Cyclic lipopeptide supramolecular ligand and its preparation method and application

Abstract

The present invention provides a cyclolipopeptide supramolecular ligand and a preparation method and application thereof, through metal ions (Ca 2+ , Fe 2+ 、Zn 2+ Mg 2+ , Cu 2+ ) coordination induced fermentation broth, including Bacillus subtilis cyclolipopeptide (CL), to self-assemble and form supramolecular coordination compounds, which greatly improved the antibacterial activity of CL and expanded its antibacterial spectrum. The Zn / CL coordination compounds in the supramolecular coordination compounds can effectively inhibit Gram bacteria such as Listeria, Escherichia coli, Staphylococcus aureus, and Salmonella typhimurium, and the Ca / CL coordination compounds can effectively inhibit fungi such as Aspergillus niger, Aspergillus flavus, Carbonaceous Aspergillus, and Fusarium moniliforme; this technology improves the antibacterial activity of natural antimicrobial peptides, strengthens the antibacterial activity of CL, and obtains metal ion / CL coordination compounds with high antibacterial properties.

Description

Technical Field

[0001] The invention relates to the technical field of antimicrobial peptide application, and in particular to a cyclolipopeptide supramolecular ligand and a preparation method and application thereof. Background Art

[0002] At present, climate warming and biological pathogens promote the growth and spread of pathogens such as bacteria, fungi and viruses, threatening public health, agricultural production and food safety. In this context, natural biological antimicrobial agents such as bacteriocins and antimicrobial peptides are becoming a new type of drug alternative due to their biodegradability. However, the development of their practical application remains challenging due to their low efficiency, solubility and especially stability. Therefore, there is an urgent need to develop low-toxic, high-efficiency and long-acting antimicrobial agents to provide better protection.

[0003] In recent years, researchers have also been committed to solving the antibacterial effect and stability problems of biological antimicrobial agents. The methods mainly focus on screening and genetically modifying high-yield strains, optimizing fermentation conditions (such as static solid fermentation, fed-batch culture, high cell density culture) to increase the secretion of antimicrobial substances, enhancing antimicrobial substances through proteolysis to increase solubility, and nano-encapsulation and release control of antimicrobial substances. There are also studies reporting the use of polymer dot technology to synthesize antimicrobial materials. However, so far, these methods have not only failed to achieve a satisfactory antibacterial effect fundamentally, but also the high temperature and high pressure preparation conditions have increased processing requirements, caused environmental, safety and cost issues, which further limited practical applications. Summary of the invention

[0004] To this end, the present invention provides a cyclolipopeptide supramolecular ligand, a preparation method and application thereof, which achieves good results in inhibiting Enterobacter, Salmonella typhimurium, Staphylococcus aureus, Listeria, Aspergillus niger, Aspergillus flavus, Aspergillus carbonarius, and Fusarium moniliforme, and solves the problems of narrow antibacterial spectrum and poor stability of antibacterial cyclolipopeptides in the prior art.

[0005] In order to achieve the above purpose, the embodiment of the present invention provides the following technical solutions:

[0006] The cyclolipopeptide supramolecular ligand is prepared by: 2+ , Fe 2+ 、Zn 2+ Mg 2+ , Cu 2+ ) The coordination-induced fermentation broth contained many active substances including Bacillus subtilis cyclolipopeptide (CL) to self-assemble and form supramolecular coordination compounds, which greatly improved the antibacterial activity of CL and expanded its antibacterial spectrum.

[0007] Specifically, the following steps are included:

[0008] Step 1, extraction of CL: centrifuge the Bacillus subtilis culture solution after culturing for 120 hours (8000r / min, 20min, 4°C), discard the bacterial precipitate, collect the supernatant to obtain the Bacillus subtilis fermentation solution, adjust the pH to 2.0 with 12mol / L concentrated hydrochloric acid, centrifuge (8000r / min, 20min, 4°C) after precipitation overnight at 4°C, discard the supernatant, take the precipitate, add appropriate amount of anhydrous methanol to extract CL in the precipitate, collect, freeze-dry and store for later use.

