WO2021135544A1 - 一种普洱茶树叶片内生芽孢杆菌及其应用 - Google Patents

一种普洱茶树叶片内生芽孢杆菌及其应用 Download PDF

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WO2021135544A1
WO2021135544A1 PCT/CN2020/123117 CN2020123117W WO2021135544A1 WO 2021135544 A1 WO2021135544 A1 WO 2021135544A1 CN 2020123117 W CN2020123117 W CN 2020123117W WO 2021135544 A1 WO2021135544 A1 WO 2021135544A1
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tea tree
surfactin
bacillus
fengycin
medium
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娄文勇
李方舟
曾英杰
宗敏华
杨继国
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华南理工大学
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    • C07K7/00—Peptides having 5 to 20 amino acids in a fully defined sequence; Derivatives thereof
    • C07K7/04—Linear peptides containing only normal peptide links
    • C07K7/06—Linear peptides containing only normal peptide links having 5 to 11 amino acids
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Definitions

  • the invention belongs to the technical field of microorganisms, and in particular relates to an endophytic bacillus from the leaves of Pu'er tea trees and applications thereof.
  • Lipopeptide is an important class of non-ionic biosurfactants, which are mainly derived from the secondary metabolic pathways of some bacteria and fungi. They have various types and complex structures, but they are generally composed of fatty acid chains and peptide chains. Amide bonds are combined to form a ring. As a molecular weapon of microorganisms, lipopeptides in the natural environment often have the natural functions of anti-virus, antagonizing bacteria and even antagonizing fungi. Therefore, they are widely used in the development of plant anti-disease agents.
  • nonionic surfactants As a class of nonionic surfactants, the antibacterial mechanism of lipopeptides may be similar to conventional antifungal surface active molecules: nonionic biosurfactants bind and separate essential proteins related to survival on the cell membrane of the target fungus, resulting in the target fungus death. Due to differences in structure and properties, lipopeptides produced by different microorganisms should theoretically have different antibacterial spectrums. A single compound often cannot achieve a wide range of antibacterial properties; and considering the adaptive evolution of microorganisms, the long-term application of a single antibiotic is very important. May make certain fungi resistant to drugs. However, if lipopeptides with more diverse structures and richer properties are used in combination, the above problems may be solved.
  • the primary purpose of the present invention is to overcome the shortcomings and deficiencies of the prior art and provide a kind of endophytic bacillus from the leaves of Pu'er tea tree.
  • Another object of the present invention is to provide the application of the above-mentioned endophytic bacillus from the leaves of Pu'er tea tree.
  • Another object of the present invention is to provide lipopeptides prepared by endophytic bacillus from the leaves of Pu'er tea tree and its application.
  • Bacillus velezensis FZ06 Bacillus velezensis FZ06
  • Bacillus velezensis FZ06 Bacillus velezensis FZ06
  • the said endophytic bacillus of Pu'er tea tree leaves is Bacillus velezensis FZ06 (Bacillus velezensis FZ06), and its preservation information is: Preserving unit: China Type Culture Collection (CCTCC), Preservation Date: October 25, 2019 , Preservation address: Wuhan University, Wuhan, China, Preservation number: CCTCC NO: M 2019854.
  • the application of the endophytic bacillus from the leaves of Pu'er tea tree in the preparation of lipopeptides preferably includes the following steps:
  • Fermentation culture the said Pu'er tea tree leaf endophytic bacillus seed liquid is connected to a fermentation medium for fermentation culture to obtain a fermentation broth;
  • step (2) Acid precipitation separation and washing: the fermentation broth obtained in step (1) is subjected to the first centrifugation to remove the bacteria, the supernatant obtained is added with acid to adjust the pH to 2.0, and the second centrifugation is carried out to remove the supernatant after standing. Clear liquid, wash the obtained precipitate, and then perform a third centrifugation to obtain an acid precipitate;
  • the aqueous extract obtained in step (3) is dried for the first time, and the obtained dry matter is reconstituted with a solvent, and then subjected to chromatographic purification to obtain a lipopeptide-containing substance
  • the eluate is dried a second time, then reconstituted with water, and finally dried a third time to obtain lipopeptides.
  • the seed liquid described in step (1) is preferably prepared by the following steps: activating the preserved Pu'er tea tree leaf endophytic bacillus and expanding the culture to obtain the seed liquid.
  • the activation step is preferably as follows: the preserved Pu'er tea tree leaf endophytic bacillus is connected to an activation medium, and the culture is carried out at 35-39°C for 24 to 48 hours to obtain activated Pu'er tea tree leaf endophytic bacillus .
  • the culture conditions are preferably 36-38°C for 24 hours.
  • the activation medium is preferably a PDA medium.
  • the step of expanding the culture is preferably as follows: the activated Pu'er tea tree leaf endophytic bacillus is inserted into a seed culture medium, and cultured at 35-39° C. and 150-170 rpm for 24 to 48 hours to obtain a seed liquid.
  • the volume ratio of the activated Bacillus endophyte in the leaves of the activated Pu'er tea tree is preferably 1% to 5% of the seed culture medium; more preferably 1% of the seed culture medium.
  • the seed culture medium is preferably liquid LB culture medium.
  • the culture conditions are preferably 36-38° C. and 160 rpm for 24 hours.
  • the volume ratio of the seed liquid is preferably 1% to 5% of the fermentation medium; more preferably, it is 1% of the fermentation medium.
  • the fermentation medium is preferably liquid LB medium.
  • the fermentation culture conditions described in step (1) are preferably 35-39°C and 150-170 rpm for 48-72 hours; more preferably 36-38°C and 160 rpm for 72 hours.
  • the conditions for the first centrifugation described in step (2) are preferably 4° C., 10000 rpm centrifugation for 10 minutes.
  • the acid described in 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 4°C for 12-24 hours; more preferably 4°C for 12 hours.
  • the conditions for the second centrifugation described in step (2) are preferably 4° C., 10000 rpm centrifugation for 5 minutes.
  • the number of washings described in step (2) is preferably 2 times.
  • the third centrifugation conditions described in step (2) are preferably 10000 rpm centrifugation for 10 minutes.
  • the solvent described in step (3) is preferably anhydrous methanol.
