Novel anti-salmonella nano preparation CCJY as well as preparation method and application thereof

Abstract

The invention discloses a novel anti-salmonella nano preparation CCJY as well as a preparation method and application thereof. The preparation comprises chitosan nano particles. The chitosan nanoparticle comprises cross-linked chitosan, and a lasso peptide MccJ25 and a lasso peptide MccY which are loaded on the chitosan. According to the invention, the synergistic antibacterial effect among different lasso peptides is exerted through a chemical cross-linking technology, on the basis of integrating the original antibacterial types of the lasso peptide MccJ25 and the lasso peptide MccY, the antibacterial types are expanded to serratia marcescens and enterococcus cloacae, and the broad-spectrum antibacterial effect is realized. Meanwhile, the invention further discloses a preparation method and application of the chitosan nano-particles and an anti-salmonella nano-preparation containing the chitosan nano-particles.

Description

Technical Field

[0001] This invention belongs to the field of biotechnology, specifically relating to a novel anti-Salmonella nanoparticle formulation CCJY, its preparation method, and its application. Background Technology

[0002] Microcins (Mcc) are small peptides secreted by intestinal bacteria that exhibit bactericidal properties against a variety of pathogens. Lasso peptides, as the simplest and most structurally stable class of microcins, are small peptides secreted by bacteria, synthesized by ribosomes, and post-translational modified. Studies have elucidated that MccJ25 and MccY are novel, structurally stable lasso peptides with antibacterial activity against Enterobacteriaceae. MccJ25, composed of 21 amino acids, is a low-molecular-weight, plasmid-encoded, ribosome-synthesized lasso peptide, initially isolated from fecal strains of *Escherichia coli*. Due to its stable lasso structure and ribosome-encoding system, it can be combined with genetic engineering to exert potent biological activity. MccY is a novel, structurally stable microprotein. MccY possesses the advantages of trace amounts, high efficiency, and complementary bactericidal activity; its combination with MccJ25 broadens the bactericidal spectrum of lasso peptides against *Salmonella* and *Shigella*. Meanwhile, chitosan (CN) is obtained by deacetylation of chitin, which is abundant in nature. Due to its good biocompatibility, biodegradability, ability to inhibit bacterial growth, and modifiability, chitosan is currently widely used in animal-derived preservatives and has been officially approved by the Chinese National Food Safety Standard GB29941-2013. Although chitosan is widely used in animal-derived preservatives, its broad-spectrum antibacterial activity has significant limitations, such as an uneven antibacterial spectrum. Existing products show strong inhibitory activity against Staphylococcus aureus, Bacillus subtilis, Salmonella enteritidis, Salmonella typhimurium, Salmonella pullorum, and ATCC 25922 Escherichia coli, but its inhibitory effect on Serratia marcescens and Enterococcus cloacae is poor.

[0003] The technical problem to be solved by this invention is: how to construct an anti-Salmonella nanoparticle formulation that achieves broad-spectrum antibacterial efficacy while ensuring its biosafety meets the application requirements of food preservation materials. Summary of the Invention

[0004] To address the aforementioned technical problems, this invention provides chitosan nanoparticles comprising cross-linked chitosan and lasso peptides loaded onto the chitosan; the lasso peptides are lasso peptide MccJ25 and lasso peptide MccY. Through the combination of cross-linking technology and lasso peptides, in addition to retaining the original antibacterial effects, it also exhibits strong inhibitory effects against Serratia marcescens and Enterococcus cloacae.

[0005] To achieve the above-mentioned objectives, a first aspect of the present invention provides chitosan (CN) nanoparticles, comprising cross-linked chitosan and lasso peptides loaded on the chitosan; wherein the lasso peptides are lasso peptide MccJ25 and lasso peptide MccY.

[0006] Furthermore, the weight ratio of chitosan to lauryl peptide is 1:0.05~0.2.

[0007] In some preferred embodiments of the present invention, the weight ratio of chitosan to lauryl peptide is 1:0.07~0.15; In some preferred embodiments of the present invention, the weight ratio of chitosan to lauryl peptide is 1:0.09~0.13; Furthermore, the weight ratio of the lasso peptide MccJ25 to the lasso peptide MccY is 1~5:1~5; In some preferred embodiments of the present invention, the weight ratio of the lasso peptide MccJ25 to the lasso peptide MccY is 1~4:1~4. In some preferred embodiments of the present invention, the weight ratio of the lasso peptide MccJ25 to the lasso peptide MccY is 1~3:1~3;

[0008] Furthermore, the crosslinking agent is genipin and / or citric acid, and the weight ratio of chitosan to the crosslinking agent is 1:1 to 10.

[0009] In some preferred embodiments of the present invention, the weight ratio of chitosan to crosslinking agent is 1:1 to 9.

[0010] In some preferred embodiments of the present invention, the weight ratio of chitosan to crosslinking agent is 1:2 to 7.

[0011] In some preferred embodiments of the present invention, the weight ratio of chitosan to crosslinking agent is 1:3 to 6.

[0012] Lasso peptides (LPs) are bacterial-derived polypeptides that are bioactive peptides synthesized via ribosomes and modified post-translationally to form a lasso-like conformation. Their structural characteristics include: the first amino acid at the N-terminus forms a large amide ring with the side chain of the 7th, 8th, or 9th amino acid, through which the C-terminal peptide chain passes, forming a typical "lasso" topology. Based on the disulfide bond linkage, lasso peptides can be classified into four types. All four types retain a carboxyl-terminal group, and their structural diversity provides a practical basis for constructing novel chitosan-lasso peptide nanoparticles through chemical cross-linking techniques. This invention innovatively employs genipin and / or citric acid (CA) crosslinking agents to construct a novel chitosan-tau peptide nanoparticle (CNCA-MccJ25-MccY, abbreviated as CCJY) through chemical crosslinking technology. The study investigates its bactericidal effect against Salmonella and its practical application in the preservation of poultry products such as eggs and chilled meat via spraying, providing a basis for the application of antimicrobial peptides in poultry products.