[0009] Step 2, synthesis of exogenous metal ion and CL coordination compound: prepare CL solution with a concentration of 1%, adjust pH with 1 mol / L sodium hydroxide solution, heat in a water bath for a certain time to reach the reaction temperature, weigh a certain mass of metal salt (anhydrous calcium chloride, ferrous chloride tetrahydrate, zinc sulfate, magnesium sulfate, anhydrous copper sulfate) and add it to the CL solution, stir quickly until dissolved, seal with plastic wrap and react in a constant temperature water bath for a certain time. After the reaction is completed, remove a certain volume of water with a rotary evaporator, add 10 times the volume of anhydrous ethanol to precipitate overnight, centrifuge (8000r / min, 15min), discard the supernatant, and freeze-dry the precipitate to obtain a powder, which is the supramolecular coordination compound of metal ions / CL.

[0010] Furthermore, Ca 2+ With Zn 2+ It can effectively enhance the antibacterial activity of CL. Specifically, Ca / CL has strong antifungal activity and Zn / CL has strong antibacterial activity, and both have long-term stability.

[0011] Furthermore, the synthesis of the exogenous metal ions and CL coordination complexes adopts a hydrothermal synthesis method, and the preparation process is at a mild temperature (below 100°C). When the reaction temperature is as low as 25-30°C, Ca / CL with high antifungal activity can be prepared, while a temperature as high as 50-70°C is conducive to the formation of Zn / CL with strong antibacterial activity.

[0012] Furthermore, the exogenous metal ion and CL coordination complex need to be synthesized under different pH conditions: Ca / CL has higher activity under slightly acidic conditions (~pH 5.0), and Zn / CL formed under neutral or slightly acidic conditions (pH 6.0-7.0) has higher activity.

[0013] Furthermore, the exogenous metal ion and CL complex have the best preparation conditions: the mass ratio of CaCl2 to CL is 0.5:1 to 2:1, the chelation rate of the complex is greater than 60.1±2.8%, and the CL and Ca 2+The molar ratio of 1:10 can produce Ca / CL with the strongest antifungal activity; the mass ratio of ZnSO4 to CL is 1:1-3:1, the chelation rate of the coordination compound is 70.9±4.2%, and the chelation rate of CL to Zn 2+ The molar ratio of 1:6.5 can produce Zn / CL with the strongest antibacterial activity.

[0014] Furthermore, the exogenous metal ion and CL complex have good acid and alkali resistance and thermal stability: Ca / CL exhibits stable and efficient antifungal activity in the pH range of 3 to 7, with an inhibition zone of 43 to 47 mm; when pH>9, the inhibition zone decreases. It can exhibit stable antifungal activity at a relatively low temperature of 0 to 40°C (inhibition zone of 43 to 46 mm), but the inhibition zone decreases at 50 to 90°C. Zn / CL can maintain stable antibacterial effects at pH 3 to 9 and below 100°C.

[0015] Furthermore, the supramolecular coordination compound is stored at a temperature of -20°C for future use.

[0016] The supramolecular ligand provided by the present invention can effectively inhibit Escherichia coli, Salmonella typhimurium, Staphylococcus aureus, Listeria, Aspergillus niger, Aspergillus flavus, Aspergillus carbonarius and Fusarium moniliforme in Bacillus subtilis fermentation broth.

[0017] The supramolecular ligand provided by the invention is used in inhibiting Escherichia coli, Salmonella typhimurium, Staphylococcus aureus, Listeria monocytogenes, Aspergillus niger, Aspergillus flavus, Aspergillus carbonarius and Fusarium moniliforme.

[0018] The present invention has the following advantages: The present invention discloses a cyclolipopeptide supramolecular ligand and a preparation method and application thereof, through metal ions (Ca 2+ , Fe 2+ 、Zn 2+ Mg 2+ , Cu 2+ ) coordination induced fermentation broth, including Bacillus subtilis cyclolipopeptide (CL), to self-assemble and form supramolecular coordination compounds, which greatly improved the antibacterial activity of CL and expanded its antibacterial spectrum. The Zn / CL coordination compounds in the supramolecular coordination compounds can effectively inhibit Gram bacteria such as Listeria, Escherichia coli, Staphylococcus aureus, and Salmonella typhimurium, and the Ca / CL coordination compounds can effectively inhibit fungi such as Aspergillus niger, Aspergillus flavus, Carbonaceous Aspergillus, and Fusarium moniliforme; this technology improves the antibacterial activity of natural antimicrobial peptides, strengthens the antibacterial activity of CL, obtains metal ion / CL coordination compounds with high antibacterial properties, and provides a new method for improving the antibacterial properties of other antimicrobial peptides. BRIEF DESCRIPTION OF THE DRAWINGS

[0019] In order to more clearly illustrate the implementation methods of the present invention or the technical solutions in the prior art, the following briefly introduces the drawings required for the implementation methods or the description of the prior art. Obviously, the drawings in the following description are only exemplary, and for ordinary technicians in this field, other implementation drawings can be derived from the provided drawings without creative work.

