Antimicrobial peptide with acid and alkali resistance and thermal stability as well as preparation method and application of antimicrobial peptide

Abstract

Provided is an acid and alkali resistant and thermostable antimicrobial peptide designed to withstand extreme pH conditions, high temperatures, and maintain its native conformation, and more specifically, to bind the antimicrobial peptide to a structural locking component through non-covalent interaction to effectively maintain its bioactive conformation. Further, the antimicrobial peptide is encapsulated with a controlled release encapsulation material to achieve controlled release and sustained release in various applications.

Description

CROSS-REFERENCE TO RELATED APPLICATIONS

[0001] This application claims priority to U.S. Application No. 18 / 506,144, filed on November 10, 2023, the disclosure of which is incorporated herein by reference in its entirety. Technical Field

[0002] The present invention relates to the field of food science. More specifically, the present invention relates to an acid- and alkali-resistant and heat-stable antimicrobial peptide. Background Art

[0003] Chemical preservatives are commonly used in a variety of products to extend shelf life, but their long-term adverse effects are often underestimated. The overuse of preservatives has also led to the emergence of resistant microorganisms, which is a major concern for industries such as food, beverages, cosmetics, personal care and pharmaceuticals, but has also stimulated the awareness of the need to develop healthier and safer preservatives for a variety of commodities.

[0004] Antimicrobial peptides (AMPs) are naturally derived peptide compounds that are potential alternatives to chemical preservatives. They exhibit strong antibacterial properties and are effective against most microorganisms with low toxicity. However, these peptide-based compounds are sensitive to changes in pH and temperature and are easily affected by enzymatic degradation and hydrophobic aggregation in different environments. These instabilities limit the application of antimicrobial peptides in a variety of products.

[0005] Many patent applications in this field introduce various technologies for protecting antimicrobial peptides. For example:

[0006] (1) US20200231638A1 proposes several methods to enhance the stability of antimicrobial peptides, including one involving hydrocarbon binding, which introduces synthetic hydrocarbon cross-links between two amino acids of the peptide to stabilize its structure; another method uses a lactam bridge, which strengthens the structure of the peptide by forming a covalent bond between two amino acid side chains. The patent also explores other stabilizers, such as disulfide bonds and unnatural amino acids, which can stabilize antimicrobial peptides and improve their effectiveness against bacterial infections.

[0007] (2) US9617309B2 provides stapled peptides with proline locks and their applications, which include a method for stabilizing peptides by introducing a stapler that can covalently bind to two amino acids, thereby limiting variability and enhancing stability. These stapled peptides have the potential to treat diseases such as cancer and viral infections. The patent also outlines the synthesis and screening methods of stapled peptides, and their pharmaceutical compositions.

[0008] (3) WO2011116963A2 provides a method for preparing lipid nanoparticles suitable as carriers for active agents (such as drugs or cosmetic ingredients). The method includes emulsifying a mixture of lipids, polymers and cationic agents in an aqueous solution, solidifying the emulsion to form solid lipid nanoparticles, and coating the nanoparticles with a second layer of polymer to enhance stability and regulate the release of the active agent. The patent includes specific examples of lipid nanoparticles and emphasizes their potential for use in drug delivery and cosmetic formulations.

[0009] (4) US20230035898A1 introduces cyclodextrin-protein-drug conjugates. This patent application focuses on the use of cyclodextrins (cyclic oligosaccharides) to conjugate drugs to proteins. This combination can enhance the pharmacokinetics of drugs, including solubility, stability and half-life in the body. The patent emphasizes the combination of cyclodextrins with antibodies, which can be used to treat various diseases.

[0010] However, these references seldom study the dual protection methods (structural restriction and encapsulation) of antimicrobial peptides and peptide-based substances in depth to resist environmental challenges and enzymatic digestion. Therefore, there is an urgent need to develop antimicrobial peptides with better stability in different environments to synergistically protect and maintain their antibacterial effects, and the present invention is intended to address these needs. Summary of the invention

[0011] The object of the present invention is to provide a compound, method or application for solving the above technical problems.

[0012] According to a first aspect of the present invention, a kind of acid and alkali resistant and thermostable antimicrobial peptide is provided.Especially, the acid and alkali resistant and thermostable antimicrobial peptide is an antimicrobial peptide bonded with a structure locking component with a non-covalent bond, to keep the antimicrobial peptide in a natural conformation, and is encapsulated by a controlled release encapsulation material.

[0013] According to one embodiment of the present invention, the non-covalent bond is selected from hydrogen bond, van der Waals bond, hydrophobic interaction or electrostatic interaction.

