CN105754917B - A kind of Plipastatin-derived cyclopentalipeptide and its producing bacteria and application - Google Patents

Abstract

The invention discloses a Plipastatin-derived cyclopentalipeptide and its producing bacteria and application. The Bacillus subtilis Best2 producing Plipastatin-derived cyclopentalipeptide is targeted to delete the ppsC and ppsD genes in the Bacillus subtilis pB2-L genome by the method of homologous recombination to obtain the Bacillus subtilis Best2 that lacks the ppsC and ppsD genes. Bacillus subtilis Best2 was fermented in a modified Landy medium to obtain a fermentation broth containing Plipastatin-derived cyclopentalipeptide, and the fermentation broth was separated and purified to obtain a crude extract containing Plipastatin-derived cyclopentalipeptide. The novel plipastatin-derived cyclopentalipeptide not only still has a certain antibacterial activity against filamentous fungi, but also has an improved antibacterial activity against Gram-positive bacteria.

Description

A kind of Plipastatin-derived cyclopentalipeptide and its producing bacteria and application

technical field

The invention belongs to the fields of molecular biology and genetic engineering, and relates to a Plipastatin-derived cyclopentalipeptide, its producing bacteria and its application.

Background technique

Secondary metabolites produced by many microorganisms contain a class of lipopeptide antibacterial substances composed of small molecular weight, cyclic short peptide backbones and β-hydroxy fatty acid chain tails. In addition to good surface activity and antibacterial activity, they also have antitumor properties , antiviral, anti-infective and other biological effects and other pharmacological activities; in addition, lipopeptides and their derivatives have diverse structures, which greatly broaden and enrich the natural compound library of drug screening sources, and have potential in the creation of new effective drugs. value.

Plipastatin is a substance very similar in structure and properties to Fengycin, produced by Bacillus subtilis 168 strain with functionally active sfp gene, and also belongs to the Fengycin family; it is a β-hydroxy fatty acid composed of 14 to 18 carbon atoms and a Cyclic decapeptide is composed of two parts. Plipastatin produced by microbial fermentation is a mixture of various homologs; Plipastatin shows good antibacterial activity against filamentous fungi, but has no effect on yeast and bacteria.

Plipastatin is a class of non-ribosomal peptides synthesized by Plipastatin synthase through the "multi-carrier thiolation template mechanism"; the Bacillus subtilis Plipastatin synthase gene cluster contains 5 ORFs, which encode the five enzyme subunits of Plipastatin synthase. The gene clusters are linearly arranged, namely ppsA, ppsB, ppsC, ppsD and ppsE; with the development of combinatorial biology, the present invention will recombine the Plipastatin synthase system at the gene level to construct a hybrid Plipastatin synthase, The use of this hybrid enzyme system to produce natural active substances provides a new idea and strategy that is different from chemical synthesis in improving the creation of microbial drugs.

SUMMARY OF THE INVENTION

The purpose of the present invention is to provide a Bacillus subtilis Best2 producing Plipastatin-derived cyclopentalipeptide.

Another object of the present invention is to provide a method for preparing Plipastatin-derived cyclopentalipeptide and the obtained Plipastatin-derived cyclopentalipeptide.

Another object of the present invention is to provide the use of Plipastatin-derived cyclopentalipeptide.

A Bacillus subtilis Best2 producing Plipastatin-derived cyclopentalipeptide is targeted to delete the ppsC and ppsD genes in the genome of Bacillus subtilis pB2-L with the accession number of CGMCC No. 11723 by the method of homologous recombination to obtain the deletion of ppsC and ppsD. Bacillus subtilis Best2 with ppsD gene producing Plipastatin-derived cyclopentalipeptide.

Described Bacillus subtilis Best2 is preferably constructed by the following method:

(1) Construction of a knockout plasmid pKS-CD that lacks the ppsC and ppsD genes:

a. Design the following primers according to the NCBI database:

P1: SEQ ID NO.1, P2: SEQ ID NO.2, P3: SEQ ID NO.3, P4: SEQ ID NO.4;

b. Using the genome of B. subtilis pB2-L as a template, use primers P1, P2 and primers P3, P4 to amplify the upstream and downstream fragments respectively, and then use the above and downstream fragments as templates, add P1 and P4 to the reaction system to carry out fusion PCR reaction , to obtain the knockout gene fragment CD;

c. Cut the knockout gene fragment CD and plasmid pKS2 with restriction endonuclease, and insert the knockout gene fragment into plasmid pKS2 through ligation reaction to obtain pKS-CD;