[0020] Figure 1A-1 This is one of the atomic force microscopy images of CL of the present invention;

[0021] Figure 1A-2 This is the second atomic force microscope image of CL of the present invention;

[0022] Figure 1B-1 This is one of the atomic force microscope images of the metal ion Ca / CL complex in the present invention;

[0023] Figure 1B-2 This is the second atomic force microscope image of the metal ion Ca / CL complex of the present invention;

[0024] Figure 1C-1 This is one of the atomic force microscope images of the metal ion Zn / CL complex in the present invention;

[0025] Figure 1C-2 This is the second atomic force microscope image of the metal ion Zn / CL complex of the present invention;

[0026] Figure 2 is the ultraviolet-visible absorption spectrum (UV-Vis) of the metal ion Ca / CL complex of the present invention;

[0027] Figure 3 is the ultraviolet-visible absorption spectrum (UV-Vis) of the metal ion Zn / CL complex of the present invention;

[0028] Figure 4 is the X-ray diffraction pattern of the metal ion / CL complex of the present invention;

[0029] Figure 5 is the Fourier infrared spectrum of the metal ion / CL complex of the present invention;

[0030] Fig. 6A Observation of the adhesion of CL(1), Ca / CL(2) and Zn / CL(3) on the surface of A. niger;

[0031] Figure 6B Observation of the adhesion of CL(1), Ca / CL(2) and Zn / CL(3) on the surface of E. coli;

[0032] Figure 6CObservation of adhesion of CL(1), Ca / CL(2) and Zn / CL(3) on the surface of S. aureus;

[0033] Fig. 7A Cell morphology of A. niger after control treatment (1), CL treatment (2), and metal ion / CL complex treatment (3);

[0034] Figure 7B The cell morphology of E. coli after control treatment (1), CL treatment (2) and metal ion Zn / CL complex treatment (3);

[0035] Figure 7C The cell morphology of S. aureus after control treatment (1), CL treatment (2) and metal ion Zn / CL complex treatment (3);

[0036] Figure 8 The inhibitory effect of the metal ion / fermentation broth (BF) of the present invention on fungi;

[0037] Fig. 9 It is the inhibitory effect of the metal ion / fermentation broth (BF) of the present invention on bacteria. DETAILED DESCRIPTION

[0038] The following is a description of the implementation of the present invention by specific embodiments. People familiar with the art can easily understand other advantages and effects of the present invention from the contents disclosed in this specification. Obviously, the described embodiments are part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of the present invention.

[0039] Example 1

[0040] This embodiment provides: by metal ions (Ca 2+ , Fe 2+ 、Zn 2+ Mg 2+ , Cu 2+ ) The coordination-induced fermentation broth contained many active substances including Bacillus subtilis cyclolipopeptide (CL) to self-assemble and form supramolecular coordination compounds, which greatly improved the antibacterial activity of CL and expanded its antibacterial spectrum.

[0041] Furthermore, this embodiment provides a modification technology for improving the antibacterial properties of antimicrobial peptides and the application of Bacillus subtilis fermentation broth with good antibacterial properties in inhibiting Escherichia coli, Salmonella typhimurium, Staphylococcus aureus, Listeria monocytogenes, Aspergillus niger, Aspergillus flavus, and Aspergillus carbonii.

[0042] In this embodiment, a number of active substances including Bacillus subtilis cyclolipopeptide (CL) in the fermentation broth are induced to self-assemble by metal ion coordination to form supramolecular coordination compounds, as shown in the microscope images of Figures 1A to 1C; Figure 2 , Figure 3 is the ultraviolet-visible absorption spectrum (UV-Vis) of the metal ion / CL complex of the present invention; Figure 4 X-ray diffraction pattern of the metal ion / CL complex of the present invention; Figure 5 Fourier infrared spectrum of the metal ion / CL complex of the present invention.

