CN110229856A - A kind of preparation method of Cyclic lipopeptide antibiotic Fusaricidin B - Google Patents
A kind of preparation method of Cyclic lipopeptide antibiotic Fusaricidin B Download PDFInfo
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- CN110229856A CN110229856A CN201910532219.9A CN201910532219A CN110229856A CN 110229856 A CN110229856 A CN 110229856A CN 201910532219 A CN201910532219 A CN 201910532219A CN 110229856 A CN110229856 A CN 110229856A
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Abstract
The present invention provides a kind of application of Paenibacillus polymyxa CGMCC No.10062 in preparation Cyclic lipopeptide antibiotic Fusaricidin B, and the method for efficiently preparing Cyclic lipopeptide antibiotic Fusaricidin B using Paenibacillus polymyxa CGMCC No.10062.This method uses Paenibacillus polymyxa CGMCC No.10062, ferments using degreasing milk medium as culture medium, and passes through centrifugation, ultrafiltration, Solid Phase Extraction, and Cyclic lipopeptide antibiotic Fusaricidin B is made in preparative HPLC purifying, vacuum drying.There is the first public Paenibacillus polymyxa CGMCC No.10062 that discloses of the present invention fermentation skimmed milk to generate the new application of Cyclic lipopeptide antibiotic Fusaricidin B, widen the source of Cyclic lipopeptide antibiotic Fusaricidin B.
Description
Technical field
The invention belongs to field of biotechnology, and in particular to a kind of to utilize Paenibacillus polymyxa CGMCC No.10062 system
The method of standby Cyclic lipopeptide antibiotic Fusaricidin B.
Background technique
Cyclic lipopeptide antibiotic belongs to microbial secondary metabolite, synthesizes through cell non-ribosomal approach.Non-ribosomal
It is template that the peptide synthesis of approach, which does not depend on mRNA, also not using tRNA as carrier, but passes through non-ribosomal peptide chain synzyme (non-
Riosomal peptide synthetases, NRPSs) it identifies specific amino acid and connects into polypeptide chain.Therefore, cyclic lipopeptide
Class antibiotic belong to non-ribosomal synthesis class antibacterial peptide, have natural broad spectrum antibacterial, heat-resisting, toxicity is low, drug residue free and
It is not likely to produce numerous features such as antibody-resistant bacterium.Cyclic lipopeptide antibiotic is made of hydrophobic fat acid chain and hydrophilic peptide chain.One
As fatty acid chain be made of 12~16 carbon atoms, and with the growth of carbochain, bacteriostatic activity and enhance.
According to the structure of Cyclic lipopeptide antibiotic and electrification property, cyclic cationic lipopeptid, cyclic annular non-cationic can be divided into
Lipopeptid and linear cationic lipopeptid.(1) end C- of cyclic cationic lipopeptid is ionized by amido bond, lipid end and acylation
-terminal amino acid connects.Cationic charge is usually from 2,4- diaminobenzoic acid residue (Dab).Structure-based difference is again
Polymyxins, Polypeptins and Octapeptins can be subdivided into.(2) common cyclic annular non-cationic lipopeptid is to kill fusarium
Rhzomorph (Fusaricidins), the ring being made of 6 amino acid residues and 15- guanidine radicals -3- hydroxypentadecanoic acid (GHPD)
Shape lipopeptid, molecular weight is in 900Da or so.(3) linear cationic lipopeptid Tridecaptins is to glue class gemma from 1978 more
It separates and obtains in bacillus NRRL B-30509 and B-30644 tunning, contain 13 amino acid residues, to gram-negative
Property bacterium have very strong activity.
Recently as the abuse of conventional antibiotic, so that the appearance of a large amount of antibody-resistant bacterium, is food safety and medical treatment neck
Domain brings serious problems.Since Cyclic lipopeptide antibiotic is not likely to produce antibody-resistant bacterium, and there is natural broad spectrum antibacterial, future
It is with higher to use prospect.Meanwhile Cyclic lipopeptide antibiotic is faced with problems, such as natural content it is low, isolate and purify
Difficulty is big, chemical synthesis is at high cost low etc. with recombinant expression yield, seriously limits it in the application of the industries such as food, medicine.
Cyclic lipopeptide antibiotic Fusaricidins may be from the secondary metabolite of series bacillus.CN106978457A
In disclose it is a kind of utilize ox series bacillus CGMCC No.8333, fermentation wheat bran culture medium prepare cyclic lipopeptide antibiosis
The method of plain Fusaricidin A.But there are no the biofermentations of Cyclic lipopeptide antibiotic Fusaricidin B in the prior art
Preparation method.This is because Fusaricidins B content is extremely low in tunning, only account for tunning 0.01% (W/W) or
Less.Tunning composition is again complex, brings larger difficulty to separation preparation.Secondly, from the point of view of culture medium angle, early period
Report all use chemical synthesis culture medium, this will directly affect the cost and use of the preparations such as object Fusaricidin B
Safety.Above-mentioned these are all those skilled in the art's urgent problems to be solved.
