CN111019866A - Endophytic bacillus of Pu' er tea tree leaves and application thereof - Google Patents
Endophytic bacillus of Pu' er tea tree leaves and application thereof Download PDFInfo
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Abstract
The invention discloses a Pu' er tea tree endophytic bacillus and application thereof. The name of the Bacillus endophytic in the leaves of the Pu 'er tea trees is Bacillus velezensis FZ06(Bacillus velezensis FZ06), the Bacillus endophytic in the leaves of the Pu' er tea trees is preserved in the China center for type culture collection of Wuhan university in Wuhan City in 2019 in 10 and 25 months, and the preservation number is CCTCC NO: m2019854. Lipopeptide substances which can be produced by the bacillus subtilis comprise 27 types of 3 types, and the abundance of the lipopeptide substances is more than that of the currently reported bacillus beilesensis; the generated lipopeptide substance has obvious inhibitory activity on pathogenic bacteria such as escherichia coli, staphylococcus aureus, salmonella, aspergillus flavus, aspergillus parasiticus and the like, and can be well applied to inhibiting the pathogenic bacteria.
Description
Technical Field
The invention belongs to the technical field of microorganisms, and particularly relates to a Pu' er tea tree endophytic bacillus and application thereof.
Background
Lipopeptides (lipopeptides) are important nonionic biosurfactants, mainly come from secondary metabolic pathways of bacteria and fungi, are various in types and complex in structure, and are generally formed by combining fatty acid chains and peptide chains into rings through lactone or amide bonds. As a molecular weapon of microorganisms, lipopeptides in the natural environment often have natural functions of resisting viruses, antagonizing bacteria and even antagonizing fungi, and therefore, the lipopeptides are widely applied to the development of plant anti-disease and insect-resistant agents. As a class of nonionic surfactants, the bacteriostatic mechanism of lipopeptides may be similar to that of conventional antifungal surface active molecules: the non-ionic biosurfactant binds and isolates essential proteins associated with survival on the cell membrane of the target fungus, resulting in death of the target fungus. Lipopeptides produced by different microorganisms theoretically have different antibacterial spectra due to differences in structure and properties, and a single compound often cannot realize wide antibacterial effect; and given the adaptive evolution of microorganisms, the long-term use of single antibiotics is likely to render certain fungi resistant. However, if lipopeptide substances with more various structures and more abundant properties are used in combination, the problems can be solved. Therefore, the method finds the microorganisms capable of generating more abundant lipopeptide substances, extracts and jointly uses the lipopeptide compounds, and has important significance for expanding the antibacterial spectrum of the lipopeptide compounds, further reducing the antibiotic adaptive evolution of target fungi and even reducing the antibacterial concentration.
Disclosure of Invention
The invention aims to overcome the defects in the prior art and provide the bacillus endophytic in the leaves of the Pu' er tea plant.
The invention also aims to provide application of the bacillus endophytic in the leaves of the Pu' er tea plant.
The invention further aims to provide the lipopeptide substance prepared from the bacillus endophytic in the leaves of the Pu' er tea trees and the application thereof.
In order to achieve the purpose, the invention is realized by the following technical scheme:
an endophytic bacillus Puer tea leaf is named as Bacillus velezensis FZ06(Bacillus velezensis FZ06) and is obtained by separating and purifying fresh Puer tea leaves of Xishuangbanna.
The bacillus endophytic to the Pu' er tea tree leaf is Bacillus beiLeisi FZ06(Bacillus velezensis FZ06), and the preservation information is as follows: the preservation unit: china Center for Type Culture Collection (CCTCC), preservation date: 25/10/2019, deposit address: wuhan university, the preservation number: CCTCC NO: m2019854.
The application of the endophytic bacillus of Pu' er tea tree leaves in the preparation of lipopeptide substances; preferably comprising the steps of:
(1) fermentation culture: inoculating the Pu' er tea tree leaf endophytic bacillus seed solution into a fermentation culture medium for fermentation culture to obtain a fermentation liquid;
(2) acid precipitation separation and washing: performing first centrifugation on the fermentation liquor obtained in the step (1) to remove thalli, adding acid into the obtained supernatant to adjust the pH to 2.0, performing second centrifugation after standing to remove the supernatant, washing the obtained precipitate, and performing third centrifugation to obtain acid precipitate;
(3) acid precipitation and extraction: adding the acid precipitate obtained in the step (2) into a solvent for extraction, then carrying out solid-liquid separation, drying the obtained liquid, and then adding water for redissolution to obtain a water-phase extraction liquid;
(4) separating and purifying lipopeptide substances: and (3) drying the aqueous phase extract obtained in the step (3) for the first time, adding a solvent to redissolve the obtained dry matter, then carrying out chromatographic purification, drying the obtained eluent containing the lipopeptide substances for the second time, then adding water to redissolve the eluent, and finally carrying out drying for the third time to obtain the lipopeptide substances.
The seed solution in the step (1) is preferably prepared by the following steps: activating and culturing the stored Pu' er tea plant endophytic bacillus to obtain seed liquid.
The activation step is preferably as follows: and (3) inoculating the stored Pu 'er tea tree leaf endophytic bacillus into an activation culture medium, and culturing for 24-48 hours at 35-39 ℃ to obtain the activated Pu' er tea tree leaf endophytic bacillus.
The culture condition is preferably 36-38 ℃ for 24 hours.
The activating culture medium is preferably PDA culture medium.
The step of the expanded culture is preferably as follows: inoculating activated Pu' er tea tree leaf endophytic bacillus into a seed culture medium, and culturing for 24-48 hours at 35-39 ℃ and 150-170 rpm to obtain a seed solution.
The inoculation amount of the activated Pu' er tea tree endophytic bacillus is preferably 1-5% of the seed culture medium according to the volume ratio; more preferably 1% of said seed medium.
The seed culture medium is preferably a liquid LB culture medium.
The preferable conditions for the culture are 36-38 ℃ and 160rpm for 24 hours.
The inoculation amount of the seed liquid in the step (1) is preferably 1-5% of the fermentation medium in volume ratio; more preferably 1% of the fermentation medium.
The fermentation medium is preferably a liquid LB medium.