(2) The recombinant plasmid pKS-CD transforms the competent cells of Bacillus subtilis pB2-L with the deposit number of CGMCC No. 11723 to obtain the Bacillus subtilis containing the knockout plasmid pKS-CD;

(3) Screening and identification of knockout strains lacking ppsC and ppsD genes: The transformed Bacillus subtilis containing the knockout plasmid pKS-CD was used to induce recombination at 37°C, and the red mold on LB solid medium plate was screened out. A single colony that is sensitive to phospholipids was extracted, the genome was extracted, the knockout gene fragment was verified by PCR, and the knockout strain was further confirmed by sequencing. .

The application of the Bacillus subtilis Best2 of the present invention in the preparation of Plipastatin-derived cyclopentalipeptide.

A method for preparing Plipastatin-derived cyclopentalipeptide by using the Bacillus subtilis Best2, comprising fermenting the Bacillus subtilis Best2 in an improved Landy medium to obtain a fermentation liquid containing the Plipastatin-derived cyclopentalipeptide, and performing fermentation on the fermented Bacillus subtilis Best2. The liquid was separated and purified to obtain a crude extract containing Plipastatin-derived cyclopentalipeptide.

The described method utilizing the Bacillus subtilis Best2 of claim 1 to prepare Plipastatin-derived cyclopentalipeptide preferably comprises the following steps:

a. Bacillus subtilis Best2 is cultivated to obtain an antibacterial substance fermentation broth;

b. Centrifuge the fermentation broth of antibacterial substances, collect the supernatant, acid-precipitate, and extract the precipitate with methanol to obtain an antibacterial extract;

c. The antibacterial extracts were separated and identified by LC-ESI-MS/MS. Plipastatin-derived cyclopentalipeptide was identified.

The described method utilizing the Bacillus subtilis Best2 of claim 1 to prepare Plipastatin-derived cyclopentalipeptide further preferably comprises the following steps:

a. Cultivate Bacillus subtilis Best2 to obtain an antibacterial substance fermentation broth

Inoculate Bacillus subtilis Best2 on the slanted PDA agar medium in a test tube, cultivate at 37°C for 24 hours, and activate the strain; then inoculate the Bacillus subtilis Best2 strain activated on the slant of the test tube into LB liquid medium, at 37°C, 180r·min ^-1 Cultivated for 18h to logarithmic growth phase to obtain fermented seed liquid; inoculate Bacillus subtilis Best2 seed liquid at 5% concentration into the improved Landy medium, cultivate at 33°C, 180r·min ^-1 for 72h to obtain fermentation liquid; Among them, the components of the modified Landy medium were 40.0g·L ^-1 glucose, 14.0g·L ^-1 L-sodium glutamate, 2.3g·L ^-1 (NH ₄ ) ₂ SO ₄ , 1.0g·L ^-1 K ₂ HPO ₄ , 0.5g·L ^-1 MgSO ₄ , 0.5g·L ^-1 KCl, 1.6mg·L ^-1 CuSO ₄ , 1.2mg·L ^-1 Fe ₂ (SO ₄ ) ₃ ^, 0.4mg·L -1 ¹ MnSO4, composed of 100mM 3-N-marphaline propanesulfonic acid (MOPS), pH 7.0～7.2;

b. Centrifuge the fermentation broth of antibacterial substances, collect the supernatant, precipitate with acid, and extract the precipitate with methanol to obtain an antibacterial extract:

The above fermentation broth was centrifuged at 5000 r/min for 15 min to remove the bacterial cells, the supernatant was adjusted to pH 2.0, overnight at a low temperature, and the precipitate was collected by centrifugation at 5000 r/min for 20 min, dissolved in methanol, adjusted to pH 7.0, and cooled on a magnetic stirrer at low temperature. After stirring for 5h and centrifuging at 10,000 r/min for 10min, the supernatant obtained is the crude extract of Plipastatin-derived cyclopentalipeptide;

c. The antibacterial extract was separated and identified by LC-ESI-MS/MS for Plipastatin-derived cyclopentalipeptide:

The antibacterial substances produced by Bacillus subtilis Best2 were determined by liquid chromatography-Fourier transform ion cyclotron resonance high-resolution mass spectrometer to determine the molecular weight and identify its structure. Column temperature: 25°C; injection volume: 10 μL; mobile phase is A phase: acetonitrile + 0.1% formic acid and B phase: water + 0.1% formic acid, flow rate is 0.2 mL/min, gradient elution conditions are: 0-20 min, A phase 5-95%, B phase 95-5%; 20-22min, A phase 95-100%, B phase 5-0%, ESI ion source, positive ion detection mode, spray voltage 3.5kV; capillary temperature 275 ℃; sheath gas (N ₂ ) flow rate 35arb; auxiliary gas (N ₂ ) flow rate 10arb; sweep tail gas (N ₂ ) flow rate 3arb; tubular lens: 110V; collision energy 35V; scanning range 100～2000u; Scanning mode; substances corresponding to m/z 903.58, m/z 875.55 and m/z 889.56 are Plipastatin-derived cyclopentalipeptides and their homologues.

The Plipastatin-derived cyclopentalipeptide or its crude extract obtained according to the method of the present invention. The Plipastatin-derived cyclopentalipeptide includes three components with molecular weights of 875.55, 889.56 and 903.58.

Application of the Plipastatin-derived cyclopentalipeptide or its crude extract of the present invention in the preparation of a preparation for inhibiting filamentous fungi and/or Gram-positive bacteria; preferably in the preparation of a preparation for inhibiting filamentous fungi and/or Gram-positive bacteria Bacterial biopesticides, food preservatives or biocontrol agents.

The beneficial effects of the present invention

The present invention uses the method of homologous recombination to transform the Plipastatin synthase system, and obtains the knockout strain Best2 by knocking out the ppsC and ppsD genes in the pps operon; the pps operon of the knockout strain is only composed of three open cells. The reading frame is composed of ppsA, ppsB and ppsE. The new synthetase system encoded by it cannot synthesize the lipopeptide Plipastatin, but can synthesize the new lipid composed of β-hydroxy fatty acid and cyclic pentapeptide composed of 16-18 carbon atoms. Peptide; this new plipastatin-derived cyclopentalipeptide is the first successful product, which not only has a certain antibacterial activity against filamentous fungi (take Fusarium oxysporum as an example), but also has antibacterial activity against Gram-positive bacteria (aureus aureus). Staphylococcus as an example) showed improved bacteriostatic activity.

The gene traceless knockout method adopted in the present invention, the obtained Bacillus subtilis Best2 has no resistance gene marker, has the stability of passage genetic, its colony morphology has no obvious difference with the original strain, and the strain can be used as the recipient strain of genetic engineering , and further study the structure and function of Plipastatin synthase by the method of combinatorial biology.

Description of drawings:

Figure 1 is a schematic diagram of the knockout plasmid pKS-CD;

Fig. 2 is a schematic diagram of the principle of homologous recombination;

Fig. 3 is fusion PCR agarose gel electrophoresis figure;

Figure 4 is an ESI-MS image of a novel plipastatin-derived cyclopentalipeptide;

Figure 5 CID map of cyclopentalipeptide m/z903.58 as a leader and its molecular structure and ion fragment analysis;

Figure 6 shows the antibacterial activity of the knockout strain Best2 cyclopentalipeptide

Biomaterial deposit information:

pB2-L, classified as Bacillus subtilis, is deposited in the General Microbiology Center of the China Microbial Culture Collection Management Committee, and the deposit address is No. 3, No. 1, Beichen West Road, Chaoyang District, Beijing, Institute of Microbiology, Chinese Academy of Sciences, preservation number It is CGMCC No.11723, and the deposit date is November 23, 2015.

Detailed ways

The present invention will be further illustrated by the following examples. The experimental methods without specifying the specific conditions in the following examples are basically carried out in accordance with the common molecular cloning manuals.

Example 1: Obtaining CD knockout gene fragments

According to the whole genome sequence of B. subtilis 168 provided by the NCBI database, the selected homology arms were 1422bp (1983998 to 1982577) in the upstream homology arm and 945bp (1964049 to 1963105) in the downstream homology arm. The specific implementation steps of this successful case are as follows:

First, primers were designed according to Prime5.0 software

P1: GTCGAC GTCGAAGGCGAGATTACTTGGACAC (SEQ ID NO. 1)

P2: CATAGTGTCTGCACCTTTCTTCACCTATAGTTTGTTGACGGCTCCCATG (SEQ ID NO. 2)

P3: CATGGGAGCCGTCAACAAACTATAGGTGAAGAAAGGTGCAGACACTATG (SEQ ID NO. 3)