  • the solid-liquid separation method described in step (3) is preferably suction filtration; more preferably, vacuum suction filtration.
  • step (3) is preferably by concentration and suspension; more preferably by vacuum concentration and suspension; most preferably, by rotary evaporator at 40° C. to dryness.
  • 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) preferably uses a Sephadex column for purification; more preferably uses a Sephadex LH-20 Sephadex column, the eluent is 100% methanol, and the flow rate is 0.5mL/ min.
  • the second drying described in step (4) is preferably by concentration and suspension; more preferably by vacuum concentration and suspension; most preferably by rotary evaporator at 40° C. to dryness.
  • a lipopeptide substance is obtained by fermentation, separation and purification of the endophytic bacillus from the leaves of the Pu'er tea tree.
  • the lipopeptide substances include Surfactin homologs, Iturin homologs and Fengycin homologs.
  • the Surfactin homologues are 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 -Val 7 -Surfactin, C 13 -Val 7 -Surfactin, C 14 -Val 7 -Surfactin, C 15 -Val 7 -Surfactin and C 14 -Val 7 -Surfactin.
  • the Iturin homologs 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.
  • the Fengycin homologues are 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 -Abu 6 -Fengycin, C 15 -Val 6 -Fengycin, C 16 -Val 6 -Fengycin, and C 17 -Val 6 -Fengycin.
  • the pathogenic bacteria include, but are not limited to, Escherichia coli, Staphylococcus aureus, Salmonella, Aspergillus flavus and Aspergillus parasiticus.
  • Said Escherichia coli is preferably Escherichia coli GIM1.708.
  • the Staphylococcus aureus is preferably Staphylococcus aureus subspecies aureus GIM1.441.
  • the Salmonella is preferably Salmonella choleraesuis subsp. cholera suis GIM1.163.
  • the Aspergillus flavus is preferably Aspergillus flavus GIM 3.493.
  • the Aspergillus parasitica is preferably Aspergillus parasitica GIM 3.395.
  • the present invention has the following advantages and effects:
  • the present invention is the first to isolate the endophytic bacillus Bacillus velezensis FZ06 from fresh Pu'er tea leaves from fresh Pu'er tea leaves.
  • the lipopeptides that the bacteria can produce include 27 species in 3 categories, and their abundance is more than the existing ones. Reported by Velez Bacillus. Among them, the amino acid sequence of the polypeptide chain of the Fengycin homologues of amino fatty acid chains containing 15-17 carbon atoms was first observed in Bacillus velezia.
  • the method for preparing lipopeptides provided by the present invention is simple, easy to implement, mild in conditions, and easy to control, which provides a new path for large-scale production of lipopeptides.
  • the lipopeptide substance produced by Bacillus velezensis FZ06 in the present invention has obvious inhibitory activity against 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 post-harvest preservation performance or disease resistance of the host plant exfoliation by improving the interaction between the endophytic bacteria and the host plant.
  • Figure 1 is a diagram showing the antibacterial phenomenon of endophytic bacteria in the leaves of Pu'er tea trees against the endophytic fungi of black spore-producing plants.
  • Figure 2 is a scanning electron microscope (SEM) image of Bacillus velezensis FZ06: where A is the SEM image of Bacillus velezensis FZ06 at 1000 ⁇ magnification; B is the SEM image of Bacillus velezensis FZ06 at 5000 ⁇ magnification.
  • SEM scanning electron microscope
  • Figure 3 is a phylogenetic tree diagram of the 16S rDNA of Bacillus velezensis FZ06.
  • Figure 4 is the total ion current diagram of the lipopeptide substance solution prepared by Bacillus velezensis FZ06: where A is the total ion current diagram of Surfactin-like homologues; B is the total ion current diagram of Iturin-like homologues; C is the Fengycin-like homologue The total ion current diagram of the substance.
  • Figure 5 shows the inhibitory effect of lipopeptides prepared by Bacillus velezensis FZ06 and sterile water on bacterial pathogens: where A is the inhibitory effect of lipopeptides prepared by Bacillus velezensis FZ06 and sterile water on E. 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. aureus GIM1.441; C is the lipopeptides prepared by Bacillus velezensis FZ06 and sterile Diagram of the inhibitory effect of water on Salmonella cholera suis subspecies GIM1.163.
  • Figure 6 shows the inhibitory effect of lipopeptides prepared by Bacillus velezensis FZ06 and sterile water on fungal pathogens: where 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.
  • test methods that do not indicate specific experimental conditions in the following examples are usually in accordance with conventional experimental conditions or in accordance with experimental conditions recommended by the manufacturer.
  • the used materials, reagents, etc., unless otherwise specified, are reagents and materials obtained from commercial sources.
  • the solid culture characteristics of the bacteria are: take a loop of purified antifungal endophytic bacteria and culture them on PDA medium at 36 ⁇ 38°C for 3 ⁇ 5 days. Initially the colonies are pale white and gradually turn to yellow; the edges of the colonies are irregular. It is fluffy.
  • the characteristics of the liquid culture of the bacteria are: take a loop of purified antifungal endophytic bacteria into 50mL potato dextrose liquid medium and culture at 36 ⁇ 38°C for 3 days: on the first day, the medium changes from clear to turbid, which is obvious Bacterial liquid culture phenomenon; on the second day, it enters a stable period, and the turbidity does not change; on the third day, wrinkles are formed on the surface of the fermentation broth.
  • Figure 2 It is a rod-shaped structure with a single cell of 0.7 ⁇ 0.8 ⁇ 2 ⁇ 3 ⁇ m.
  • the 16S rDNA sequence was analyzed in NCBI using Blast for homology analysis, and several strains were selected for phylogenetic tree analysis. The results are shown in Figure 3: It shows that the isolated antifungal endophytic bacteria strain is Bacillus velez ( Bacillus velezensis), named as Bacillus velezensis FZ06 (Bacillus velezensis FZ06).
