Preparation method and application of zanthoxylum seed antibacterial peptide-chitosan nanoparticles

Abstract

The invention provides a preparation method and application of zanthoxylum seed antibacterial peptide-chitosan nanoparticles. Based on an ionic cross-linking method, chitosan is selected as a carrier, sodium tripolyphosphate is selected as a cross-linking agent, zanthoxylum seed antibacterial peptide is used as an active substance to be embedded in the chitosan nanoparticles, and the antibacterial activity of the zanthoxylum seed antibacterial peptide is protected; and the chitosan nanoparticles with small particle size, uniform dispersion and good stability can be obtained. After optimization, the zanthoxylum seed antibacterial peptide-chitosan nanoparticles with the embedding rate of 48.03 + / -1.71%, the particle size of 137.5333 + / -2.4173 nm, the PDI value of 0.3890 + / -0.0044 and the Zeta potential of 21.4333 + / -0.8145 mV can be prepared. The method has the advantages of simple steps, mild reaction conditions, easily available raw materials and low requirements on equipment, and the prepared zanthoxylum seed antibacterial peptide-chitosan nanoparticles have a good antibacterial effect, and have a wide application prospect in the field of food preservation as a novel preservative.

Description

Technical Field

[0001] The invention belongs to the field of biotechnology, and specifically relates to a Zanthoxylum bungeanum seed antibacterial peptide-chitosan nanoparticle and a preparation method and application thereof. Background Art

[0002] Antimicrobial peptides are a class of small molecule polypeptides with antimicrobial activity, generally composed of 12 to 100 amino acid residues. They are an important part of the natural immune system of microorganisms, plants and animals. They have effects on bacteria, fungi, viruses and tumors, especially on drug-resistant bacteria. They have a broad spectrum of activity. Therefore, antimicrobial peptides are natural preservatives and potential alternatives to antibiotics. They have broad application prospects in clinical medicine, animal husbandry, food industry and aquaculture. Zanthoxylum bungeanum antimicrobial peptide is a plant-derived antimicrobial peptide that has good antibacterial effects on Escherichia coli, Staphylococcus aureus, Pseudomonas aeruginosa, Bacillus subtilis and Saccharomyces cerevisiae. However, antimicrobial peptides are subject to many restrictions in practical applications due to their own characteristics. They may be highly sensitive to production processes and storage conditions, or easily interact with certain matrix components, resulting in a decrease in antimicrobial efficacy and hindering the scope of application. Chitosan has a relatively simple composition and structure, and has good biophysical and chemical properties such as film-forming, biocompatibility, biodegradability and non-toxicity. Chitosan nanocarriers have the ability to deliver bioactive ingredients. They can isolate bioactive ingredients from the external environment and keep their structure and activity stable. Therefore, nanotechnology can be used to encapsulate Zanthoxylum bungeanum antimicrobial peptides into chitosan nanoparticles to protect and enhance the stability and antibacterial properties of Zanthoxylum bungeanum antimicrobial peptides.

