A multi-effect polymyxin chitosan nanoparticle, a preparation method and application thereof

Abstract

The application discloses a multi-effect microcin chitosan nanoparticle and a preparation method and application thereof. The chitosan nanoparticle comprises one or more than one lariat peptide, has a multi-effect synergistic antibacterial effect, and realizes broad-spectrum antibacterial effect. The chitosan nanoparticle prepared by the application can further comprise arginine, so that the acid-base tolerance of the lariat peptide is improved, and the application bottleneck of adding the lariat peptide in livestock and poultry feeding is broken. In addition, the chitosan nanoparticle prepared by the application has high safety.

Description

Technical Field

[0001] This invention belongs to the field of biomedicine, specifically relating to a multifunctional microbial chitosan nanoparticle, its preparation method, and its application. Background Technology

[0002] The problem of antibiotic resistance in Enterobacteriaceae caused by antibiotic overuse has attracted international research attention. To reduce dependence on antibiotics and protect livestock health, antibiotic-free farming has gradually become the future development direction of my country's animal husbandry. Previously, there were no ideal antibiotic alternatives to combat the common Enterobacteriaceae bacteria in livestock farming, but microcins (Mcc) have shown great potential in addressing antibiotic resistance. Lasso peptides (LPs) are a class of small molecule microcins (<10kDa) assembled from ribosomes and post-translational modified with a "lasso" structure, possessing stable and highly efficient bactericidal biological activity. Common lasso peptides mainly include MccJ25, MccY, and Klebsidin. After entering bacterial cells, they exert highly efficient bactericidal effects by inhibiting RNA polymerase activity (MIC = 0.001-1.0 μg / mL). Previous studies have found that the novel natural lasso peptide Microcin Y (MccY) exhibits a significant complementary bactericidal advantage against Salmonella and Shigella compared to MccJ25. While both MccJ25 and MccY show no sensitivity to Klebsiella, the lasso peptide Klebsidin does exhibit some inhibitory activity against Klebsiella. Compared to other lasso peptides such as Citrocin, MccY, MccJ25, and Klebsidin demonstrate strong antibacterial activity against Enterobacteriaceae and Gram-positive bacteria (MIC = 0.001-20.0 μg / mL). Despite the high bactericidal efficacy of lasso peptides, the bacteria targeted by a single lasso peptide are specific; furthermore, lasso peptide microindices generally exhibit acid-base instability and sensitivity to digestive enzymes, which pose significant obstacles to their use in animal feed. Summary of the Invention

[0003] This invention aims to at least solve one of the aforementioned technical problems existing in the prior art. To this end, this invention provides a pleiotropic microbeta chitosan nanoparticle, wherein the nanoparticle contains multiple different lasso peptides simultaneously coupled together, synergistically enhancing the specific antibacterial effects of these different lasso peptides to achieve broad-spectrum antibacterial activity. Furthermore, this nanoparticle can improve the acid-base tolerance and safety of lasso peptides, thereby overcoming the application bottleneck of lasso peptides in livestock and poultry feed.

[0004] To achieve the above-mentioned objectives, a first aspect of the present invention provides chitosan (CN) nanoparticles, wherein the chitosan nanoparticles comprise chitosan and one or more lauroyl peptides, wherein the lauroyl peptides have carboxyl groups, and the amino groups in the chitosan structure are connected to the carboxyl groups of the lauroyl peptides via amide bonds.

[0005] Lasso peptides (LPs) are naturally occurring polypeptide products found in bacteria. They are bioactive peptides with a lasso conformation, synthesized by ribosomes and modified post-translationally. Lasso peptides typically contain 14-33 amino acid residues. The first amino acid at the N-terminus forms a large amide ring with the 7th, 8th, or 9th amino acid side chain, while the C-terminal amino acid passes through this large amide ring, forming the characteristic "lasso" structure. Based on the number and position of disulfide bonds, lasso peptides can be classified into four classes (e.g., ...). Figure 8 (As shown). Lasso peptides whose C-terminal peptide chain is connected to the macropeptide ring by two disulfide bonds are defined as Class I lasso peptides. If, after the peptide tail extends out of the peptide ring, there is no disulfide bond between the C-terminal peptide chain and the macropeptide ring, but the lasso topology is stabilized by spatial interactions, it is called a Class II lasso peptide. If a lasso peptide forms only one disulfide bond between the C-terminal sequence of the peptide tail and the peptide ring, it is classified into Class III or Class IV lasso peptides (Class III or Class IV) depending on the position of the disulfide bond. Specifically, in Class III lasso peptides, the disulfide bond connects the peptide ring to the peptide tail, while in Class IV, the disulfide bond exists only at the peptide tail. All four classes of lasso peptides shown have a carboxyl group.

[0006] In some embodiments of the present invention, the molar ratio of chitosan to tau peptide is 5:(3-4).