The condition of fermentation culture in the step (1) is preferably 35-39 ℃ and 150-170 rpm for 48-72 hours; more preferably, the culture is carried out at 36 to 38 ℃ and 160rpm for 72 hours.
The first centrifugation in step (2) is preferably performed at 4 ℃ and 10000rpm for 10 min.
The acid in the step (2) is preferably hydrochloric acid; more preferably hydrochloric acid having a concentration of 6 mol/L.
The preferred standing condition in the step (2) is standing for 12-24 hours at 4 ℃; more preferably, it is left standing at 4 ℃ for 12 hours.
The second centrifugation in step (2) is preferably performed at 10000rpm at 4 ℃ for 5 min.
The washing in step (2) is preferably washing with dilute hydrochloric acid; more preferably, the washing is carried out with dilute hydrochloric acid having a pH of 2.0.
The number of washing times described in step (2) is preferably 2.
The third centrifugation condition in the step (2) is preferably 10000rpm centrifugation for 10 min.
The solvent in step (3) is preferably anhydrous methanol.
The solid-liquid separation mode in the step (3) is preferably suction filtration; more preferably vacuum filtration.
The drying in step (3) is preferably suspended by concentration; more preferably by vacuum concentration suspension drying; most preferably by rotary evaporation at 40 ℃ to dryness.
The water in step (3) is preferably deionized water.
The first drying and the third drying in the step (4) are preferably freeze-drying.
The solvent in step (4) is preferably anhydrous methanol.
The chromatographic purification in the step (4) is preferably carried out by using a sephadex chromatographic column; more preferably, Sephadex LH-20 sepharose column is used, the eluent is 100% methanol, and the flow rate is 0.5 mL/min.
The second drying in step (4) is preferably suspended by concentration; more preferably by vacuum concentration suspension drying; most preferably by rotary evaporation at 40 ℃ to dryness.
A lipopeptide is prepared from the endophytic bacillus of Pu' er tea tree leaves by fermenting, separating and purifying.
The lipopeptide substances comprise Surfactin similar substances, Iturin similar substances and Fengycin similar substances.
The Surfactin analogue is preferably C12-Leu7-Surfactin、C13-Leu7-Surfactin、C14-Leu7-Surfactin、C15-Leu7-Surfactin、C16-Leu7-Surfactin、C12-Val7-Surfactin、C13-Val7-Surfactin、C14-Val7-Surfactin、C15-Val7Surfactin and C14-Val7-Surfactin。
The Iturin analog is preferably C14-Bacillomycin F、C15-Bacillomycin F、C16-Bacillomycin F、C17-Bacillomycin F、C14-Bacillomycin D、C15-Bacillomycin D、C16Bacillus bacteria D and C17-Bacillomycin D。
The Fengycin analog is preferably C15-Ala6-Fengycin、C16-Ala6-Fengycin、C17-Ala6-Fengycin、C15-Abu6-Fengycin、C16-Abu6-Fengycin、C17-Abu6-Fengycin、C15-Val6-Fengycin、C16-Val6-Fengycin and C17-Val6-Fengycin。
The lipopeptide substance is applied to inhibiting pathogenic bacteria.
Such pathogens include, but are not limited to, Escherichia coli, Staphylococcus aureus, Salmonella, Aspergillus flavus, and Aspergillus parasiticus.
The Escherichia coli is preferably Escherichia coli GIM 1.708.
The staphylococcus aureus is preferably staphylococcus aureus subspecies aureobasidium GIM 1.441.
The salmonella is preferably salmonella choleraesuis subsp.
The aspergillus flavus is preferably aspergillus flavus GIM 3.493.
The Aspergillus parasiticus is preferably Aspergillus parasiticus GIM 3.395.
Compared with the prior art, the invention has the following advantages and effects:
(1) the invention separates the endophytic Bacillus velezensis FZ06 from the fresh leaves of the Pu' er tea tree for the first time, lipopeptide substances which can be produced by the Bacillus velezensis FZ06 comprise 27 kinds of 3 kinds, and the abundance of the lipopeptide substances is more than that of the presently reported Bacillus belgii. Among them, the amino acid sequence of the polypeptide chain of Fengycin-like homologues having amino fatty acid chains of 15 to 17 carbon atoms was first observed in Bacillus belgii.
(2) The method for preparing the lipopeptide substance is simple and easy to implement, mild in condition and easy to control, and provides a new path for large-scale production of the lipopeptide substance.
(3) The lipopeptide substance generated by the Bacillus velezensis FZ06 has obvious activity inhibition effect on pathogenic bacteria such as escherichia coli, staphylococcus aureus, salmonella, aspergillus flavus, aspergillus parasiticus and the like, can be well applied to inhibiting the pathogenic bacteria, and provides a new idea for improving the postharvest storage performance or disease resistance of host plant desquamate by improving the interaction of endophytic bacteria and host plants.
Drawings
FIG. 1 is a bacteriostatic phenomenon diagram of endophytic fungi of black spore plants generated by antagonism of plant endophytic bacteria in Pu' er tea tree leaves.
FIG. 2 is a Scanning Electron Microscope (SEM) image of Bacillus velezensis FZ 06: wherein A is an SEM picture of Bacillus velezensis FZ06 under 1000 times of magnification; b is an SEM image of Bacillus velezensis FZ06 at 5000 Xmagnification.
FIG. 3 is a phylogenetic tree diagram of 16S rDNA of Bacillus velezensis FZ 06.
FIG. 4 is a total ion flow diagram of a lipopeptide solution prepared by Bacillus velezensis FZ 06: wherein A is a total ion flow diagram of Surfactin similar congeners; b is a total ion flow diagram of Iturin analog isogeny; c is the total ion flow diagram of Fengycin analog congener.
FIG. 5 is a graph showing the effect of lipopeptides prepared by Bacillus velezensis FZ06 and the effect of sterile water on the inhibition of bacterial pathogens: wherein A is an effect diagram of the lipopeptide substance prepared by Bacillus velezensis FZ06 and the inhibition effect of sterile water on Escherichia coli GIM 1.708; b is a diagram of the inhibitory effect of lipopeptide substances prepared by Bacillus velezensis FZ06 and sterile water on staphylococcus aureus subspecies aureobasidium GIM 1.441; c is a figure of the inhibitory effect of lipopeptide substances prepared by Bacillus velezensis FZ06 and sterile water on Salmonella choleraesuis subsp suis GIM 1.163.