Summary of the invention
Based on above-mentioned technical problem, Paenibacillus polymyxa CGMCC No.10062, height are utilized the present invention provides a kind of
The method of effect preparation Cyclic lipopeptide antibiotic Fusaricidin B, using skimmed milk as somatomedin, it is anti-that fermentation obtains cyclic lipopeptide
Raw element Fusaricidin B.
Specifically, the present invention provides a kind of Paenibacillus polymyxa CGMCC No.10062 efficiently to prepare cyclic lipopeptide
Application in antibiotic Fusaricidin B.
Cyclic lipopeptide antibiotic is prepared using Paenibacillus polymyxa CGMCC No.10062 the present invention also provides a kind of
The method of Fusaricidin B, method includes the following steps:
(a) Paenibacillus polymyxa CGMCC No.10062 is inoculated in skimmed milk and is fermented, obtain fermentation liquid；
(b) the fermentation liquid centrifuging and taking supernatant for obtaining step (a), inactivated proteases use molecular cut off after cooling
The ultrafiltration membrane of 1000Da obtains crude product I；
(c) the crude product I for obtaining step (b) collects active component, vacuum drying by reverse phase solid phase extraction post separation
Afterwards, crude product II is obtained；
(d) the crude product II for obtaining step (c) is purified by preparative HPLC, collects bacteriostatic activity fraction, vacuum drying
Afterwards, Cyclic lipopeptide antibiotic Fusaricidin B is obtained.
Further, in step (a), the inoculum concentration of the Paenibacillus polymyxa CGMCC No.10062 is 1.6x106
~8.0x106CFU/mL。
In step (a), the degreasing milk medium includes skimmed milk and water, the mass concentration of skimmed milk be 60g/L~
100g/L。
Further, in step (a), the fermentation is shake culture, and concussion speed is 100rpm~300rpm；Described
Fermentation temperature is 25 DEG C~35 DEG C, and fermentation time is 48h~96h.
Further, in step (b), the speed of the centrifugation is 6000g~10000g, and the time is 10min~20min.
Further, in step (b), the temperature of the high temperature bath is 60 DEG C~90 DEG C, and the time is 10min~30min.
Further, in step (c), separation condition are as follows: the methanol/water solution for being 0%~100% (V/V) with volume ratio
Carry out gradient elution.
Further, in step (d), the described preparative HPLC separation, using the reverse-phase chromatographic column of 30mm × 100mm,
Packing material size is 5 μm~10 μm in column；Mobile phase: with H2O and containing 0.01%~0.1% (V/V) trifluoroacetic acid be A phase, methanol/
Acetonitrile simultaneously containing 0.01%~0.1% (V/V) trifluoroacetic acid be B phase, carry out gradient elution, elution speed be 30mL/min~
50mL/min, the gathering speed of fraction are that 0.1min/ pipe~0.2min/ manages (every pipe 6mL).
Further, retention time is collected in the fraction of 7.90min~8.10min, to get cyclic lipopeptide after vacuum drying
Antibiotic Fusaricidin B.
In above-mentioned technical proposal, makes public for the first time Paenibacillus polymyxa CGMCC No.10062 and prepare cyclic lipopeptide antibiosis
The new application of plain Fusaricidin B.Present invention firstly discloses Paenibacillus polymyxa CGMCC No.10062 is used, with de-
Rouge cream culture medium ferments as culture medium, and passes through centrifugation, ultrafiltration, Solid Phase Extraction, and preparative HPLC purifying, vacuum are dry
It is dry, Cyclic lipopeptide antibiotic Fusaricidin B is made, disclosing Paenibacillus polymyxa CGMCC No.10062 has fermentation
Degreasing milk medium generates the new application of Cyclic lipopeptide antibiotic Fusaricidin B, has widened Cyclic lipopeptide antibiotic
The source of Fusaricidin B.Degreasing milk medium is used simultaneously, avoids the ring grease made using chemical synthesis culture medium
Peptide antibiotics Fusaricidin B has higher safety in utilization, lower preparation cost.
Detailed description of the invention
Fig. 1 is crude product I point after the separating methanol by reverse phase solid phase extraction column various concentration in the embodiment of the present invention 1
The effect of kind method detection bacteriostatic activity；
Fig. 2 is that crude product I is obtained after the separating methanol by reverse phase solid phase extraction column various concentration in the embodiment of the present invention 1
The inhibition zone result figure arrived；
Fig. 3 is purification effect of the crude product II in preparative HPLC in the embodiment of the present invention 1；
Fig. 4 is the first mass spectrometric figure of bacteriostatic activity component in the embodiment of the present invention 1；
Fig. 5 is the second order ms figure of bacteriostatic activity component in the embodiment of the present invention 1.