  • Seed solution culture Under aseptic conditions, take 1mL of activated Bacillus velezensis FZ06 and transfer it to 100mL liquid LB medium, and cultivate it at 36 ⁇ 38°C, 160rpm shaker for 24 hours to obtain seed solution;
  • Fermentation culture Under aseptic conditions, take 20 mL of the seed liquid obtained in step (2) into 2L liquid LB medium, culture at 36-38°C and 160rpm shaker for 72 hours to obtain fermentation broth;
  • Acid precipitation extraction The acid precipitation is extracted with 200 mL of anhydrous methanol, and the extract is vacuum filtered. The resulting suction filtrate is evaporated to dryness using a rotary evaporator at 40°C, and re-dissolved in deionized water to obtain water Phase extract
  • Chromatographic separation conditions Chromatographic column: Waters ACQUITY UPLC BEHC18 column (2.1mm ⁇ 100mm, 1.7 ⁇ m); column temperature: 30°C; flow rate: 0.4mL/min; mobile phase: A--ultra pure water containing 0.1% formic acid, B--Acetonitrile containing 0.1% formic acid; gradient elution conditions: 0 ⁇ 0.5min, 60%A; 0.5 ⁇ 3.5min, 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 ⁇ L.
  • Mass spectrometry conditions select Agilent G6545A QTOF mass spectrometer, positive ion scan mode, set the mass scan range of the primary mass spectrum to 110 ⁇ 1600 amu, set the mass scan range of the secondary mass spectrum to 50 ⁇ 1600 amu; ion source temperature 150°C, capillary voltage 3.26 kV; N 2 flow rate is 600L/h, Ar 2 flow rate is 50L/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 homologs with molecular weights of 994, 1008, 1022, 1036, and 1050, which differ by one methylene (-CH2) ( Figure 4-) A); Iturin homologues with molecular weights of 1043, 1057, 1071, and 1085 differing by one methylene group (-CH2) ( Figure 4-B) and molecular weights of 1435, 1449, 1463, 1477, 1491, 1505, etc. differ by one methylene group (-CH2) Fengycin-like homologues (Figure 4-C).
  • Surfactin homologues are a cyclic structure formed by a hydroxy fatty acid chain containing 12-16 carbon atoms and a polypeptide chain containing 7 amino acids. There are two types of polypeptide chains, the 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 homolog is a ring 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 Acid (Asn)-Tyrosine (Tyr)-Glutamine (Gln)-Aspartic acid (Asn)-Proline (Pro)-Aspartic acid (Asn)-Threonine (Thr); Fengycin homologues are a cyclic structure formed by an amino fatty acid chain containing 15-17 carbon atoms and a polypeptide chain containing 10 amino acids.
  • the polypeptide chain consists of three types, and its amino acid sequence is: glutamic acid (Glu)- Ornithine (Orn)-Tyrosine (Tyr)-Threonine (Thr)-Glutamate (Glu)-Valine (Val)-Proline (Pro)-Glutamine (Gln)-Tyrosine Tyrosine (Tyr)-isoleucine (Ile), glutamic acid (Glu)-ornithine (Orn)-tyrosine (Tyr)-threonine (Thr)-glutamic acid (Glu)-butan Amino Acid (Abu)-Proline (Pro)-Glutamine (Gln)-Tyrosine (Tyr)-Isoleucine (Ile) or Glutamic Acid (Glu)-Ornithine (Orn)-Tyrosine Tyrosine (Tyr)-Threonine (Thr)-Glutamic acid (Glu)-Alanine (Ala)-Proline (Pro)
  • Surfactin A Surfactin B, Bacilomycin F, Bacilomycin D, Fengycin A, Fengycin B, Fengycin C have the following structural formulas:
  • Escherichia coli GIM1.708 purchasedd from Guangdong Microbial Culture Collection
  • Staphylococcus aureus subsp.aureus GIM1.441 purchasedd) In Guangdongzhou Microbial Culture Collection
  • Salmonella choleraesuis subsp.Choleraesuis GIM1.163 purchasedd from Guangdongzhou Microbial Culture Collection
  • use an inoculation needle to pick A small amount of Escherichia coli GIM1.708, Staphylococcus aureus subsp. aureus GIM1.441, and Salmonella choleraesuis subsp.
  • cholera suis GIM1.163 stored at -80°C were respectively connected to a solid LB medium test tube and activated at 36 ⁇ 38°C for 24 hours , To obtain activated E. coli GIM1.708, Staphylococcus aureus subsp. aureus GIM1.441, and Salmonella choleraesuis subsp. cholera GIM1.163;
  • the obtained seed liquid was diluted with sterile water to about 1 ⁇ 10 8 cfu/mL, and then spread on LB agar solid medium to obtain E. coli GIM1.708 Petri dishes, petri dishes containing Staphylococcus aureus subsp. aureus GIM1.441, and petri dishes containing Salmonella choleraesuis subsp. cholera suis GIM1.163;
  • step (3) Dilute the Aspergillus flavus GIM 3.493 spore suspension and Aspergillus parasiticus GIM 3.395 spore suspension obtained in step (3) to 1 ⁇ 10 6 cfu/mL with sterile saline, and then apply them to PDA agar.
