CN111019866B - Endophytic bacillus of Pu' er tea tree leaves and application thereof - Google Patents

Abstract

The invention discloses an endophytic bacillus of Pu'er tea leaves and its application. The name of the endophyte bacillus in Pu'er tea leaves is Bacillus velezensis FZ06 (Bacillus velezensis FZ06). : M 2019854. The lipopeptide substances produced by this bacteria include 27 species in 3 categories, and its abundance is more than that of Bacillus velesi which has been reported so far; Aspergillus flavus and Aspergillus parasiticus have obvious inhibitory activity and can be well applied to inhibit pathogenic bacteria.

Description

A kind of Pu'er tea leaf endophyte bacillus and its application

technical field

The invention belongs to the technical field of microorganisms, in particular to an endophyte bacillus of Pu'er tea leaves and applications thereof.

Background technique

Lipopeptides are an important class of non-ionic biosurfactants, mainly derived from the secondary metabolic pathways of some bacteria and fungi. They are diverse and complex in structure, but are generally composed of fatty acid chains and peptide chain lactones or oligosaccharides. The amide bond forms a ring. As a molecular weapon of microorganisms, lipopeptides in the natural environment often have natural functions of anti-virus, antagonizing bacteria and even antagonizing fungi. Therefore, they are widely used in the development of plant anti-disease and insect pest reagents. As a class of nonionic surfactants, the antibacterial mechanism of lipopeptides may be similar to that of conventional antifungal surfactants: nonionic biosurfactants bind and separate essential proteins for survival on the target fungal cell membrane, resulting in die. Due to the differences in structure and properties, lipopeptides produced by different microorganisms should theoretically have different antibacterial spectra, and a single compound often cannot achieve broad bacteriostasis; and considering the adaptive evolution of microorganisms, the long-term application of a single antibiotic is very important. May make some fungi resistant. However, if the lipopeptide substances with more diverse structures and richer properties are used in combination, the above problems may be solved. Therefore, looking for microorganisms that can produce more abundant lipopeptide substances, extracting and using their lipopeptide compounds in combination, can expand the antibacterial spectrum of lipopeptide compounds, further reduce the adaptive evolution of antibiotics of target fungi, and even reduce the inhibition of lipopeptide compounds. The bacterial concentration is of great significance.

SUMMARY OF THE INVENTION

The primary purpose of the present invention is to overcome the shortcomings and deficiencies of the prior art, and to provide an endophyte bacillus of Pu'er tea leaves.

Another object of the present invention is to provide the application of the above-mentioned endophyte Bacillus puer tea leaves.

Another object of the present invention is to provide the lipopeptide substances prepared by the above-mentioned Bacillus endophytes of Pu'er tea leaves and their application.

To achieve the above object, the present invention is achieved through the following technical solutions:

An endophytic bacillus of Pu'er tea leaves, named Bacillus velezensis FZ06 (Bacillus velezensis FZ06), is isolated and purified from fresh Pu'er tea leaves in Xishuangbanna.

The described endophytic bacillus in Pu'er tea leaves is Bacillus velezensis FZ06 (Bacillus velezensis FZ06), and its preservation information is: preservation unit: China Center for Type Culture Collection (CCTCC), preservation date: October 25, 2019, Deposit address: China. Wuhan. Wuhan University, deposit number: CCTCC NO: M 2019854.

The application of the endophyte bacillus in Pu'er tea leaves in the preparation of lipopeptide substances; preferably comprises the following steps:

(1) fermentation culture: the described Pu'er tea leaf endophyte bacillus seed liquid is inserted into the fermentation medium for fermentation and culture, to obtain fermentation liquid;

(2) acid precipitation separation and washing: the fermentation broth obtained in step (1) is centrifuged for the first time to remove bacterial cells, the obtained supernatant is added with acid to adjust the pH to 2.0, and the second centrifugation is performed after standing to remove the supernatant. clear liquid, wash the obtained precipitate, and then carry out the third centrifugation to obtain acid precipitate;

(3) acid precipitation extraction: the acid precipitation obtained in step (2) is added to a solvent for extraction, then solid-liquid separation is performed, the obtained liquid is dried, and then water is added to reconstitute to obtain an aqueous extract;

(4) Separation and purification of lipopeptide substances: the aqueous extract obtained in step (3) is first dried, the obtained dry substance is reconstituted with a solvent, and then purified by chromatography, and the obtained lipopeptide-containing substances have The eluate is dried for the second time, then reconstituted with water, and finally dried for the third time to obtain lipopeptides.