Specific embodiment
Technical solution of the present invention is illustrated to be clearer, below in conjunction with specific embodiment to skill of the invention
Art scheme is further elaborated:
When facing " Fusaricidins B content is extremely low in the tunning of bacterial strain " this technical problem, the present inventor
By creative labor, a kind of Paenibacillus polymyxa CGMCC No.10062 is provided in preparation Cyclic lipopeptide antibiotic
Application in Fusaricidin B.It is anti-that the present invention prepares cyclic lipopeptide using Paenibacillus polymyxa CGMCC No.10062
When raw element Fusaricidin B, Fusaricidins B content is higher in obtained tunning, suitable Cyclic lipopeptide antibiotic
The large-scale production application of Fusaricidins B.
Facing " source of Cyclic lipopeptide antibiotic Fusaricidin B how is widened, and avoids training using chemical synthesis
When this technical problem of feeding base ", the present inventor passes through creative work, provides a kind of Cyclic lipopeptide antibiotic
The preparation method of Fusaricidin B, comprising the following steps:
(a) Paenibacillus polymyxa CGMCC No.10062 is inoculated in degreasing milk medium and is fermented, sent out
Zymotic fluid；
(b) by fermentation liquid centrifuging and taking supernatant, inactivated proteases are after cooling the super of 1000Da using molecular cut off
Filter membrane obtains crude product I；
(c) crude product I is collected bacteriostatic activity fraction, after vacuum drying, obtains crude product by reverse phase solid phase extraction post separation
II；
(d) crude product II is purified by preparative HPLC, collects bacteriostatic activity fraction, after vacuum drying, obtains cyclic lipopeptide
Class antibiotic Fusaricidin B.
Preparation method in above-mentioned technical proposal uses Paenibacillus polymyxa CGMCC No.10062, for the first time with degreasing
Newborn culture medium ferments as culture medium, and by centrifugation, ultrafiltration, Solid Phase Extraction, preparative HPLC purifying, vacuum is dry
It is dry, Cyclic lipopeptide antibiotic Fusaricidin B is prepared, disclosing Paenibacillus polymyxa CGMCC No.10062 has
The degreasing milk medium that ferments generates the new application of Cyclic lipopeptide antibiotic Fusaricidin B, has widened Cyclic lipopeptide antibiotic
The source of Fusaricidin B.In addition, from a wealth of sources, the natural safe and stable property of skimmed milk is strong；In addition fermenting microbe use is
A kind of this single culture of Paenibacillus polymyxa CGMCC No.10062, the combination of above-mentioned raw material, strain are conducive to product product
The standardization of matter and the cost control of industrial mass production.
Further, in step (a), the inoculum concentration of Paenibacillus polymyxa CGMCC No.10062 is 1.6x106~
8x106CFU/mL；Preferably 3.2x106~6.4x106CFU/mL is more preferably 4.8x106CFU/mL。
Further, in above-mentioned steps (a), the degreasing milk medium includes skimmed milk and water, and the quality of skimmed milk is dense
Degree is 60g/L~100g/L.Preparation method may comprise steps of: skimmed milk is added in distilled water, after mixing sufficiently, and 95
DEG C~118 DEG C of sterilizing 5min~20min, cooling to obtain the final product.
Further, in above-mentioned steps (a), preferred fermentation method be shake culture, concussion speed for 100rpm~
300rpm；Preferably 150rpm~250rpm；It is more preferably 200rpm.
Further, in above-mentioned steps (a), preferred fermentation temperature is 25 DEG C~35 DEG C；Preferably 27 DEG C~33 DEG C；
It is more preferably 31 DEG C.
Further, in above-mentioned steps (a), preferred fermentation time is 48h~96h；Preferably 60h~84h；More preferably
Ground is 72h.
In conjunction with comparative example 1 also it is found that when except the range of preferred fermentation parameter, Paenibacillus polymyxa CGMCC
No.10062 fermentation degreasing milk medium, using the skim-milk fermentation liquid supernatant obtained after inactivated proteases, centrifugation, to golden yellow
The staphylococcic fungistatic effect of color is decreased obviously.And within preferred scope, inoculum concentration, degreasing milk content, hunting speed, hair
Ferment temperature and time cooperates, and a complete preferred embodiment is formed, so that Paenibacillus polymyxa CGMCC No.10062
The fungistatic effect generated ferment more preferably.
Further, in above-mentioned steps (b), preferred centrifugal speed be 6000g~10000g, preferably 7000g~
9000g is more preferably 8000g；Preferred centrifugation time is 10min~20min, preferably 13min~17min, more preferably
For 15min.
Further, in above-mentioned steps (b), the temperature of high temperature bath is preferably 60 DEG C~90 DEG C, is more preferably 65 DEG C
~85 DEG C, be optimally 80 DEG C；Soaking time is preferably 10min~30min, is more preferably 15min~25min, optimally
For 20min.
Further, in above-mentioned steps (b), when ultrafiltration, the molecular cut off of the ultrafiltration membrane of selection is 1000Da.