FIG. 6 is a graph showing the effect of lipopeptides prepared by Bacillus velezensis FZ06 and the effect of sterile water on the inhibition of fungal pathogens: wherein A is an effect diagram of the lipopeptide substance prepared from Bacillus velezensis FZ06 and the inhibition effect of sterile water on Aspergillus flavus GIM 3.493; b is a diagram of the inhibition effect of lipopeptide substances prepared by Bacillus velezensis FZ06 and sterile water on Aspergillus parasiticus GIM 3.395.
Detailed Description
The present invention will be described in further detail with reference to examples and drawings, but the present invention is not limited thereto.
The test methods in the following examples, in which specific experimental conditions are not specified, are generally performed according to conventional experimental conditions or according to the experimental conditions recommended by the manufacturer. The materials, reagents and the like used are, unless otherwise specified, reagents and materials obtained from commercial sources.
Example 1 isolation and identification of antifungal endophytic bacteria (Bacillus endophytic Puer tea plant leaf)
(1) Pretreating fresh Pu' er tea leaves: transporting collected fresh and complete Pu' er tea leaves (from Xishuangbanna Meng Hai county of Yunnan province) to a laboratory within 24 hours, and carrying out surface disinfection operation, wherein the method specifically comprises the following steps: soaking fresh and complete Pu' er tea tree leaves in 75% (volume fraction) ethanol solution for 15 seconds in a sterile operating platform, then washing with sterile water and wiping with sterile cotton; subsequently, it was immersed in a 3.25% (effective chlorine content) sodium hypochlorite solution for 15 seconds, rinsed with sterile water and wiped dry with sterile cotton; the above surface sterilization operation was repeated three times. The last wash water was spread in potato dextrose agar medium (PDA medium) and then incubated at 37 ℃ for 2 weeks, and if no microorganisms were present, the leaf surface was considered to be completely disinfected.
Placing the fresh Pu' er tea tree leaves with completely sterilized surfaces into a breathable sterile bag, and storing for 30 days at 4 ℃. After 30 days, selecting complete Pu' er tea tree leaves which are green and have no obvious decomposition phenomenon of plant leaves caused by growth of endophytic fungi. The above surface sterilization operation was repeated three times. The last wash water was plated in PDA medium and then incubated at 37 ℃ for 2 weeks, and if no microorganisms were present, the leaf surface was considered to be completely disinfected.
(2) Culturing and separating antifungal endophytic bacteria:
and (2) carrying out sterile slicing on the leaves of the Pu' er tea tree which is kept green and has a secondarily sterilized surface and obtained in the step (1) to obtain plant slices with the diameter of about 0.5 cm, placing the plant slices in a PDA culture medium, and culturing for one week at the temperature of 30 ℃, wherein the result is shown in figure 1: there is a clear phenomenon that endophytic bacteria antagonize endophytic fungi of plants producing black spores (upper right part of culture medium). Selecting endophytic bacteria colony inhibiting black spore plant endophytic fungi, streaking, repeating for 3 times, and separating and purifying to obtain antifungal endophytic bacteria.
The solid culture characteristics of the thallus are as follows: culturing a ring of purified antifungal endophytic bacteria on a PDA culture medium at 36-38 ℃ for 3-5 days, wherein the initial colony is pale and gradually changed into yellow; the colony has irregular edge and is villous.
The liquid culture characteristics of the thallus are as follows: and (3) culturing the antifungal endophytic bacteria obtained by one-ring purification in 50mL of potato glucose liquid culture medium at 36-38 ℃ for 3 days: on day 1, the culture medium turns turbid from clear, and the phenomenon of liquid culture of bacteria is obvious; on day 2, the turbidity did not change any more when the solution entered the stabilization phase; on day 3, skin forming occurs on the surface of the fermentation broth.
The form of the cells under a scanning electron microscope is shown in FIG. 2: the single cell is in a rod-shaped structure, and the size of the single cell is 0.7-0.8 multiplied by 2-3 mu m.
(3) 16S identification of single colonies: the separated strain is inoculated in 50mL LB culture medium, shaking culture is carried out at 37 ℃ and 160rpm for 24 hours, 10mL of bacterial liquid is taken and sent to Shanghai biological engineering company Limited for strain sequencing. The 16SrDNA sequence of the strain is shown as SEQ ID NO: 1: (1483bp)
TCAGGACGAACGCTGGCGGCGTGCCTAATACATGCAAGTCGAGCGGACAGATGGGAGCTTGCTCCCTGATGTTAGCGGCGGACGGGTGAGTAACACGTGGGTAACCTGCCTGTAAGACTGGGATAACTCCGGGAAACCGGGGCTAATACCGGATGGTTGTCTGAACCGCATGGTTCAGACATAAAAGGTGGCTTCGGCTACCACTTACAGATGGACCCGCGGCGCATTAGCTAGTTGGTGAGGTAACGGCTCACCAAGGCGACGATGCGTAGCCGACCTGAGAGGGTGATCGGCCACACTGGGACTGAGACACGGCCCAGACTCCTACGGGAGGCAGCAGTAGGGAATCTTCCGCAATGGACGAAAGTCTGACGGAGCAACGCCGCGTGAGTGATGAAGGTTTTCGGATCGTAAAGCTCTGTTGTTAGGGAAGAACAAGTGCCGTTCAAATAGGGCGGCACCTTGACGGTACCTAACCAGAAAGCCACGGCTAACTACGTGCCAGCAGCCGCGGTAATACGTAGGTGGCAAGCGTTGTCCGGAATTATTGGGCGTAAAGGGCTCGCAGGCGGTTTCTTAAGTCTGATGTGAAAGCCCCCGGCTCAACCGGGGAGGGTCATTGGAAACTGGGGAACTTGAGTGCAGAAGAGGAGAGTGGAATTCCACGTGTAGCGGTGAAATGCGTAGAGATGTGGAGGAACACCAGTGGCGAAGGCGACTCTCTGGTCTGTAACTGACGCTGAGGAGCGAAAGCGTGGGGAGCGAACAGGATTAGATACCCTGGTAGTCCACGCCGTAAACGATGAGTGCTAAGTGTTAGGGGGTTTCCGCCCCTTAGTGCTGCAGCTAACGCATTAAGCACTCCGCCTGGGGAGTACGGTCGCAAGACTGAAACTCAAAGGAATTGACGGGGGCCCGCACAAGCGGTGGAGCATGTGGTTTAATTCGAAGCAACGCGAAGAACCTTACCAGGTCTTGACATCCTCTGACAATCCTAGAGATAGGACGTCCCCTTCGGGGGCAGAGTGACAGGTGGTGCATGGTTGTCGTCAGCTCGTGTCGTGAGATGTTGGGTTAAGTCCCGCAACGAGCGCAACCCTTGATCTTAGTTGCCAGCATTCAGTTGGGCACTCTAAGGTGACTGCCGGTGACAAACCGGAGGAAGGTGGGGATGACGTCAAATCATCATGCCCCTTATGACCTGGGCTACACACGTGCTACAATGGACAGAACAAAGGGCAGCGAAACCGCGAGGTTAAGCCAATCCCACAAATCTGTTCTCAGTTCGGATCGCAGTCTGCAACTCGACTGCGTGAAGCTGGAATCGCTAGTAATCGCGGATCAGCATGCCGCGGTGAATACGTTCCCGGGCCTTGTACACACCGCCCGTCACACCACGAGAGTTTGTAACACCCGAAGTCGGTGAGGTAACCTTTTAGGAGCCAGCCGCCGAAGGTGGGACAGATGATTGGGGTGAAGTCGT
The 16S rDNA sequence was listed in NCBI for homology analysis using Blast, and several species were selected for evolutionary tree analysis, the results are shown in FIG. 3: the separated antifungal endophytic bacterial strain is Bacillus subtilis FZ06(Bacillus velezensis FZ 06).