[0013] In the preliminary experiments of this invention, it has been confirmed that after sufficient acetylation, chitosan can exhibit a superior antibacterial effect compared to the uncrosslinked product within a weight ratio of 0.05 to 0.2 with lauryl peptide. Meanwhile, preliminary experiments of this invention have also confirmed that whether MccJ25 and MccY are used alone or in combination, they can achieve a superior antibacterial effect compared to the uncrosslinked product. Therefore, within the above-mentioned proportion range, the present invention can achieve a relatively excellent antibacterial effect.

[0014] Secondly, the present invention provides a method for preparing the above-mentioned chitosan nanoparticles. The method specifically involves dispersing azathiopeptide in a chitosan solution containing chitosan, allowing the azathiopeptide and chitosan to react, and then adding a crosslinking agent to allow the chitosan to undergo a crosslinking reaction, thereby obtaining the chitosan nanoparticles.

[0015] Furthermore, the method specifically includes the following steps: 1) Dissolve chitosan particles in glacial acetic acid solution to obtain chitosan solution; 2) Disperse the tau peptide in the chitosan solution, mix, and obtain a mixed solution; 3) Add a crosslinking agent to the mixed solution and react to obtain the product solution; 4) The product solution is subjected to ultrasonic and dialysis treatment to obtain the chitosan nanoparticles.

[0016] Furthermore, in step 4) of the above preparation method, the ultrasonic frequency is 40 kHz, the ultrasonic duration is 30 min, and the ultrasonic temperature is 25 ℃.

[0017] Furthermore, in step 4) of the above preparation method, the dialysis treatment time is 24 hours, and the dialysis bag used for the dialysis treatment is a 3kDa dialysis bag.

[0018] Thirdly, the present invention provides the use of the above-mentioned chitosan nanoparticles in the preparation of products with antibacterial effects.

[0019] Furthermore, the above-mentioned products have an inhibitory effect on Serratia marcescens and Enterococcus cloacae.

[0020] A novel anti-Salmonella nanoparticle formulation, characterized in that the formulation comprises chitosan nanoparticles as described in any one of claims 1 to 4.

[0021] The beneficial effects of this invention are as follows: 1. This invention prepares chitosan nanoparticles comprising cross-linked chitosan and lassotide MccJ25 and lassotide MccY loaded onto the chitosan. Through chemical cross-linking technology, the synergistic antibacterial effect between lassotide MccJ25 and lassotide MccY is achieved. Building upon the original antibacterial species of lassotide MccJ25 and lassotide MccY, the antibacterial range is expanded to include Serratia marcescens and Enterococcus cloacae, achieving a broad-spectrum antibacterial effect.

[0022] 2. The CCJY prepared by this invention has high safety, does not cause red blood cell lysis at high concentrations, and cells can survive at high concentrations.

[0023] 3. The CCJY prepared by this invention can be applied to eggs and chicken in a spray state, and has the effects of sterilization and preservation, which can extend the shelf life of livestock and poultry products. Attached Figure Description

[0024] To make the objectives, technical solutions, and beneficial effects of this invention clearer, the following figures are provided for illustration: Figure 1 This is the CCJY synthesis route design diagram in Example 1; Figure 2 These are the FTIR analysis results of the nanomaterial CCJY and its derivatives CCJ, CCY, CNCA, and CA; Figure 3A This is a graph showing the particle size analysis results of CCJY. Figure 3B This is a graph showing the particle size analysis results of CCJ. Figure 3C This is a graph showing the particle size analysis results of CCY. Figure 3D This is a graph showing the particle size analysis results from CNCA; Figure 3EThis is a graph showing the particle size analysis results for CA. Figure 4A This is a characterization and analysis diagram of CCJY's Zeta potential; Figure 4B This is a characterization analysis diagram of the zeta potential in CCJ; Figure 4C This is a characterization analysis diagram of the Zeta potential of CCY; Figure 4D This is a characterization analysis diagram of the zeta potential of CNCA; Figure 4E This is a graph representing the Zeta potential characterization of CA; Figure 5 These are transmission electron microscope (TEM) images of two types of CN nanoparticles, CCJY and CNCA. Figure 6A The effect of different CCJY ratios on the encapsulation efficiency of the tau peptide; Figure 6B This is a comparison chart of the antibacterial effects of MccJ25+MccY(JY), JY-CN, JY-CN-Genipin, and JY-CNCA on SE63. Figure 7A The MTT assay was used to evaluate the changes in the survival rate of DF-1 cells after 24 hours of treatment with different concentrations of CCJY and its derivatives (CCJ, CCY, CNCA, CN). Figure 7B The effects of different concentrations of CCJY and its derivatives (CCJ, CCY, CNCA, CN) on the hemolytic activity of chicken erythrocytes were investigated. Figure 8A The nanomaterial CCJY, bacteriophage, and control group were used to investigate the effect of SE63 (1.0 × 10⁻⁶) on the surface of eggs. 8 The bactericidal effect diagram (CFU / mL) is shown, where S1 represents CKT1 bacteriophage (Shandong Agricultural University, Sun Shuhong) and S2 represents mixed bacteriophages ФPLL1 and ФF118P13 (Shandong Agricultural University, Sun Shuhong). Figure 8B This is a graph showing the bactericidal effect of the nanomaterial CCJY, bacteriophage, and control group on ST53 (1.0×108 CFU / mL) on the surface of eggs. S1 represents CKT1 bacteriophage (Shandong Agricultural University, Sun Shuhong), and S2 represents a mixed bacteriophage of ФPLL1 and ФF118P13 (Shandong Agricultural University, Sun Shuhong). Figure 8C The nanomaterial CCJY, bacteriophage, and control group were used to investigate the effect of SE63 (1.0 × 10⁻⁶) on the surface of chicken meat. 8 The bactericidal effect diagram (CFU / mL) is shown, where S1 represents CKT1 bacteriophage (Shandong Agricultural University, Sun Shuhong) and S2 represents mixed bacteriophages ФPLL1 and ФF118P13 (Shandong Agricultural University, Sun Shuhong). Figure 8D The nanomaterial CCJY, bacteriophage, and control group were used to investigate the effect of 1.0 × 10⁻⁶ on ST53 (1.0 × 10⁻⁶) on the surface of chicken meat. 8 The bactericidal effect diagram (CFU / mL) is shown, where S1 represents CKT1 bacteriophage (Shandong Agricultural University, Sun Shuhong) and S2 represents mixed bacteriophages ФPLL1 and ФF118P13 (Shandong Agricultural University, Sun Shuhong). Figure 9A Sensory images of eggs treated with 125 μg / ml CCJY and a water-treated control group at 0 d, 5 d, 10 d, 15 d, and 20 d. Figure 9B The weight loss rate of eggs treated with different concentrations of CCJY from 0 to 20 days; Figure 9C The yolk index of eggs treated with different concentrations of CCJY from 0 to 20 days. Figure 9D The protein index of eggs treated with different concentrations of CCJY from 0 to 20 days is shown. Figure 10A The images show the sample states of chicken meat at 0, 2, 4, and 8 days after treatment with different concentrations of CCJY. Figure 10B These are HE staining images of chicken meat treated with 125 μg / ml CCJY and the blank control group at 0 d, 2 d, 4 d, and 8 d. Detailed Implementation