[0003] Methods for preparing chitosan nanoparticles include ion crosslinking, emulsion crosslinking, spray drying, reverse micelle method, polyelectrolyte composite method, self-assembly method, etc. A study used the emulsion crosslinking method to embed tea polyphenols. After adding surfactants and tea polyphenols to the chitosan solution and dispersing them evenly, glutaraldehyde was added for crosslinking. Chitosan microcapsules with an embedding rate of 80.91% were successfully prepared, and the average particle size was 8.721 μm. However, the main problem with the emulsion crosslinking method is that more substances are introduced during the preparation process, and some crosslinkers are toxic and harmful to the body. Spray drying was used to embed orange oil in chitosan microparticles. The chitosan microparticles formed had a regular morphology and an average particle size of less than 20 μm. The disadvantage was that the particles formed were larger in size and were not suitable for temperature-sensitive substances. The reverse micelle method can synthesize highly monodispersed chitosan nanoparticles. Studies have shown that the nanoparticles can also significantly improve the solubility of curcumin in water. The nanoparticles prepared by the reverse micelle method have a small particle size and uniform distribution, but the operation is complicated and a large amount of organic solvents will be introduced. Chitosan and sodium carboxymethyl cellulose can form polyelectrolyte particles with certain antibacterial effects on Gram-positive and Gram-negative bacteria by utilizing the strong electrostatic interaction of opposite charges. Amphiphilic chitosan derivatives were synthesized using carboxymethyl chitosan as raw material, and nanoliposomes were prepared by thin film evaporation combined with dynamic high-pressure microfluidization. After adding dextran sulfate, epigallocatechin gallate can be successfully embedded in it by self-assembly, which can effectively slow down the degradation of epigallocatechin gallate in simulated intestinal fluid. The principle of the ionic crosslinking method is that when chitosan is dissolved in an acidic medium, the amino groups on the chitosan are converted into positively charged NH 3 ＋ and attract each other with negatively charged sodium tripolyphosphate, and the two cross-link each other through intermolecular electrostatic interaction to form chitosan nanoparticles. The method of preparing chitosan nanoparticles by ion cross-linking has simple operation steps and mild reaction conditions, which can effectively protect the biological activity of the coating and can be applied to the food field. Studies have shown that when bovine serum albumin is used as a model protein, the protein structure and integrity are not damaged during the synthesis of chitosan nanoparticles; the chitosan nanoparticles encapsulated with lactoferrin have a strong antibacterial effect on Staphylococcus aureus, and can be used as a strawberry coating to delay bacterial growth and effectively extend the shelf life of strawberries; the antimicrobial peptide LL37 is loaded in chitosan nanoparticles, and the nanoparticles show excellent antibacterial and anti-biofilm activity and in vitro sustained release performance; chitosan nanoparticles loaded with bighead carp peptides are prepared by ion cross-linking, which improves the thermal stability and retention rate of bighead carp peptides during simulated digestion.

[0004] The present invention aims to prepare nanoparticles with good antibacterial activity which use chitosan as a carrier and load antibacterial peptides from Zanthoxylum bungeanum seeds, and provides a method for preparing a novel preservative. Summary of the invention

[0005] The invention aims to provide a Zanthoxylum bungeanum seed antimicrobial peptide-chitosan nanoparticle, which has good antibacterial performance and can be used as a novel preservative.

[0006] The technical solution adopted by the present invention is as follows:

[0007] S1: Dissolve chitosan in 1% (m / v) acetic acid aqueous solution, stir magnetically overnight, adjust the pH to 4.0-6.0, filter insoluble impurities, and obtain a chitosan solution.

[0008] S2: Dissolve sodium tripolyphosphate in the aqueous solution, and filter using a 0.22 μm filter membrane after complete dissolution to obtain a sodium tripolyphosphate solution.

[0009] S3: dissolving the Zanthoxylum bungeanum seed antimicrobial peptide into the aqueous solution to obtain a Zanthoxylum bungeanum seed antimicrobial peptide solution.

[0010] S4: Under magnetic stirring and room temperature conditions, slowly add the Zanthoxylum bungeanum antimicrobial peptide solution to the chitosan solution and stir for 20 min to fully mix the two. Then add the sodium tripolyphosphate solution to the mixed solution of Zanthoxylum bungeanum antimicrobial peptide and chitosan, and continue stirring for 30 min to obtain the Zanthoxylum bungeanum antimicrobial peptide-chitosan nanoparticle solution.

[0011] Preferably, in step S1, the concentration of the chitosan solution is 1 mg / mL-5 mg / mL.

[0012] Preferably, in step S1, the pH of the chitosan solution is 4.0-6.0.

[0013] Preferably, in step S4, the concentration of sodium tripolyphosphate is 0.8 mg / mL-4.0 mg / mL.

[0014] Preferably, in step S4, the mass ratio of chitosan to sodium tripolyphosphate is 2.5 mg / mL-12 mg / mL.

[0015] Preferably, in the step S4, after the sodium tripolyphosphate solution is added dropwise to the mixed solution of chitosan and Zanthoxylum bungeanum seed antimicrobial peptide, stirring is required at room temperature for 15 min to 75 min.

[0016] Chitosan nanoparticles loaded with Zanthoxylum bungeanum antimicrobial peptide are prepared by the method for preparing Zanthoxylum bungeanum antimicrobial peptide-chitosan nanoparticles as described above.

[0017] The use of the Zanthoxylum bungeanum seed antimicrobial peptide-chitosan nanoparticles as described above in inhibiting Staphylococcus aureus or Escherichia coli.