Further, in above-mentioned steps (b), above-mentioned freeze-drying is vacuum freeze drying, vacuum freeze drying condition
Preferably: plate layer limiting temperature≤- 60 DEG C, -70 DEG C of cold-trap limiting temperature, plate layer charging thickness 0.5mm~2.0mm, vacuum
Spend 10Pa~30Pa.
Further, in above-mentioned steps (c), the solid-phase extraction column is reversed-phase column；Separation condition are as follows: with volume ratio
(V/V) gradient elution is carried out for 0%~100% methanol/water solution.
Further, in above-mentioned steps (d), the preparation chromatographic column that HPLC is used for reversed-phase column, specification be 30mm ×
100mm, 5 μm~10 μm of packing material size in column, mobile phase: with H2O simultaneously contains 0.05% (V/V) trifluoroacetic acid for A phase, methanol/second
Nitrile simultaneously contains 0.05% (V/V) trifluoroacetic acid for B phase, carries out gradient elution, elution speed is preferably 45mL/min, eluent
Gathering speed is 4mL/ pipe~10mL/ pipe, is more preferably 5mL/ pipe~8mL/ pipe, is optimally 6mL/ pipe.
Above-mentioned step (a), (b), (c) and (d) between front and back or step, in the base for not influencing core of the invention thought
Those skilled in the art can also increase other reasonable steps on plinth, also include within protection scope of the present invention.
Above-mentioned specific embodiment is further illustrated below by embodiment, but is not therefore limited the present invention to described
Scope of embodiments among.In the following examples, the experimental methods for specific conditions are not specified, according to conventional methods and conditions, or presses
It is selected according to product manual.It is analytical reagents if reagent used in embodiment does not add explanation, buys from traditional Chinese medicines collection
Group.Other test apparatuses, strain are not illustrated such as, can directly be bought by commercial sources.
Embodiment 1
1, materials and methods
(a) preparation of seed (fermenting microbe): by Paenibacillus polymyxa CGMCC No.10062, (source of the bacterial strain is asked
Referring to the Chinese patent of Publication No. CN106701610A) cryopreservation tube taken out from ultra low temperature freezer, line mass concentration
In the skimmed milk agar plate of 20g/L, 30 DEG C of aerobic culture 2d；After activating for 2 generations, picking single colonie is inoculated in mass concentration
In the skimmed milk fluid nutrient medium of 80g/L (loading amount be 20mL/100mL triangular flask), 30 DEG C, 180rpm shake culture for 24 hours after, will
Fermentation liquid 10000g is centrifuged 20min, abandons supernatant, takes thallus sterile water wash 2 times, to remove remaining culture medium, finally will
Thallus is suspended in former volume of culture, and mass concentration is in 80g/L skimmed milk fluid nutrient medium, and the bacteria concentration of seed liquor is
1.6x108CFU/mL。
(b) preparation of degreasing milk medium: the skimmed milk that mass concentration is 80g/L is mixed well with distilled water, 118
DEG C, sterilize 15min, is cooled to room temperature to get the degreasing milk medium of required concentration.
(c) Solid phase extraction separation: sample to be separated (following crude product I) is dissolved in deionized water and is configured to concentration and is
The solution of 200mg/mL collects the fraction of various concentration methanol extraction in reverse phase solid phase extraction column, each with Odontothrips loti measurement
The bacteriostatic activity of pipe merges bacteriostatic activity component, vacuum drying.
(d) preparative HPLC is analyzed: sample to be purified (following crude product II) is dissolved in the methanol solution of 80% (V/V)
In be configured to concentration be 200mg/mL solution, be 30mm × 100mm, the preparative HPLC that 5 μm of partial size, using H with specification2O
And be A phase, methanol/acetonitrile containing 0.05% (V/V) trifluoroacetic acid and be B phase containing 0.05% (V/V) trifluoroacetic acid, with 45mL/min
Flow velocity carry out gradient elution (gradient elution step is as shown in table 1 below), with 6mL/ pipe collect eluent, measured with Odontothrips loti
The bacteriostatic activity of each pipe, merges bacteriostatic activity component, and concentration is evaporated and is dried in vacuo.
1 gradient elution step of table
(e) detection method of sample bacteriostatic activity: Odontothrips loti.Make being covered with staphylococcus aureus (ATCC 6538)
To place Oxford cup on the nutrient agar panel of indicator bacteria, 200 μ L samples to be tested are added in cup, are placed in 37 DEG C of culture 16h, see
Examine the presence or absence of inhibition zone and size.Antibacterial circle diameter is bigger, and fungistatic effect is better.
2, the preparation of Cyclic lipopeptide antibiotic FusaricidinB
It is in mass concentration with inoculum concentration 3% (V/V) aseptic inoculation by Paenibacillus polymyxa CGMCC No.10062
In 80g/L degreasing milk medium, 31 DEG C, 200rpm culture 72h obtain fermentation liquid, amount to 1L.