The seed liquid described in step (1) is preferably prepared by the following steps: activating the Bacillus endophytes on the preserved Pu'er tea leaves, and expanding the culture to obtain the seed liquid.

The activation step is preferably as follows: insert the preserved endophyte bacillus of Pu'er tea leaves into the activation medium, and cultivate under the conditions of 35 to 39 ° C for 24 to 48 hours to obtain the activated endophyte bacillus of Pu'er tea leaves. .

The culture conditions are preferably cultured at 36-38°C for 24 hours.

The activation medium is preferably a PDA medium.

The step of expanding the culture is preferably as follows: insert the activated endophyte bacillus from Pu'er tea leaves into the seed medium, and cultivate at 35-39° C. and 150-170 rpm for 24-48 hours to obtain the seed liquid.

The amount of the activated endophyte bacillus in Pu'er tea leaves is preferably 1-5% of the seed medium by volume; more preferably 1% of the seed medium.

The seed medium is preferably a liquid LB medium.

The culturing conditions are preferably cultured at a temperature of 36-38° C. and 160 rpm for 24 hours.

In step (1), the feeding amount of the seed liquid is preferably 1-5% of the fermentation medium by volume; more preferably 1% of the fermentation medium.

The fermentation medium is preferably a liquid LB medium.

The conditions of the fermentation culture described in step (1) are preferably 35-39°C, 150-170 rpm for 48-72 hours; more preferably 36-38°C, 160 rpm for 72 hours.

The first centrifugation conditions described in step (2) are preferably centrifugation at 4° C. and 10,000 rpm for 10 min.

The acid described in the step (2) is preferably hydrochloric acid; more preferably, it is hydrochloric acid with a concentration of 6 mol/L.

The standing conditions described in step (2) are preferably 12-24 hours at 4°C; more preferably 12 hours at 4°C.

The second centrifugation conditions described in step (2) are preferably centrifugation at 4° C. and 10,000 rpm for 5 min.

The washing described in step (2) is preferably washed with dilute hydrochloric acid; more preferably, washed with dilute hydrochloric acid with pH=2.0.

The washing times described in step (2) is preferably 2 times.

The third centrifugation condition described in step (2) is preferably 10000rpm centrifugation for 10min.

The solvent described in step (3) is preferably anhydrous methanol.

The solid-liquid separation method described in the step (3) is preferably suction filtration; more preferably, vacuum suction filtration.

The drying described in step (3) is preferably suspended by concentration; more preferably suspended by vacuum concentration; most preferably by rotary evaporation at 40° C. to dryness by rotary evaporator.

The water in step (3) is preferably deionized water.

The first drying and the third drying described in step (4) are preferably freeze drying.

The solvent described in step (4) is preferably anhydrous methanol.

The chromatographic purification described in step (4) is preferably carried out using Sephadex chromatographic column; more preferably, Sephadex LH-20 Sephadex column is used, the eluent is 100% methanol, and the flow rate is 0.5mL/ min.

The second drying described in step (4) is preferably suspended by concentration; more preferably suspended by vacuum concentration; most preferably by rotary evaporation at 40° C. to dryness.

A lipopeptide substance is obtained by fermenting, separating and purifying the endophytic bacillus of the above-mentioned Pu'er tea leaves.

The lipopeptide substances include Surfactin-like homologues, Iturin-like homologues and Fengycin-like homologues.

Described Surfactin class homologue is preferably C ₁₂ -Leu ⁷ -Surfactin, C ₁₃ -Leu ⁷ -Surfactin, C ₁₄ -Leu ⁷ -Surfactin, C ₁₅ -Leu ⁷ -Surfactin, C ₁₆ -Leu ⁷ -Surfactin, C ₁₂ -Val7 ^- Surfactin, _C13 -Val7 ^- Surfactin, _C14 -Val7 ^- Surfactin, _C15 -Val7 ^- Surfactin and _C14 -Val7 ^- Surfactin.

The iturin class homologues are preferably C ₁₄ -Bacillomycin F, C ₁₅ -Bacillomycin F, C ₁₆ -Bacillomycin F, C ₁₇ -Bacillomycin F, C ₁₄ -Bacillomycin D, C ₁₅ -Bacillomycin D, C ₁₆ -Bacillomycin D and C ₁₇ -Bacillomycin D.