Example 2 preparation of lipopeptides from Bacillus velezensis FZ06
(1) Activating strains: under the aseptic condition, taking 1-ring Bacillus velezensis FZ06 from a glycerol freezing storage tube, inoculating into a PDA culture medium, and performing activation culture at 36-38 ℃ for 24 hours;
(2) seed liquid culture: under the aseptic condition, taking the activated Bacillus velezensis FZ 061 mL to transfer into 100mL of liquid LB culture medium, and carrying out shake cultivation for 24 hours at 36-38 ℃ and 160rpm to obtain seed liquid;
(3) fermentation culture: under the aseptic condition, taking 20mL of the seed liquid obtained in the step (2) to be inoculated into 2L of liquid LB culture medium, and carrying out shake cultivation for 72 hours at 36-38 ℃ and 160rpm to obtain fermentation liquid;
(4) acid precipitation separation and washing: centrifuging the fermentation liquor in a low-temperature high-speed centrifuge with the rotation speed of 10000rpm at the temperature of 4 ℃ for 10min to remove thalli, then adding 6mol/L HCl into the centrifugal supernatant to adjust the pH value to 2.0, and standing for 12 hours at the temperature of 4 ℃ to obtain acid precipitation fermentation liquor; centrifuging the acid precipitation fermentation liquor in a low-temperature high-speed centrifuge at 4 ℃ and 10000rpm for 5min, removing supernatant, collecting precipitate, washing the precipitate with 200mL of diluted hydrochloric acid with the pH value of 2.0 twice, centrifuging at 10000rpm for 10min, and collecting precipitate to obtain acid precipitate;
(5) acid precipitation and extraction: extracting the acid precipitate with 200mL of anhydrous methanol, vacuum filtering the extract, evaporating the obtained filtrate at 40 ℃ by using a rotary evaporator to dryness, and re-dissolving the filtrate in deionized water to obtain a water-phase extract;
(6) and (3) separating and purifying lipopeptide substances: freeze-drying the aqueous phase extract to obtain a freeze-dried substance, adding 50mL of anhydrous methanol for redissolving, carrying out Sephadex chromatography LH-20 column chromatography purification on the obtained redissolved solution (eluent is 100% methanol, and the flow rate is 0.5mL/min), carrying out vacuum concentration on the obtained eluent containing the lipopeptide substances until the eluent is dried, then adding water for redissolving, and carrying out freeze-drying on the redissolved solution to obtain the lipopeptide substances generated by Bacillus velezensis FZ06, wherein the yield is 1.03 g/L.
Example 3 structural characterization of lipopeptide species prepared by Bacillus velezensis FZ06
Re-dissolving 50mg of lipopeptide powder obtained in the extraction example 2 in 50mL of anhydrous methanol, diluting the solution by 1000 times with the anhydrous methanol, and performing ultra performance liquid chromatography-electrospray-secondary mass spectrometry UPLC-ESI-MS/MS structural determination under the following specific analysis conditions:
chromatographic separation conditions: a chromatographic column: waters ACQUITY UPLC BEHC18 column (2.1 mm. times.100 mm, 1.7 μm); column temperature: 30 ℃; flow rate: 0.4 mL/min; mobile phase: a- -ultrapure water containing 0.1% formic acid, B- -acetonitrile containing 0.1% formic acid; gradient elution conditions: 0-0.5 min, 60% A; 60-20% of A for 0.5-3.5 min; . 3.5-4 min, 20% A; 4-6 min, 20-5% A; 6-7 min, 5-2% A; 7-10 min, 2% A; 10-10.5 min, 2-60% A; 10.5-15 min, 60% A; sample introduction amount: 1 μ L.
Mass spectrometry conditions: selecting an Agilent G6545A QTOF mass spectrometer and a cation scanning mode, wherein the mass scanning range of a primary mass spectrum is set to be 110-1600 amu, and the mass scanning range of a secondary mass spectrum is set to be 50-1600 amu; the ion source temperature is 150 ℃, and the capillary voltage is 3.26 kV; n is a radical of2Flow rate 600L/h, Ar2The flow rate is 50L/h; the mass spectrometry data acquisition and processing software is Agilent MassHunter.
The UPLC-MS results are shown in fig. 4: lipopeptides produced by Bacillus velezensis FZ06 comprise Surfactin analogs with molecular weights of 994, 1008, 1022, 1036, 1050, etc. differing by one methylene group (-CH2) (FIG. 4-A); iturin-like homologues having a molecular weight difference of 1043, 1057, 1071 and 1085, etc. by one methylene group (-CH2) (FIG. 4-B) and Fengycin-like homologues having a molecular weight difference of 1435, 1449, 1463, 1477, 1491, 1505, etc. by one methylene group (-CH2) (FIG. 4-C).