[0007] In some embodiments of the present invention, the lasso peptides include MccJ25, MccY, Klebsidin, Citrocin, Lihuanodin, Synthetase B1, Astexin 1, Koveensin, MccS, and MccC7.

[0008] In some embodiments of the present invention, the lasso peptide includes MccY and MccJ25.

[0009] In some embodiments of the present invention, the lasso peptide includes McCY and Klebsidin.

[0010] In some embodiments of the present invention, the lasso peptide includes MccJ25 and Klebsidin.

[0011] In some embodiments of the present invention, the lasso peptides include MccY, MccJ25, and Klebsidin.

[0012] In this invention, CN nanoparticles comprising a single MccY lasso peptide (CN-MccY) have no antibacterial activity against Serratia marcescens and Klebsiella pneumoniae; CN nanoparticles comprising a single MccJ25 lasso peptide (CN-MccJ25) have no antibacterial activity against Salmonella typhimurium; and CN nanoparticles comprising a single Klebsidin lasso peptide (CN-Klebsidin) have no antibacterial activity against Salmonella typhimurium.

[0013] Furthermore, in this invention, the combined use of MccY and MccJ25 exhibits additive antibacterial activity against *Salmonella enteritidis*, *Salmonella typhimurium*, *Serratia marcescens*, and *Klebsiella pneumoniae*, and synergistic antibacterial activity against *Staphylococcus aureus*; the combined use of MccY and Klebsidin exhibits additive antibacterial activity against *Salmonella enteritidis*, *Salmonella typhimurium*, and *Serratia marcescens*, and synergistic antibacterial activity against *Klebsiella pneumoniae* and *Staphylococcus aureus*; the combined use of MccJ25 and Klebsidin exhibits additive antibacterial activity against *Salmonella enteritidis*, *Salmonella typhimurium*, and *Serratia marcescens*, and synergistic antibacterial activity against *Klebsiella pneumoniae* and *Staphylococcus aureus*; Klebsiella pneumoniae and Escherichia coli showed additive antibacterial activity against Klebsiella pneumoniae, and synergistic antibacterial activity against Serratia marcescens. Therefore, CN nanoparticles (CN-LPs) including MccY, MccJ25, and Klebsidin showed synergistic activity against Klebsiella pneumoniae, Staphylococcus aureus, and Serratia marcescens, and additive antibacterial activity against Salmonella enteritidis and Salmonella typhimurium.

[0014] In this invention, CN nanoparticles (CN-LPs) comprising MccY, MccJ25 and Klebsidin extend the antibacterial activity of microbes to major foodborne pathogens Salmonella, Serratia, and Klebsiella.

[0015] In some embodiments of the present invention, the molar ratio of CN, MccY, MccJ25 and Klebsidin is 5:(1-2):1:1.

[0016] In some embodiments of the present invention, the chitosan nanoparticles further include arginine (Arg), wherein the amino group in the chitosan structure is connected to the carboxyl group of arginine via an amide bond.

[0017] In some embodiments of the present invention, the molar ratio of CN to Arg is (1-10):1.

[0018] In this invention, the arginine is coupled to chitosan via an amide bond, thereby improving the acid-base stability of the chitosan nanoparticles.

[0019] In some embodiments of the present invention, the chitosan nanoparticles have a particle size of 100-200 nm.

[0020] In a second aspect of the present invention, a method for preparing chitosan nanoparticles is provided, wherein the method prepares chitosan nanoparticles according to the first aspect of the present invention.

[0021] In some embodiments of the present invention, the preparation method includes the following steps:

[0022] 1) Mix 1-ethyl-(3-dimethylaminopropyl)carbodiimide (EDC) solution and N-hydroxysuccinimide solution (NHS) to obtain an EDCN mixed solution;

[0023] 2) Dissolve chitosan and lazotocin separately in the acetylation solution and mix them evenly to obtain chitosan acetylation solution and lazotocin acetylation solution;

[0024] 3) Add the EDCN mixed solution from step 1) to the chitosan acetylation solution and the laurocapeptide acetylation solution from step 2) respectively, and mix them evenly to obtain the chitosan-EDCN mixed solution and the laurocapeptide-EDCN mixed solution.

[0025] 4) The chitosan-EDCN mixed solution from step 3) is mixed sequentially with the tau peptide-EDCN mixed solution, and the esterification reaction is completed by stirring thoroughly to prepare the chitosan nanoparticles.

[0026] In some embodiments of the present invention, step 2) further includes the preparation of an acetylated arginine (Arg) solution, the preparation steps of which include: dissolving arginine in the acetylation solution, mixing evenly, to obtain an acetylated arginine solution.

[0027] In some embodiments of the present invention, step 3) further includes the preparation of an arginine-EDCN mixed solution, the preparation steps of which include: adding the EDCN mixed solution from step 1) to the arginine acetylation solution from step 2), mixing evenly to obtain the arginine-EDCN mixed solution.

[0028] In some embodiments of the present invention, step 4) further includes mixing with the arginine-EDCN mixed solution in step 3).