Described Fengycin class homologue is preferably C ₁₅ -Ala ⁶ -Fengycin, C ₁₆ -Ala ⁶ -Fengycin, C ₁₇ -Ala ⁶ -Fengycin, C ₁₅ -Abu ⁶ -Fengycin, C ₁₆ -Abu ⁶ -Fengycin, C ₁₇ ^{- Abu6 -} Fengycin, _C15 ^- Val6-Fengycin, C16- _Val6 -Fengycin and _C17 -Val6-Fengycin.

The application of the lipopeptide substance in inhibiting pathogenic bacteria.

The pathogenic bacteria include, but are not limited to, Escherichia coli, Staphylococcus aureus, Salmonella, Aspergillus flavus and Aspergillus parasiticus.

The Escherichia coli is preferably Escherichia coli GIM1.708.

The Staphylococcus aureus is preferably Staphylococcus aureus subsp. GIM1.441.

Said Salmonella is preferably Salmonella choleraesuis subsp. choleraesuis GIM1.163.

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 present invention has the following advantages and effects:

(1) The present invention isolates Bacillus velezensis FZ06 from fresh Pu'er tea leaves for the first time, and the lipopeptide substances that the bacteria can produce include 27 species of 3 categories, and its abundance is more than the existing reported Bacillus veles. Among them, the amino acid sequence of the polypeptide chain of Fengycin-like homologs containing amino fatty acid chains of 15-17 carbon atoms was observed in Bacillus velesi for the first time.

(2) The method for preparing lipopeptide substances provided by the present invention is simple and easy to implement, with mild conditions and easy control, and provides a new path for large-scale production of lipopeptide substances.

(3) The present invention adopts the lipopeptide substances produced by Bacillus velezensis FZ06 to have obvious inhibitory activity on pathogenic bacteria such as Escherichia coli, Staphylococcus aureus, Salmonella, Aspergillus flavus and Aspergillus parasiticus, and can be well applied to inhibit pathogenic bacteria It provides a new idea for improving the postharvest preservation performance or disease resistance of host plant exfoliants by improving the interaction between endophytic bacteria and host plants.

Description of drawings

Fig. 1 is a graph showing the antibacterial phenomenon of endophytic bacteria in the leaves of Pu'er tea tree antagonizing black spore-producing endophytic fungi.

Figure 2 is a scanning electron microscope (SEM) image of Bacillus velezensis FZ06: wherein, A is the SEM image of Bacillus velezensis FZ06 under 1000× magnification; B is the SEM image of Bacillus velezensis FZ06 under 5000× magnification.

Figure 3 is a phylogenetic tree diagram of 16S rDNA of Bacillus velezensis FZ06.

Figure 4 is the total ion current diagram of the lipopeptide solution prepared by Bacillus velezensis FZ06: wherein, A is the total ion current diagram of the Surfactin-like congeners; B is the total ion current diagram of the Iturin-like congeners; C is the Fengycin-like congeners The total ion chromatogram of the substance.

Figure 5 is a graph showing the inhibitory effect of lipopeptides prepared by Bacillus velezensis FZ06 and sterile water on bacterial pathogens: wherein, A is the inhibition of lipopeptides prepared by Bacillus velezensis FZ06 and sterile water on Escherichia coli GIM1.708 Effect diagram; B is the inhibitory effect diagram of lipopeptides prepared by Bacillus velezensis FZ06 and sterile water on Staphylococcus aureus subsp. GIM1.441; C is the lipopeptide prepared by Bacillus velezensis FZ06 and sterile water Graph of the inhibitory effect on Salmonella choleraesuis subsp. cholera suis GIM1.163.

Figure 6 is a graph showing the inhibitory effect of lipopeptides prepared by Bacillus velezensis FZ06 and sterile water on fungal pathogens: A is the inhibitory effect of lipopeptides prepared by Bacillus velezensis FZ06 and sterile water on Aspergillus flavus GIM 3.493 Effect diagram; B is the effect diagram of the inhibitory effect of lipopeptides prepared by Bacillus velezensis FZ06 and sterile water on Aspergillus parasiticus GIM 3.395.

Detailed ways

The present invention will be described in further detail below with reference to the embodiments and the accompanying drawings, but the embodiments of the present invention are not limited thereto.

The test methods that do not specify specific experimental conditions in the following examples are usually in accordance with conventional experimental conditions or in accordance with experimental conditions suggested by the manufacturer. The materials, reagents, etc. used, unless otherwise specified, are the reagents and materials obtained from commercial sources.

