CN110951667B - Fenogen element high-yield strain LPB-18N and breeding and application thereof - Google Patents

Abstract

The invention discloses a high-yield Fenugreesu bacterial strain LPB-18N, and breeding and application thereof, which is identified as Bacillus amyloliquefaciens (Bacillus amyloliquefaciens) which is preserved in the common microorganism center of the China Committee for culture Collection of microorganisms, wherein the preservation time is 2019, 10 and 22 days, and the preservation number is CGMCC NO.18720. The high-yield Fenogen strain LPB-18N can effectively produce Fenogen in large quantity, saves cost, improves industrial production and commercial potential, has the characteristics of wide antibacterial spectrum, difficult generation of drug resistance, safety, degradability and the like, is widely applied to the field of food, and has huge economic benefit and potential for developing novel food preservatives.

Description

Fenogen element high-yield strain LPB-18N and breeding and application thereof

Technical Field

The invention belongs to the technical field of microorganisms, and relates to a high-yield Fengycin strain LPB-18N and breeding and application thereof.

Background

Fengycin is a non-ribosomally synthesized antibacterial cyclic lipopeptide produced by the metabolism of Bacillus amyloliquefaciens and composed of 10 amino acids and beta-hydroxy fatty acids containing 14-18C. Fengycin as a novel biological source antibacterial substance has proved to have strong function of inhibiting filamentous fungi, and can be applied to the aspects of biological control of plant diseases and the like; in addition, fengycin has the characteristics of wide antibacterial spectrum, difficult generation of drug resistance, safety, degradability and the like, and has better effects on the aspects of prevention and treatment of food pathogenic bacteria, food processing, medicines and the like. As a secondary metabolite, fengycin is a synthetic process catalyzed by intracellular enzymes, and has the regulation and control effects of various factors and factors, and the regulation and control mechanism is relatively complex.

In recent years, with the intensive research on RNA, researchers have found that sRNA plays an important role in regulation and control of life activities such as prokaryotic metabolic processes, environmental stress response, and quorum sensing. In the process of cell adversity stress response, sRNA regulates and controls cell physiological functions through complementary pairing with mRNA molecules, and simultaneously sRNA can regulate and control transcription level to influence translation of mRNA in cells, respond to environmental changes, and influence growth and reproduction of bacteria.

With the knowledge of the synthetic pathway of fengycin and the success of positioning, cloning and expression of synthetase genes, the genetic structure of the strain is changed by using a genetic engineering means, the signal molecule regulation mechanism of sRNA for fengycin synthesis is clarified, and the improvement of the molecular level so as to improve the yield of fengycin becomes possible.

Due to the lack of modification of the molecular level of the existing wild strain, the yield of fengycin is low, the cost is high, the price is high, and the industrial production and large-scale application of fengycin are greatly limited.

Disclosure of Invention

The purpose of the invention is as follows: aiming at the problems in the prior art, the invention provides a high-yield bacterial strain LPB-18N of fengycin, which is identified as Bacillus amyloliquefaciens (Bacillus amyloliquefaciens), the high-yield bacterial strain LPB-18N of fengycin can effectively produce a large amount of fengycin in large quantity, and the fengycin has the characteristics of wide antibacterial spectrum, difficult generation of drug resistance, safety, degradability and the like, is widely applied in the field of food, has huge economic benefit and has the potential of being developed into a novel food preservative.

The invention also provides breeding and application of the high-yield fengycin strain LPB-18N.

The technical scheme is as follows: in order to achieve the purpose, the high-yield Fenugreek strain LPB-18N is identified as Bacillus amyloliquefaciens (Bacillus amyloliquefaciens) and is preserved in the general microbiological center of China Committee for culture Collection of microorganisms, the preservation time is 2019, 10 and 22 days, and the preservation number is CGMCC NO.18720. Address: west road No.1, north west of the republic of kyo, yang, institute of microbiology, academy of sciences of china, zip code: 100101.

the application of the strain LPB-18N in the promotion of the increase in the yield of the fengycin is disclosed.

The fermentation liquor containing the fengycin is produced by utilizing the strain LPB-18N.