[0029] In some embodiments of the present invention, the acetylation solution includes sodium acetate solution, acetyl chloride solution, acetic anhydride solution, and glacial acetic acid solution.

[0030] In some embodiments of the present invention, the acetylation solution comprises sodium acetate buffer (SAB).

[0031] In a third aspect of the invention, the use of chitosan nanoparticles of the first aspect of the invention in the preparation of products having antibacterial properties is provided.

[0032] In some embodiments of the present invention, the bacteria include Salmonella, Serratia, and Klebsiella.

[0033] In some embodiments of the present invention, the Salmonella genus includes Salmonella enteritidis and Salmonella typhimurium.

[0034] In some embodiments of the present invention, the Serratia genus includes Serratia marcescens.

[0035] In some embodiments of the present invention, the Klebsiella genus includes Klebsiella pneumoniae.

[0036] In some embodiments of the present invention, the product includes feed, preservatives, disinfectants, and drugs for external wound infections.

[0037] The beneficial effects of this invention are:

[0038] 1) The chitosan nanoparticles prepared by this invention, which include chitosan and one or more tau peptides, have a multi-effect synergistic antibacterial effect against a variety of bacteria, and synergistically enhance the specific antibacterial effect between a variety of different tau peptides, thereby achieving a broad-spectrum antibacterial effect.

[0039] 2) The chitosan nanoparticles prepared by this invention are also linked to arginine (Arg) through amide bonds, thereby improving the acid and alkali tolerance of the tau peptide and breaking through the application bottleneck of tau peptide in livestock and poultry feed.

[0040] 3) The chitosan nanoparticles prepared by this invention have high safety and will not cause hemolysis even at high concentrations. Attached Figure Description

[0041] Figure 1 This is the design diagram of the CN-LPs-Arg synthesis route in Example 1.

[0042] Figure 2These are the characterization and analysis results of the nanomaterial CN-LPs-Arg in Example 2; whereby... Figure 2 A represents the FTIR analysis results of CN. Figure 2 B represents the FTIR analysis results of CN-LPs-Arg. Figure 2 C represents the particle size analysis result of CN-LPs-Arg. Figure 2 D is a graph representing the Zeta potential characterization analysis of CN-LPs-Arg.

[0043] Figure 3 CN in Example 3 Figure 3 A), CN-Arg( Figure 3 B) and CN-LPs-Arg( Figure 3 C) Transmission electron micrographs of three types of CN nanoparticles.

[0044] Figure 4 These are the physicochemical stability test results of the CN nanoparticles in Example 4; wherein Figure 4 A-4C represent the UPLC-Q-TOF mass spectrometry analysis results for McCY, Klebsidin, and McCJ25, respectively. Figure 4 D-4E represents the stability analysis results of McY at 95℃, pH=2.0, and pH=12.0. Figure 4 F represents the stability analysis results of CN-LPs-Arg at 95℃, pH=2.0, and pH=12.0. Figure 4 G represents the antibacterial activity of CN-LPs-Arg after treatment at 95℃, pH=2.0, and pH=12.0, as determined by the dot method.

[0045] Figure 5 The results are from spot test of CN, Arg and various CN nanoparticles (CN-Arg, CN-MccY, CN-MccJ25, CN-Klebsidin, CN-LPs and CN-LPs-Arg), showing the antibacterial effect determined by the spot method; **p<0.01, ***p<0.001, ****p<0.0001.

[0046] Figure 6 This describes the experimental procedure for the combined FICI antibacterial analysis of the lasso peptides MccY, MccJ25, and Klebsidin.

[0047] Figure 7 These are the results of the biocompatibility analysis of CN nanoparticles; among which... Figure 7 A shows the morphology of erythrocytes after treatment with CN nanoparticles; Figure 7 B represents the evaluation of the cytotoxicity of CN nanoparticles against HEK293T cells using the MTT assay. Figure 7 C represents the hemolytic effect of CN nanoparticles on erythrocytes.

[0048] Figure 8 This is a schematic diagram of the structure of four types of lasso peptides. Detailed Implementation

[0049] The present invention will be further described in detail below through specific embodiments. Unless otherwise specified, the raw materials, reagents, or apparatus used in the embodiments and comparative examples are all available from conventional commercial sources or can be obtained by existing technical methods. Unless otherwise specified, the test or experimental methods are conventional methods in the art.

[0050] The instrument information used in this embodiment is shown in Table 1 below:

[0051] Table 1. Information on the instruments used

[0052]

[0053]

[0054] Statistical analysis was performed using GraphPad Prism 8.0 software. Within-group statistical results are expressed as Mean ± SD. One-way ANOVA was used for comparisons between two and three or more groups. Student's t-test was used to compare significant differences between groups, with each treatment performed in three independent replicates.