Example 1 Isolation and identification of antifungal endophytic bacteria (Bacillus endophyte from Pu'er tea leaves)

(1) Pretreatment of fresh Pu’er tea leaves: The collected fresh and complete Pu’er tea leaves of the same leaf age (from Menghai County, Xishuangbanna, Yunnan Province) will be transported to the laboratory within 24 hours, and surface disinfection operations are carried out, as follows: In a sterile operation bench, soak fresh and intact Pu'er tea leaves in 75% (volume fraction) ethanol solution for 15 seconds, then rinse with sterile water and dry with sterile cotton; then, in 3.25% (available chlorine) content) in sodium hypochlorite solution for 15 seconds, rinse with sterile water and dry with sterile cotton; repeat the above surface disinfection operation three times. The last rinse water was spread on potato dextrose agar medium (PDA medium), and then cultured at 37°C for 2 weeks. If no microorganisms appeared, it was considered that the leaf surface was completely disinfected.

The fresh Pu'er tea leaves with complete surface disinfection were placed in a ventilated sterile bag and stored at 4°C for 30 days. After 30 days, the intact Pu'er tea leaves that remained green and had no obvious endophytic fungal growth leading to the decomposition of plant leaves were selected. Repeat the above surface disinfection operation three times. The last rinse water was spread in PDA medium, and then incubated at 37°C for 2 weeks. If no microorganisms appeared, it was considered that the leaf surface was completely disinfected.

(2) Culture and isolation of antifungal endophytic bacteria:

The aseptic sections of the Pu'er tea leaves that keep green, surface secondary disinfection that step (1) obtains, obtain the plant slices of about 0.5 centimeters in diameter, the plant slices are placed in PDA medium, cultivated for one week at 30°C, the results are as shown in Figure 1. Shows: the phenomenon that endophyte bacteria antagonize black spore-producing endophyte fungi obviously (the upper right part of the medium). The colony of endophytic bacteria that inhibited the endophytic fungi of black spore-producing plants was selected and streaked, and the separation and purification were repeated three times to obtain antifungal endophytic bacteria.

The characteristics of the solid culture of the bacteria are as follows: take a ring of purified antifungal endophytic bacteria and culture them on PDA medium at 36-38°C for 3-5 days. The initial colony is light white and gradually turns yellow; Fluffy.

The characteristics of the liquid culture of the bacteria are as follows: take the antifungal endophytic bacteria obtained by one-ring purification into 50 mL of potato dextrose liquid medium and cultivate for 3 days at 36-38 °C: on the first day, the medium changed from clarification to turbidity, and there were obvious Bacterial liquid culture phenomenon; on the 2nd day, it entered the stable period, and the turbidity did not change; on the 3rd day, the surface of the fermentation broth formed wrinkles.

The morphology of the bacteria under the scanning electron microscope is shown in Figure 2: it has a rod-like structure, and a single cell is 0.7-0.8 × 2-3 μm.

(3) 16S identification of single colony: The isolated strain was inoculated into 50 mL of LB medium, cultured with shaking at 160 rpm at 37° C. for 24 hours, and 10 mL of bacterial liquid was taken and sent to Shanghai Sangon Bioengineering Co., Ltd. for strain sequencing. The 16SrDNA sequence of the strain is shown in SEQ ID NO: 1: (1483bp)