1L fermentation liquid centrifugation 15min is obtained into supernatant, is placed in 80 DEG C of water-bath heat preservation 20min inactivated proteases, it is to be cooled, amount to
950mL, mass concentration 75mg/mL amount to 71.25g.The ultrafiltration membrane for reusing cutoff value 1000Da carries out ultrafiltration behaviour to fermentation liquid
Make, lesser group of detection molecules amount is divided into active constituent, and freeze-drying obtains crude product I, amounts to 53.44g.
Crude product I is first subjected to Solid Phase Extraction, using various concentration methanol/water, the antibacterial work of different component obtained after separation
Property it is as shown in Figs. 1-2, collect active component, freeze-drying obtain crude product II, amount to 2.67g.It is prepared again through HPLC, purification effect
As shown in fig. 3, it was found that component of the retention time in 8.1min has bacteriostatic activity, the fraction is collected, concentration is evaporated and vacuum is dry
It is dry to get Cyclic lipopeptide antibiotic Fusaricidin B, amount to 0.134g.So Cyclic lipopeptide antibiotic Fusaricidin
B total yield is 0.188% (W/W).
3, the determination of Cyclic lipopeptide antibiotic Fusaricidin B structure
Bacteriostatic activity component after above-mentioned HPLC preparation is subjected to high resolution mass spectrum analysis, as a result as shown in figure 4, the activity
The mass-to-charge ratio (m/z) of component is 897.58137, identical as Cyclic lipopeptide antibiotic Fusaricidin B.
For the structure for further confirming that antibacterial substance in this patent, continue to have carried out the component second mass analysis, tie
Fruit is as shown in figure 5, active component has a stable quasi-molecular ions (m/z) 256.23767, in Fusaricidin B structure
15- guanidine radicals -3- hydroxypentadecanoic acid (GHPD) quasi-molecular ions is identical, and quasi-molecular ions (m/z) 879.57073 is [M+H-H2O]+, i.e., female
Core sloughs the corresponding quasi-molecular ions of a hydrone.
In conclusion being Cyclic lipopeptide antibiotic by Substance obtained by above-mentioned preparation method
Fusaricidin B。
Embodiment 2
1, materials and methods
(a) preparation of seed (fermenting microbe): with embodiment 1.
(b) preparation of degreasing milk medium: the skimmed milk that mass concentration is 90g/L being mixed well with distilled water, 95 DEG C,
Sterilize 20min, is cooled to room temperature to get the degreasing milk medium of required concentration.
(c) ultrafiltration, reverse phase solid phase extraction post separation and preparative HPLC and sample bacteriostatic activity detection method: with implement
Example 1.
2, the preparation of lipopeptide antibiotic Fusaricidin B
It is in mass concentration with inoculum concentration 2% (V/V) aseptic inoculation by Paenibacillus polymyxa CGMCC No.10062
In the degreasing milk medium of 100g/L, 35 DEG C, 120rpm culture 96h obtain fermentation liquid.
1L fermentation liquid centrifugation 15min is obtained into supernatant, is placed in 80 DEG C of water-bath heat preservation 10min inactivated proteases, it is to be cooled, amount to
960mL, mass concentration 85mg/mL amount to 81.6g.
Further ultrafiltration, Solid phase extraction separation and preparative HPLC step are same as Example 1, are concentrated into dry doubling vacuum
It is dry, lipopeptide antibiotic Fusaricidin B is made, amounts to 0.228g, total yield is 0.280% (W/W).
3, the determination method of lipopeptide antibiotic Fusaricidin B structure: with embodiment 1.
Embodiment 3
1, materials and methods
(a) preparation of seed (fermenting microbe): by Paenibacillus polymyxa CGMCC No.10062 seed (fermenting microbe)
Preparation: with embodiment 1.
(b) preparation of degreasing milk medium: the skimmed milk that mass concentration is 60g/L is mixed well with distilled water, 100
DEG C, sterilize 15min, is cooled to room temperature to get the degreasing milk medium of required concentration.
(c) ultrafiltration, reverse phase solid phase extraction post separation and preparative HPLC and sample bacteriostatic activity detection method: with implement
Example 1.
2, the preparation of lipopeptide antibiotic Fusaricidin B
It is in mass concentration with inoculum concentration 5% (V/V) aseptic inoculation by Paenibacillus polymyxa CGMCC No.10062
In the degreasing milk medium of 60g/L, 25 DEG C, 300rpm culture 48h obtain fermentation liquid.
1L fermentation liquid centrifugation 15min is obtained into supernatant, is placed in 80 DEG C of water-bath heat preservation 10min inactivated proteases, it is to be cooled, amount to
920mL, mass concentration 65mg/mL amount to 59.8g.
Further ultrafiltration, Solid phase extraction separation and preparative HPLC step are same as Example 1, are concentrated into dry doubling vacuum
It is dry, lipopeptide antibiotic Fusaricidin B is made, amounts to 0.075g, total yield is 0.125% (W/W).