  • a culture dish containing Aspergillus flavus GIM 3.493 and a culture dish containing Aspergillus parasiticus GIM 3.395 were obtained;

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Abstract

一种普洱茶树叶片内生芽孢杆菌及其应用。该普洱茶树叶片内生芽孢杆菌的名称为贝莱斯芽孢杆菌FZ06(Bacillus velezensis FZ06)，于2019年10月25日保藏于中国武汉市武汉大学的中国典型培养物保藏中心，保藏编号为CCTCC NO：M 2019854。该菌能产生的脂肽类物质，包括3大类27种，其丰富度多于目前已有报道的贝莱斯芽孢杆菌；产生的脂肽类物质对大肠杆菌、金黄色葡萄球菌、沙门氏菌、黄曲霉和寄生曲霉等病原菌具有明显的抑制活性作用，能应用于抑制病原菌中。

Description

一种普洱茶树叶片内生芽孢杆菌及其应用 技术领域
本发明属于微生物技术领域，尤其涉及一种普洱茶树叶片内生芽孢杆菌及其应用。
背景技术
脂肽(Lipopeptide)是一类重要的非离子型生物表面活性剂，主要来源于一些细菌、真菌的次级代谢途径，其种类多样、结构复杂，但一般均由脂肪酸链和肽链以内酯或酰胺键结合成环而成。作为微生物的分子武器，自然环境中的脂肽往往具有抗病毒、拮抗细菌甚至拮抗真菌的天然功能，因此，被广泛应用于植物抗病虫害试剂开发。作为一类非离子型表面活性剂，脂肽的抑菌机理可能与常规抗真菌表面活性分子近似：非离子型生物表面活性剂结合并分离了目标真菌细胞膜上生存相关的必需蛋白质，导致目标真菌死亡。不同微生物产生的脂肽由于结构与性质上的差异，理论上应具有不同的抑菌谱，单一化合物往往不能做到广泛抑菌；且考虑到微生物的适应性进化，单一抗菌素的长期应用很有可能使得某些真菌产生抗药性。而若将结构更为多样、性质更为丰富的脂肽类物质联合使用，可能能够解决以上问题。因此，寻找能够产生更为丰富的脂肽类物质的微生物并提取、联合应用其脂肽类化合物，对于扩大脂肽类化合物抑菌谱、进一步减小目标真菌的抗生素适应性进化、甚至降低抑菌浓度方面具有重要的意义。
发明内容
本发明的首要目的在于克服现有技术的缺点与不足，提供一种普洱茶树叶片内生芽孢杆菌。
本发明的另一目的在于提供上述普洱茶树叶片内生芽孢杆菌的应用。
本发明的再一目的在于提供上述普洱茶树叶片内生芽孢杆菌制备的脂肽类物质及其应用。
为实现上述目的，本发明通过下述技术方案实现：
一种普洱茶树叶片内生芽孢杆菌，名称为贝莱斯芽孢杆菌FZ06(Bacillus velezensis FZ06)，从西双版纳新鲜普洱茶树叶片中分离、纯化得到。
所述的普洱茶树叶片内生芽孢杆菌为贝莱斯芽孢杆菌FZ06(Bacillus velezensis FZ06)，其保藏信息为：保藏单位：中国典型培养物保藏中心(CCTCC)，保藏日期：2019年10月25日，保藏地址：中国.武汉.武汉大学，保藏编号：CCTCC NO：M 2019854。
所述的普洱茶树叶片内生芽孢杆菌在制备脂肽类物质中的应用；优选包括如下步骤：
(1)发酵培养：将所述的普洱茶树叶片内生芽孢杆菌种子液接入发酵培养基中发酵培养，得到发酵液；
(2)酸沉淀分离、洗涤：将步骤(1)中得到的发酵液进行第一次离心去除菌体，得到的上清液添加酸调节pH至2.0，静置后进行第二次离心去除上清液，洗涤得到的沉淀，接着进行第三次离心，得到酸沉淀；
(3)酸沉淀萃取：将步骤(2)得到的酸沉淀加入溶剂中萃取，然后进行固液分离，得到的液体干燥，接着加水复溶，得到水相萃取液；
(4)脂肽类物质分离、纯化：将步骤(3)得到的水相萃取液进行第一次干燥，得到的干物加溶剂复溶，然后进行层析纯化，得到的含脂肽类物质的洗脱液进行第二次干燥，接着加水复溶，最后进行第三次干燥，得到脂肽类物质。
步骤(1)中所述的种子液优选通过如下步骤制备得到：将保存的普洱茶树叶片内生芽孢杆菌活化，扩大培养，得到种子液。
所述的活化的步骤优选如下：将保存的普洱茶树叶片内生芽孢杆菌接入活化培养基中，在35～39℃的条件下培养24～48小时，得到活化的普洱茶树叶片内生芽孢杆菌。
所述的培养的条件优选为36～38℃下培养24小时。
所述的活化培养基优选为PDA培养基。
所述的扩大培养的步骤优选如下：将活化的普洱茶树叶片内生芽孢杆菌接入种子培养基中，在35～39℃、150～170rpm的条件下培养24～48小时，得到种子液。
所述的活化的普洱茶树叶片内生芽孢杆菌的接入量按体积比优选为所述的种子培养基的1～5％；更优选为所述的种子培养基的1％。
所述的种子培养基优选为液体LB培养基。
所述的培养的条件优选为温度为36～38℃、160rpm下培养24小时。
步骤(1)中所述的种子液的接入量按体积比优选为所述的发酵培养基的1～5％；更优选为所述的发酵培养基的1％。
所述的发酵培养基优选为液体LB培养基。
步骤(1)中所述的发酵培养的条件优选为35～39℃、150～170rpm培养 48～72小时；更优选为36～38℃、160rpm培养72小时。
步骤(2)中所述的第一次离心条件优选为4℃、10000rpm离心10min。
步骤(2)中所述的酸优选为盐酸；更优选为浓度为6mol/L的盐酸。
步骤(2)中所述的静置条件优选为4℃静置12～24小时；更优选为4℃静置12小时。
步骤(2)中所述的第二次离心条件优选为4℃、10000rpm离心5min。
步骤(2)中所述的洗涤优选用稀盐酸洗涤；更优选用pH＝2.0的稀盐酸洗涤。
步骤(2)中所述的洗涤次数优选为2次。
步骤(2)中所述的第三次离心条件优选为10000rpm离心10min。
步骤(3)中所述的溶剂优选为无水甲醇。
步骤(3)中所述的固液分离方式优选为抽滤；更优选为真空抽滤。
步骤(3)中所述的干燥优选通过浓缩悬干；更优选通过真空浓缩悬干；最优选通过旋转蒸发仪于40℃旋转蒸发至干燥。
步骤(3)中的水优选为去离子水。
步骤(4)中所述的第一次干燥和第三次干燥优选为冷冻干燥。