TCAGGACGAACGCTGGCGGCGTGCCTAATACATGCAAGTCGAGCGGACAGATGGGAGCTTGCTCCCTGATGTTAGCGGCGGACGGGTGAGTAACACGTGGGTAACCTGCCTGTAAGACTGGGATAACTCCGGGAAACCGGGGCTAATACCGGATGGTTGTCTGAACCGCATGGTTCAGACATAAAAGGTGGCTTCGGCTACCACTTACAGATGGACCCGCGGCGCATTAGCTAGTTGGTGAGGTAACGGCTCACCAAGGCGACGATGCGTAGCCGACCTGAGAGGGTGATCGGCCACACTGGGACTGAGACACGGCCCAGACTCCTACGGGAGGCAGCAGTAGGGAATCTTCCGCAATGGACGAAAGTCTGACGGAGCAACGCCGCGTGAGTGATGAAGGTTTTCGGATCGTAAAGCTCTGTTGTTAGGGAAGAACAAGTGCCGTTCAAATAGGGCGGCACCTTGACGGTACCTAACCAGAAAGCCACGGCTAACTACGTGCCAGCAGCCGCGGTAATACGTAGGTGGCAAGCGTTGTCCGGAATTATTGGGCGTAAAGGGCTCGCAGGCGGTTTCTTAAGTCTGATGTGAAAGCCCCCGGCTCAACCGGGGAGGGTCATTGGAAACTGGGGAACTTGAGTGCAGAAGAGGAGAGTGGAATTCCACGTGTAGCGGTGAAATGCGTAGAGATGTGGAGGAACACCAGTGGCGAAGGCGACTCTCTGGTCTGTAACTGACGCTGAGGAGCGAAAGCGTGGGGAGCGAACAGGATTAGATACCCTGGTAGTCCACGCCGTAAACGATGAGTGCTAAGTGTTAGGGGGTTTCCGCCCCTTAGTGCTGCAGCTAACGCATTAAGCACTCCGCCTGGGGAGTACGGTCGCAAGACTGAAACTCAAAGGAATTGACGGGGGCCCGCACAAGCGGTGGAGCATGTGGTTTAATTCGAAGCAACGCGAAGAACCTTACCAGGTCTTGACATCCTCTGACAATCCTAGAG ATAGGACGTCCCCTTCGGGGGCAGAGTGACAGGTGGTGCATGGTTGTCGTCAGCTCGTGTCGTGAGATGTTGGGTTAAGTCCCGCAACGAGCGCAACCCTTGATCTTAGTTGCCAGCATTCAGTTGGGCACTCTAAGGTGACTGCCGGTGACAAACCGGAGGAAGGTGGGGATGACGTCAAATCATCATGCCCCTTATGACCTGGGCTACACACGTGCTACAATGGACAGAACAAAGGGCAGCGAAACCGCGAGGTTAAGCCAATCCCACAAATCTGTTCTCAGTTCGGATCGCAGTCTGCAACTCGACTGCGTGAAGCTGGAATCGCTAGTAATCGCGGATCAGCATGCCGCGGTGAATACGTTCCCGGGCCTTGTACACACCGCCCGTCACACCACGAGAGTTTGTAACACCCGAAGTCGGTGAGGTAACCTTTTAGGAGCCAGCCGCCGAAGGTGGGACAGATGATTGGGGTGAAGTCGT

Its 16S rDNA sequence was used for homology analysis in NCBI with Blast, and several bacterial species were selected to do phylogenetic tree analysis, the results were as shown in Figure 3: it was shown that the isolated antifungal endophytic bacterial species was Bacillus velesi ( Bacillus velezensis), named Bacillus velezensis FZ06.

Example 2 Preparation of lipopeptides by Bacillus velezensis FZ06

(1) Activation of strains: Under aseptic conditions, take 1 ring of Bacillus velezensis FZ06 from a glycerol cryopreservation tube and insert it into PDA medium, and activate and culture at 36-38°C for 24 hours;

(2) Seed liquid culture: Under sterile conditions, 1 mL of activated Bacillus velezensis FZ06 was transferred into 100 mL of liquid LB medium, and cultured on a shaker at 36-38° C. and 160 rpm for 24 hours to obtain seed liquid;

(3) Fermentation culture: under aseptic conditions, take 20 mL of the seed liquid obtained in step (2) and insert it into 2 L of liquid LB medium, and cultivate at 36-38° C. and 160 rpm shaker for 72 hours to obtain a fermentation broth;

(4) Acid precipitation separation and washing: centrifuge the fermentation broth in a low-temperature high-speed centrifuge at 4°C and 10000rpm for 10min to remove bacteria, then add 6mol/L HCl to the centrifugation supernatant to adjust the pH to 2.0, and let stand at 4°C After 12 hours, the acid-precipitated fermentation broth was obtained; the acid-precipitated fermentation broth was centrifuged in a low-temperature high-speed centrifuge at 4°C and 10000 rpm for 5 min, the supernatant was discarded and the precipitate was collected, and the precipitate was washed twice with 200 mL of pH=2.0 dilute hydrochloric acid water , the precipitate was collected after centrifugation at 10000 rpm for 10 min to obtain an acid precipitate;

(5) Acid precipitation extraction: the acid precipitation was extracted with 200 mL of anhydrous methanol, the extract was subjected to vacuum suction filtration, and the obtained suction filtrate was evaporated to dryness at 40° C. using a rotary evaporator, and redissolved in deionized water to obtain water phase extract;

(6) Separation and purification of lipopeptides: freeze-dry the aqueous phase extract to obtain a freeze-dried product, add 50 mL of anhydrous methanol to reconstitute, and the obtained reconstituted solution is subjected to Sephadex LH-20 column chromatography Purification (eluent is 100% methanol, flow rate is 0.5mL/min), the obtained eluent containing lipopeptides is vacuum concentrated to dryness, then reconstituted with water, and the reconstituted solution is freeze-dried to obtain Bacillus velezensis FZ06 The yield of lipopeptides was 1.03 g/L.