[0025] The technical solutions in the embodiments of the present invention will be clearly and completely described below. Obviously, the described embodiments are only some embodiments of the present invention, and not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention.

[0026] The instrument information used in this embodiment is shown in Table 1 below: Table 1. Information on the instruments used Instrument Name company ZHJH-C1214C Clean Bench Suzhou Purification Equipment Co., Ltd. DK-8D Electric Heating Constant Temperature Water Bath Shanghai Yiheng Technology Co., Ltd. GDS8000PC Gel Imaging and Analysis System UVP Company ND-1000 spectrophotometer NanoDrop T3000 Thermocycler PCR instrument Whatman Biometra Micro-adjustable pipette Eppendorf, Germany Power Pac™ Basic Electrophoresis System BIO-RAD Company ordinary centrifuge Eppendorf, Germany Thermostatic Shaker Shanghai Zhichu Instrument Co., Ltd. Ultrasonic cleaning machine Clean Alliance Company 3 kDa dialysis bag Shanghai Yuanye Biotechnology Co., Ltd. Chitosan CN Sigma-Aldrich Citric acid (CA) Merck glacial acetic acid Merck Penicillin-Streptomycin Gibco™ ThermoFisher Technologies DMEM Gibco™ ThermoFisher Technologies Fetal bovine serum FBS Gibco™ ThermoFisher Technologies Kinnipin Merck M9 culture medium Coolaber Company

[0027] Unless otherwise specified, all experimental materials and reagents used are commercially available consumables and reagents.

[0028] Statistical analysis was performed using GraphPad Prism 10.0 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.

[0029] Example 1: Chemical Synthesis of CCJY, CCJ, CCY, and CNCA In this embodiment, secreted proteins were obtained using an E. coli prokaryotic expression system, and two lasso peptides (MccJ25 and MccY) were prepared by HPLC. These peptides were then cross-linked with chitosan (CN) via chemical reaction to obtain multifunctional nanomaterials.

[0030] Figure 1 The synthesis process was described in detail, in which the amino groups of CN and the carboxyl groups of lauryl peptide were linked by amide bonds to synthesize various CN nanoparticles. Among them, the carboxyl groups of MccJ25 and MccY were sequentially crosslinked with CN under the action of crosslinking agent CA to form CCJ, CCY, and CCJY. The optimized formulation of these nanomaterials achieved an encapsulation efficiency of over 90% w / w in CN (e.g., ...). Figure 6A The synthesis process of CCJY is as follows: The expression and preparation of the two lasso peptides involved introducing the vector pET-32a(+) into BL21(DE3) competent cells via heat shock. After incubation at 37 ℃ for 16 h, BL21(DE3) bacteria containing pET-32a(+) were obtained. Single colonies were picked and inoculated onto LB agar plates containing 50 µg / mL kanamycin. The plates were incubated upside down at 37 ℃ for 12 h to activate the bacterial strain. Activated single colonies were then picked with a sterile inoculating loop and transferred to a 2 L Erlenmeyer flask containing 800 mL of LB liquid medium. Kanamycin was added to a final concentration of 50 µg / mL, and the flasks were incubated at 37 ℃ and 200 rpm on a shaker until the bacterial density reached OD. 600 When the concentration reached 0.6-0.8, isopropyl-β-D-thiogalactoside (IPTG) was added to a final concentration of 0.2 mM. The culture was then continued at 37 ℃ and 200 rpm for 48 hours. The expressed bacterial culture was collected, centrifuged at 6000 r / min for 10 min, and the supernatant was collected. The supernatant was filtered through a 0.22 µm filter to obtain a crude extract containing the target protein. The crude extract was then enriched with the target protein using a C18 reversed-phase solid-phase extraction column at a flow rate of 1 mL / min. The enriched sample was sequenced using Q-TOF mass spectrometry, and after matching the theoretical molecular weight, it proceeded to the purification stage. Preparative high-performance liquid chromatography (HPLC) was used to separate and purify the target protein (mobile phase: acetonitrile-water gradient elution, flow rate 5 mL / min), and the main peak fraction was collected. The protein was concentrated under reduced pressure at 40 °C using a rotary evaporator, and then dried using vacuum freeze-drying technology to obtain a white powdery target protein. After quantification by the BCA method, the protein was stored at -20 °C.