步骤(4)中所述的溶剂优选为无水甲醇。
步骤(4)中所述的层析纯化优选使用葡聚糖凝胶色谱柱进行纯化；更优选使用Sephadex LH-20葡聚糖凝胶柱，洗脱液为100％甲醇，流速为0.5mL/min。
步骤(4)中所述的第二次干燥优选通过浓缩悬干；更优选通过真空浓缩悬干；最优选通过旋转蒸发仪于40℃旋转蒸发至干燥。
一种脂肽类物质，通过上述普洱茶树叶片内生芽孢杆菌发酵、分离、纯化得到。
所述的脂肽类物质包括Surfactin类同系物，Iturin类同系物及Fengycin类同系物。
所述的Surfactin类同系物优选为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-Val 7-Surfactin、C 13-Val 7-Surfactin、C 14-Val 7-Surfactin、C 15-Val 7-Surfactin和C 14-Val 7-Surfactin。
所述的Iturin类同系物优选为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和C 17-Bacillomycin D。
所述的Fengycin类同系物优选为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-Abu 6-Fengycin、C 15-Val 6-Fengycin、C 16-Val 6-Fengycin和C 17-Val 6-Fengycin。
所述的脂肽类物质在抑制病原菌中的应用。
所述的病原菌包括但不限于大肠杆菌、金黄色葡萄球菌、沙门氏菌、黄曲霉和寄生曲霉。
所述的大肠杆菌优选为大肠杆菌GIM1.708。
所述的金黄色葡萄球菌优选为金黄色葡萄球菌金黄亚种GIM1.441。
所述的沙门氏菌优选为猪霍乱沙门氏菌猪霍乱亚种GIM1.163。
所述的黄曲霉优选为黄曲霉GIM 3.493。
所述的寄生曲霉优选为寄生曲霉GIM 3.395。
本发明相对于现有技术具有如下的优点及效果：
(1)本发明首次从新鲜普洱茶树叶片中分离得到普洱茶树叶片内生芽孢杆菌Bacillus velezensis FZ06，该菌能产生的脂肽类物质，包括3大类27种，其丰富度多于目前已有报道的贝莱斯芽孢杆菌。其中，含有15-17个碳原子的氨基脂肪酸链的Fengycin类同系物的多肽链氨基酸序列为首次在贝莱斯芽孢杆菌中观察到。
(2)本发明提供的制备脂肽类物质的方法，简单易行、条件温和、易于控制，为大规模生产脂肽类物质提供了新路径。
(3)本发明采用芽孢杆菌Bacillus velezensis FZ06产生的脂肽类物质对大肠杆菌、金黄色葡萄球菌、沙门氏菌、黄曲霉和寄生曲霉等病原菌具有明显的抑制活性作用，能很好地应用于抑制病原菌中，为通过改进内生细菌与宿主植物的相互作用提高宿主植物脱落物的采后保存性能或抗病能力提供了新的思路。
附图说明
图1为普洱茶树叶片中的植物内生细菌拮抗产黑色孢子植物内生真菌的抑菌现象图。
图2为Bacillus velezensis FZ06的扫描电子显微镜(SEM)图：其中，A为Bacillus velezensis FZ06在1000×放大倍数下的SEM图；B为Bacillus velezensis FZ06在5000×放大倍数下的SEM图。
图3为Bacillus velezensis FZ06的16S rDNA的系统发育树图。
图4为Bacillus velezensis FZ06制备的脂肽类物质溶液的总离子流图：其中，A为Surfactin类同系物的总离子流图；B为Iturin类同系物的总离子流图；C为Fengycin类同系物的总离子流图。
图5为Bacillus velezensis FZ06制备的脂肽类物质及无菌水对细菌病原菌的抑制作用效果图：其中，A为Bacillus velezensis FZ06制备的脂肽类物质及无菌 水对大肠杆菌GIM1.708的抑制作用效果图；B为Bacillus velezensis FZ06制备的脂肽类物质及无菌水对金黄色葡萄球菌金黄亚种GIM1.441的抑制作用效果图；C为Bacillus velezensis FZ06制备的脂肽类物质及无菌水对猪霍乱沙门氏菌猪霍乱亚种GIM1.163的抑制作用效果图。
图6为Bacillus velezensis FZ06制备的脂肽类物质及无菌水对真菌病原菌的抑制作用效果图：其中，A为Bacillus velezensis FZ06制备的脂肽类物质及无菌水对黄曲霉GIM 3.493的抑制作用效果图；B为Bacillus velezensis FZ06制备的脂肽类物质及无菌水对寄生曲霉GIM 3.395的抑制作用效果图。
具体实施方式
下面结合实施例及附图对本发明作进一步详细的描述，但本发明的实施方式不限于此。
下列实施例中未注明具体实验条件的试验方法，通常按照常规实验条件或按照制造厂所建议的实验条件。所使用的材料、试剂等，如无特别说明，为从商业途径得到的试剂和材料。
实施例1抗真菌内生细菌(普洱茶树叶片内生芽孢杆菌)的分离、鉴定
(1)新鲜普洱茶树叶片预处理：将采集的同一叶龄新鲜、完整普洱茶树叶片(来源于云南省西双版纳勐海县)于24小时内运抵实验室，并进行表面消毒操作，具体如下：在无菌操作台中，将新鲜、完整的普洱茶树叶片先在75％(体积分数)乙醇溶液中浸泡15秒，然后用无菌水冲洗并用无菌棉擦干；随后，在3.25％(有效氯含量)次氯酸钠溶液中浸泡15秒，用无菌水冲洗并用无菌棉擦干；重复以上表面消毒操作三次。最后一次冲洗水在马铃薯葡萄糖琼脂培养基(PDA培养基)中涂布，然后在37℃温度下培养2周，若未出现微生物，则认为叶片表面消毒完全。
将表面消毒完全的新鲜普洱茶树叶片放置于透气的无菌袋中，于4℃保存30天。30天后，选择仍保持绿色、未有明显内生真菌生长导致植物叶片分解现象的完整普洱茶树叶片。重复以上表面消毒操作三次。最后一次冲洗水在PDA培养基中涂布，然后在37℃温度下培养2周，若未出现微生物，则认为叶片表面消毒完全。
(2)抗真菌内生细菌的培养与分离：