3, the determination method of lipopeptide antibiotic Fusaricidin B structure: with embodiment 1.
Embodiment 4
1, materials and methods
(a) preparation of seed (fermenting microbe): with embodiment 1.
(b) preparation of degreasing milk medium: the skimmed milk that mass concentration is 100g/L is mixed well with distilled water, 120
DEG C, sterilize 10min, is cooled to room temperature to get the degreasing milk medium of required concentration.
(c) ultrafiltration, reverse phase solid phase extraction post separation and preparative HPLC and sample bacteriostatic activity detection method: with implement
Example 1.
2, the preparation of lipopeptide antibiotic Fusaricidin B
It is in mass concentration with inoculum concentration 2% (V/V) aseptic inoculation by Paenibacillus polymyxa CGMCC No.10062
In the degreasing milk medium of 90g/L, 32 DEG C, 150rpm culture 84h obtain fermentation liquid.
1L fermentation liquid centrifugation 15min is obtained into supernatant, is placed in 80 DEG C of water-bath heat preservation 10min inactivated proteases, it is to be cooled, amount to
965mL, mass concentration 95mg/mL amount to 91.7g.
Further ultrafiltration, Solid phase extraction separation and preparative HPLC step are same as Example 1, are concentrated into dry doubling vacuum
It is dry, lipopeptide antibiotic Fusaricidin B is made, amounts to 0.293g, total yield is 0.320% (W/W).
3, the determination method of lipopeptide antibiotic Fusaricidin B structure: with embodiment 1.
Embodiment 5
1, materials and methods
(a) preparation of seed (fermenting microbe): with embodiment 1.
(b) preparation of degreasing milk medium: the skimmed milk that mass concentration is 70g/L is mixed well with distilled water, 110 DEG C
Sterilize 12min, is cooled to room temperature to get the degreasing milk medium of required concentration.
(c) ultrafiltration, reverse phase solid phase extraction post separation and preparative HPLC and sample bacteriostatic activity detection method: with implement
Example 1.
2, the preparation of lipopeptide antibiotic Fusaricidin B
It is in mass concentration with inoculum concentration 4% (V/V) aseptic inoculation by Paenibacillus polymyxa CGMCC No.10062
In the degreasing milk medium of 70g/L, 28 DEG C, 250rpm culture 60h obtain fermentation liquid.
1L fermentation liquid centrifugation 15min is obtained into supernatant, is placed in 80 DEG C of water-bath heat preservation 10min inactivated proteases, it is to be cooled, amount to
940mL, mass concentration 60mg/mL amount to 56.4g.
Further ultrafiltration, Solid phase extraction separation and preparative HPLC step are same as Example 1, are concentrated into dry doubling vacuum
It is dry, lipopeptide antibiotic Fusaricidin B is made, amounts to 0.068g, total yield is 0.121% (W/W).
3, the determination method of lipopeptide antibiotic Fusaricidin B structure: with embodiment 1.
Comparative example 1
By the inoculum concentration in embodiment 1, degreasing milk content, fermentation temperature, the speed of fermentation time and fermentation concussion by
One is adjusted, and obtains the skim-milk fermentation liquid of following set of distinct methods preparation, is placed in 80 DEG C of water-bath heat preservation 10min inactivations
Protease measures the minimum inhibitory concentration (MIC) to staphylococcus aureus, as a result such as 2 institute of table after to be cooled and freeze-drying
Show.
Inhibitory effect of 2 distinct methods of the table preparation gained skim-milk fermentation liquid supernatant to staphylococcus aureus
The above method equally be used to measure the skim-milk fermentation liquid supernatant of Examples 1 to 5 the method preparation, antibacterial
Effect is as shown in table 3.
The skim-milk fermentation liquid supernatant of 3 Examples 1 to 5 of table preparation is to the MIC value for killing Fusariumsp
Referring to shown in table 3 as a result, in the result shown in table 2 it can be concluded that, by the Cyclic lipopeptide antibiotic
The speed of inoculum concentration in the preparation method of Fusaricidin B, degreasing milk content, cultivation temperature, fermentation time and fermentation oscillation
Degree is when be adjusted to except preferred scope, and Paenibacillus polymyxa CGMCC No.10062 can still ferment skimmed milk, produces
Raw Cyclic lipopeptide antibiotic Fusaricidin B, but its yield is decreased obviously.