[0055] Example 1: Chemical Synthesis of CN-LPs-Arg

[0056] In this embodiment, secreted proteins were obtained using an E. coli prokaryotic expression system, and then three lasso peptides (MccY, MccJ25, and Klebsidin) were prepared by HPLC. The preparation method is described in reference 1. Multifunctional nanomaterials were obtained by chemically coupling these peptides with chitosan (CN). Figure 1 The synthesis process is described in detail. Various CN nanoparticles were synthesized by linking the amino groups of CN with the carboxyl groups of lasso peptides via amide bonds. The carboxyl groups of MccY, MccJ25, and Klebsidin were activated by EDC and then sequentially coupled to CN to form CN-LPs. Furthermore, to improve acid and alkali resistance, Arg was introduced to combine with CN-LPs to form CN-LPs-Arg. The amino groups of CN-LPs and the carboxyl groups of Arg react in the presence of water-soluble EDC to form new amide bonds. The optimized formulation of these nanomaterials achieved an encapsulation efficiency of over 60% w / w in CN and encapsulated approximately 90% of the lasso peptides. The specific synthesis process of CN-LPs-Arg is shown below:

[0057] The expression and preparation of the three lasso peptides involved introducing the vector pET-28a(+) into BL21(DE3) competent cells to obtain BL21(DE3) bacteria containing pET-28a(+). These pET-28a(+) BL21(DE3) bacteria were then revived in LB agar plates containing 30 μg / mL kanamycin. One loopful of bacteria was then inoculated onto 100 mL of... In a 250 ml Erlenmeyer flask of LB medium, kanamycin and isopropyl-β-D-thiogalactoside (IPTG) were added (the final concentration of kanamycin was 30 μg / mL, and the final concentration of IPTG was 0.1 M). The Erlenmeyer flask was placed in a shaker and cultured (culture conditions: 200 r / min, 37 ℃, 14 h). The expressed bacterial culture was collected, centrifuged at 5000 r / min for 20 min, and the supernatant was collected. The supernatant was filtered through a 0.22 μm filter membrane to obtain the supernatant filtrate. The supernatant was then enriched with protein using reverse solid-phase extraction with an SPE C18 column. The expressed target protein was then identified using Q-TOF, and the target protein was collected and purified using preparative HPLC. The protein was concentrated by vacuum distillation, and finally dried, weighed, and quantified by freezing.

[0058] The synthesis of CN-LPs-Arg mainly consists of two steps: First, MccY, MccJ25, and Klebsidin undergo stepwise coupling reactions with CN. Reagents are prepared: 0.5 mM 1-ethyl-(3-dimethylaminopropyl)carbodiimide (EDC) solution and 0.25 mM N-hydroxysuccinimide (NHS) solution are mixed in a 1:1 volume ratio to form an EDCN solution. 0.1 M sodium acetate buffer (SAB, pH = 6.4) is prepared. CN is weighed and dissolved in SAB to prepare a 1 mg / mL CN acetylated solution. 100 μL of EDCN is added dropwise to activate the amino groups of CN, with the volume ratio of EDCN solution to CN acetylated solution being 1:10, to generate a CN-EDCN solution. 1 mg of each of the three lasso peptides is dissolved in SAB to obtain a 1 mg / mL lasso peptide SAB solution. 100 μL of EDCN solution was added to a lassotide SAB solution to activate the carboxyl group of lassotide. The volume ratio of EDCN solution to lassotide SAB solution was 1:10, resulting in MccY-EDCN solution, MccJ25-EDCN solution, and Klebsidin-EDCN solution, respectively. 1 mL of MccY-EDCN solution was added to 1 mL of CN-EDCN solution, and the mixture was stirred thoroughly to complete the esterification reaction, generating a CN-MccY EDCN solution. 1 mL of MccJ25-EDCN solution was added to 2 mL of CN-MccY EDCN solution, and the mixture was stirred to initiate the esterification reaction, generating a CN-MccY-MccJ25 EDCN solution. Finally, 1 mL of Klebsidin-EDCN solution was added to 3 mL of CN-MccY-MccJ25 EDCN solution, and the mixture was stirred thoroughly to complete the esterification reaction, generating a CN-LPs EDCN solution.

[0059] In the second step, Arg and the amino group of CN are coupled under certain conditions to form a new amide bond, enabling CN-LPs to complete amino acid modification and synthesize CN-LPs-Arg. The introduction of Arg further improves the acid and alkali resistance of CN-LPs. Specifically, 1 mg of Arg is dissolved in 1 mL of SAB solution to prepare a 1 mg / mL Arg SAB solution. 100 μL of the EDCN solution prepared in the first step is added dropwise to activate the carboxyl group of Arg. The volume ratio of EDCN solution to Arg SAB solution is 1:10, generating an Arg-EDCN solution. 1 mL of Arg-EDCN solution is added to 1 mL of CN-LPs EDCN solution, and the solution is dialyzed three times continuously for 24 hours using a MWCO 2000 benzoylation dialysis tube filled with distilled water to remove excess reactants and byproducts. Finally, CN-LPs-Arg is obtained, and the resulting solution is freeze-dried for later use.