[0031] It should be noted that the above is only one of the methods for preparing lasso peptides. MccJ25 and MccY are very common lasso peptides in the field, and their preparation methods have been disclosed in various literatures. The expression of lasso peptides in this invention is not limited to the above methods.

[0032] Synthesis of CCJY: 0.1 g chitosan particles were completely dissolved in 10 ml of 1 wt% glacial acetic acid solution and stirred continuously until a homogeneous and transparent solution was formed, yielding a chitosan acetylated solution. 10 ml of cassopeptide (formed by mixing 5 ml MccJ25 solution and 5 ml MccY solution, with a cassopeptide concentration of 1 mg / ml in both MccJ25 and MccY solutions) was added separately to the chitosan acetylated solution. The mixture was stirred continuously at 300 rpm for 2 hours using a magnetic stirrer until homogeneous, yielding a chitosan and cassopeptide acetylated solution. 3 wt% citric acid crosslinking agent, equivalent to the total weight of the aforementioned chitosan and cassopeptide acetylated solution, was slowly added dropwise to this solution to promote the formation of the chitosan-cassopeptide network structure through non-covalent interactions. The prepared chitosan nanoparticles were transferred to an ultrasonic processor, with the working frequency set to 40 kHz and the temperature precisely controlled at 25 ± 1 ℃, for segmented ultrasonic treatment: first, treatment with 30% amplitude for 10 minutes, followed by treatment with 50% amplitude... Amplitude treatment for 20 minutes promotes uniform dispersion of nanoparticles; using a MWCO 3000 benzoyl cellulose dialysis tube, the sonicated solution is loaded into the dialysis tube and completely immersed in distilled water, and three cycles of dialysis are performed at room temperature: fresh distilled water is replaced after 8 hours of each dialysis, with a total dialysis time of 24 hours, effectively removing unreacted small molecule impurities and byproducts; finally, CCJY is obtained, and the obtained solution is freeze-dried for later use.

[0033] The preparation methods of CN-MccJ25 (CCJ), CN-MccY (CCY), and CNCA, which serve as control samples, are similar to those of CCJY.

[0034] It should be noted that: CN-MccJ25 (CCJ) refers to MccJ25 loaded on cross-linked chitosan; CN-MccY (CCY) essentially means MccY loaded on cross-linked chitosan; CNCA refers to cross-linked chitosan.

[0035] Meanwhile, the present invention also prepared uncrosslinked samples, namely JY (CCJ+CCY) and CN-JY (CCJ+CCY+CN, which are basically the same as the above experiments except that no crosslinking agent was used); at the same time, samples JY-CN-genipin (CCJ+CCY+CN+3wt% genipin crosslinking agent) and JY-CNCA (CCJY mentioned above) were also used when the gradient dilution method was used for detection.

[0036] JY (CCJ+CCY) refers to a 1 mg / ml solution of tau peptide.

[0037] Example 2: Characterization analysis of CCJY, CCJ, CCY, and CNCA The conjugation efficiencies of CCJY, CCJ, CCY, CNCA, and CN were investigated using Vertex 70 Fourier Transform Infrared Spectrometer (FTIR). Figure 2 As shown, in the CN infrared spectrum, 3346 cm⁻¹ -1 The position corresponds to the stretching vibration absorption of OH and NH on chitosan. At 2925 cm⁻¹ -1 and 2875 cm -1 The nearby bimodal absorption is due to the asymmetric stretching of CH. Chitosan exhibits three main amide characteristic peaks, located at 1635 cm⁻¹, 1541 cm⁻¹, and 1377 cm⁻¹. -1 Nearby, there are tensile vibrations of CO (amide I band), bending vibrations of NH (amide II band), and bending vibrations of CN (amide III band). The range is 10¹³–10⁵⁸ cm⁻¹. -1 The nearby peak is the absorption peak of CO stretching vibration. In CNCA, CCJ, CCY, and CCJY, it is at 3345 cm⁻¹. -1 The NH stretching vibration absorption peak at [value] is broadened and moderately shifted, due to the stretching vibration of the secondary amine (-NH). [Value] 2875-2925 cm⁻¹ on protochitosan. -1 The stretching vibration absorption peak at CH was moderately shifted and broadened, confirming the simultaneous presence of CA, MccJ25, MccY, MccJ25, and MccY. (1640 cm⁻¹) -1 The absorption peak near the O stretching vibration of the C double bond is at 1401 cm⁻¹. -1 The vicinity is near the bending absorption vibration peak of CN, at 1060 cm⁻¹. -1 The absorption peak is near the stretching vibration of CO. Additionally, NH has an absorption peak at 1535 cm⁻¹. -1 The broadening of the absorption peak at the bending vibration further confirms the consumption of the primary amine and the formation of the secondary amine. In summary, CN, MccJ25, and MccY successfully crosslinked via amide bonds to form CCJY.

[0038] The particle size and zeta potential of the synthesized compound were determined using a Malvern ZS-90 zetaizer at 25 °C. Figures 3A to 3E As shown, after modification, CCJY ( Figure 3A ), CCJ ( Figure 3B ), CCY Figure 3C ), CNCA ( Figure 3D ) and CA ( Figure 3E The static number average particle size increased slightly, ranging from 400 to 800 nm; like Figures 4A to 4E As shown, CCJY ( Figure 4A ), CCJ ( Figure 4B ), CCY Figure 4C ), CNCA ( Figure 4D ) and CA ( Figure 4E The lower molecular weight 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.