P4: GGTACC TTTAATTCGAATCGGTATGGT (SEQ ID NO. 4)

The 5' end of primer P1 introduces a Sal restriction site; the 5' end of primer P4 introduces a Kpn restriction site. Using the B.subtilispB2-L genome as a template, the upstream and downstream fragments were amplified with primers P1, P2 and P3, P4, respectively. Gel purification. Then the above and downstream fragments are used as templates, and P1 and P4 are added to the reaction system for fusion PCR reaction to obtain CD gene knockout fragments. The agarose gel electrophoresis of each PCR product is shown in Figure 3, M is 1kb ladder Marker, and lane 1 is upstream Homology arm, lane 2 is the downstream homology arm, lane 3 is the fusion PCR product. The gene was cloned into pMD19-T vector and sequenced.

Example 2: Construction of Knockout Plasmids

Use restriction enzymes Sal and Kpn to knock out the CD gene fragment and plasmid pKS2 (the full sequence of the plasmid is shown in SEQ ID NO.5, Yang Huilin, Wang Kun, Liao Yuling, Wang Bin, Lin Ying, Pan Li. Starch solution Knockout of Bacillus ptsGHI gene and growth characteristics of defective strains DH5α competent, pick positive transformants, extract plasmids, sequence verification, the results show that the connection is successful. The plasmid pKS-CD was obtained.

Example 3: Knockout plasmid pKS-CD transformed B. subtilis pB2-L

Similar to the chemical transformation method of Bacillus subtilis 168 reported in domestic and foreign literature, specific successful examples are as follows:

(1) The starting strain B.subtilis pB2-L was streaked on LB medium at 37°C and cultured for 2 days;

(2) pick single colony and inoculate in 10mL GM1 solution, 30 ℃, ^100r ·min overnight culture;

(3) The cell culture was transferred to GMII solution at 1:10 (volume ratio), 37° C., 200 r·min ⁻¹ , and cultivated for 3.5 h;

(4) The cell culture was transferred to GMII solution at 1:10 (volume ratio) again, 30° C., 100 r·min ⁻¹ , and cultivated for 90 min;

(5) Take out the cell culture, centrifuge at 5000 r·min ^-1 for 10 min to collect the cells, retain the supernatant, resuspend the cells with the supernatant at 1:10 (volume ratio), and divide the cells into 200 μL per tube. Current use.

(6) Add about 1 μg of plasmid pKS-CD to 200 μL of the prepared competent cells, culture at 30°C, 80 r·min ^-1 for 20 to 40 min; spread evenly on LB solid medium containing 10 μg/mL erythromycin Plate and culture at 30°C for 36-48 hours to obtain Bacillus subtilis containing the knockout plasmid pKS-CD.

Wherein, GM1: 10×T baase 100mL, 100g·L ^-1 glucose 50mL, 50g·L ^-1 yeast extract 20mL, 10g·L ^-1 acid hydrolyzed casein 20mL, 2.5g·L ^-1 tryptophan 20mL, add water to 1000mL;

GMΙΙ: 10×T baase 100mL, 100g·L ^-1 glucose 50mL, 50g·L ^-1 yeast extract 0.4mL, 10g·L ^-1 acid hydrolyzed casein·5mL, 0.5mol·L ^-1 MgCl ₂ 5mL, 0.05mol ·L ^-1 CaCl ₂ 10mL, add water to 1000mL;

10×T baase: (NH ₄ ) ₂ SO ₄ 20.0 g, K ₂ HPO ₄ 140.0 g, KH ₂ PO ₄ 60.0 g, Na ₃ C ₆ H ₅ O ₇ · 2H ₂ O 10.0 g, MgSO ₄ · 7H ₂ O 2.0g, add water to 1000mL;

Each component in GM1 and GMII was sterilized separately, and mixed in proportion when used, wherein amino acids were sterilized by filtration, glucose was sterilized at 115° C. for 30 minutes, and the rest were all sterilized at 121° C. for 20 minutes.