[0060] The preparation methods of CN-MccY, CN-Arg, CN-MccJ25, and CN-Klebsidin as control samples are similar to those of CN-LPs-Arg. In the first step, EDCN activation and esterification reaction with CN-EDCN solution are used to prepare EDCN solutions of CN-MccY, CN-Arg, CN-MccJ25, and CN-Klebsidin. Then, CN-MccY, CN-Arg, CN-MccJ25, and CN-Klebsidin are obtained by dialysis and freeze-drying in the second step.

[0061] Example 2: Characterization Analysis of CN-LPs-Arg

[0062] The conjugation efficiencies of CN-LPs-Arg, CN-Arg, and CN were investigated using Vertex 70 Fourier Transform Infrared Spectrometer (FTIR). The spectral range was 4000–15000 cm⁻¹. -1 Between, with a resolution of 4cm -1 The scans were performed 64 times. The structures of CN and CN-LPs-Arg were analyzed using FT-IR, and the results are as follows: Figure 2 As shown in A and 2B, the characteristic peaks of NH in CN are located at 2850 and 2930 cm⁻¹. -1 Nearby, CN-LPs are at 2921.99 and 2858.79 cm. -1 At this location, CN-LPs-Arg is between 2923.30 and 2871.22 cm. -1 An NH stretching band was observed. The stretching bands of -COOH in CN-LPs and CN-LPs-Arg were observed to be approximately 3359.96 cm⁻¹. -1 and 3351.45cm -1 At 1735cm -1 A new peak related to the CN structure and carboxyl group reaction appears, represented by the characteristic peak of -C=O, confirming the successful reaction between the amino group of CN and the carboxyl group of the lazolide. The infrared absorption peak of -C=O generally appears at 1700-1750 cm⁻¹. -1 Within the range, however, due to the high degree of deacetylation of CN, the infrared absorption peak is relatively weak. The vibrational peak of the amide group in CN-LPs-Arg is obvious during the reaction. The particle size and zeta potential of the synthesized compound were determined using a Malvern ZS-90 Zetasizer at 25℃. The static number-average particle size of the modified CN-LPs-Arg increased slightly, remaining between 200-400 nm. Figure 2As shown in Figure C, the lower molecular weight of CN-LPs-Arg results in a more uniform pore morphology and lower polydispersity of the pores. The zeta potential of the prepared nanomaterials is between -22.0 and 22.0 mV. Figure 2 As shown in D.

[0063] Example 3: Transmission electron microscopy analysis of CN-LPs-Arg

[0064] CN-LPs-Arg nanoparticles were resuspended in 0.1M sodium acetate solution to obtain a 1 mg / mL CN-LPs-Arg nanoparticle suspension. 20 μL of this suspension was added dropwise to a 200-mesh grid and incubated at room temperature for 10 min. Then, the nanoparticles were negatively stained with 2% phosphotungstic acid for 3 min, and the remaining liquid was filtered off with filter paper. The samples were observed using an HT7700 and a Hitachi Su-8010 transmission electron microscope. The structures and particle sizes of the three types of nanoparticles (CN, CN-Arg, and CN-LPs-Arg) are shown below. Figure 3 As shown. In Figure 3 In A, the particle size of CN is between 10-50 nm; Figure 4 B and Figure 4 In C, the particle sizes of CN-LPs and CN-LPs-Arg are between 100-200 nm.

[0065] Example 4: Physicochemical stability test of CN-LPs-Arg

[0066] To investigate the stability of chitosan nanoparticles, acid-base polarity tests and high-temperature stability tests were conducted.

[0067] First, the molecular weights of the three lasso peptides were analyzed by UPLC-Q-TOF mass spectrometry. These three lasso peptides were expressed and prepared according to Example 1. UPLC-Q-TOF mass spectrometry analysis results showed that the precise molecular weight of MccY was 2224.0165 m / z, corresponding to (M+3H). +3 and (M+2H) +2 The ionized molecular weights are 742.6794 m / z and 1113.5188 m / z, respectively. Figure 4 A). Klebsidin consists of only 19 amino acids, with a molecular weight of 2032.0015 m / z, corresponding to (M+2H). +2 The ionized molecular weight is 1017.5097 m / z ( Figure 4 B). By Figure 4 According to C, the precise molecular weight of MccJ25 is 2106.0223 m / z, corresponding to (M+3H). +3 Its ionized molecular weight is 703.3494 m / z.

[0068] The experimental steps for the high temperature stability test are as follows: MccY was dissolved in 0.1M SAB solution to prepare a 1mg / mL MccY SAB solution. Then, MccY was divided into two groups. The experimental group was treated at 95℃ for 2h, and the control group was treated at room temperature for 2h.