The preparation method of the fermentation liquor containing the fengycin is prepared by the following steps:

inoculating the strain LPB-18N to a seed culture medium, performing shaking culture on a shaker to a logarithmic phase to obtain a seed solution, inoculating the seed solution to a fermentation culture medium, and performing shaking culture on the shaker to obtain a fermentation broth; centrifuging the fermentation liquid at 4 deg.C, collecting supernatant, standing at 4 deg.C overnight, centrifuging the solution and precipitate at 4 deg.C, collecting thallus, dissolving with methanol at 4 deg.C, and centrifuging the solution at 4 deg.C to obtain supernatant as crude extract of fengycin.

The breeding method of the strain LPB-18N comprises the following steps:

collecting a soil sample, breeding a strain of bacillus amyloliquefaciens with a strain code of LPB-18, wherein the preservation number is CGMCC No.18719, the preservation date is 2019, 10 and 22 days, and performing target gene analysis on the transcriptome data of the strain LPB-18 through bioinformatics analysis to obtain a non-coding RNA (sRNA) segment with the length of 355bp and the basic group complementary relation with the Bacillus amyloliquefaciens fengogen synthetase, wherein the segment is named as FenSr3, and the nucleotide sequence is SEQ ID NO.1; connecting the recombinant fragment with a linearized vector through DNA homologous recombination to construct a FenSr3 gene knockout vector, and transforming the gene knockout vector into the competence of the bacillus amyloliquefaciens LPB-18 through an electric transformation system to construct a gene knockout mutant strain, namely the high-yield fengycin strain LPB-18N.

Wherein, the primer of the FenSr3 gene knockout carrier is shown by SEQ ID NO. 2-5.

In order to solve the technical problems that the yield of fengycin of a wild strain is low and cannot be suitable for industrial production and the like, the invention also provides a method for modifying bacillus amyloliquefaciens from a molecular level and improving the yield of fengycin. The invention constructs a recombinant bacillus amyloliquefaciens for knocking out the FenSr3 gene, and the FenSr3 gene of the bacillus amyloliquefaciens is knocked out by a homologous recombination technology. Specifically, the method comprises the following steps: (1) Constructing a recombinant fragment through DNA overlapping extension, connecting the recombinant fragment with a linearized vector, and constructing FenSr3 deletion mutation to obtain a FenSr3 gene knockout plasmid; (2) The plasmid is transformed into an original Bacillus amyloliquefaciens competent strain through an electric transformation system to obtain the fengycin high-yield strain.

The Fenugreek anabolism regulation gene FenSr3 is a key gene for regulating and producing antibacterial lipopeptide Fenugreek, is cloned in Bacillus amyloliquefaciens LPB-18, and the used strain Bacillus amyloliquefaciens LPB-18 is obtained by self breeding of an inventor, is preserved in the common microorganism center of China Committee for culture Collection of microorganisms and management, has the preservation number of CGMCC No.18719 and the preservation date of 2019 and 10 and 22 days, can produce fengycin to a certain extent, is internationally recognized as that the Bacillus amyloliquefaciens is a food safety strain, and is widely applied to the field of food.

Has the advantages that: compared with the prior art, the invention has the following advantages:

the invention successfully clones a fengycin anabolism gene FenSr3 from a strain of Bacillus amyloliquefaciens LPB-18, wherein the gene is a non-coding RNA gene (FenSr 3) related to the fengycin anabolism and is a brand new gene related to the fengycin anabolism. The invention relates to a recombinant expression vector FenSr3 gene knockout vector constructed by using a Fenugoniin anabolic gene FenSr3, wherein the plasmid is transformed into Bacillus amyloliquefaciens LPB-18 competence through an electric transformation system, a gene knockout mutant is constructed by utilizing the deletion of the gene, so that a Fenugoniin high-yield strain LPB-18N is obtained and is identified as Bacillus amyloliquefaciens (Bacillus amyloliquefaciens), the Fenugoniin high-yield strain is preserved in the general microbiological culture Collection center of China Committee for culture Collection of microorganisms for 22 days in 2019, and the preservation number is CGMCC NO.18720. The fengycin high-yield strain LPB-18N Feng Yuansu fengycin has high yield and high efficiency, the fengycin content in fermentation liquor after fermentation for 72 hours is up to 327.598mg/L, and the yield is 1.72 times of the yield of an original wild strain; the strain LPB-18N obtained by the invention not only has high fengycin yield, but also saves the cost and improves the industrial production and commercial potential.