The Surfactin-like homologues is a ring structure formed by a hydroxy fatty acid chain containing 12-16 carbon atoms and a polypeptide chain containing 7 amino acids, and the polypeptide chains have two kinds, and the amino acid sequences are as follows: glutamic acid (Glu) -leucine (Leu) -valine (Val) -aspartic acid (Asp) -leucine (Leu) or glutamic acid (Glu) -leucine (Leu) -aspartic acid (Asp) -leucine (Leu) -valine (Val); the Iturin analog is a ring structure formed by an amino fatty acid chain containing 14-17 carbon atoms and a polypeptide chain containing 7 amino acids, wherein the polypeptide chains are composed of two polypeptide chains, and the amino acid sequences are as follows: aspartic acid (Asn) -tyrosine (Tyr) -aspartic acid (Asn) -glutamine (Gln) -proline (Pro) -aspartic acid (Asn) -threonine (Thr) or aspartic acid (Asn) -tyrosine (Tyr) -glutamine (Gln) -aspartic acid (Asn) -proline (Pro) -aspartic acid (Asn) -threonine (Thr); the Fengycin analog is a ring structure formed by an amino fatty acid chain containing 15-17 carbon atoms and a polypeptide chain containing 10 amino acids, wherein the polypeptide chain comprises three polypeptide chains, and the amino acid sequences are as follows: glutamic acid (Glu) -ornithine (Orn) -tyrosine (Tyr) -threonine (Thr) -glutamic acid (Glu) -valine (Val) -proline (Pro) -glutamine (Gln) -tyrosine (Tyr) -isoleucine (Ile), glutamic acid (Glu) -ornithine (Orn) -tyrosine (Tyr) -threonine (Thr) -glutamic acid (Glu) -threonine (Abu) -proline (Pro) -glutamine (Gln) -tyrosine (Tyr) -isoleucine (Ile) or glutamic acid (Glu) -ornithine (Orn) -tyrosine (Tyr) -threonine (Thr) -glutamic acid (Glu) -alanine (Ala) -proline (Pro) -glutamine (Gln) -tyrosine (Tyr) -isoleucine (Ile).
The specific molecular formula and structural formula of the lipopeptide substance are shown in the table 1:
TABLE 1 chemical name, formula and formula of lipopeptide species obtained in example 2
Wherein the structural formulas of Surfactin A, Surfactin B, Bacillus F, Bacillus D, Fengycin A, Fengycin B and Fengycin C are as follows:
Surfactin A
Surfactin B
Bacillomycin F
Bacillomycin D
Fengycin A
Fengycin B
Fengycin C
example 4 testing of the bacterial inhibitory Effect of lipopeptides prepared by Bacillus velezensis FZ06
(1) Selecting 3 common bacterial pathogens: escherichia coli (Escherichia coli) GIM1.708 (purchased from Guangdong province microbial strain collection center), Staphylococcus aureus aureobasis aureus (Staphylococcus aureus subsp. aureobasis) GIM1.441 (purchased from Guangdong province microbial strain collection center) and Salmonella choleraesuis GIM1.163 (purchased from Guangdong province microbial strain collection center), wherein a small amount of Escherichia coli GIM1.708, Staphylococcus aureus aureobasis aureus GIM1.441 and Salmonella choleraesuis GIM1.163 (purchased from Guangdong province microbial strain collection center) preserved at-80 ℃ are picked up by an inoculating needle under aseptic conditions, and are respectively inoculated into a solid LB culture tube and activated at 36-38 ℃ for 24 hours to obtain activated Escherichia coli GIM1.708, Staphylococcus aureus aureobasis GIM1.441 and Salmonella choleraesuis GIM 1.163;
(2) respectively inoculating a ring of activated escherichia coli GIM1.708, staphylococcus aureus golden subspecies GIM1.441 and salmonella choleraesuis swine choleraesuis GIM1.163 into 50mL of liquid LB seed culture medium under an aseptic condition, and carrying out shake cultivation at 36-38 ℃ and 160rpm for 24 hours to obtain escherichia coli GIM1.708 seed liquid, staphylococcus aureus golden subspecies GIM1.441 seed liquid and salmonella choleraesuis swine choleraesuis GIM1.163 seed liquid;
(3) under aseptic conditions, the obtained seed liquid is respectively diluted to about 1 × 10 with sterile water8cfu/mL, and then coated on LB agar solid medium, respectively, to obtain a petri dish containing Escherichia coli GIM1.708, a petri dish containing Staphylococcus aureus golden subspecies GIM1.441, and a petri dish containing Salmonella choleraesuis and Salmonella choleraesuis subspecies sui GIM 1.163;
(4) taking 10mg of the lipopeptide freeze-dried powder prepared in the embodiment 2, and adding 2mL of deionized water to obtain a lipopeptide solution of 5 mg/mL;
(5) placing oxford cups (with the inner diameter of 6mm and sterilized) into the culture dishes containing the bacterial pathogens obtained in the step (3), respectively, adding 200 muL of 5mg/mL lipopeptide solution into the oxford cups in the experimental group, adding 200 muL of sterile water into the sterile oxford cups in the blank control group, placing the oxford cups in an incubator at 37 ℃ for constant-temperature culture for 24 hours, observing the bacteriostatic effect, wherein the result is shown in figure 5, and meanwhile, the size of the bacteriostatic circle is measured, and the result is shown in table 2; the lipopeptide substance prepared from Bacillus velezensis FZ06 has obvious effect of inhibiting bacterial pathogenic bacteria.