[0039] Example 3: Transmission electron microscopy analysis of CCJY, CCJ, CCY, and CNCA CCJY nanoparticles were resuspended in 0.1 M acetic acid solution to prepare a 1 mg / mL CCJY nanoparticle dispersion system using precise weighing. The dispersion was then treated with a vortex mixer at 2500 rpm for 5 minutes to ensure uniform dispersion. 20 μL of the suspension was precisely added dropwise to a 200-mesh copper mesh support using a micropipette and allowed to stand at room temperature for 10 minutes. Negative staining was then performed: the copper mesh was immersed in a 2% (w / v) phosphotungstic acid aqueous solution for staining for 3 minutes. Excess staining solution was gently blotted away with filter paper to avoid damaging the sample structure. High-resolution imaging of the samples was performed using an HT7700 and a Hitachi Su-8010 transmission electron microscope. The structures and particle sizes of the CNCA and CCJY nanoparticles are shown below. Figure 5 As shown. In Figure 5 Among them, CCJY has a near-spherical regular structure with high stability and a particle size between 400-800 nm, while CNCA exhibits an irregular network structure with a rough and porous surface.

[0040] Example 4: Antibacterial activity of CCJY, CCJ, CCY, CNCA, and CN The synthesized samples (CCJY, CCJ, CCY, CNCA, CN) were subjected to MIC testing. The minimum inhibitory concentration (MIC) was determined using a 96-well plate serial dilution method for CCJY, CCJ, CCY, MccJ25-MccY, MccJ25, and MccY. MIC testing allows for accurate quantification of the antibacterial activity of microinfectants against different types of bacteria. The tested bacterial strains included Gram-negative bacteria (such as Salmonella enteritidis, Salmonella typhimurium, Salmonella pullorum, Escherichia coli, and Serratia marcescens) and Gram-positive bacteria (such as Staphylococcus aureus and Bacillus subtilis).

[0041] Table 2. Strains used in Example 4 strain Related information source SE63 Salmonella enteritidis, wide type strain Reference 1 ST53 Salmonella typhimurium, wide type strain Reference 1 SP284 Salmonella pullorum Reference 1 25922 Escherichia coli ATCC 25922 ATCC Seromorpha Serratia marcescens ATCC Enterobacteriaceae Enterobacter cloacae ATCC Staphylococcus aureus Staphylococcus aureus, S. aureus ATCC Bacillus subtilis Bacillus subtilis ATCC

[0042] As shown in Table 3, CCJY effectively inhibits both Salmonella (pullorum disease / typhoid / enteritis) and Escherichia coli, with MIC values ​​consistently ranging from 25 to 50 μg / mL. Its inhibitory effect on Enterococcus cloacae (25 μg / mL) is superior to CCJ (12.5 μg / mL), forming a concentration gradient coverage with CCY (1.56 μg / mL). CCJY's structural optimization expands its antibacterial spectrum, resulting in a broad-spectrum and highly effective inhibitory effect.

[0043] Table 3 MIC results of CN nanoparticles

[0044] Simultaneously, this application also conducted concurrent detection on JY, CN-JY, JY-CN-genipin, and JY-CNCA, and the detection results are referenced. Figure 6B , Figure 6B The figures show the inhibition zone sizes of JY, ​​CN-JY, JY-CN-genipin, and JY-CNCA against SE63 bacteria (diluted one well at a time using a serial dilution method). Figure 6B It is evident that JY-CN-genipin and JY-CNCA are significantly superior to JY and CN-JY, indicating that cross-linking treatment is the core factor in improving antibacterial efficacy.

[0045] Example 5: Biocompatibility analysis of CCJY, CCJ, CCY, CNCA, and CN To assess the effect of CCJY on cellular metabolic activity, an MTT assay was performed. The specific procedures were as follows: DF-1 cells were first cultured at 37°C and 5% CO2, with the addition of 10% fetal bovine serum and 1% penicillin / streptomycin. 200 μL of cell suspension was seeded into 96-well plates and cultured for 24 h. Cells were then treated with different concentrations of CCJY, CCJ, CCY, CNCA, and CN, with 200 μL of diluted CN derivative solution added to each well, and incubated for 24 h. 20 μL of MTT solution (5 mg / mL, DPBS) was added to all wells, and incubation continued at 37°C and 5% CO2 for 3.5 h. The absorbance at 560 nm was measured, and the background absorbance at 670 nm was simultaneously measured. The background absorbance was subtracted from the former to obtain the corrected absorbance value. The corrected absorbance values ​​were normalized by combining absorbance values ​​from 1% Triton X-100 treatment (representing 100% cell death, as a positive control) and no treatment (representing 0% cell death, as a negative control).

[0046] like Figure 7AAs shown, the results indicate that within the range of minimum inhibitory concentration (MIC) to high-level MIC, the survival rate of DF-1 cells gradually decreased with increasing concentration of CCJY and its derivatives. In the low concentration range (1×MIC~8×MIC), the cell survival rate of CCJY and its derivatives remained between 95% and 100%, showing no significant difference from the negative control (untreated group, survival rate ≈100%) (p>0.05). In the medium-to-high concentration range (16×MIC~32×MIC), the cell survival rate of CCJY and its derivatives showed a slight concentration-dependent decrease, but the decrease was limited, and the survival rate was less than 80%, indicating significantly better safety than the positive control. This suggests that CCJY and its derivatives have no significant toxicity to host cells within the effective antibacterial concentration range. Specifically, at the high-level MIC (32×MIC), the cell survival rate of CCJY was 90%±2%, higher than other derivative groups, indicating that CCJY has superior cell compatibility, providing a safety basis for its application as a potential antibacterial agent.