[0014] According to one embodiment of the present invention, the structure-locking component is bonded to the hydrophobic residue of the antimicrobial peptide.

[0015] According to one embodiment of the present invention, the antimicrobial peptide is selected from nisin, polylysine, caecal peptide, defensin, bacitracin, lactic acid bacteria, their salts or any combination thereof.

[0016] According to one embodiment of the present invention, the structure-locking component is selected from cyclodextrin, polylysine, zein, silk fibroin, silk sericin, salts thereof or any combination thereof.

[0017] According to one embodiment of the present invention, the encapsulating material is a polymer or lipid component, which includes polyvinyl pyrrolidone, solid lipids, natural waxes and salts thereof.

[0018] According to one embodiment of the present invention, the acid-base resistant and heat-stable antimicrobial peptide has at least 99% antibacterial effect on Escherichia coli, Staphylococcus aureus and Candida albicans.

[0019] According to one embodiment of the present invention, the acid-base resistant and heat-stable antimicrobial peptide is stable and can function normally at a pH value of 4-10.

[0020] According to one embodiment of the present invention, the acid-base resistant and heat-stable antimicrobial peptide is stable and can function normally at a temperature of 0-120°C.

[0021] According to a second aspect of the present invention, a method for preparing an acid- and alkali-resistant and heat-stable antimicrobial peptide is provided, the method comprising capping and / or grafting a structure-locking component onto a hydrophobic residue of the antimicrobial peptide to obtain a structure-locked peptide, and homogenizing the structure-locked peptide with an encapsulating material and a membrane permeation enhancer in a solvent to form an acid- and alkali-resistant and heat-stable antimicrobial peptide.

[0022] According to one embodiment of the present invention, the weight percentages of the antimicrobial peptide, the structure-locking component, the encapsulating material, the membrane permeation enhancer and the solvent are 0.5-38wt%, 0.5-38wt%, 0.01-5wt%, 0.01-47wt% and 0-98.98% respectively.

[0023] According to another embodiment of the present invention, the antimicrobial peptide is 0.5-1wt%, the structure-locking component is 0.5-1wt.%, the encapsulating material is 0.01-0.1wt%, the membrane permeation enhancer is 0.01-0.55wt%, and the solvent is 97.35-98.98wt%.

[0024] According to one embodiment of the present invention, the antimicrobial peptide is 25-38 wt%, the structure-locking component is 25-38 wt.%, the encapsulating material is 3-5 wt%, and the membrane permeation enhancer is 19-47 wt%.

[0025] According to one embodiment of the present invention, the size of the structure-locked peptide is in the range of 100-1000 nm.

[0026] According to one embodiment of the present invention, the antimicrobial peptide is selected from nisin, polylysine, caecal peptide, defensin, bacitracin, lactic acid bacteria, their salts or any combination thereof.

[0027] According to one embodiment of the present invention, the structure-locking component is selected from cyclodextrin, polylysine, zein, silk fibroin, silk sericin, salts thereof or any combination thereof.

[0028] According to one embodiment of the present invention, the encapsulating material is a polymer or lipid component, which includes polyvinyl pyrrolidone, solid lipids, natural waxes and salts thereof.

[0029] According to one embodiment of the present invention, the membrane permeation enhancer is selected from citric acid, phytic acid, myristic acid, fumaric acid, mandelic acid, succinic acid, lauric acid, honokiol, poly-L-lysine or a salt thereof.

[0030] According to one embodiment of the present invention, the solvent is selected from water, isopropanol or ethanol.

[0031] According to a third aspect of the present invention, there is provided an acid-base resistant, heat-stable and non-toxic preservative, wherein the preservative comprises the acid-base resistant and heat-stable antimicrobial peptide mentioned above.

[0032] According to a fourth aspect of the present invention, there is provided an acid- and alkali-resistant, heat-stable and non-toxic food additive. BRIEF DESCRIPTION OF THE DRAWINGS

[0033] Embodiments of the present invention will be described in more detail below with reference to the accompanying drawings, in which:

[0034] Figure 1 A schematic diagram showing capping and / or grafting of structural locking components onto hydrophobic residues of antimicrobial peptides;

[0035] Figure 2 Demonstrating encapsulation of structure-locked antimicrobial peptides and their applications;

[0036] Figure 3 Shows the appearance of different ratios of nisin dissolved in β-cyclodextrin;

[0037] Figures 4A-4B Shows the experimental design and results of thin layer chromatography (TLC), where Figure 4A Design showing TLC, Figure 4B Shows the results of a thin layer chromatography plate stained with iodine and vanillin, respectively, after UV visualization;