Example 5 testing of the fungus-inhibiting Effect of lipopeptides prepared by Bacillus velezensis FZ06
(1) 2 common fungal pathogens were selected: aspergillus flavus (Aspergillus flavus) GIM3.493 (purchased from Guangdong province culture collection center) and Aspergillus parasiticus (Aspergillus parasiticus special) GIM3.395 (purchased from Guangdong province culture collection center), picking a small amount of Aspergillus flavus GIM3.493 and Aspergillus parasiticus GIM3.395 stored at-80 ℃ into a PDA culture medium test tube by using an inoculating needle under the aseptic condition, respectively, and activating at 36-38 ℃ for 24 hours to obtain activated Aspergillus flavus GIM3.493 and Aspergillus parasiticus GIM 3.395;
(2) inoculating the activated aspergillus flavus GIM3.493 and aspergillus parasiticus GIM3.395 into a sterilized PDA culture medium, and culturing for 96 hours in an incubator at 36-38 ℃ to obtain an aspergillus flavus GIM3.493 plate with spores and an aspergillus parasiticus GIM3.395 with spores;
(3) scraping Aspergillus flavus GIM3.493 spores and Aspergillus parasiticus GIM3.395 spores into sterile physiological saline respectively, and performing sterile filtration by using sterile filter paper after shaking for 3min by using a vortex oscillator to obtain uniform Aspergillus flavus GIM3.493 spore suspension and Aspergillus parasiticus GIM3.395 spore suspension;
(4) diluting the obtained Aspergillus flavus GIM3.493 spore suspension and Aspergillus parasiticus GIM3.395 spore suspension obtained in the step (3) to 1 × 10 with sterile physiological saline respectively6cfu/mL, and then respectively coating the cfu/mL into a PDA agar solid culture medium to obtain a culture dish containing Aspergillus flavus GIM3.493 and a culture dish containing Aspergillus parasiticus GIM 3.395;
(5) taking 10mg of the lipopeptide freeze-dried powder prepared in the embodiment 2, and adding 2mL of deionized water to obtain a lipopeptide solution of 5 mg/mL;
(6) placing oxford cups (with the inner diameter of 6mm and sterilized) into the culture dishes containing the fungal pathogens obtained in the step (4), respectively, adding 200 mu L of 5mg/mL lipopeptide solution into the oxford cups for experimental groups, adding 200 mu L of sterile water into the sterile oxford cups for blank control groups, then placing the oxford cups into an incubator at 37 ℃ for constant-temperature culture for 5 days, and observing the bacteriostatic effect, wherein the result is shown in figure 6; meanwhile, the size of the inhibition zone is measured, and the result is shown in table 2; the lipopeptide substance prepared by Bacillus velezensis FZ06 has obvious effect of inhibiting fungal pathogenic bacteria.
TABLE 2 antibacterial Activity of the strains extracted in example 2 for the production of lipopeptides
The above embodiments are preferred embodiments of the present invention, but the present invention is not limited to the above embodiments, and any other changes, modifications, substitutions, combinations, and simplifications which do not depart from the spirit and principle of the present invention should be construed as equivalents thereof, and all such changes, modifications, substitutions, combinations, and simplifications are intended to be included in the scope of the present invention.
Sequence listing
<110> university of southern China's science
<120> endophytic bacillus of Pu' er tea tree leaves and application thereof
<160>1
<170>SIPOSequenceListing 1.0
<210>1
<211>1483
<212>DNA
<213> Artificial Sequence (Artificial Sequence)
<220>
<223> Bacillus velezensis FZ 0616S rDNA sequence
<400>1
tcaggacgaa cgctggcggc gtgcctaata catgcaagtc gagcggacag atgggagctt 60
gctccctgat gttagcggcg gacgggtgag taacacgtgg gtaacctgcc tgtaagactg 120
ggataactcc gggaaaccgg ggctaatacc ggatggttgt ctgaaccgca tggttcagac 180
ataaaaggtg gcttcggcta ccacttacag atggacccgc ggcgcattag ctagttggtg 240
aggtaacggc tcaccaaggc gacgatgcgt agccgacctg agagggtgat cggccacact 300
gggactgaga cacggcccag actcctacgg gaggcagcag tagggaatct tccgcaatgg 360
acgaaagtct gacggagcaa cgccgcgtga gtgatgaagg ttttcggatc gtaaagctct 420
gttgttaggg aagaacaagt gccgttcaaa tagggcggca ccttgacggt acctaaccag 480
aaagccacgg ctaactacgt gccagcagcc gcggtaatac gtaggtggca agcgttgtcc 540
ggaattattg ggcgtaaagg gctcgcaggc ggtttcttaa gtctgatgtg aaagcccccg 600
gctcaaccgg ggagggtcat tggaaactgg ggaacttgag tgcagaagag gagagtggaa 660
ttccacgtgt agcggtgaaa tgcgtagaga tgtggaggaa caccagtggc gaaggcgact 720
ctctggtctg taactgacgc tgaggagcga aagcgtgggg agcgaacagg attagatacc 780
ctggtagtcc acgccgtaaa cgatgagtgc taagtgttag ggggtttccg ccccttagtg 840
ctgcagctaa cgcattaagc actccgcctg gggagtacgg tcgcaagact gaaactcaaa 900
ggaattgacg ggggcccgca caagcggtgg agcatgtggt ttaattcgaa gcaacgcgaa 960
gaaccttacc aggtcttgac atcctctgac aatcctagag ataggacgtc cccttcgggg 1020
gcagagtgac aggtggtgca tggttgtcgt cagctcgtgt cgtgagatgt tgggttaagt 1080
cccgcaacga gcgcaaccct tgatcttagt tgccagcatt cagttgggca ctctaaggtg 1140
actgccggtg acaaaccgga ggaaggtggg gatgacgtca aatcatcatg ccccttatga 1200
cctgggctac acacgtgcta caatggacag aacaaagggc agcgaaaccg cgaggttaag 1260
ccaatcccac aaatctgttc tcagttcgga tcgcagtctg caactcgact gcgtgaagct 1320
ggaatcgcta gtaatcgcgg atcagcatgc cgcggtgaat acgttcccgg gccttgtaca 1380
caccgcccgt cacaccacga gagtttgtaa cacccgaagt cggtgaggta accttttagg 1440
agccagccgc cgaaggtggg acagatgatt ggggtgaagt cgt 1483
Claims (10)
1. An endophytic bacillus of Pu' er tea tree leaves is characterized in that: the Bacillus subtilis strain is named as Bacillus belgii FZ06(Bacillus velezensis FZ06), is preserved in China center for type culture collection of Wuhan university in Wuhan City in 2019, 10 and 25 months, and has the preservation number of CCTCC NO: m2019854.