[0047] The effect of CCJY on mammalian cell activity was evaluated to assess its biocompatibility. The specific steps were as follows: Chicken erythrocytes were collected and added to a PBS solution containing CCJY, CCJ, CCY, CNCA, and CN at a volume ratio of 1:1. Simultaneously, 10% Triton X-100 was used as a positive control, and pure PBS as a negative control. The mixture was incubated at 37°C for 45 min, followed by centrifugation at 2000×g for 10 min, and the supernatant was collected. The absorbance of the supernatant at 540 nm was measured in a 96-well plate to calculate the hemolysis percentage. The hemolysis percentage was calculated using the formula: Erythrocyte hemolysis % = (Absorbance of the microbial treatment group - Absorbance of the PBS treatment group) / (Absorbance of the Triton X-100 treatment group - Absorbance of the PBS treatment group) × 100%. This hemolysis assay method can be used to evaluate the lysing effect of microbial agents on erythrocytes.

[0048] Experimental results are as follows Figure 7B As shown, the hemolytic activity results of erythrocytes indicate that the hemolysis rate of erythrocytes gradually increases with the increase of CCJY and its derivatives concentration, but the hemolysis rate is less than 5% in all cases. This indicates that CCJY and its derivatives have no significant hemolytic toxicity to erythrocytes within the effective antibacterial concentration range, providing safety support for their clinical application as potential antibacterial agents.

[0049] Example 6: Disinfection effect of CCJY on eggshells and chilled meat surfaces Experimental Salmonella contamination of eggs and its treatment: In a laminar flow hood, the surface of the eggs was treated with a 1.0 × 10⁻⁶ solution. 8The eggs were challenged with a spray of CFU / mL Salmonella enteritidis SE63, with 1 mL of bacteria per egg. The eggs were then air-dried in a clean bench for 30 min to allow the Salmonella to adhere to the egg surface. After contamination, the eggs were treated in three groups: the experimental group (equal volumes of 250 μg / mL, 125 μg / mL, and 62.5 μg / mL CCJY sprayed onto the egg surface); the phage group (8.0 × 10⁻⁶ CFU / mL CCJY sprayed onto the egg surface); and the phage group (equal volumes of CCJY sprayed onto the egg surface). 10 Equal volumes of PFU / mL S1 and S2 bacteriophages were sprayed onto the egg surface; in the control group, equal volumes of CNCA and sterile water were sprayed onto the egg surface. Salmonella bacterial suspension, CCJY treatment group, bacteriophage suspension, and sterile water were sprayed manually at a rate of approximately 0.125 mL / spray. Eggs from each group were placed in separate 25 ℃ incubators. At 1 h, 6 h, 12 h, 24 h, 36 h, and 48 h, three eggs from each group were collected and placed in sterile sampling bags pre-filled with 100 mL of sterile water. The bags were then treated with an ultrasonic vibrator for 30 s to obtain the egg washing solution. The potency of the egg washing solution was tested. The treatment method for the challenged Salmonella typhimurium ST53 was similar. The experiment evaluated the bactericidal ability of CCJY on the eggshell surface.

[0050] Figure 8A The numbers represent different concentrations of CCJY treatment groups, 8.0 × 10⁻⁶. 10 Line graph showing the bactericidal effect of PFU / mL phage treatment group, CNCA control group and blank control group on SE63 on egg surface within 48 h. Figure 8B The results show the changing trends of the bactericidal effects of each treatment group on the ST53 strain. The results also show that the colony counts in the CCJY treatment groups (250 μg / mL, 125 μg / mL, 62.5 μg / mL) showed a slow upward trend over time, but the colony counts in the high-concentration groups (250 μg / mL, 125 μg / mL) were significantly lower than those in the low-concentration group (62.5 μg / mL) at all time points. Figure 8AIt can be seen that the colony count of 250 μg / mL CCJY was stable at 6.3±0.5 lg cfu / mL at 48 h, which was significantly lower than that of the 62.5 μg / mL group (8.0±0.7 lg cfu / mL, p<0.01), but slightly higher than that of the phage group; the colony count of 125 μg / mL CCJY at 48 h was 6.7±0.4 lg cfu / mL, which was not significantly different from that of the 250 μg / mL group (p>0.05); the colony count of 62.5 μg / mL CCJY at 48 h reached 8.0±0.7 lg cfu / mL, which was lower than that of the blank control (8.6±0.6 lg cfu / mL), but the difference was not significant (p=0.08), indicating that the bactericidal ability of low concentration CCJY is limited, and 125 μg / mL is the optimal concentration of CCJY for egg surface disinfection. The colony counts in the phage treatment groups (S1, S2) remained lower than those in the control group and higher than those in the CCJY treatment group over 48 hours, with S1 showing a slightly better effect than S2. The colony counts in the control groups (Ctrl, CNCA) increased significantly over time, with the CNCA group showing a colony count change highly consistent with the blank control (8.3 ± 0.5 lg cfu / mL at 48 h), indicating that CNCA had no significant bactericidal activity. Figure 8B Similarly, it can be concluded that 125 μg / mL is the optimal concentration of CCJY for egg surface disinfection.

[0051] Fresh chilled meat samples were purchased from a local supermarket and stored at 4 °C. The ability of CCJY to reduce Salmonella (Salmonella enteritidis and Salmonella typhimurium) contamination in fresh chilled meat was assessed using the following procedures:

[0052] Cut the chicken into pieces with a surface area of ​​1 g using a sterile knife. Sterilize both sides under a UV lamp for 30 minutes each to obtain a sterile meat sample. Place the meat sample in the center of a petri dish and take a 1.0 × 10⁻⁶ cm² piece. 8 CFU / mL Salmonella enteritidis SE63 was sprayed onto the surface of chicken meat and spread evenly using a sterile swab to obtain artificially contaminated Salmonella. The sample was then air-dried in a laminar flow hood for 30 min to allow the host bacteria to fully adsorb onto the sample surface. Next, the meat samples were treated in three groups: the experimental group was sprayed with equal volumes of 250 μg / mL, 125 μg / mL, and 62.5 μg / mL CCJY onto the chicken meat surface; the phage group was treated with a concentration of 8.0 × 10⁻⁶ CFU / mL... 10 Equal volumes of PFU / mL S1 and S2 phages were sprayed onto the surface of eggs; for the control group, equal volumes of CNCA and sterile water were sprayed onto the surface of eggs. Spraying was done manually at a rate of approximately 0.125 mL / spray. After spraying, the chilled meat was refrigerated at 4 °C, and the total bacterial count was measured at 1 h, 2 h, 12 h, 24 h, 48 h, and 72 h. The treatment method for challenging the ST53 Salmonella typhimurium was the same.