将步骤(1)得到的保持绿色、表面二次消毒的普洱茶树叶片无菌切片，得 到直径约0.5厘米的植物切片，将植物切片置于PDA培养基，30℃培育一周，结果如图1所示：明显有植物内生细菌拮抗产黑色孢子植物内生真菌的现象(培养基右上部分)。挑选出抑制产黑色孢子植物内生真菌的内生细菌菌落划线，继续重复3次进行分离纯化，得到抗真菌内生细菌。
菌体的固体培养特征为：取一环纯化得到的抗真菌内生细菌至PDA培养基上36～38℃下培养3～5天，最初菌落呈浅白色，渐变为黄色；菌落边缘不整齐，呈绒毛状。
菌体的液体培养特征为：取一环纯化得到的抗真菌内生细菌至50mL马铃薯葡萄糖液体培养基中36～38℃下培养3天：第1天，培养基由澄清变为浑浊，有明显细菌液体培养现象；第2天，进入稳定期，浑浊度不再变化；第3天，发酵液表面形成皱醭。
菌体在扫描电子显微镜下的形态如图2所示：呈杆状结构，单个细胞0.7～0.8×2～3μm。
(3)单菌落的16S鉴定：将分离得到的菌株接种于50mL的LB培养基中，37℃160rpm振荡培养24小时，取10mL菌液送至上海生工生物工程有限公司进行菌株测序。菌株的16S rDNA序列如SEQ ID NO:1所示：(1483bp)
Figure PCTCN2020123117-appb-000001
Figure PCTCN2020123117-appb-000002
将其16S rDNA序列于NCBI中用Blast进行同源性分析，并选取若干菌种做进化树分析，结果如图3所示：表明分离所得抗真菌内生细菌菌种为贝莱斯芽孢杆菌(Bacillus velezensis)，命名为贝莱斯芽孢杆菌FZ06(Bacillus velezensis FZ06)。
实施例2 Bacillus velezensis FZ06制备脂肽类物质
(1)菌种活化：在无菌条件下，从甘油冻存管中取1环Bacillus velezensis FZ06接入PDA培养基，于36～38℃活化培养24小时；
(2)种子液培养：在无菌条件下，取活化后的Bacillus velezensis FZ06 1mL转接入100mL液体LB培养基中，于36～38℃、160rpm摇床培养24小时，得种子液；
(3)发酵培养：在无菌条件下，取20mL步骤(2)得到的种子液接入2L液体LB培养基中，于36～38℃、160rpm摇床培养72小时，得到发酵液；
(4)酸沉淀分离、洗涤：将发酵液于4℃、10000rpm低温高速离心机中离心10min去除菌体，然后向离心上清液中添加6mol/L HCl调整pH至2.0，4℃环境静置12小时，得到酸沉发酵液；将酸沉发酵液于4℃、10000rpm低温高速离心机中离心5min，弃去上清液并收集沉淀，沉淀用200mL的pH＝2.0的稀盐酸水洗涤两次，10000rpm离心10min后收集沉淀，得到酸沉淀；
(5)酸沉淀萃取：将酸沉淀用200mL无水甲醇萃取，萃取液进行真空抽滤，得到的抽滤液采用旋转蒸发仪于40℃转蒸发至干燥，并复溶于去离子水，得到水相萃取液；
(6)脂肽类物质分离纯化：对水相萃取液进行冷冻干燥，得到冻干物，加入50mL无水甲醇复溶，所得到的复溶液进行葡聚糖凝胶色谱LH-20柱层析纯化(洗脱液为100％甲醇，流速为0.5mL/min)，得到的含脂肽类物质的洗脱液进行真空浓缩至悬干，然后加水复溶，复溶液冷冻干燥，得到Bacillus velezensis FZ06产生的脂肽类物质，产量为1.03g/L。
实施例3 Bacillus velezensis FZ06制备得到的脂肽类物质的结构鉴定
将提取实施例2得到的脂肽粉末50mg复溶于50mL无水甲醇，再用无水甲醇稀释1000倍后，进行超高效液相色谱-电喷雾-二级质谱UPLC-ESI-MS/MS结构测定，具体分析条件如下：
色谱分离条件：色谱柱：Waters ACQUITY UPLC BEHC18柱(2.1mm×100mm，1.7μm)；柱温：30℃；流速：0.4mL/min；流动相：A--含0.1％甲酸的超纯水，B--含0.1％甲酸的乙腈；梯度洗脱条件：0～0.5min，60％A；0.5～3.5min，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；进样量：1μL。
质谱分析条件：选择Agilent G6545A QTOF质谱仪，阳离子扫描模式，一级质谱质量扫描范围设定为110～1600amu，二级质谱质量扫描范围设定为50～1600amu；离子源温度150℃，毛细管电压3.26kV；N 2流流速600L/h，Ar 2流流速50L/h；质谱数据采集及处理软件为Agilent MassHunter。
UPLC-MS结果如图4所示：Bacillus velezensis FZ06产生的脂肽类物质包含分子量为994、1008、1022、1036和1050等相差一个亚甲基(-CH2)的Surfactin类同系物(图4-A)；分子量1043、1057、1071和1085等相差一个亚甲基(-CH2)的Iturin类同系物(图4-B)和分子量1435、1449、1463、1477、1491、1505等相差一个亚甲基(-CH2)的Fengycin类同系物(图4-C)。
通过UPLC-ESI-MS/MS测定，Surfactin类同系物是由含有12-16个碳原子的羟基脂肪酸链与含有7个氨基酸的多肽链形成的环状结构，其多肽链有两种，其氨基酸序列为：谷氨酸(Glu)-亮氨酸(Leu)-亮氨酸(Leu)-缬氨酸(Val)-天冬氨酸(Asp)-亮氨酸(Leu)-亮氨酸(Leu)或谷氨酸(Glu)-亮氨酸(Leu) -亮氨酸(Leu)-亮氨酸(Leu)-天冬氨酸(Asp)-亮氨酸(Leu)-缬氨酸(Val)；Iturin类同系物是由含有14-17个碳原子的氨基脂肪酸链与含有7个氨基酸的多肽链形成的环状结构，其多肽链由两种，其氨基酸序列为：天冬氨酸(Asn)-酪氨酸(Tyr)-天冬氨酸(Asn)-谷氨酰胺(Gln)-脯氨酸(Pro)-天冬氨酸(Asn)-苏氨酸(Thr)或天冬氨酸(Asn)-酪氨酸(Tyr)-谷氨酰胺(Gln)-天冬氨酸(Asn)-脯氨酸(Pro)-天冬氨酸(Asn)-苏氨酸(Thr)；Fengycin类同系物是由含有15-17个碳原子的氨基脂肪酸链与含有10个氨基酸的多肽链形成的环状结构，其多肽链由三种，其氨基酸序列为：谷氨酸(Glu)-鸟氨酸(Orn)-酪氨酸(Tyr)-苏氨酸(Thr)-谷氨酸(Glu)-缬氨酸(Val)-脯氨酸(Pro)-谷氨酰胺(Gln)-酪氨酸(Tyr)-异亮氨酸(Ile)、谷氨酸(Glu)-鸟氨酸(Orn)-酪氨酸(Tyr)-苏氨酸(Thr)-谷氨酸(Glu)-丁氨酸(Abu)-脯氨酸(Pro)-谷氨酰胺(Gln)-酪氨酸(Tyr)-异亮氨酸(Ile)或谷氨酸(Glu)-鸟氨酸(Orn)-酪氨酸(Tyr)-苏氨酸(Thr)-谷氨酸(Glu)-丙氨酸(Ala)-脯氨酸(Pro)-谷氨酰胺(Gln)-酪氨酸(Tyr)-异亮氨酸(Ile)。