Drawings

FIG. 1 is a morphological diagram (65X) of Bacillus amyloliquefaciens LPB-18N;

FIG. 2 is an observation picture of Bacillus amyloliquefaciens LPB-18N colonies;

FIG. 3 is an image of a gel of the PCR product of the knock-out plasmid (Lanem: DNAker, lane1: single colony PCR amplification product);

FIG. 4 is a gel image of the PCR products of the electrotransformation single colony (LaneM: DNAmaker, lane1-4: PCR amplification products of the single colony);

FIG. 5 is an HPLC chromatogram of an original Bacillus amyloliquefaciens LPB-18 fengycin sample;

FIG. 6 is an HPLC chromatogram of a sample of Bacillus amyloliquefaciens LPB-18N plumping element.

Detailed Description

The following further description is made in conjunction with the accompanying drawings and examples.

The microorganisms and reagent sources used in the examples are as follows:

kpn I, bgl II, and BamH I were obtained from Fermentas;

the Escherichia coli-Bacillus subtilis shuttle plasmid pCBS is provided by Nanjing university of agriculture; synthesis method (knock out of rapC improvements of the Bacillus subtilis D Yield Based on De Novo Genome Sequencing of Bacillus amyloliquefaciens fmbJ, J.Agric.food chem.2018,66, 4422-4430.)

Coli DH 5. Alpha. Competent cells, trans110 competent cells were purchased from Takara Biotech.

Example 1

Separating and screening a strain of microorganism with the code of LPB-18 in earthworm cultivation soil of university city of Jiangsu Huaian, wherein the strain is a gram-positive bacterium and an aerobic bacterium, has the length of about 1.7-2.6 microns and the width of about 0.6-0.9 micron; the colonies grow well on solid LB plates and are round, milky white and opaque.

The 16S rDNA sequencing sequence of the strain LPB-18 is amplified by a molecular biological method and sequenced, the LPB-18 16S rDNA obtained by PCR amplification is sequenced, as shown in SEQ ID No.6, the comparison on genebank shows that the homology of the strain LPB-18 and the strain of Bacillus amyloliquefaciens (Bacillus amyloliquefaciens B10 and Bacillus amyloliquefaciens HYM 32) reaches 98.41 recently. The strain LPB-18 is preliminarily identified as Bacillus amyloliquefaciens (Bacillus amyloliquefaciens) by combining morphological and physiological and biochemical characteristics, and is named as the Bacillus amyloliquefaciens LPB-18. The strain Bacillus amyloliquefaciens LPB-18 is delivered to the China general microbiological culture Collection center (CGMCC for short) of China institute of microbiology, beijing, china, and the collection number is CGMCC No.18719, and the collection date is 2019, 10 months and 22 days.

Example 2

Through bioinformatics analysis, target gene analysis is carried out on the obtained Bacillus amyloliquefaciens LPB-18 transcriptome data to obtain a segment of non-coding RNA (sRNA) segment with the length of 355bp and the base complementary relation with Bacillus amyloliquefaciens fengycin synthetase, the segment is named as FenSr3, and the nucleotide sequence of the gene FenSr3 is shown in SEQ ID NO. 1.

SEQ ID NO.1

GGAGCTGTGTACGCGGTTTGAAGCGCGAGCTCCGGTTCGGGCAGATTTTTCCGATCCAATTTACCGTTCGGCGTAAGCGGCAGCTTCTCAAGTTCCATCATATAGGCCGGGACCATGTAATTCGGCAGTTGCTTAGAAAGTGATGAACGCACTTTTTCTGTATCCATGTCCGTCTGCAGACTCATATATGCGATCAGTTCTTTATTGCCGGACTGCCCGGTTCTGACTGACACGGCAGCTTCTTTCACGCCGTCCAGGCTCCTTAGTGCGGCTTCAATCTCTCCCAGCTCGACCCGATAACCGCGGATTTTCACTTGATCATCCATCCGTCCGGCATATTCGATCGTTCCGTCCG