[0043] The supramolecular complex greatly improves the antibacterial activity of CL and expands its antibacterial spectrum. The Zn / CL complex in the supramolecular complex can effectively inhibit Gram bacteria such as Listeria, Escherichia coli, Staphylococcus aureus, and Salmonella typhimurium, and the Ca / CL complex can effectively inhibit fungi such as Aspergillus niger, Aspergillus flavus, and Aspergillus carbonarius; the method improves the antibacterial activity of natural antimicrobial peptides, strengthens the antibacterial activity of CL, and obtains a metal ion / CL complex with high antibacterial performance.

[0044] Example 2

[0045] This embodiment provides a method for preparing a supramolecular ligand, comprising the following steps:

[0046] Step 1, extraction of CL: centrifuge the Bacillus subtilis culture solution after culturing for 120 hours (8000r / min, 20min, 4°C), discard the bacterial precipitate, collect the supernatant to obtain the Bacillus subtilis fermentation solution, adjust the pH to 2.0 with 12mol / L concentrated hydrochloric acid, centrifuge (8000r / min, 20min, 4°C) after precipitation overnight at 4°C, discard the supernatant, take the precipitate, add appropriate amount of anhydrous methanol to extract CL in the precipitate, collect, freeze-dry and store for later use.

[0047] Step 2, synthesis of exogenous metal ion and CL coordination compound: prepare CL solution with a concentration of 1%, adjust pH with 1 mol / L sodium hydroxide solution, heat in a water bath for a certain time to reach the reaction temperature, weigh a certain mass of metal salt (anhydrous calcium chloride, ferrous chloride tetrahydrate, zinc sulfate, magnesium sulfate, anhydrous copper sulfate) and add it to the CL solution, stir quickly until dissolved, seal with plastic wrap and react in a constant temperature water bath for a certain time. After the reaction is completed, remove a certain volume of water with a rotary evaporator, add 10 times the volume of anhydrous ethanol to precipitate overnight, centrifuge (8000r / min, 15min), discard the supernatant, and freeze-dry the precipitate to obtain a powder, which is the metal ion / CL coordination compound.

[0048] In the modification technology for improving the antibacterial properties of antimicrobial peptides, Ca was found by screening 2+ With Zn2+ It can effectively enhance the antibacterial activity of CL. Specifically, Ca / CL has strong antifungal activity and Zn / CL has strong antibacterial activity, and both have long-term stability.

[0049] The synthesis of the exogenous metal ion and CL coordination complex adopts a hydrothermal synthesis method, and the preparation process is at a mild temperature (below 100°C). When the reaction temperature is as low as 25-30°C, Ca / CL with higher antifungal activity can be prepared, while a temperature as high as 50-70°C is conducive to the formation of Zn / CL with stronger antibacterial activity.

[0050] The exogenous metal ion and CL coordination complex need to be synthesized under different pH conditions: Ca / CL has higher activity under slightly acidic conditions (~pH5.0), and Zn / CL formed under neutral or slightly acidic conditions (pH6.0-7.0) has higher activity.

[0051] The exogenous metal ion and CL complex have the best preparation conditions: when the mass ratio of CaCl2 to CL is 1:1, the chelation rate of the complex is 86.6±2.8%, and the chelation rate of CL to Ca 2+ The molar ratio of 1:10 can produce Ca / CL with the strongest antifungal activity; when the mass ratio of ZnSO4 to CL is 2:1, the chelation rate of the complex is 82.9±4.2%, and the molar ratio of CL to Zn2+ is 1:6.5, which can produce Zn / CL with the strongest antibacterial activity.

[0052] The exogenous metal ion and CL complex have good acid and alkali resistance and thermal stability: Ca / CL exhibits stable and efficient antifungal activity in the pH range of 3-7, with an inhibition zone of 43-47 mm; when pH>9, the inhibition zone decreases. It can exhibit stable antifungal activity at a relatively low temperature of 0-40°C (inhibition zone of 43-46 mm), but the inhibition zone decreases at 50°C-90°C. Zn / CL can maintain stable antibacterial effects at pH 3-9 and below 100°C.

[0053] like Figure 6A to Figure 6C Observation of the adhesion of CL(1), Ca / CL(2) and Zn / CL(3) on the surfaces of A. niger, E. coli and S. aureus; Figure 7A to Figure 7C Cell morphology of A. niger, S. aureus after control treatment (1), CL treatment (2), and metal ion / CL complex treatment (3).