Comparative example 2
The method of reference implementation example 1 compares by Paenibacillus polymyxa CGMCC No.10062, Lactobacillus casei
(L.casei) ATCC 393, lactobacillus bulgaricus (L.bulgaricus) LB340), streptococcus thermophilus
(S.thermophilus) inhibitory effect of the skim-milk fermentation liquid supernatant of ST-BODY-3 preparation to staphylococcus aureus, tool
Gymnastics is made as follows:
1, materials and methods
(a) preparation of seed (fermenting microbe):
The preparation of Lactobacillus casei and lactobacillus bulgaricus seed: by Lactobacillus casei BTCC 393 and bulgarian milk
The freeze-dried powder of bacillus LB340 is dissolved with a small amount of sterile distilled water respectively, and each personal oese takes a ring to line MRS solid culture
On base (purchase is German from Merck), 37 DEG C of Anaerobic culturels take out for 24 hours, are put into 1mL MRS liquid with oese picking single colonie
Bacterium colony, is dispersed in fluid nutrient medium, 37 DEG C of Anaerobic culturels are for 24 hours by (purchase is German from Merck) with vortex oscillator
It takes out, 50mL MRS liquid is inoculated in 2% (V/V) inoculum concentration, after 37 DEG C of cultures for 24 hours, culture 9000g is centrifuged 10min, abandons
After going supernatant, thallus to wash 2 times with sterile distilled water, is suspended with the sterile distilled water of former volume of culture, fermented accordingly
Seed.
The preparation of streptococcus thermophilus seed: the freeze-dried powder of streptococcus thermophilus ST-BODY-3 is molten with a small amount of sterile distilled water
Solution takes a ring to line on M17 solid medium (purchase is German from Merck) with oese, and 40 DEG C of Anaerobic culturels take out for 24 hours,
It is put into 1mL M17 liquid (purchase is German from Merck) with oese picking single colonie, uniformly divides bacterium colony with vortex oscillator
It dissipating in fluid nutrient medium, 40 DEG C of Anaerobic culturels take out for 24 hours, 50mL M17 liquid is inoculated in 2% (V/V) inoculum concentration, 40 DEG C
After culture for 24 hours, culture 9000g is centrifuged 10min, discards supernatant, and after thallus washs 2 times with sterile distilled water, cultivates body with original
Long-pending sterile distilled water suspends, and obtains the seed of fermentation.
(b) preparation of degreasing milk medium: with embodiment 1.
2, the preparation of skim-milk fermentation liquid supernatant
By each bacterial strain with 3% (V/V) inoculum concentration aseptic inoculation in the degreasing milk medium that mass concentration is 80g/L, point
It Pei Yang not (37 DEG C of Anaerobic culturels of lactobacillus bulgaricus and Lactobacillus casei, 40 DEG C of Anaerobic culturels of streptococcus thermophilus, mostly viscous class bud
30 DEG C of spore bacillus, 180rpm shaken cultivation) 72h, obtain corresponding skim-milk fermentation liquid.
Above-mentioned fermentation liquid is subjected to inactivated proteases according to method described in embodiment 1, degreasing suppurative mastitis is prepared in centrifugation
Zymotic fluid supernatant.
3, the measurement of bacteriostatic activity
After fermented liquid supernatant pH obtained by different strains is adjusted to 7.0,200 μ L addition is taken to be covered with staphylococcus aureus work
In the Oxford cup on the nutrient agar panel of indicator bacteria, to be placed in 37 DEG C of culture 24min, inhibition zone size is observed, as a result such as table 4
Show:
The fungistatic effect of the skim-milk fermentation liquid supernatant of 4 different strains of table preparation
As shown in Table 4, other normal fermentation bacterial strains do not have the energy that fermentation degreasing milk medium generates lipopeptide antibiotic
Power, the degreasing milk medium and Paenibacillus polymyxa CGMCC No.10062 can ferment, finally obtains Cyclic lipopeptide antibiotic
Fusaricidin B, to the inhibitory activity highly significant of staphylococcus aureus.
Comparative example 3
1, materials and methods
(a) preparation of Paenibacillus polymyxa CGMCC No.10062 seed liquor: with embodiment 1.
(b) sucrose the preparation of the M17 fluid nutrient medium containing 1% (W/V) sucrose: is added to M17 liquid with 1% (W/V)
Mixed well in culture medium, sterilize 15min at 121 DEG C, be cooled to room temperature to get.
(c) preparation of wheat bran fluid nutrient medium: the wheat bran that mass percent is 4% (W/V) is mixed well with distilled water,
Sterilize 15min at 121 DEG C, be cooled to room temperature to get.
(d) ultrafiltration, reverse phase solid phase extraction post separation and preparative HPLC and sample bacteriostatic activity detection method: with implement
Example 1.
2, the preparation of fermented liquid supernatant
By Paenibacillus polymyxa CGMCC No.10062 with 3% (V/V) inoculum concentration difference aseptic inoculation in sucrose 1%
(W/V) in M17 fluid nutrient medium, wheat bran fluid nutrient medium, after 30 DEG C, 180rpm shaken cultivation 72h, fermentation liquid is obtained.
Above-mentioned fermentation liquid is subjected to inactivated proteases according to method described in embodiment 1, corresponding hair is prepared in centrifugation
Zymotic fluid supernatant.
