograph) chromatography
Condition: chromatographic column: Agilent SB-C18 column (2.1mm × 100mm, 2.7 μm)；Column temperature: 25 DEG C；Sample volume: 10 μ L；Mobile phase
It is divided into A, B two parts, wherein A is acetonitrile, and B is water (containing 0.1% formic acid), and condition of gradient elution is as shown in table 2:
Table 2
| Time (min) | A (%) | B (%) |
| 0.0 | 95 | 5 |
| 6.0 | 100 | 0 |
| 7.0 | 100 | 0 |
| 9.0 | 95 | 5 |
Mass Spectrometry Conditions: ion source: electric spray ion source (ESI)；Scanning mode: cation (ESI+) scanning；Detection mode:
Multiple-reaction monitoring (Multiple Reaction Monitor, MRM)；100 DEG C of ion source temperature；250 DEG C of desolventizing temperature；Electricity
Ionization voltage: 3kV-5kV.
It is as shown in Figure 11 A that nystatin A1 marks product result, it can be seen that 1mg/mL, target substance relative molecular mass are
926.5192；
Methanol extraction liquid result is as shown in Figure 11 B, and the substance for being 926.5106 with the presence of relative molecular mass shows methanol
Contain nystatin A1 in extract liquor.
Claims (9)
1. S. ahygroscopicus (Streptomyces ahygroscopicus) W-273 CGMCC NO. 9604 or its fermentation
The extract of product or its tunning is preparing the application in nystatin.
2. a kind of method for preparing nystatin, includes the following steps:
1) ferment S. ahygroscopicus (Streptomyces ahygroscopicus) W-273 CGMCC NO. 9604, collects
Tunning；
2) tunning described in methanol extraction obtains methanolic extract to get nystatin is arrived.
3. according to the method described in claim 2, it is characterized by:
Further include following steps after obtaining methanol extract liquid in step 2: the methanol extract liquid described in liquid chromatography purification is received
Collect retention time and the consistent product of nystatin standard items, obtains nystatin；
The condition of the liquid chromatography purification are as follows:
Mobile phase is acetonitrile and the isometric mixed solution of water；
Sample introduction speed: 0.5mLmin-1Or 1.0mLmin-1。
4. according to the method in claim 2 or 3, it is characterised in that:
The tunning described in methanol extraction includes the following steps:
1) precipitating in the tunning is collected；
2) methanol is added into the precipitating to mix, reaction, the supernatant in collecting reaction product obtains methanol extraction
Object.
5. according to the method described in claim 4, it is characterized by: the additional amount of the methanol is by the tunning and institute
The amount that methanol volume ratio is 1: 1 is stated to be added；
Or the time of the reaction is 30min-60min.
6. according to the method described in claim 2, it is characterized by:
It is described fermentation use culture medium, every L by 3.0% soy meal of mass percentage, 0.8% corn flour of mass percentage,
2.0% oatmeal of mass percentage, mass percentage 0.02%NaCl, mass percentage 0.02%MgSO4, quality percentage
Content 0.02%K2HPO4, mass percentage 0.02%FeSO4, mass percentage 0.5%CaCO3, mass percentage 0.25%
(NH4)2SO4It is formed with water.
7. a kind of prepare S. ahygroscopicus (Streptomyces ahygroscopicus) W-273 CGMCC NO. 9604
The method of the methanolic extract of tunning includes the steps that any described 1) -2) in claim 2-6: obtaining methanol extraction
Object.
8. the methanolic extract of method of claim 7 preparation.
9. methanolic extract according to any one of claims 8 has the application in bacteria resistance function product in preparation.
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