[0038] FIG5 shows the results of the inhibition zone test to detect the antibacterial effect before and after protease digestion;

[0039] Figure 6 The solution appearance of the encapsulated antimicrobial peptide is shown;

[0040] Figure 7A-7BThe scanning electron microscope (SEM) image of the mixed freeze-dried powder of nisin / cyclodextrin / poly-L-lysine / propolis is shown, wherein Fig. 7A This is a scanning electron microscope image magnified 5000X. Figure 7B This is a scanning electron microscope image magnified 10000X;

[0041] Figure 8 Display the particle distribution of sample AMP_NAMECDPLLHKP_05-05-1;

[0042] Fig. 9 Shows the scanning electron microscope image of sample AMP_NAMECDPLLHKP_05-05-1;

[0043] Fig.10 Showing clear inhibition zones for various formulations at different pH values ​​after treatment with Staphylococcus aureus and Escherichia coli; and

[0044] Fig.11 Nisin showing conjugation of honokiol and poly-L-lysine: antimicrobial evaluation of cyclodextrin formulations. DETAILED DESCRIPTION

[0045] In the following description, the compounds, preparation methods and / or applications of the antimicrobial peptides etc. that are acid-resistant and heat-stable are listed as preferred examples. It will be apparent to those skilled in the art that modifications including additions and / or substitutions may be made without departing from the scope and spirit of the invention, and specific details may be omitted to avoid confusing the invention; however, the disclosure is written to enable those skilled in the art to practice the disclosure herein without excessive experimentation.

[0046] As used herein, the term "controlled release" refers to the controlled delivery of a substance, such as an active ingredient or component, in a specific manner over an extended period of time. Controlled release mechanisms are intended to release a substance at a predetermined rate and duration, ensuring that it maintains a therapeutic or functional effect in the target environment. This controlled and sustained release is typically achieved through encapsulation, coating, or other delivery techniques to optimize the effectiveness of the substance while minimizing potential side effects or waste. Controlled release systems are valuable in a variety of fields, such as pharmaceuticals and food science, where maintaining consistent levels of the active ingredient is critical to the desired outcome.

[0047] As used herein, the term "structural locking" refers to a mechanism or component for stabilizing or maintaining a specific structural arrangement or conformation of a molecule (such as an antimicrobial peptide). This locking mechanism can be formed by non-covalent bonds such as hydrogen bonds, van der Waals interactions, hydrophobic interactions, or electrostatic interactions, which helps to maintain the molecular structure and function of the antimicrobial peptide. The structural locking component can be strategically applied to the hydrophobic residues of the antimicrobial peptide to ensure that it can maintain its expected conformation even under adverse conditions.

[0048] As used herein, the term "native conformation" refers to the natural and biologically active three-dimensional structure of a molecule (such as a protein or peptide) when it is in its unchanged state, which represents a specific arrangement of atoms and functional groups so that the molecule can effectively perform its expected biological function. In the present invention, it is essential to maintain the antimicrobial peptide in its "native conformation" to ensure its antimicrobial activity. The structural locking component helps to maintain this native conformation so that the antimicrobial peptide can perform its best function, particularly under challenging conditions such as acidity, alkalinity and high temperature.

[0049] As used herein, the term "encapsulation" refers to encapsulating a protective material onto the outer shell of the antimicrobial peptide to form nanoparticles, in which way the antimicrobial peptide will be gradually released and remain active during contact with microorganisms.

[0050] According to a first aspect of the present invention, there is provided an acid- and alkali-resistant and heat-stable antimicrobial peptide.

[0051] Acid-resistant, alkali-resistant and heat-stable antimicrobial peptides, that is, they have a certain robustness under different pH and temperature conditions. This excellent adaptability is achieved by strategically combining the components. The antimicrobial peptide is non-covalently bonded to the structural locking component. This bonding is the key to maintaining the natural conformation of the antimicrobial peptide; in addition, the entire structure is encapsulated in a controlled release encapsulation material, which also enhances its stability and controlled release performance.

[0052] The non-covalent bonds that ensure the native conformation of antimicrobial peptides can take various forms, including hydrogen bonds, van der Waals bonds, hydrophobic interactions, or electrostatic interactions, and this binding plays a key role in maintaining the antimicrobial efficacy of the peptides.

[0053] Notably, the structural locking component forms this noncovalent bond primarily by interacting with the hydrophobic residues of the antimicrobial peptides, an interaction that acts as a structural lock and thus prevents the loss of antimicrobial activity even under challenging conditions.