[0054] The metal ion / CL complex is stored at -20°C for future use.

[0055] Example 3

[0056] This embodiment provides the use of a cyclolipopeptide supramolecular ligand in a Bacillus subtilis fermentation broth, comprising the following steps: in a Bacillus subtilis fermentation broth (BF) (~1×10 7 cfu / mL) were directly added with Ca 2+ (4%) and Zn 2+ (8%), heated in a constant temperature water bath at 30℃ and 70℃, respectively, and stirred for 20min, the obtained Ca / BF and Zn / BF greatly improved the inhibitory activity of BF against fungi and bacteria;

[0057] The inhibition zone of 100 μL of Ca / BF against Aspergillus niger reached 65 mm, and the inhibition zone of 100 μL of Zn / BF against Escherichia coli and Staphylococcus aureus reached 55 mm and 48 mm, respectively. Importantly, the inhibition zone did not shrink with the extension of the culture time. Figure 8 , the inhibitory effects of BF (A1-A4) and Ca / BF (B1-B4) on A. niger (1-4 days); Fig. 9 , Inhibitory effects of BF(1) and Zn / BF(2) on E. coli(A) and S. aureus(B) (24h).

[0058] Although the present invention has been described in detail above by general description and specific embodiments, it is obvious to those skilled in the art that some modifications or improvements can be made to the present invention. Therefore, these modifications or improvements made without departing from the spirit of the present invention all belong to the scope of protection claimed by the present invention.

Claims

1. A method for preparing a cyclolipopeptide supramolecular ligand, characterized in that: The active substances including Bacillus subtilis cyclolipopeptide CL in the fermentation broth were induced to self-assemble by metal ion coordination to form supramolecular coordination compounds. The metal ions include Ca 2+ and Zn 2+ ; The specific steps include: Step 1, extraction of CL: centrifuge the Bacillus subtilis culture solution after culturing for 120 h at 8000 r / min, 20 min, 4°C, discard the bacterial precipitate, collect the supernatant to obtain the Bacillus subtilis fermentation solution, adjust the pH to 2.0 with 12 mol / L concentrated hydrochloric acid, centrifuge overnight at 4°C, at 8000 r / min, 20 min, 4°C, discard the supernatant, take the precipitate, add an appropriate amount of anhydrous methanol to extract CL in the precipitate, collect, freeze-dry and store for later use; Step 2, synthesis of exogenous metal ion and CL coordination complex: prepare 1% CL solution, adjust pH with 1 mol / L sodium hydroxide solution, heat in a water bath to reach the reaction temperature, weigh metal salt and add it to the CL solution, stir rapidly until dissolved, seal with plastic wrap and react in a constant temperature water bath; after the reaction, remove moisture with a rotary evaporator, add 10 times the volume of anhydrous ethanol to precipitate overnight, centrifuge at 8000 r / min for 15 min, discard the supernatant, and freeze-dry the precipitate to obtain a powder, which is the metal ion / CL coordination complex.

2. The method for preparing the cyclolipopeptide supramolecular ligand according to claim 1, characterized in that: The metal ion is Ca 2+ The reaction temperature of step 2 is 25 ~ 30 ° C; the pH is not higher than 5.0; anhydrous calcium chloride is used, and the mass ratio of CaCl2 to CL is 0.5:1~2:

1.

3. The method for preparing the cyclolipopeptide supramolecular ligand according to claim 1, characterized in that: The metal ion is Zn 2+ The reaction temperature of step 2 is 50 ~ 70°C; pH 6.0-7.0; zinc sulfate is used, and the mass ratio of ZnSO4 to CL is 1:1~3:

1.

4. A cyclolipopeptide supramolecular ligand obtained by the preparation method according to any one of claims 1 to 3.

5. Use of the cyclolipopeptide supramolecular ligand according to claim 4 as a raw material for preparing an antibacterial agent.

6. The use of the cyclolipopeptide supramolecular ligand according to claim 5, characterized in that: It can be used as a raw material for preparing antibacterial agents for inhibiting Escherichia coli, Salmonella typhimurium, Staphylococcus aureus, Listeria, Aspergillus niger, Aspergillus flavus, Aspergillus carbonarius, and Fusarium moniliforme.

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