Example 3

1. Construction of sRNA Gene knockout vector

Designing primers for constructing knock-out vectors by 520bp sequences of the upstream and downstream of the FenSr3 gene:

L-FenSr3-F：(SEQ ID NO.2)

atcgatgcatgccatggtaccCCAAGTATCTGTCTCAGAC(Kpn Ⅰ)

L-FenSr3-R：(SEQ ID NO.3)

GTTCTTCAAGCTCATTGCGGGCAGCCAGCGGGCTA

R-FenSr3-F：(SEQ ID NO.4)

GATTTAGCCCGCTGGCTGCCCGCAATGAGCTTGAA

R-FenSr3-R：(SEQ ID NO.5)

gcgtcgggcgatatcagatctTTATATGTAAGTGACCAGG(Bgl Ⅱ)

(1) Amplification of upstream and downstream sequences of sRNA Gene

Using LPB-18 genome DNA as template, primers L-FenSr3-F/R and R-FenSr3-F/R are adopted to amplify homologous arm sequences between upstream and downstream genes of sRNA respectively.

PCR reaction (50. Mu.L): 2 × Phanta Master Mix 25 μ L, upstream primer 2 μ L, downstream primer 2 μ L, template DNA 1 μ L, ddH ₂ O20 mu L; PCR amplification conditions: pre-denaturation at 95 ℃ for 5min, denaturation at 95 ℃ for 30s, annealing at 55-65 ℃ for 30s, extension at 72 ℃ for 1min, circulation for 30 times, and extension at 72 ℃ for 10min. And respectively purifying the PCR products to recover the target products. And amplifying a target product by adopting overlapping extension primers L-FenSr3-F and R-FenSr3-R, and obtaining a recombinant target fragment according to the electrophoretic detection result shown in figure 3.

(2) The recombinant fragment is ligated to a linearized vector

The shuttle plasmid pCBS of the escherichia coli-bacillus subtilis is subjected to double enzyme digestion for 3h by using restriction enzymes Kpn I and BamH I, the digestion site of the BamH I is eliminated by utilizing the characteristic that Bgl II and BamH I are isocaudarner, and a linearized vector is recovered.

A recombination reaction system: 5 XCE II Buffer 4. Mu.L, linearized vector 150ng, target fragment 160ng, exnase II 2. Mu.L, ddH ₂ O Up to 20. Mu.L. Reacting in a water bath at 37 ℃ for 30min, and cooling in an ice water bath for 5min. Transforming the reaction product into E.coli DH5 alpha competent cells, screening LB resistant flat plate blue white spots (IPTG, X-gaL, amp), and selecting positive single colony PCR to detect target fragments. The plasmid is extracted and verified by double digestion with restriction enzymes Kpn I and Bgl II. And transforming the correctly verified knock-out plasmid into a Trans110 competent cell, an LB resistance plate (Amp), extracting the plasmid, and obtaining the demethylated knock-out plasmid pCBS-delta sRNA through PCR and double enzyme digestion verification.

2. Construction of Bacillus amyloliquefaciens LPB-18 knockout sRNA strain

(1) Preparation of Bacillus amyloliquefaciens LPB-18 competence

LBS medium was grown overnight at 1:25 (v/v) diluting (250 mL shake flask, liquid loading 100 mL), shaking and culturing for 2h in shaking table (37 deg.C, 180 r/min), during which time detecting with ultraviolet spectrophotometer when OD is ₆₀₀ If =0.5 or so, glycine solution was added to the flask so that the final system concentration was 10mg/mL, and the shaking culture was continued until OD reached ₆₀₀ And when the concentration is about =1.0, placing the shake flask on ice and standing for 30min, centrifuging the shake flask by a precooled low-temperature centrifuge at 4 ℃ and 8000rpm for 5min, discarding the supernatant, collecting the somatic cells, repeatedly washing the somatic cells by a precooled electric transformation liquid for 3 times, resuspending the competent cells by a precooled resuspension liquid according to a ratio of 1 (v/v) to 100 (v/v) to obtain the LPB-18 competent cells, and placing the competent cells in a precooled centrifuge tube and storing the competent cells at-80 ℃ for later use. Note: sterile and low-temperature precooling operation in the whole process. The composition of the electrotransformation liquid is as follows: 0.5M trehalose, 0.5M sorbitol, 0.5M mannitol, and 10% glycerol, sterilized at 115 deg.C for 30min. Composition of the heavy suspension: the electrotransformation liquor is supplemented with 14% PEG6000 polyethylene glycol.