3, the measurement of bacteriostatic activity
After gained fermented liquid supernatant pH is adjusted to 7.0,200 μ L addition is taken to be covered with staphylococcus aureus as indicator bacteria
Nutrient agar panel on Oxford cup in, be placed in 37 DEG C of culture 16h, observe inhibition zone size, the results are shown in Table 5.By table 5
It is found that Paenibacillus polymyxa CGMCC No.10062 is in other culture mediums, as sucrose 1% (W/V) M17 fluid nutrient medium and
It ferments, the component for having obvious inhibitory activity of staphylococcus aureus cannot not generated in wheat bran fluid nutrient medium
Cyclic lipopeptide antibiotic Fusaricidin B, showing Paenibacillus polymyxa CGMCC No.10062 of the present invention can be using de-
The metabolism of rouge cream culture medium generates Cyclic lipopeptide antibiotic Fusaricidin B.
The fungistatic effect for the fermented liquid supernatant that 5 different culture medium of table is fermented
Cyclic lipopeptide antibiosis is prepared using Paenibacillus polymyxa CGMCC No.10062 to provided by the present invention above
The method of plain Fusaricidin B is described in detail.Specific case used herein is to the principle of the present invention and implementation
Mode is expounded, and the above description of the embodiment is only used to help understand the method for the present invention and its core ideas.It should
It points out, it for those skilled in the art, without departing from the principle of the present invention, can also be to this hair
Bright some improvement and modification can also be carried out, and these improvements and modifications also fall within the scope of protection of the claims of the present invention.
Claims (10)
1. application of the Paenibacillus polymyxa CGMCC No.10062 in preparation Cyclic lipopeptide antibiotic Fusaricidin B.
2. using the method for Paenibacillus polymyxa CGMCC No.10062 preparation Cyclic lipopeptide antibiotic Fusaricidin B,
It is characterized in that, method includes the following steps:
(a) Paenibacillus polymyxa CGMCC No.10062 is inoculated in degreasing milk medium and is fermented, obtain fermentation liquid；
(b) the fermentation liquid centrifuging and taking supernatant for obtaining step (a), inactivated proteases use molecular cut off 1000Da after cooling
Ultrafiltration membrane obtain crude product I；
(c) the crude product I for obtaining step (b) collects bacteriostatic activity component, vacuum drying by reverse phase solid phase extraction post separation
Afterwards, crude product II is obtained；
(d) the crude product II for obtaining step (c) is purified by preparative HPLC, is collected bacteriostatic activity fraction, after vacuum drying, is obtained
To Cyclic lipopeptide antibiotic Fusaricidin B after purification.
3. the preparation method of Cyclic lipopeptide antibiotic Fusaricidin B according to claim 2, which is characterized in that step
Suddenly in (a), the inoculum concentration of the Paenibacillus polymyxa CGMCC No.10062 is 1.6x106~8.0x106CFU/mL。
4. the preparation method of Cyclic lipopeptide antibiotic Fusaricidin B according to claim 2, which is characterized in that step
Suddenly in (a), the degreasing milk medium includes skimmed milk and water, and the mass concentration of skimmed milk is 60g/L~100g/L.
5. the preparation method of Cyclic lipopeptide antibiotic Fusaricidin B according to claim 2, which is characterized in that step
Suddenly in (a), the fermentation is shake culture, and concussion speed is 100rpm~300rpm；The fermentation temperature is 25 DEG C~35
DEG C, fermentation time is 48h~96h.
6. the preparation method of Cyclic lipopeptide antibiotic Fusaricidin B according to claim 2, which is characterized in that step
Suddenly in (b), the speed of the centrifugation is 6000g~10000g, and the time is 10min~20min.
7. the preparation method of Cyclic lipopeptide antibiotic Fusaricidin B according to claim 2, which is characterized in that step
Suddenly in (b), the temperature of the high temperature bath is 60 DEG C~90 DEG C, and the time is 10min~30min.
8. the preparation method of Cyclic lipopeptide antibiotic Fusaricidin B according to claim 2, which is characterized in that step
Suddenly in (c), separation condition are as follows: gradient elution is carried out with the methanol/water solution that volume ratio is 0%~100%.
9. the preparation method of Cyclic lipopeptide antibiotic Fusaricidin B according to claim 2, which is characterized in that step
Suddenly in (d), the HPLC prepares the reversed column that column is 30mm × 100mm specification, and packing material size is 5 μm~10 μm in column；Stream
Dynamic phase: with H2O is simultaneously A phase, methanol/acetonitrile containing 0.01%~0.1% trifluoroacetic acid and containing 0.01%~0.1% trifluoroacetic acid is
B phase, carries out gradient elution, and elution speed is 30mL/min~50mL/min, the gathering speed of fraction be 0.1min/ pipe~
0.2min/ pipe.
10. the preparation method of Cyclic lipopeptide antibiotic Fusaricidin B according to claim 2, which is characterized in that receive
Collect retention time in the fraction of 7.90min~8.10min, to get Cyclic lipopeptide antibiotic Fusaricidin after vacuum drying
B。
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