脂肽类物质的具体分子式及结构式如表1所示：
表1实施例2得到的脂肽类物质的化学名称、分子式及结构式
Figure PCTCN2020123117-appb-000003
Figure PCTCN2020123117-appb-000004
其中，Surfactin A、Surfactin B、Bacillomycin F、Bacillomycin D、Fengycin A、Fengycin B、Fengycin C结构式如下：
Figure PCTCN2020123117-appb-000005
Figure PCTCN2020123117-appb-000006
Figure PCTCN2020123117-appb-000007
实施例4 Bacillus velezensis FZ06制备得到的脂肽类物质的细菌抑制效果测试
(1)选取3种常见细菌病原菌：大肠杆菌(Escherichia coli)GIM1.708(购于广东省微生物菌种保藏中心)、金黄色葡萄球菌金黄亚种(Staphylococcus aureus subsp.aureus)GIM1.441(购于广东省微生物菌种保藏中心)和猪霍乱沙门氏菌猪霍乱亚种(Salmonella choleraesuis subsp.Choleraesuis)GIM1.163(购于广东省微生物菌种保藏中心)，在无菌条件下，用接种针挑取少量-80℃保存的大肠杆菌GIM1.708、金黄色葡萄球菌金黄亚种GIM1.441和猪霍乱沙门氏菌猪霍乱亚种GIM1.163分别接入固体LB培养基试管，于36～38℃活化24小时，得到活化的大肠杆菌GIM1.708、金黄色葡萄球菌金黄亚种GIM1.441和猪霍乱沙门氏菌猪霍乱亚种GIM1.163；
(2)在无菌条件下，取一环活化后的大肠杆菌GIM1.708、金黄色葡萄球菌金黄亚种GIM1.441和猪霍乱沙门氏菌猪霍乱亚种GIM1.163分别转接入50mL液体LB种子培养基中，于36～38℃、160rpm摇床培养24小时，得大肠杆菌GIM1.708种子液、金黄色葡萄球菌金黄亚种GIM1.441种子液和猪霍乱沙门氏 菌猪霍乱亚种GIM1.163种子液；
(3)在无菌条件下，将得到的种子液分别用无菌水稀释至约1×10 8cfu/mL，接着分别涂布于LB琼脂固体培养基中，得到含有大肠杆菌GIM1.708的培养皿、含有金黄色葡萄球菌金黄亚种GIM1.441的培养皿和含有猪霍乱沙门氏菌猪霍乱亚种GIM1.163的培养皿；
(4)取实施例2制备的脂肽类物质冻干粉10mg，加入去离子水2mL，得到5mg/mL的脂肽溶液；
(5)将牛津杯(内径6mm，已灭菌)分别置于步骤(3)得到的含有细菌病原菌的培养皿中，实验组于牛津杯中添加200μL 5mg/mL脂肽溶液，而空白对照组则于无菌牛津杯中添加200μL无菌水，然后置于37℃的培养箱中恒温培养24小时，观察其抑菌效果，结果如图5所示，同时，测量其抑菌圈大小，结果如表2所示；表明Bacillus velezensis FZ06制备得到的脂肽类物质具有明显的抑制细菌病原菌的作用。
实施例5 Bacillus velezensis FZ06制备得到的脂肽类物质的真菌抑制效果测试
(1)选取2种常见的真菌病原菌：黄曲霉(Aspergillus flavus)GIM 3.493(购于广东省微生物菌种保藏中心)和寄生曲霉(Aspergillus parasiticus Speare)GIM 3.395(购于广东省微生物菌种保藏中心)，在无菌条件下，用接种针挑取少量-80℃保存的黄曲霉GIM 3.493和寄生曲霉GIM 3.395分别接入PDA培养基试管，于36～38℃活化24小时，得到活化的黄曲霉GIM 3.493和寄生曲霉GIM 3.395；
(2)将活化的黄曲霉GIM 3.493和寄生曲霉GIM 3.395接入已灭菌的PDA培养基中，于36～38℃培养箱培养96小时，得到带有孢子的黄曲霉GIM 3.493平板和带有孢子的寄生曲霉GIM 3.395；
(3)刮取黄曲霉GIM 3.493孢子和寄生曲霉GIM 3.395孢子分别置于无菌生理盐水中，漩涡振荡器震荡3min后用无菌滤纸无菌过滤，得到均匀的黄曲霉GIM 3.493孢子悬浮液和寄生曲霉GIM 3.395孢子悬浮液；
(4)将步骤(3)得到的得到的黄曲霉GIM 3.493孢子悬浮液和寄生曲霉GIM 3.395孢子悬浮液分别用无菌生理盐水稀释至1×10 6cfu/mL，然后分别涂布于PDA琼脂固体培养基中，得到含黄曲霉GIM 3.493的培养皿和含寄生曲霉GIM 3.395的培养皿；
(5)取实施例2制备的脂肽类物质冻干粉10mg，加入去离子水2mL，得到5mg/mL的脂肽溶液；
(6)将牛津杯(内径6mm，已灭菌)分别置于步骤(4)得到的含有真菌病原菌的培养皿中，实验组于牛津杯中添加200μL的5mg/mL脂肽溶液，而空白对照组则于无菌牛津杯中添加200μL无菌水，然后置于37℃的培养箱中恒温培养5天，观察其抑菌效果，结果如图6所示；同时，测其抑菌圈的大小，结果如表2所示；表明Bacillus velezensis FZ06制备得到的脂肽类物质具有明显的抑制真菌病原菌的作用。
表2实施例2提取得到的菌株产生脂肽类物质的抗菌活性
Figure PCTCN2020123117-appb-000008