Example 4: Screening and identification of knockout strains with deleted genes ppsC and ppsD

(1) The Bacillus subtilis strain containing the knockout plasmid pKS-CD was inoculated into 20 mL of LB liquid medium at 37°C, cultured at 180 r·min ^-1 for 24 h, diluted and spread on LB solid culture containing 10 μg/mL erythromycin basal plate, cultured overnight at 37°C;

(2) Pick a single colony and inoculate it in 20 mL of LB liquid medium at 30°C, culture at 180 r·min ^-1 for 48h, dilute it and spread it on a LB solid medium plate, and incubate at 37°C overnight;

(3) Screen out the erythromycin-sensitive single colonies on the LB solid medium plate, extract the genome as a template, use primers P1 and P4 PCR to obtain the knockout gene fragment CD, purify and sequence, and identify the genes ppsC and ppsD as missing Bacillus subtilis, hereinafter referred to as the knockout strain Best2.

Example 5: Production and Molecular Structure Identification of Novel Plipastatin-Derived Cyclic Pentalipopeptides

(1) A single colony of the knockout strain Best2 was inoculated into LB medium, and cultured at 37°C for 18h at 180r·min ^-1 to obtain a fermented seed liquid;

(2) The fermented seed liquid was transferred to the modified Landy medium, and cultured at 33°C for 72 hours at 180r·min ^-1 to obtain a fermentation liquid; the modified Landy medium component was 40.0g·L ^-1 glucose, 14.0g· L ^-1 L-Sodium Glutamate, 2.3g·L ^-1 (NH ₄ ) ₂ SO ₄ , 1.0g·L ^-1 K ₂ HPO ₄ , 0.5g·L ^-1 MgSO ₄ , 0.5g·L ^-1 KCl, 1.6 mg·L ^-1 CuSO ₄ , 1.2 mg·L ^{- 1} Fe ₂ (SO ₄ ) ₃ , 0.4 mg·L ^-1 MnSO ₄ , 100 mM 3-N-marpheline propanesulfonic acid (MOPS) , pH 7.0～7.2.

(3) centrifuge the fermentation at 5000g for 15min, collect the supernatant, and treat it with 6N HCl acid precipitation (pH=2.0) overnight, collect the precipitate by centrifugation, redissolve the precipitate with methanol, adjust pH=7.0, and obtain an antibacterial crude extract;

After the antibacterial crude extract was treated with a 0.45 μm membrane, liquid chromatography-Fourier transform ion cyclotron resonance high-resolution mass spectrometry (LC-FTICR-MS) was used to determine the theoretically predicted new Plipastatin-derived lipopeptides in the antibacterial crude extract molecular weight and identify its molecular structure.

(4) LC-ESI-MS/MS conditions:

a) Mass spectrometer column: C18-MS-II 2.6mmФ×250mm, TSK;

b) Column temperature: 25°C;

c) Injection volume: 10 μL;

The mobile phase is acetonitrile + 0.1% formic acid (A) and water + 0.1% formic acid (B), the flow rate is 0.2 mL/min, and the gradient elution conditions are: 0-20 min, A 5-95%, B 95-5%; 20-22min, A 95-100%, B 5-0%.

ESI ion source, positive ion detection mode; spray voltage 3.5kV; capillary temperature 275°C; sheath gas (N ₂ ) flow rate 35arb; auxiliary gas (N ₂ ) flow rate 10arb; sweep gas (N ₂ ) flow rate 3arb; tubular lens: 110V; collision energy 35V; scanning range 100～2000u; scanning mode: LTQ adopts full scanning mode.

(5) Figure 4 is the ESI-MS image of the knockout strain Best2 producing cyclopentalipeptide. It can be seen from Figure 4 that m/z 875.55, m/z 889.56 and m/z 903.58 are consistent with the theoretically predicted cyclopentalipide The mass-to-charge ratios of the peptide homologs are the same, and the molecular weights between the homologs differ by 14 Da; in order to further determine the structure of the lipopeptide, m/z 903.58 was selected as the leader for CID analysis, and the ions obtained from Figure 5 The fragments are consistent with the predicted molecular structure of cyclopentalipeptide, so it can be inferred that the substance with m/z 903.58 is the predicted cyclopentalipeptide. Similarly, m/z 875.55 and m/z 889.56 are their homologues, respectively.

Example 6 Bacteriostatic test of a new type of Plipastatin-derived cyclopentalipeptide produced by knockout strain Best2

1) The bacterial indicator bacteria used in the antibacterial test is Staphylococcus aureus AS1.2465, LB medium, cultured at 37°C; fungal indicator bacteria: Fusarium oxysporumf.sp.cucumerinum 37438 ), PDA medium, cultured at 28°C; all purchased from the Culture Collection Center, Institute of Microbiology, Chinese Academy of Sciences.