Example 3 Structure identification of lipopeptides prepared by Bacillus velezensis FZ06

50 mg of the lipopeptide powder obtained in the extraction example 2 was redissolved in 50 mL of anhydrous methanol, and then diluted 1000 times with anhydrous methanol, and the structure of ultra-high performance liquid chromatography-electrospray-secondary mass spectrometry UPLC-ESI-MS/MS was carried out. The specific analysis conditions are as follows:

Chromatographic separation conditions: Chromatographic column: Waters ACQUITY UPLC BEHC18 column (2.1mm×100mm, 1.7μm); column temperature: 30°C; flow rate: 0.4mL/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; 0.5-3.5 min, 60-20% A;. 3.5～4min, 20%A; 4～6min, 20～5%A; 6～7min, 5～2%A; 7～10min, 2%A; 10～10.5min, 2～60%A; 10.5～15min , 60% A; injection volume: 1 μL.

Mass spectrometry analysis conditions: select Agilent G6545A QTOF mass spectrometer, cation scanning mode, the mass scanning range of primary mass spectrometry is set to 110-1600 amu, and the mass scanning range of secondary mass spectrometry is set to 50-1600 amu; ion source temperature is 150 °C, capillary voltage is 3.26 kV; N ₂ flow rate of 600 L/h, Ar ₂ flow rate of 50 L/h; mass spectrometry data acquisition and processing software is Agilent MassHunter.

The UPLC-MS results are shown in Figure 4: The lipopeptides produced by Bacillus velezensis FZ06 contain Surfactin-like homologues with molecular weights of 994, 1008, 1022, 1036, and 1050 that differ by one methylene group (-CH2) (Figure 4- A); Iturin-like homologues whose molecular weights 1043, 1057, 1071, and 1085 differ by one methylene group (-CH2) (Figure 4-B) and molecular weights 1435, 1449, 1463, 1477, 1491, 1505, etc., differ by one methylene group Fengycin-like homologs of the base (-CH2) (Fig. 4-C).

As determined by UPLC-ESI-MS/MS, Surfactin-like homologues are cyclic structures formed by a hydroxy fatty acid chain containing 12-16 carbon atoms and a polypeptide chain containing 7 amino acids. The sequence is: glutamic acid (Glu)-leucine (Leu)-leucine (Leu)-valine (Val)-aspartic acid (Asp)-leucine (Leu)-leucine ( Leu) or glutamic acid (Glu)-leucine (Leu)-leucine (Leu)-leucine (Leu)-aspartic acid (Asp)-leucine (Leu)-valine ( Val); Iturin-like homologues are a cyclic structure formed by an amino fatty acid chain containing 14-17 carbon atoms and a polypeptide chain containing 7 amino acids. The polypeptide chain consists of two types, and its amino acid sequence is: aspartic acid (Asn)-Tyrosine (Tyr)-Aspartic acid (Asn)-Glutamine (Gln)-Proline (Pro)-Aspartic acid (Asn)-Threonine (Thr) or Aspartic acid Amino acid (Asn)-tyrosine (Tyr)-glutamine (Gln)-aspartic acid (Asn)-proline (Pro)-aspartic acid (Asn)-threonine (Thr); Fengycin homologues are cyclic structures formed by amino fatty acid chains containing 15-17 carbon atoms and polypeptide chains containing 10 amino acids. There are three types of polypeptide chains, and their amino acid sequences are: glutamic acid (Glu)- Ornithine (Orn) - Tyrosine (Tyr) - Threonine (Thr) - Glutamic acid (Glu) - Valine (Val) - Proline (Pro) - Glutamine (Gln) - Tyrosine Amino acid (Tyr)-Isoleucine (Ile), Glutamate (Glu)-Ornithine (Orn)-Tyrosine (Tyr)-Threonine (Thr)-Glutamate (Glu)-Butyl Amino acid (Abu)-proline (Pro)-glutamine (Gln)-tyrosine (Tyr)-isoleucine (Ile) or glutamic acid (Glu)-ornithine (Orn)-tyrosine Amino acid (Tyr) - Threonine (Thr) - Glutamic acid (Glu) - Alanine (Ala) - Proline (Pro) - Glutamine (Gln) - Tyrosine (Tyr) - Isoleucine Amino acid (Ile).