[0069] The experimental steps for the acid-base polarity test are as follows: 1 mg / mL McY SAB solution was divided into three groups. The first group was treated in hydrochloric acid solution with pH=2.0 for 2 h, the second group was treated in sodium hydroxide solution with pH=12.0 for 2 h, and the third group, the control group, was not treated in any way.

[0070] Then, the tau peptide MccY was detected using 280 nm ultraviolet scanning spectroscopy, and the results are as follows: Figure 4 As shown in D and 4E, the degradation of the lasso peptide MccY was mainly caused by acid-base treatment at pH=2.0 and pH=12.0, while high-temperature treatment had no effect on the MccY protein. Acid treatment weakened the characteristic absorption peak of MccY, indicating that acid treatment led to partial peptide degradation. In addition, alkaline treatment showed additional degradation peaks in the spectrum from 12.7 to 13.3 min.

[0071] The stability of CN-LPs-Arg nanoparticles was further tested using high-temperature stability and acid-base polarity tests. CN-LPs-Arg nanoparticles were dissolved in 0.1M SAB solution to prepare a 1 mg / mL CN-LPs-Arg SAB solution; the remaining experimental steps were as described above. Then, the CN-LPs-Arg nanoparticles were detected using 280 nm UV-Vis scanning spectroscopy, and the results are as follows. Figure 4 As shown in Figure F, the CN-LPs-Arg nanoparticles treated with acid and alkali, treated with high temperature, and the control group all showed characteristic peaks at 12.2 min, 12.7 min, and 13.6 min, respectively. These results indicate that CN-LPs-Arg nanoparticles can improve the stability of locust peptides in acid, alkali, and high temperature environments. CN-LPs-Arg nanoparticles are stable in acid, alkali, and high temperature environments and have acid and alkali resistance and high temperature resistance.

[0072] After treating 0.1 mg / mL CN-LPs-Arg for 2 h at 95 °C, pH = 2.0, and pH = 12.0 respectively, its bactericidal activity was determined by the dot assay. The OD values ​​were then calculated. 600=0.8 The test bacteria (Salmonella typhimurium (ST53, information as shown in Table 2)) 10 μL was cultured in LB solid medium, and then the treated lasso peptide was diluted to 6 concentrations and spotted into 6 sector regions. The drug concentrations in wells 1 to 6 were: region 1: 10.0 μM, region 2: 5.0 μM, region 3: 1.0 μM, region 4: 0.5 μM, region 5: 0.2 μM, region 6: 0.04 μM. The culture was carried out at 37℃ for 12 h. The results are as follows. Figure 4 As shown in G, high-temperature treatment led to a 5-fold decrease in activity (0.2 μM), while the activities of the two acid-base treated groups were comparable to the control group (0.04 μM). This indicates that chitosan-arginine nanoparticles CN-LPs-Arg possess nanoparticle stability and acid-base buffering properties, and the acid and alkali resistance of the three lasso peptides in CN-LPs-Arg is improved.

[0073] Example 5: Antibacterial activity of CN-LPs-Arg

[0074] The synthesized samples (CN, Arg, CN-Arg, CN-MccY, CN-MccJ25, CN-Klebsidin, CN-LPs, and CN-LPs-Arg) were subjected to spot plating tests to demonstrate their antibacterial effects as determined by the spot method. The tested strains included *Salmonella enterica* (SE63), *Salmonella typhimurium* (ST53), *Serratia marcescens* (SMATCC14756), and *Klebsiella pneumoniae* (KP07), with specific strain information shown in Table 2. The specific steps included: culturing the strains overnight on LB agar plates, then transferring the strains to LB broth and culturing at 37°C until OD (dose elapsed). 600 The value reached 0.8. Subsequently, the bacterial suspension was diluted 1:1000 and added to LB solid medium containing 0.5% agar. Plates were prepared at 40°C, and each LB plate was divided into eight sector regions. From sector 1 to sector 8, 10-20 μL of the following drugs were inoculated at 0.05 μM: 1. CN, 2. Arg, 3. CN-Arg, 4. CN-MccY, 5. CN-MccJ25, 6. CN-Klebsidin, 7. CN-LPs, and 8. CN-LPs-Arg. After 24 h of incubation, the inhibition zone diameter was observed and recorded. The inhibition zone determination results of CN nanoparticles for each strain in the plate test are shown in Table 3.

[0075] Table 2. Strains used in Example 5

[0076]

[0077] Table 3 shows the determination of the inhibition zone of CN nanoparticles in the spot test.