2. The application of the endophytic bacillus puerh tea tree leaves in the preparation of lipopeptide substances in claim 1 is characterized by comprising the following steps:
(1) fermentation culture: inoculating the seed solution of the bacillus subtilis in the puer tea tree leaves of claim 1 into a fermentation culture medium for fermentation culture to obtain a fermentation liquid;
(2) acid precipitation separation and washing: performing first centrifugation on the fermentation liquor obtained in the step (1) to remove thalli, adding acid into the obtained supernatant to adjust the pH to 2.0, performing second centrifugation after standing to remove the supernatant, washing the obtained precipitate, and performing third centrifugation to obtain acid precipitate;
(3) acid precipitation and extraction: adding the acid precipitate obtained in the step (2) into a solvent for extraction, then carrying out solid-liquid separation, drying the obtained liquid, and then adding water for redissolution to obtain a water-phase extraction liquid;
(4) separating and purifying lipopeptide substances: and (3) drying the aqueous phase extract obtained in the step (3) for the first time, adding a solvent to redissolve the obtained dry matter, then carrying out chromatographic purification, drying the obtained eluent containing the lipopeptide substances for the second time, then adding water to redissolve the eluent, and finally carrying out drying for the third time to obtain the lipopeptide substances.
3. The application of the endophytic bacillus puerh tea tree leaves in the preparation of lipopeptide substances according to claim 2 is characterized in that:
the inoculation amount of the seed liquid in the step (1) is 1-5% of the fermentation medium in volume ratio; further 1% of said fermentation medium;
the fermentation medium is a liquid LB medium;
the fermentation culture conditions in the step (1) are 35-39 ℃ and 150-170 rpm for 48-72 hours; further, the culture was carried out at 36 to 38 ℃ and 160rpm for 72 hours.
4. The application of the endophytic bacillus puerh tea tree leaves in the preparation of lipopeptide substances according to claim 3 is characterized in that:
the seed solution is prepared by the following steps: activating and carrying out enlarged culture on stored Pu' er tea tree leaf endophytic bacillus to obtain seed liquid;
the activating steps are as follows: inoculating the stored Pu 'er tea tree leaf endophytic bacillus into an activation culture medium, and culturing for 24-48 hours at 35-39 ℃ to obtain activated Pu' er tea tree leaf endophytic bacillus;
the activation culture medium is a PDA culture medium;
the steps of the expanded culture are as follows: inoculating activated Pu' er tea tree leaf endophytic bacillus into a seed culture medium, and culturing for 24-48 hours at 35-39 ℃ and 150-170 rpm to obtain a seed solution;
the inoculation amount of the activated Pu' er tea tree leaf endophytic bacillus is 1-5% of the seed culture medium according to the volume ratio; further 1% of said seed medium;
the seed culture medium is a liquid LB culture medium.
5. The application of the endophytic bacillus puerh tea tree leaves in the preparation of lipopeptide substances according to claim 2 is characterized in that:
the first centrifugation condition in the step (2) is centrifugation at 10000rpm for 10min at 4 ℃;
the acid in the step (2) is hydrochloric acid; further hydrochloric acid with the concentration of 6 mol/L;
the standing condition in the step (2) is standing for 12-24 hours at 4 ℃; further standing for 12 hours at 4 ℃;
centrifuging for 5min at 10000rpm under the second centrifugation condition of 4 ℃ in the step (2);
the washing in the step (2) is washing with dilute hydrochloric acid; further washing with dilute hydrochloric acid having a pH of 2.0;
the washing times in the step (2) are 2 times;
centrifuging at 10000rpm for 10min for the third time in the step (2);
the solvent in the step (3) is absolute methanol;
the solid-liquid separation mode in the step (3) is suction filtration; further carrying out vacuum filtration;
the drying in the step (3) is suspended by concentration; further concentrating by vacuum and suspending; further rotary evaporating to dryness at 40 deg.C by rotary evaporator;
the water in the step (3) is deionized water.
6. The application of the endophytic bacillus puerh tea tree leaves in the preparation of lipopeptide substances according to claim 2 is characterized in that:
the first drying and the third drying in the step (4) are freeze drying;
the solvent in the step (4) is absolute methanol;
the chromatographic purification in the step (4) is purification by using a sephadex chromatographic column; further using Sephadex LH-20 Sephadex column, the eluent is 100% methanol, the flow rate is 0.5 mL/min;
the second drying in step (4) is suspended by concentration; further concentrating and drying in vacuum; further, the solution was evaporated to dryness by rotary evaporation at 40 ℃.
7. A lipopeptide species, characterized by: the bacillus Puer tea plant endophytic bacteria is obtained by fermenting, separating and purifying the bacillus Puer tea plant leaves according to claim 1.
8. The lipopeptide-like substance of claim 7, wherein: the lipopeptide substances comprise Surfactin similar substances, Iturin similar substances and Fengycin similar substances.
9. The lipopeptide-like substance of claim 8, wherein:
the Surfactin similar congener is C12-Leu7-Surfactin、C13-Leu7-Surfactin、C14-Leu7-Surfactin、C15-Leu7-Surfactin、C16-Leu7-Surfactin、C12-Val7-Surfactin、C13-Val7-Surfactin、C14-Val7-Surfactin、C15-Val7Surfactin and C14-Val7-Surfactin；
The Iturin analog is C14-Bacillomycin F、C15-Bacillomycin F、C16-Bacillomycin F、C17-Bacillomycin F、C14-Bacillomycin D、C15-Bacillomycin D、C16Bacillus bacteria D and C17-Bacillomycin D；
The Fengycin analog is C15-Ala6-Fengycin、C16-Ala6-Fengycin、C17-Ala6-Fengycin、C15-Abu6-Fengycin、C16-Abu6-Fengycin、C17-Abu6-Fengycin、C15-Val6-Fengycin、C16-Val6-Fengycin and C17-Val6-Fengycin。
10. Use of a lipopeptide according to any one of claims 7 to 9 for the inhibition of pathogenic bacteria, wherein: such pathogens include, but are not limited to, Escherichia coli, Staphylococcus aureus, Salmonella, Aspergillus flavus, and Aspergillus parasiticus.