2) Bacteriostatic test method: Dilute the bacteria cultured to the logarithmic phase to 10 ⁶ cfu/L with sterile physiological saline, mix 1.0 mL of bacterial liquid into the LB agar medium, pour it into a petri dish and wait for it to cool down. , using the agar hole diffusion method, take 100 μL of the sample and add it to the agar hole, incubate at 37°C overnight, and observe the antibacterial effect.

3) Fungal antibacterial test method: the method is the same as that of bacteria. Using the agar hole diffusion method, 1.0 mL of indicator bacteria with a concentration of 10 ⁶ cfu/L was mixed into the PDA medium, and 100 μL of the sample was added to the agar hole and incubated at 28°C for more than 72 hours to observe the bacteriostatic effect.

It can be seen from Figure 6 that the lipopeptide sample has an obvious inhibition zone against Fusarium oxysporum, indicating that the new Plipastatin-derived cyclopentalipeptide produced by strain Best2 still has bacteriostatic activity against fungi; The diameter of the bacterial circle was larger than that of the original strain pB2-L, indicating that the strain Best2 produced a new type of Plipastatin-derived cyclopentalipeptide, and its antibacterial activity against Staphylococcus aureus was improved.

Since the new Plipastatin-derived cyclopentalipeptide produced by Bacillus subtilis Best2 has obvious inhibitory effect on a variety of crop pests and exhibits high efficiency and broad-spectrum characteristics, it can be developed into a new type of natural food preservatives and biocontrol agents, which are useful for reducing crop diseases and insect pests, etc. of great value.

Claims (10)

1. a kind of bacillus subtilis (Bacillus subtilis) Best2 for producing antibacterial material, it is characterised in that logical The method targeting missing deposit number for crossing homologous recombination is in the bacillus subtilis pB2-L genome of CGMCC No.11723 PpsC and ppsD gene obtains the bacillus subtilis Best2 that missing ppsC and ppsD gene produces antibacterial material.

2. bacillus subtilis Best2 according to claim 1, it is characterised in that construct by the following method:

(1) the knockout plasmid pKS-CD of building missing ppsC and ppsD gene:

A. following primer is designed according to ncbi database:

P1:SEQ ID NO.1, P2:SEQ ID NO.2, P3:SEQ ID NO.3, P4:SEQ ID NO.4；

B. using B.subtilis pB2-L genome as template, upstream and downstream piece is expanded respectively with primer P1, P2 and primer P3, P4 Section, then above, segments downstream are template, and P1, P4 are added in the reaction system and carries out fusion DNA vaccine reaction, obtains and knocks out gene piece Section CD；

C. digestion is carried out to genetic fragment CD and plasmid pKS2 is knocked out with restriction enzyme, and base will be knocked out by connecting reaction Because segment insertion plasmid pKS2 obtains pKS-CD；

(2) recombinant plasmid pKS-CD converts the bacillus subtilis pB2-L competent cell that deposit number is CGMCC No.11723, It obtains containing the bacillus subtilis for knocking out plasmid pKS-CD；

(3) screening and identification of the knock-out bacterial strain of ppsC and ppsD gene have been lacked: with the transformed plasmid pKS-CD containing knockout Bacillus subtilis carry out 37 DEG C of temperature-induced recombinations, filter out the single bacterium of erythromycin-sensitive in LB solid medium tablets It falls, extracts its genome, verified with PCR method and knock out genetic fragment, and further confirmed that by sequencing as knock-out bacterial strain, it should Knock-out bacterial strain is the bacillus subtilis Best2 of the production antibacterial material.

3. bacillus subtilis Best2 described in claim 1 is preparing the application in antibacterial material；The antibacterial material packet Include 3 kinds of components that molecular weight is 875.55,889.56 and 903.58.

4. a kind of method for preparing antibacterial material using bacillus subtilis Best2 described in claim 1, it is characterised in that packet It is contained in the Landy culture medium of improvement and ferments to bacillus subtilis Best2, obtain derivative ring containing Plipastatin five The fermentation liquid of lipopeptid, isolates and purifies fermentation liquid, obtains crude extract containing antibacterial material；Wherein, the Landy of the improvement Nutrient media components 40.0gL^-1Glucose, 14.0gL^-1L-sodium, 2.3gL^-1(NH₄)₂SO₄, 1.0gL^{- 1}K₂HPO₄, 0.5gL^-1MgSO₄, 0.5gL^-1KCl, 1.6mgL^-1CuSO₄, 1.2mgL^-1Fe₂(SO₄)₃, 0.4mgL^{- 1}What MnSO4,100mM 3-N- Ma coffee quinoline propane sulfonic acid were constituted, pH 7.0~7.2.