[0078]

[0079]

[0080] Furthermore, the statistical results of the plate test are as follows: Figure 5 As shown, individual CN and Arg have no inhibitory effect on these microorganisms. CN nanoparticles containing single taure peptides (CN-MccY, CN-MccJ25, CN-Klebsidin) exhibit specific inhibitory effects against specific strain types. Furthermore, CN nanoparticles containing three taure peptides (CN-LPs) show antibacterial activity against *Salmonella enteritidis* (SE63), *Salmonella typhimurium* (ST53), *Serratia marcescens* (SMATCC14756), and *Klebsiella pneumoniae* (KP07), demonstrating that CN-LPs achieve a synergistic antibacterial effect against different bacterial species. Compared to CN nanoparticles containing single taure peptides (CN-MccY, CN-MccJ25, CN-Klebsidin), the concentration of the corresponding single taure peptide in CN-LPs at the same concentration is reduced to approximately one-third of that in CN nanoparticles containing single taure peptides, resulting in a smaller inhibition zone diameter for some strains. Compared to CN-LPs, CN nanoparticles further coupled with Arg (CN-LPs-Arg) can further improve the antibacterial effect against different types of bacteria.

[0081] Example 6: FICI-based antibacterial analysis of MccY, MccJ25, and Klebsidin

[0082] To confirm the synergistic antibacterial relationship among the three individual tau peptides, the relationships between MccY and MccJ25, MccY and Klebsidin, and MccJ25 and Klebsidin were further investigated. The experimental procedure is as follows: Figure 6 As shown in Table 4-6, the experimental results are as follows. FICI experiment: In each well of a 96-well plate, equal volumes of 50 μL of a specific concentration of lassotide A and 50 μL of a specific concentration of lassotide B were added. The concentrations of lassotide were as follows: Figure 6 As shown. The well plates were incubated at 37°C for 24 h to observe and record the MIC of each lasso peptide. Each experimental concentration was repeated three times to ensure the accuracy of the results. The interaction between lasso peptide A and lasso peptide B was evaluated according to the formula for calculating the FICI (Combined Inhibition Fraction). The formula for calculating the FICI for combined inhibition is as follows:

[0083]

[0084] When FICI > 2, the antagonistic effect of lasso peptides A and B is shown; when 1 < FICI ≤ 2, an irrelevant effect is shown; when 0.5 < FICI ≤ 1, the additive effect of lasso peptides A and B is shown; when FICI ≤ 0.5, the synergistic effect of lasso peptides A and B is shown.

[0085] Table 4 FICI numerical results of MccY and MccJ25

[0086]

[0087] Table 5 FICI numerical results of MccY and Klebsidin

[0088]

[0089] Table 6 FICI numerical results of MccJ25 and Klebsidin

[0090]

[0091] The results in Table 4, Table 5 and Table 6 show that: (1) In the drug detection of MccY and MccJ25, Salmonella enteritidis (SE63), Salmonella typhimurium (ST53), Serratia marcescens (SMATCC14756) and Klebsiella pneumoniae (KP07) showed an additive effect. (2) In the drug detection of MccY and Klebsidin, Salmonella enteritidis (SE63), Salmonella typhimurium (ST53), Serratia marcescens (SMATCC14756) showed an additive effect, and Klebsiella pneumoniae (KP07) showed a synergistic effect. (3) In the drug detection of MccJ25 and Klebsidin, Salmonella enteritidis (SE63), Klebsiella pneumoniae (KP07) showed an additive effect, Salmonella typhimurium (ST53) showed an irrelevant effect, and Serratia marcescens (SMATCC14756) showed a synergistic effect.

[0092] Example 7: Biocompatibility analysis of CN1.25-LPs-Arg

[0093] Evaluate the effect of CN-LPs-R on the activity of mammalian cells to evaluate its biocompatibility. The specific steps are as follows: Collect human red blood cells in a heparin-containing collection tube. The steps are as follows: Centrifuge to separate the serum at 750g for 15 minutes, wash the red blood cells 3 times with sterile PBS, and then dilute the red blood cells with PBS to make a red blood cell suspension of 1%; then treat the cells with samples (CN-Arg, CN-LPs, CN-LPs-Arg) at a concentration of 10 μg / mL, and observe their morphological changes. Triton X-100 diluted with PBS is used as a positive control, and cells treated with PBS are used as a negative control.

[0094] Experimental results are as follows Figure 7 As shown in Figure A, the negative control cells had normal morphology, while the positive control cells treated with Triton X-100 showed cell membrane lysis and cell death. Cells treated with CN, CN-Arg, CN-LPs, and CN-LPs-Arg did not cause significant cell membrane lysis and hemolysis.

[0095] Next, the cytotoxicity of CN nanoparticles to HEK293T cells was evaluated using the MTT assay. 293T cells were cultured at 37°C and 5% CO2 in DMEM supplemented with 10% fetal bovine serum and 1% penicillin / streptomycin. 200 μL of the above 1% erythrocyte suspension (adjusted to a concentration of 1.0 × 10⁻⁶) was taken. 5 Cells were seeded in 96-well plates and cultured for 24 h. CN nanoparticles (CN-LPs-Arg and CN) were then resuspended and diluted in DMEM. CN nanoparticles were serially diluted to 0.5%, 1.0%, 5.0%, 10.0%, and 20.0% concentrations (20 mg / mL); CN-LPs-Arg was serially diluted to 10 μM, 20 μM, 40 μM, 80 μM, 160 μM, and 320 μM. 200 μL of the diluted CN nanoparticle solution was added to each well, and the cells were incubated at 37 °C and 5% CO2 for 24 h. Triton X-100 diluted in PBS was used as a positive control, and PBS-treated cells were used as a negative control.