The specific molecular formula and structural formula of lipopeptides are shown in Table 1:

The chemical name, molecular formula and structural formula of the lipopeptide substances obtained in Table 1 Example 2

Among them, the structural formulas of Surfactin A, Surfactin B, Bacillomycin F, Bacillomycin D, FengycinA, Fengycin B, and Fengycin C are as follows:

Surfactin A

Surfactin B

Bacillomycin F

Bacillomycin D

Fengycin A

Fengycin B

Fengycin C

Example 4 Bacterial inhibitory effect test of lipopeptides prepared by Bacillus velezensis FZ06

(1) Three common bacterial pathogens were selected: Escherichia coli GIM1.708 (purchased from the Guangdong Provincial Microbial Culture Collection Center), Staphylococcus aureus subsp.aureus GIM1.441 (purchased in Guangdong Provincial Microbial Culture Collection Center) and Salmonella choleraesuis subsp. Choleraesuis GIM1.163 (purchased from Guangdong Provincial Microorganism Culture Collection Center), pick a small amount with an inoculating needle under sterile conditions Escherichia coli GIM1.708, Staphylococcus aureus subsp. GIM1.441 and Salmonella choleraesuis subsp. cholera suis GIM1.163 stored at -80°C were placed in test tubes of solid LB medium, respectively, and activated at 36-38°C for 24 hours. Activated Escherichia coli GIM1.708, Staphylococcus aureus subsp. aureus GIM1.441 and Salmonella choleraesuis subsp. cholera suis GIM1.163 were obtained;

(2) Under sterile conditions, take a loop of activated Escherichia coli GIM1.708, Staphylococcus aureus subsp. GIM1.441 and Salmonella choleraesuis subsp. cholera suis GIM1.163 and transfer them into 50 mL of liquid LB seeds respectively In the culture medium, culture at 36-38° C. and 160 rpm shaker for 24 hours to obtain Escherichia coli GIM1.708 seed liquid, Staphylococcus aureus subsp. GIM1.441 seed liquid and Salmonella choleraesuis subsp. cholera suis GIM1.163 seed liquid;

(3) Under sterile conditions, the obtained seed liquids were diluted with sterile water to about 1×10 ⁸ cfu/mL, respectively, and then spread on LB agar solid medium respectively to obtain Escherichia coli GIM1.708-containing seed liquids. Petri dishes, petri dishes containing Staphylococcus aureus subsp. GIM1.441 and petri dishes containing Salmonella choleraesuis subsp. cholera suis GIM1.163;

(4) Take 10 mg of the lyophilized lipopeptide powder prepared in Example 2, add 2 mL of deionized water to obtain a 5 mg/mL lipopeptide solution;

(5) The Oxford cup (inner diameter 6mm, sterilized) was placed in the petri dish containing bacterial pathogenic bacteria obtained in step (3), the experimental group added 200 μL 5mg/mL lipopeptide solution in the Oxford cup, and the blank control group Then add 200 μL of sterile water to a sterile Oxford cup, and then place it in a 37°C incubator for 24 hours at a constant temperature to observe its antibacterial effect. The results are shown in Figure 5. As shown in Table 2; it shows that the lipopeptides prepared by Bacillus velezensis FZ06 have obvious inhibitory effect on bacterial pathogens.

Example 5 Fungal inhibitory effect test of lipopeptides prepared by Bacillus velezensis FZ06

(1) Two common fungal pathogens were selected: Aspergillus flavus GIM 3.493 (purchased from Guangdong Provincial Culture Collection Center for Microorganisms) and Aspergillus parasiticus Speare GIM 3.395 (purchased from Guangdong Provincial Culture Collection Center for Microorganisms) ), under aseptic conditions, pick a small amount of Aspergillus flavus GIM3.493 and Aspergillus parasiticus GIM 3.395 stored at -80°C with an inoculating needle and insert them into PDA medium test tubes respectively, and activate them at 36-38°C for 24 hours to obtain activated yellow Aspergillus GIM 3.493 and Aspergillus parasiticus GIM3.395;

(2) The activated Aspergillus flavus GIM 3.493 and Aspergillus parasiticus GIM 3.395 were inserted into the sterilized PDA medium, and cultured in a 36-38° C. incubator for 96 hours to obtain the Aspergillus flavus GIM 3.493 plate with spores and the Aspergillus parasiticus GIM 3.395 of spores;