(2) Electric conversion

About 500ng of plasmid pCBS-delta sRNA to be transformed is put into 100 mu L of LPB-18 competent cells, the tube wall is flicked and mixed evenly, the competent cells and the plasmid are transferred into an electric rotating cup (2 mm) precooled on ice, and the electric rotating cup is placed on ice for 5min. Setting up electric transformation parameters, immediately adding 1mL LBS culture medium into an electric rotating cup after electric shock at 2800v and 4.5ms, gently and uniformly mixing, slowly sucking into a 2mL centrifuge tube, carrying out mild shaking culture for 3h by using a shaking table (30 ℃,120 r/min), centrifuging for 3min at the room temperature of 2500 Xg, discarding 900 mu L of supernatant, carrying out heavy suspension on thalli in the centrifuge tube by using the remaining culture medium, uniformly coating the thalli on an LB resistant plate (Amp and erythromycin), and carrying out culture for 72h at the temperature of 30 ℃. And (3) verifying the single colony by adopting PCR amplification and electrophoresis detection, wherein amplification primers L-FenSr3-F and R-FenSr3-R are the same as the PCR reaction system, and the electrophoresis detection result is as shown in figure 4 and is consistent with the electrophoresis detection result in figure 3. The strain which is successfully verified is a strain which is correctly knocked out, namely the strain high-yield fengycin strain LPB-18N, the strain is gram-positive bacteria and aerobic, the length of the strain is about 1.7-2.6 microns, and the width of the strain is about 0.6-0.9 microns, as shown in figure 1; the colonies grew well on solid LB plates and appeared round, milky, opaque as shown in FIG. 2. Identified as Bacillus amyloliquefaciens (Bacillus amyloliquefaciens), named as Bacillus amyloliquefaciens LPB-18N, which is preserved in the common microorganism center of China Committee for culture Collection of microorganisms for 10 and 22 days in 2019 and with the preservation number of CGMCC NO.18720.

Original wild strain LPB-18 and strain LPB-18N are picked up and respectively inoculated in a seed culture medium (250 mL shake flask, liquid loading capacity 100 mL), and shaking cultured for 24h to logarithmic phase in a shaking table (37 ℃,180 r/min). Respectively inoculating the seed liquid into a fermentation medium (1L shake flask, liquid containing amount 400 mL) according to the inoculation amount of 3% (v/v), and shake-culturing for 72h by using a shaking table (33 ℃,180 r/min) to obtain fermentation liquid; the fermentation broth was centrifuged at 5000 Xg for 20min at 4 ℃ and the supernatant collected and adjusted to pH 2.0,4 ℃ with 6M HCl and left overnight. The solution and precipitate were centrifuged at 5000 Xg for 20min at 4 deg.C, the cells were collected, dissolved thoroughly in 5mL of methanol at 4 deg.C, and the pH was adjusted to 7.0. Finally, the solution was centrifuged at 1000 Xg for 20min at 4 ℃ to obtain the supernatant, i.e. the crude extract of fengycin. The seed culture medium comprises the following components: 5g of beef extract, 10g of peptone, 5g of yeast extract, 5g of NaCl5g of glucose, and 10g of distilled water with constant volume of 1L, pH7.0, and sterilizing at 115 ℃ for 15min; the fermentation medium comprises the following components: 1g of yeast extract, 14g of sodium glutamate, 0.5g of KCL ₂ PO ₄ 1g，MgSO ₄ 0.5g，MnSO ₄ 5mg，CuSO ₄ 0.16mg，FeSO ₄ ·7H ₂ 0.15g of O, distilled water to a volume of 1L, pH7.0, and sterilization at 115 ℃ for 30min.