[0018] The present invention provides a method for preparing Zanthoxylum bungeanum antimicrobial peptide-chitosan nanoparticles. The present invention is based on an ion crosslinking method, selects chitosan as a carrier, and sodium tripolyphosphate as a crosslinking agent. The Zanthoxylum bungeanum antimicrobial peptide is used as an active substance and is embedded in the chitosan nanoparticles, so as to protect the antibacterial activity and stability of the Zanthoxylum bungeanum antimicrobial peptide, and can obtain chitosan nanoparticles with small particle size, uniform dispersion and good stability.

[0019] The Zanthoxylum bungeanum seed antimicrobial peptide-chitosan nanoparticles prepared by the present invention have good antibacterial performance, can be used for food preservation, and have broad application prospects. BRIEF DESCRIPTION OF THE DRAWINGS Figure 1 The Zanthoxylum bungeanum seed antimicrobial peptide-chitosan nanoparticles prepared in Examples 1 to 5 of the present invention; Figure 2 This is a transmission electron microscopy image of the Zanthoxylum bungeanum antimicrobial peptide-chitosan nanoparticles prepared in Example 2 of the present invention; Figure 3 This is the antibacterial zone diagram of the Zanthoxylum bungeanum antimicrobial peptide-chitosan nanoparticles prepared in Example 2 of the present invention; Figure 4 Figure A is a time-killing curve of the antimicrobial peptide-chitosan nanoparticles of Zanthoxylum bungeanum prepared in Example 2 of the present invention against Escherichia coli, and Figure B is a time-killing curve of the antimicrobial peptide-chitosan nanoparticles of Zanthoxylum bungeanum prepared in Example 2 of the present invention against Staphylococcus aureus.

Claims

1. A method for preparing Zanthoxylum bungeanum antimicrobial peptide-chitosan nanoparticles, characterized in that: The Zanthoxylum bungeanum antimicrobial peptide-chitosan nanoparticles are prepared by using chitosan as a carrier and sodium tripolyphosphate as a crosslinking agent through an ion crosslinking method.

2. The Zanthoxylum bungeanum seed antimicrobial peptide according to claim 1, characterized in that The amino acid sequence of the Zanthoxylum bungeanum seed antimicrobial peptide NP-6 is: AGDKKIKIGINGFGRIGRL.

3. The Zanthoxylum bungeanum seed antimicrobial peptide according to claim 1, characterized in that When preparing Zanthoxylum bungeanum antimicrobial peptide-chitosan nanoparticles, the amount of Zanthoxylum bungeanum antimicrobial peptide corresponding to every 10-15 mg of chitosan is 5-10 mg.

4. The method for preparing Zanthoxylum bungeanum antimicrobial peptide-chitosan nanoparticles according to claim 1, characterized in that: The preparation method specifically comprises the following steps: S1: Dissolve chitosan in 1% (m / v) acetic acid aqueous solution to a concentration of 1-5 mg / mL. After magnetic stirring overnight, adjust the pH to 4.0-6.0, filter insoluble impurities, and obtain chitosan solution; S2: Dissolve sodium tripolyphosphate in aqueous solution to a concentration of 0.5-1.5 mg / mL. After complete dissolution, filter using a 0.22 μm filter membrane to obtain a sodium tripolyphosphate solution; dissolve the antimicrobial peptide from Zanthoxylum bungeanum in aqueous solution to a concentration of 1-3 mg / mL; S3: Under magnetic stirring and room temperature, slowly add the antimicrobial peptide solution from Zanthoxylum bungeanum to the chitosan solution and stir for 20 min to fully mix the two. Then add the sodium tripolyphosphate solution to the mixed solution of Zanthoxylum bungeanum and chitosan, and continue stirring for 30 min to obtain a Zanthoxylum bungeanum-chitosan nanoparticle solution.

5. The method for preparing Zanthoxylum bungeanum antimicrobial peptide-chitosan nanoparticles according to claim 1, characterized in that: The mass ratio of chitosan to sodium tripolyphosphate is 3:1, and the mass ratio of chitosan to Zanthoxylum bungeanum seed antimicrobial peptide is 2:

1.

6. Use of the Zanthoxylum bungeanum seed antimicrobial peptide-chitosan nanoparticles as described in claims 1-6 in preserving chilled fresh pork.

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