(3) scrape the Aspergillus flavus GIM 3.493 spores and the Aspergillus parasiticus GIM 3.395 spores and place them in sterile saline, respectively, and filter them with sterile filter paper after oscillating with a vortex shaker for 3 minutes to obtain a uniform Aspergillus flavus GIM 3.493 spore suspension and Aspergillus parasiticus GIM 3.395 spore suspension;

(4) Dilute the obtained Aspergillus flavus GIM 3.493 spore suspension and Aspergillus parasiticus GIM 3.395 spore suspension obtained in step (3) with sterile physiological saline to 1×10 ⁶ cfu/mL, respectively, and then spread on PDA agar respectively In the solid medium, a petri dish containing Aspergillus flavus GIM 3.493 and a petri dish containing Aspergillus parasiticus GIM 3.395 were obtained;

(5) Take 10 mg of the lyophilized lipopeptide powder prepared in Example 2, add 2 mL of deionized water to obtain a 5 mg/mL lipopeptide solution;

(6) The Oxford cups (inner diameter 6mm, sterilized) were placed in the petri dishes containing fungal pathogens obtained in step (4), respectively. The experimental group added 200 μL of 5 mg/mL lipopeptide solution to the Oxford cups, while the blank control The group added 200 μL of sterile water to a sterile Oxford cup, and then placed it in a 37°C incubator for 5 days at a constant temperature to observe its antibacterial effect. The results are shown in Figure 6; , the results are shown in Table 2; it shows that the lipopeptides prepared by Bacillus velezensis FZ06 have obvious inhibitory effect on fungal pathogens.

Table 2 Antibacterial activity of lipopeptide substances produced by strains extracted in Example 2

The above-mentioned embodiments are preferred embodiments of the present invention, but the embodiments of the present invention are not limited by the above-mentioned embodiments, and any other changes, modifications, substitutions, combinations, The simplification should be equivalent replacement manners, which are all included in the protection scope of the present invention.

sequence listing

<110> South China University of Technology

<120> An endophytic bacillus of Pu'er tea leaves and its application

<160> 1

<170> SIPOSequenceListing 1.0

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<211> 1483

<212> DNA

<213> Artificial Sequence

<220>

<223> Bacillus velezensis FZ06 16S rDNA sequence

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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: named Bacillus belgii: (Bacillus velezensis) FZ06, which is preserved in China center for type culture Collection of Wuhan university in Wuhan City in 2019, 10 months and 25 days, with the preservation number of CCTCC NO: m2019854.

2. The application of the endophytic bacillus puerh tea tree leaves in preparing lipopeptide substances 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;

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.

4. The application of the endophytic bacillus puerh tea tree leaves in preparing lipopeptide substances according to claim 3 is characterized in that:

the inoculation amount of the seed liquid in the step (1) is 1 percent of the fermentation medium according to the volume ratio;

the fermentation culture conditions in the step (1) are 36-38 ℃ and 160rpm for 72 hours.

5. The application of the endophytic bacillus puerh tea tree leaves in preparing 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;

the seed culture medium is a liquid LB culture medium.

6. The application of the endophytic bacillus puerh tea tree leaves in the preparation of lipopeptide substances according to claim 5 is characterized in that: the inoculation amount of the activated Pu' er tea tree endophytic bacillus is 1% of the seed culture medium according to the volume ratio.

7. The application of the endophytic bacillus puerh tea tree leaves in preparing 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;

the standing condition in the step (2) is standing for 12-24 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;

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;

the drying in the step (3) is suspended by concentration;

the water in the step (3) is deionized water.

8. The application of the endophytic bacillus puerh tea tree leaves in the preparation of lipopeptide substances according to claim 7 is characterized in that:

the acid in the step (2) is hydrochloric acid with the concentration of 6 mol/L;

the standing condition in the step (2) is standing for 12 hours at 4 ℃;

the washing in the step (2) is washing with dilute hydrochloric acid having pH = 2.0;

the solid-liquid separation mode in the step (3) is vacuum filtration;

the drying described in step (3) is suspended by vacuum concentration.

9. 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;

the second drying described in step (4) is suspended by concentration.

10. The application of the bacillus endophytic to puer tea tree leaves in the preparation of lipopeptide substances according to claim 9 is characterized in that:

performing chromatographic purification in the step (4) by using a Sephadex LH-20 Sephadex column, wherein the eluent is 100% methanol, and the flow rate is 0.5 mL/min;

the second drying described in step (4) is suspended by vacuum concentration.

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