5. according to the method described in claim 4, it is characterized in that comprising the steps of:

A. bacillus subtilis Best2 is cultivated, obtains antibacterial material fermentation liquid；

B. antibacterial material fermentation liquid is centrifuged, and collects supernatant, and acid is heavy, and precipitating is carried out methanol extracting, obtains antimicrobial extract；

C. antimicrobial extract separates identification antibacterial material by LC-ESI-MS/MS.

6. according to the method described in claim 5, it is characterized by comprising the following steps:

A. bacillus subtilis Best2 is cultivated, obtains antibacterial material fermentation liquid

Bacillus subtilis Best2 is inoculated on test tube slant PDA agar medium, 37 DEG C are cultivated for 24 hours, by actication of culture； The bacillus subtilis Best2 strain that test tube slant activates is inoculated in LB liquid medium again, 37 DEG C, 180rmin^-1Culture 18h obtains fermentation seed liquid to logarithmic growth phase；Bacillus subtilis Best2 seed liquor is seeded to improvement with 5% concentration In Landy culture medium, 33 DEG C, 180rmin^-172h is cultivated, fermentation liquid is obtained；Wherein, the Landy nutrient media components of improvement 40.0g·L^-1Glucose, 14.0gL^-1L-sodium, 2.3gL^-1(NH₄)₂SO₄, 1.0gL^-1K₂HPO₄, 0.5gL^{- 1}MgSO₄, 0.5gL^-1KCl, 1.6mgL^-1CuSO₄, 1.2mgL^-1Fe₂(SO₄)₃, 0.4mgL^-1MnSO4,100mM3-N- What Ma coffee quinoline propane sulfonic acid was constituted, pH 7.0~7.2；

B. antibacterial material fermentation liquid is centrifuged, and collects supernatant, and acid is heavy, and precipitating is carried out methanol extracting, obtains antimicrobial extract:

It taking above-mentioned fermentation liquid to be centrifuged 15min at 5000r/min and removes thallus, supernatant tune pH to 2.0, low temperature is overnight, 5000r/min centrifugation 20min takes precipitating, is dissolved with methanol, adjusts pH to 7.0, stirs 5h in low temperature on magnetic stirring apparatus, The supernatant obtained after 10000r/min centrifugation 10min is antibacterial material crude extract；

C. antimicrobial extract separates identification antibacterial material by LC-ESI-MS/MS:

Bacillus subtilis Best2 produces antibacterial material and uses liquid chromatogram-Fourier transformation ion cyclotron resonance high resolution mass spectrum Instrument measures its molecular weight and identifies its structure, condition are as follows: mass spectrum column: C18-MS- II 2.6mm Ф × 250mm, TSK；Column temperature: 25℃；Sampling volume: 10 μ L；Mobile phase is A phase :+0.1% formic acid of acetonitrile and B phase :+0.1% formic acid of water, flow velocity 0.2mL/ Min, condition of gradient elution are as follows: 0-20min, A phase 5-95%, B phase 95-5%；20-22min, A phase 95-100%, B phase 5-0%, ESI ion source, using positive ion detection mode, spray voltage 3.5kV；275 DEG C of capillary temperature；Sheath gas N₂Flow velocity 35arb； Assist gas N₂Flow velocity 10arb；Wind up gas N₂Flow velocity 3arb；Tube lens: 110V；Collision energy 35V；Scanning range 100~ 2000u；Scanning mode: LTQ uses full scan mode；M/z 903.58 and m/z 875.55, the corresponding substance of m/z 889.56 As antibacterial material and homologue.

7. the antibacterial material or its crude extract that are obtained according to method described in any one of claim 4~6.

8. antibacterial material according to claim 7 or its crude extract, it is characterised in that the antibacterial material includes molecule 3 kinds of components that amount is 875.55,889.56 and 903.58.

9. antibacterial material as claimed in claim 7 or its crude extract inhibit filamentous fungi and/or gram-positive bacterium in preparation Preparation in application.

10. application according to claim 9, it is characterised in that antibacterial material as claimed in claim 7 or its crude extract exist Application in the biological pesticide of preparation inhibition filamentous fungi and/or gram-positive bacterium, food preservative.