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Cited By (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN111349588A (en) * | 2020-04-29 | 2020-06-30 | 福建省农业科学院畜牧兽医研究所 | Bacillus belgii and application thereof |
| WO2021135544A1 (en) * | 2019-12-30 | 2021-07-08 | 华南理工大学 | Endophytic bacillus from pu'er tea tree leaves and application thereof |
| CN115651857A (en) * | 2022-07-20 | 2023-01-31 | 广西科学院 | Bacillus belgii strain HS1 and application thereof |
| CN116082470A (en) * | 2022-12-12 | 2023-05-09 | 南京工业大学 | Bacillus bailii antibacterial lipopeptide and preparation method and application thereof |
Citations (6)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN107245457A (en) * | 2017-06-29 | 2017-10-13 | 华南理工大学 | A kind of extracting method and application of dendrobium candidum endogenetic fungal bacterial strain and its exocellular polysaccharide of generation and the exocellular polysaccharide |
| US20180020676A1 (en) * | 2014-12-29 | 2018-01-25 | Fmc Corporation | Bacillus velezensis rti301 compositions and methods of use for benefiting plant growth and treating plant disease |
| CN108865930A (en) * | 2018-06-27 | 2018-11-23 | 浙江大学 | RADIX CURCUMAE endogenetic bacteria bacterial strain ZJU-C612-2 and its application |
| CN110117556A (en) * | 2019-03-27 | 2019-08-13 | 海南大学 | A kind of efficient biocontrol microorganisms HN-2 and zymotechnique for producing lipopeptid substance |
| CN110790820A (en) * | 2019-10-29 | 2020-02-14 | 福建省农业科学院农业生物资源研究所 | Lipopeptide produced by bacillus strain FJAT-52631 and preparation method thereof |
| US20200100504A1 (en) * | 2018-09-28 | 2020-04-02 | Fmc Corporation | Bacillus amyloliquefaciens fcc1256 compositions and methods of controlling plant pathogens |
Family Cites Families (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO2010006235A1 (en) * | 2008-07-11 | 2010-01-14 | Novozymes A/S | Bacillus velezensis strain |
| KR101073932B1 (en) * | 2008-11-11 | 2011-10-17 | 한국화학연구원 | Bacillus velezensis pb3 strain and method for the biological control of plant diseases using same |
| KR100995286B1 (en) * | 2009-12-29 | 2010-11-19 | 장병일 | Bacillus velezensis strain, growth medium for the same and microbial agent containing the same |
| CN110564646B (en) * | 2019-09-20 | 2020-05-05 | 湖北省生物农药工程研究中心 | Bacillus belgii CY30 and application thereof in prevention and treatment of tea round spot |
| CN111019866B (en) * | 2019-12-30 | 2022-08-16 | 华南理工大学 | Endophytic bacillus of Pu' er tea tree leaves and application thereof |
-
2019
- 2019-12-30 CN CN201911396583.3A patent/CN111019866B/en active Active
-
2020
- 2020-10-23 WO PCT/CN2020/123117 patent/WO2021135544A1/en active Application Filing
- 2020-10-23 AU AU2020418980A patent/AU2020418980A1/en active Pending
Patent Citations (6)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US20180020676A1 (en) * | 2014-12-29 | 2018-01-25 | Fmc Corporation | Bacillus velezensis rti301 compositions and methods of use for benefiting plant growth and treating plant disease |
| CN107245457A (en) * | 2017-06-29 | 2017-10-13 | 华南理工大学 | A kind of extracting method and application of dendrobium candidum endogenetic fungal bacterial strain and its exocellular polysaccharide of generation and the exocellular polysaccharide |
| CN108865930A (en) * | 2018-06-27 | 2018-11-23 | 浙江大学 | RADIX CURCUMAE endogenetic bacteria bacterial strain ZJU-C612-2 and its application |
| US20200100504A1 (en) * | 2018-09-28 | 2020-04-02 | Fmc Corporation | Bacillus amyloliquefaciens fcc1256 compositions and methods of controlling plant pathogens |
| CN110117556A (en) * | 2019-03-27 | 2019-08-13 | 海南大学 | A kind of efficient biocontrol microorganisms HN-2 and zymotechnique for producing lipopeptid substance |
| CN110790820A (en) * | 2019-10-29 | 2020-02-14 | 福建省农业科学院农业生物资源研究所 | Lipopeptide produced by bacillus strain FJAT-52631 and preparation method thereof |
Non-Patent Citations (4)
| Title |
|---|
| FANG-ZHOU LI等: "Bioprospecting of a novel endophytic Bacillus velezensis FZ06 from leaves of Camellia assamica: Production of three groups of lipopeptides and the inhibition against food spoilage microorganisms", 《JOURNAL OF BIOTECHNOLOGY》 * |
| Y. LIU等: "Antimicrobial Bacillus velezensis HC6: production of three kinds of lipopeptides and biocontrol potential in maize", 《JOURNAL OF APPLIED MICROBIOLOGY》 * |
| 李方舟: "普洱茶树叶片内生细菌Bacillus velezensis FZ06的基因组测序及抗菌活性代谢产物研究", 《中国优秀博硕士学位论文全文数据库(硕士)工程科技Ⅰ辑》 * |
| 王若琳等: "桑树内生拮抗菌的分离鉴定及其对桑断枝烂叶病的生防初探", 《微生物学报》 * |
Cited By (6)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO2021135544A1 (en) * | 2019-12-30 | 2021-07-08 | 华南理工大学 | Endophytic bacillus from pu'er tea tree leaves and application thereof |
| CN111349588A (en) * | 2020-04-29 | 2020-06-30 | 福建省农业科学院畜牧兽医研究所 | Bacillus belgii and application thereof |
| CN111349588B (en) * | 2020-04-29 | 2022-01-11 | 福建省农业科学院畜牧兽医研究所 | Bacillus belgii and application thereof |
| CN115651857A (en) * | 2022-07-20 | 2023-01-31 | 广西科学院 | Bacillus belgii strain HS1 and application thereof |
| CN116082470A (en) * | 2022-12-12 | 2023-05-09 | 南京工业大学 | Bacillus bailii antibacterial lipopeptide and preparation method and application thereof |
| CN116082470B (en) * | 2022-12-12 | 2023-08-15 | 南京工业大学 | Bacillus bailii antibacterial lipopeptide and preparation method and application thereof |
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