Purifying the crude extract sample by Waters 600 preparative reverse phase high performance liquid chromatograph and C18 reverse phase chromatographic column, wherein each sample introduction is 2-3mL, and the mobile phase is deionized water and acetonitrile added with 0.1% trifluoroacetic acid. And (3) identifying the purified product by using HPLC (high performance liquid chromatography), wherein a chromatographic column is an Agilent C18 column, gradient elution is carried out by using acetonitrile and deionized water as mobile phases, ultraviolet detection is carried out at 205nm, a liquid phase diagram is shown as a wild strain in figure 5, a fengycin target peak is 1-6, the concentration is 190.908mg/L, a strain LPB-18N is shown in figure 6, a fengycin target peak is 1-6, the concentration is 327.598mg/L, and the yield of the fengycin of the knockout strain is 1.72 times of the yield of the fengycin of the original wild strain.

Sequence listing

<110> Huaiyin institute of Industrial and research

<120> high-yield Fengyin strain LPB-18N and breeding and application thereof

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ggagctgtgt acgcggtttg aagcgcgagc tccggttcgg gcagattttt ccgatccaat 60

ttaccgttcg gcgtaagcgg cagcttctca agttccatca tataggccgg gaccatgtaa 120

ttcggcagtt gcttagaaag tgatgaacgc actttttctg tatccatgtc cgtctgcaga 180

ctcatatatg cgatcagttc tttattgccg gactgcccgg ttctgactga cacggcagct 240

tctttcacgc cgtccaggct ccttagtgcg gcttcaatct ctcccagctc gacccgataa 300

ccgcggattt tcacttgatc atccatccgt ccggcatatt cgatcgttcc gtccg 355

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gatttagccc gctggctgcc cgcaatgagc ttgaa 35

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gcgtcgggcg atatcagatc tttatatgta agtgaccagg 40

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tggggagtgc gggcgctata catgcagtcg agcggacaga tgggagcttg ctccctgatg 60

ttagcggcgg acgggtgagt aacacgtggg taacctgcct gtaagactgg gataactccg 120

ggaaaccggg gctaataccg gatggttgtt tgaaccgcat ggttcagaca taaaaggtgg 180

cttcggctac cacttacaga tggacccgcg gcgcattagc tagttggtga ggtaacggct 240

caccaaggcg acgatgcgta gccgacctga gagggtgatc ggccacactg ggactgagac 300

acggcccaga ctcctacggg aggcagcagt agggaatctt ccgcaatgga cgaaagtctg 360

acggagcaac gccgcgtgag tgatgaaggt tttcggatcg taaagctctg ttgttaggga 420

agaacaagtg ccgttcaaat agggcggcac cttgacggta cctaaccaga aagccacggc 480

taactacgtg ccagcagccg cggtaatacg taggtggcaa gcgttgtccg gaattattgg 540

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gagggtcatt ggaaactggg gaacttgagt gcagaagagg agagtggaat tccacgtgta 660

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gcattaagca ctccgcctgg ggagtacggt cgcaagactg aaactcaaag gaattgacgg 900

gggcccgcac aagcggtgga gcatgtggtt taattcgaag caacgcgaag aaccttacca 960

ggtcttgaca tcctctgaca atcctagaga taggacgtcc ccttcggggg cagagtgaca 1020

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caatcggagt aagggtgggg atgacgtcat cattcatgcc ctaatgaact ggccatacac 1200

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Claims (3)

1. The bacterial strain LPB-18N with high yield of the fengycin is identified as bacillus amyloliquefaciens (Bacillus amyloliquefaciens: (A)Bacillus amyloliquefaciens) The microbial inoculum is preserved in the China general microbiological culture Collection center, the preservation time is 2019, 10 and 22 months, and the preservation number is CGMCC NO.18720.

2. Use of the strain LPB-18N according to claim 1 for promoting the production of fengycin.

3. A preparation method of fermentation liquor containing fengycin is characterized by comprising the following steps:

inoculating the strain LPB-18N of claim 1 to a seed culture medium, performing shake culture on a shaker to logarithmic phase to obtain a seed solution, inoculating the seed solution to a fermentation culture medium, and performing shake culture on the shaker to obtain a fermentation broth; centrifuging the fermentation liquid at 4 deg.C, collecting supernatant, standing at 4 deg.C overnight, centrifuging the solution and precipitate at 4 deg.C, collecting thallus, dissolving with methanol at 4 deg.C, and centrifuging the solution at 4 deg.C to obtain supernatant as crude extract of fengycin.

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