上述实施例为本发明较佳的实施方式，但本发明的实施方式并不受上述实施例的限制，其他的任何未背离本发明的精神实质与原理下所作的改变、修饰、替代、组合、简化，均应为等效的置换方式，都包含在本发明的保护范围之内。

Claims (10)

  1. 一种普洱茶树叶片内生芽孢杆菌，其特征在于：名称为贝莱斯芽孢杆菌FZ06(Bacillus velezensis FZ06)，于2019年10月25日保藏于中国武汉市武汉大学的中国典型培养物保藏中心，保藏编号为CCTCC NO：M 2019854。
  2. 权利要求1所述的普洱茶树叶片内生芽孢杆菌在制备脂肽类物质中的应用，其特征在于包括如下步骤：
    (1)发酵培养：将权利要求1所述的普洱茶树叶片内生芽孢杆菌的种子液接入发酵培养基中发酵培养，得到发酵液；
    (2)酸沉淀分离、洗涤：将步骤(1)中得到的发酵液进行第一次离心去除菌体，得到的上清液添加酸调节pH至2.0，静置后进行第二次离心去除上清液，洗涤得到的沉淀，接着进行第三次离心，得到酸沉淀；
    (3)酸沉淀萃取：将步骤(2)得到的酸沉淀加入溶剂中萃取，然后进行固液分离，得到的液体干燥，接着加水复溶，得到水相萃取液；
    (4)脂肽类物质分离、纯化：将步骤(3)得到的水相萃取液进行第一次干燥，得到的干物加溶剂复溶，然后进行层析纯化，得到的含脂肽类物质的洗脱液进行第二次干燥，接着加水复溶，最后进行第三次干燥，得到脂肽类物质。
  3. 根据权利要求2所述的普洱茶树叶片内生芽孢杆菌在制备脂肽类物质中的应用，其特征在于：
    步骤(1)中所述的种子液的接入量按体积比为所述的发酵培养基的1～5％；进一步为所述的发酵培养基的1％；
    所述的发酵培养基为液体LB培养基；
    步骤(1)中所述的发酵培养的条件为35～39℃、150～170rpm培养48～72小时；进一步为36～38℃、160rpm培养72小时。
  4. 根据权利要求3所述的普洱茶树叶片内生芽孢杆菌在制备脂肽类物质中的应用，其特征在于：
    所述的种子液通过如下步骤制备得到：将保存的普洱茶树叶片内生芽孢杆菌活化，扩大培养，得到种子液；
    所述的活化的步骤如下：将保存的普洱茶树叶片内生芽孢杆菌接入活化培养基中，在35～39℃的条件下培养24～48小时，得到活化的普洱茶树叶片内生芽孢杆菌；
    所述的活化培养基为PDA培养基；
    所述的扩大培养的步骤如下：将活化的普洱茶树叶片内生芽孢杆菌接入种 子培养基中，在35～39℃、150～170rpm的条件下培养24～48小时，得到种子液；
    所述的活化的普洱茶树叶片内生芽孢杆菌的接入量按体积比为所述的种子培养基的1～5％；进一步为所述的种子培养基的1％；
    所述的种子培养基为液体LB培养基。
  5. 根据权利要求2所述的普洱茶树叶片内生芽孢杆菌在制备脂肽类物质中的应用，其特征在于：
    步骤(2)中所述的第一次离心条件为4℃、10000rpm离心10min；
    步骤(2)中所述的酸为盐酸；进一步为浓度为6mol/L的盐酸；
    步骤(2)中所述的静置条件为4℃静置12～24小时；进一步为4℃静置12小时；
    步骤(2)中所述的第二次离心条件为4℃、10000rpm离心5min；
    步骤(2)中所述的洗涤为用稀盐酸洗涤；进一步为用pH＝2.0的稀盐酸洗涤；
    步骤(2)中所述的洗涤次数为2次；
    步骤(2)中所述的第三次离心条件为10000rpm离心10min；
    步骤(3)中所述的溶剂为无水甲醇；
    步骤(3)中所述的固液分离方式为抽滤；进一步为真空抽滤；
    步骤(3)中所述的干燥通过浓缩悬干；进一步通过真空浓缩悬干；更进一步通过旋转蒸发仪于40℃旋转蒸发至干燥；
    步骤(3)中的水为去离子水。
  6. 根据权利要求2所述的普洱茶树叶片内生芽孢杆菌在制备脂肽类物质中的应用，其特征在于：
    步骤(4)中所述的第一次干燥和第三次干燥为冷冻干燥；
    步骤(4)中所述的溶剂为无水甲醇；
    步骤(4)中所述的层析纯化为使用葡聚糖凝胶色谱柱进行纯化；进一步为使用Sephadex LH-20葡聚糖凝胶柱，洗脱液为100％甲醇，流速为0.5mL/min；
    步骤(4)中所述的第二次干燥通过浓缩悬干；进一步为通过真空浓缩悬干；更进一步为通过旋转蒸发仪于40℃旋转蒸发至干燥。
  7. 一种脂肽类物质，其特征在于：通过权利要求1所述的普洱茶树叶片内生芽孢杆菌发酵、分离、纯化得到。
  8. 根据权利要求7所述的脂肽类物质，其特征在于：所述的脂肽类物质包括Surfactin类同系物，Iturin类同系物及Fengycin类同系物。
  9. 根据权利要求8所述的脂肽类物质，其特征在于：
    所述的Surfactin类同系物为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-Val 7-Surfactin、C 13-Val 7-Surfactin、C 14-Val 7-Surfactin、C 15-Val 7-Surfactin和C 14-Val 7-Surfactin；
    所述的Iturin类同系物为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和C 17-Bacillomycin D；
    所述的Fengycin类同系物为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-Abu 6-Fengycin、C 15-Val 6-Fengycin、C 16-Val 6-Fengycin和C 17-Val 6-Fengycin。
  10. 权利要求7～9中任一项所述的脂肽类物质在抑制病原菌中的应用，其特征在于：所述的病原菌包括但不限于大肠杆菌、金黄色葡萄球菌、沙门氏菌、黄曲霉和寄生曲霉。
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