[0096] Qualitative results of CN nanoparticle cytotoxicity against HEK293T cells are as follows: Figure 7 As shown in B. Figure 7 B reflects the color change of red blood cells at different concentrations. The smaller the color change, the lower the degree of hemolysis. The color change of red blood cells is not obvious in the concentration ranges of 0.5%-20.0% and 10μM-80μM, while the color change is larger at concentrations of 160μM and 320μM, which represent red blood cell lysis.

[0097] The 1% erythrocyte suspension prepared above was used. For the experimental group, 100 μL of erythrocyte suspension and 100 μL of CN-LPs-Arg sample (concentrations: 10 μM, 20 μM, 40 μM, 80 μM, 160 μM, 320 μM) were added to microcentrifuge tubes, respectively. The positive control was 100 μL of erythrocyte suspension treated with Triton X-100 diluted in sterile PBS (final concentration = 0.5 mg / mL), and the negative control was erythrocyte suspension treated with PBS. Both the control and experimental groups were incubated at 37℃ for 2 h. After centrifugation, 50 μL of the supernatant was transferred to a 96-well plate. The absorbance of the supernatant in both the control and experimental groups at 540 nm was measured spectrophotometrically to determine the hemolysis rate. The percentage calculation formula for the hemolysis rate is as follows:

[0098] Hemolysis rate % = (Abs) Compound -Abs Positive ) / (Abs Negative -Abs Blank )×100%,

[0099] Abs Compound Abs represents the absorbance of the CN nanoparticle sample treated (experimental group). Positive Abs represents the absorbance of the positive control. Negative Abs represents the absorbance of the negative control. Blank This indicates the absorbance of the blank control.

[0100] The results of the hemolysis rate are as follows Figure 7 As shown in Figure C, CN-LPs-Arg had no effect on erythrocyte viability at concentrations below 80 μM, and compared to the complete erythrocyte lysis induced by Triton X-100, CN nanoparticles exhibited no hemolytic toxicity. This indicates that CN-LPs-Arg nanoparticles are insufficient to cause significant host side effects.

[0101] The above embodiments are preferred embodiments of the present invention, but the embodiments of the present invention are not limited to the above embodiments. Any changes, modifications, substitutions, combinations, or simplifications made without departing from the spirit and principle of the present invention shall be considered equivalent substitutions and shall be included within the protection scope of the present invention.

[0102] References

[0103] 1.Li,Y.,Han,Y.,Zeng,Z.,Li,W.,Feng,S.,&Cao,W.(2021).Discovery andBioactivity of the Novel Lasso Peptide Microcin Y.Journal of agricultural andfood chemistry,69(31),8758–8767.https: / / doi.org / 10.1021 / acs.jafc.1c02659.

Claims

1. Use of chitosan nanoparticles in the manufacture of a product having an antibacterial effect, characterized in that, The chitosan nanoparticles comprise chitosan and one or more than one lariat peptide, the carboxyl group of the lariat peptide is connected to the amino group in the chitosan structure through an amide bond, the lariat peptide comprises MccJ25, MccY and Klebsidin, the chitosan nanoparticles further comprise arginine, the carboxyl group of the arginine is connected to the amino group in the chitosan structure through an amide bond; the product with antibacterial effect is feed.

2. Use according to claim 1, characterized in that, The molar ratio of the chitosan to the lariat peptide is 5:(3-4).

3. Use according to claim 1, characterized in that, The bacteria comprise Salmonella, Serratia and Klebsiella.

4. Use according to any one of claims 1 to 3, characterized in that, The chitosan nanoparticles are prepared by the following preparation method, which comprises the following steps: 1) mixing 1-ethyl-(3-dimethylaminopropyl) carbodiimide solution and N-hydroxysuccinimide solution to obtain an EDCN mixed solution; 2) dissolving chitosan, lariat peptide and arginine in acetylation solution respectively, mixing uniformly to obtain chitosan acetylation solution, lariat peptide acetylation solution and arginine acetylation solution; 3) adding the EDCN mixed solution in step 1) into the chitosan acetylation solution, the lariat peptide acetylation solution and the arginine acetylation solution in step 2) respectively, mixing uniformly to obtain chitosan-EDCN mixed solution, lariat peptide-EDCN mixed solution and arginine-EDCN mixed solution; 4) mixing the chitosan-EDCN mixed solution in step 3) with the lariat peptide-EDCN mixed solution and the arginine-EDCN mixed solution in turn, fully stirring to complete esterification reaction, thereby preparing the chitosan nanoparticles.

5. Use according to claim 4, characterized in that, The acetylation solution comprises sodium acetate solution, acetyl chloride solution, acetic anhydride solution and glacial acetic acid solution.

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