[0053] Figure 8C The graph shows the bactericidal effect of different treatment groups on the surface of chilled fresh meat against SE63 within 72 hours. Figure 8D The graph shows the bactericidal effect of different treatment groups on ST53 on the surface of chilled meat during the same period. The results showed that the colony count of the CCJY treatment groups (250 μg / mL, 125 μg / mL, 62.5 μg / mL) increased over time. The colony count of the high concentration groups (250 μg / mL, 125 μg / mL) was significantly lower than that of the low concentration group (62.5 μg / mL) at all time points. Moreover, the colony count of the 125 μg / mL group was 1.5~2.0 lg cfu / g lower than that of the 62.5 μg / mL group at 72 h. The colony count of the phage treatment groups (S1, S2) was consistently lower than that of the control group and higher than that of the CCJY treatment group within 72 h. The colony count of the control group (Ctrl, CNCA) changed less over time. The blank control group maintained a high level (11.6 lg cfu / g), while the CNCA group was slightly lower (11.0 lg cfu / g). The results show that the bactericidal effect of CCJY on the surface of chilled fresh meat is concentration-dependent. Under the condition of balancing bactericidal efficiency and cost-effectiveness, 125 μg / mL is the optimal concentration of CCJY for the bactericidal effect on the surface of chilled fresh meat.

[0054] Example 7: CCJY's evaluation of the quality preservation of eggs and fresh meat CCJY's evaluation of egg quality preservation includes: ① Sensory evaluation, through observation and recording of the egg white, chalaza, and yolk condition. ② The egg weight loss rate is positively correlated with storage time. Weight loss mainly refers to the rate of moisture evaporation, which is related to the eggshell, shell coating, and internal components, and is also affected by storage temperature and humidity. Weight loss rate = (G1-G2) / G1×100%, where G1 is the egg weight before storage and G2 is the egg weight after storage. ③ Yolk index determination and ④ Albumen index determination: using yolk index and albumen index determination methods, the significant effects of CCJY on extending egg shelf life, maintaining albumen consistency, and yolk stability are explored, thus demonstrating CCJY's protective effect on the internal structure of eggs.

[0055] The results are as follows Figures 9A to 9D As shown, Figure 9A In the 125 μg / mL CCJY treatment group, as the storage time increased, the concentrated albumen gradually decreased until it mixed with the thin albumen after 20 days. The chalaza also gradually thinned and fell off, but the yolk remained intact. In the control group, the concentrated albumen almost disappeared after 10 days, the chalaza disappeared after 15 days, and the yolk ruptured after 20 days. This shows that CCJY has a certain preservation effect on eggs and can extend the shelf life of eggs. Figure 9BC, D, and C represent the weight loss rate of eggs, yolk index, and albumen index of CCJY at different concentrations, respectively, over time. Figure 9B It can be seen that the weight loss rate in the 250 μg / mL group increased from 0.32% on day 5 to 0.82% on day 20, significantly higher than the blank control (p < 0.01). The weight loss rate in the 125 μg / mL group increased from 0.31% on day 5 to 0.78% on day 20, with no significant difference from the 250 μg / mL group (p > 0.05). The weight loss rate in the 62.5 μg / mL group increased from 0.3% on day 5 to 1.2% on day 20, significantly lower than the high concentration group (p < 0.01) and lower than the blank control group, indicating that CCJY can reduce the weight loss of eggs. Figure 9C As can be seen, the protein index of the 250 μg / mL group decreased from 0.598 on day 5 to 0.437 on day 20, significantly lower than that of the blank control (p < 0.01); the protein index of the 125 μg / mL group decreased from 0.581 on day 5 to 0.431 on day 20, with no significant difference from the 250 μg / mL group (p > 0.05); the protein index of the 62.5 μg / mL group decreased from 0.581 on day 5 to 0.397 on day 20, significantly higher than that of the high-concentration control group, while the protein index of the blank control decreased from 0.587 to 0.351, indicating that CCJY can effectively maintain protein consistency. Figure 9D As can be seen, the yolk index of the 250 μg / mL group decreased from 0.457 on day 5 to 0.358 on day 20, significantly lower than that of the blank control (p < 0.01). The yolk index of the 125 μg / mL group decreased from 0.455 on day 5 to 0.352 on day 20, with no significant difference from the 250 μg / mL group (p > 0.05). The yolk index of the 62.5 μg / mL group decreased from 0.461 on day 5 to 0.319 on day 20, significantly higher than that of the control group. The yolk index of the blank control decreased from 0.451 to 0.255, indicating that CCJY can significantly inhibit yolk flattening and maintain yolk stability. In conclusion, the preservation effects of 250 μg / mL and 125 μg / mL CCJY on eggs are better than those of 62.5 μg / mL CCJY, and both are superior to the blank control group.

[0056] CCJY's evaluation of chilled meat quality preservation: ① Sensory evaluation: According to the sensory requirements stipulated in GB 2707-2016 "National Food Safety Standard for Fresh (Frozen) Livestock and Poultry Products", the samples were placed on white trays, and sensory evaluators evaluated the color, odor, and state of the samples in sequence under natural light. ② The state of the meat under hematoxylin-eosin (HE) staining was used to preliminarily determine the improvement effect of CCJY on chilled meat preservation.

[0057] The results are as follows Figure 10A and Figure 10B As shown, Figure 10A The sensory images show the effects of different concentrations of CCJY on chicken meat from 0 to 8 days. It can be seen that in the 250 μg / mL CCJY group, the meat color gradually deepened from an initial light pink (0 days) to a deep orange-yellow after 8 days of storage, but the overall color was uniform, with no localized blackening or greening. Furthermore, the odor remained slightly gamey at 8 days, without any putrid or sour smell. In the 125 μg / mL CCJY group, the meat color turned deep yellow at 8 days, with a smaller color difference compared to the 250 μg / mL group, indicating that medium to high concentrations of CCJY can effectively delay meat color deterioration. In the 62.5 μg / mL CCJY group, the meat color was yellow at 8 days, with a more significant color difference than the blank control, but the deterioration rate was slightly faster than the high-concentration group. In the blank control group, the meat color was yellow and uneven at 8 days, with localized brown spots, a strong odor, a noticeable sour smell, and a sticky surface. Figure 10B As shown in the HE staining images of chilled meat treated with 125 μg / mL CCJY, at 0 days, cells were tightly packed with small intercellular spaces (<5 μm) and clearly visible nuclei. At 4 days, the intercellular spaces increased to 10-15 μm, and the nuclei showed slight condensation but intact structure. At 8 days, the intercellular spaces further increased to 20-25 μm, and the nuclei's edges became blurred but not completely disappeared. In the blank control group, cells were tightly packed at 0 days, showing no difference from the treated group. At 4 days, the intercellular spaces increased to 15-20 μm, and the nuclei began to dissolve, with some areas showing nuclei disappearing. At 8 days, the intercellular spaces disappeared (>30 μm), the nuclei were completely dissolved, and the tissue became loose. In conclusion, CCJY can significantly delay the deterioration of the sensory quality of chilled meat, and 125 μg / mL is the optimal preservation concentration for CCJY. While extending shelf life (≥8 days), it minimizes sensory deterioration and cell structure damage, providing a practical solution for the preservation of chilled meat.

[0058] In summary, this invention innovatively uses citric acid as a cross-linking agent to promote the chemical reaction between cetylpyridinium MccY, cetylpyridinium MccJ25, and chitosan to form chitosan nanoparticles. The chemical cross-linking technology leverages the synergistic antibacterial effect between cetylpyridinium MccJ25 and cetylpyridinium MccY, expanding the antibacterial spectrum to include Serratia marcescens and Enterococcus cloacae, achieving a broad-spectrum antibacterial effect. CCJY prepared using the aforementioned nucleopolysaccharide nanoparticles exhibits significant safety advantages: experiments show that even at high concentrations, CCJY does not induce erythrocyte lysis, and cell viability remains good. The provided CCJY can be sprayed onto eggs and chicken for efficient sterilization and extended shelf life, significantly delaying spoilage of poultry products.

[0059] In summary, CCJY, as a novel nano-spray formulation, combines broad-spectrum antibacterial properties, high safety, and convenient application, providing an innovative solution for the preservation and shelf-life extension of livestock and poultry products.

[0060] The above-described embodiments prepared by the present invention are preferred embodiments of the present invention. However, the embodiments of the present invention are not limited to the above-described 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.

[0061] References

[0062] 1. Li, Y., Han, Y., Zeng, Z., Li, W., Feng, S., & Cao, W. (2021). Discovery and Bioactivity of the Novel Lasso Peptide Microcin Y. Journal of agricultural and food chemistry, 69(31), 8758–8767. https: / / doi.org / 10.1021 / acs.jafc.1c02659.

Claims

1. A chitosan nanoparticle, characterized in that, It includes cross-linked chitosan and lasso peptides loaded on chitosan; the lasso peptides are lasso peptide MccJ25 and lasso peptide MccY.

2. The chitosan nanoparticles according to claim 1, characterized in that, The weight ratio of chitosan to lauryl peptide is 1:0.05~0.

2.

3. The chitosan nanoparticles according to claim 1, characterized in that, The weight ratio of the lasso peptide MccJ25 to the lasso peptide MccY is 1~5:1~5.

4. The chitosan nanoparticles according to claim 1, characterized in that, The crosslinking agent is genipin and / or citric acid, and the weight ratio of chitosan to crosslinking agent is 1:1~10.

5. A method for preparing chitosan nanoparticles as described in any one of claims 1 to 4, characterized in that, The method specifically involves dispersing tau peptides in a chitosan solution containing chitosan, allowing the tau peptides and chitosan to react, and then adding a crosslinking agent to allow the chitosan to undergo a crosslinking reaction, thereby obtaining the chitosan nanoparticles.

6. The preparation method according to claim 5, characterized in that, The method specifically includes the following steps: 1) Dissolve chitosan particles in glacial acetic acid solution to obtain chitosan solution; 2) Disperse the tau peptide in the chitosan solution, mix, and obtain a mixed solution; 3) Add a crosslinking agent to the mixed solution and react to obtain the product solution; 4) The product solution is subjected to ultrasonic and dialysis treatment to obtain the chitosan nanoparticles.

7. The preparation method according to claim 6, characterized in that, In step 4), the ultrasound frequency is 40 kHz, the ultrasound duration is 30 min, and the ultrasound temperature is 25℃; the dialysis treatment time in step 4) is 24 hours, and the dialysis bag used in the dialysis treatment is a 3 kDa dialysis bag.

8. Use of the chitosan nanoparticles as described in any one of claims 1 to 5 in the preparation of products with antibacterial properties.

9. The use according to claim 8, characterized in that, The product has an inhibitory effect on Serratia marcescens and Enterococcus cloacae.

10. A novel anti-Salmonella nanoparticle formulation, characterized in that, The formulation comprises chitosan nanoparticles as described in any one of claims 1 to 4.

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