[0054] The selection of antimicrobial peptides is also relatively broad, and various peptides can be used, including but not limited to nisin, polylysine, caecal peptide, defensin, bacitracin and lactic acid bacteria, or their salts. This diversity enables antimicrobial peptides to effectively meet specific needs or target a wide range of pathogens.

[0055] The structure-locking component is a key element to maintain the native conformation of the antimicrobial peptide, and can be selected from cyclodextrin, polylysine, zein, silk fibroin, sericin or their salts, depending on the specific requirements of different applications.

[0056] In order to further enhance stability and realize controlled release, the antimicrobial peptide is encapsulated in an encapsulating material. The encapsulating material can be a polymer or a lipid component, which can include materials such as polyvinyl pyrrolidone, solid lipids, natural waxes or their salts, to provide a shielding effect for the peptide, and to extend its sustainability before reacting with microorganisms. This technology also compensates for the shortcoming of structural lock protection, i.e., wherein the antimicrobial peptide is still susceptible to the influence of enzyme degradation and / or hydrophobic aggregation, and therefore, by the synergistic effect of structural lock protection and encapsulation technology, the antimicrobial activity of the antimicrobial peptide can be maintained under different conditions, and its stability has been improved, which is beneficial to developing and using in various commercial products. Generally speaking, this encapsulation strategy extends the duration of the antimicrobial action of the peptide, and has ensured long-term effectiveness.

[0057] One of the most notable features of acid-, alkali-resistant and heat-stable antimicrobial peptides is their excellent antibacterial efficacy. They have shown significant effectiveness against many pathogens, especially Escherichia coli, Staphylococcus aureus and Candida albicans, with an antibacterial effect of at least 99%.

[0058] The adaptability of the acid-base-resistant and heat-stable antimicrobial peptides to different environmental conditions is also worth emphasizing. They can remain stable and fully functional in the pH range of 4-10. In addition, they can withstand temperatures from 0 to 120°C without losing their efficacy.

[0059] In summary, acid-, alkali-, and thermostable antimicrobial peptides represent a tremendous advancement in antimicrobial technology, with enhanced structure, stability, and broad-spectrum antimicrobial efficacy, showing potential for a variety of applications ranging from healthcare to food preservation.

[0060] According to a second aspect of the present invention, a method for preparing an acid- and alkali-resistant and heat-stable antimicrobial peptide is provided. The method will be described below to reveal the preparation process.

[0061] First, the structure-locking component is bound to the hydrophobic residues of the antimicrobial peptide by capping and / or grafting. This step produces a structure-locked peptide and is a key factor in ensuring the integrity and efficacy of the peptide because by binding to the hydrophobic residues of the peptide, the structure-locking component can play a protective role and maintain the native conformation of the antimicrobial peptide even under adverse conditions.

[0062] In one embodiment, the preparation method involves two simple steps that can be industrially mass-produced. The first step is to cap and / or graft structure-locking components, such as cyclodextrins on the hydrophobic residues of the peptide by physical mixing methods under controlled temperature and time; the second step is to encapsulate lipids and / or polysaccharide materials (such as hexadecyl palmitate (solid lipids)) on the structure-locking peptide by homogenization methods (including pressurized homogenization methods under controlled temperature and time), and subsequently separate the antimicrobial peptides produced by centrifugation or filtration processes.

[0063] The structure-locked peptide obtained after the combination process is then homogenized with two components that can be mixed in a solvent, namely the encapsulation material and the membrane permeation enhancer. Through this synthetic step, the foundation is laid for the stable and controlled release of the acid-resistant, alkali-resistant and heat-stable antimicrobial peptide with enhanced antibacterial activity.

[0064] This method is applicable to a range of weight percentages, ensuring flexibility in tailoring antimicrobial peptides for specific applications. The weight percentages of antimicrobial peptides, structure-locked peptides, encapsulating materials, membrane permeation enhancers, and solvents, with the combined range of the remaining ingredients, excluding the solvent, ranging from approximately 0.01% to 38%, provide the adaptability required for different applications.

[0065] As an illustrative example, one of the formulations may include 0.5-1% antimicrobial peptides, 0.5-1% structural locking components, 0.01-0.1% encapsulating materials, 0.01-0.55% membrane permeation enhancers, and 97.35-98.98% solvents. In addition, another formulation may consist of 25-38% antimicrobial peptides, 25-38% structural locking components, 3-5% encapsulating materials, and 19-47% membrane permeation enhancers. This wide range of combinations enables formulations to meet specific needs and optimize performance.

[0066] The size of the structure-locked peptide is a key factor affecting its effectiveness, and this method ensures that the size of the structure-locked peptide is in the range of 100-1000 nanometers, which is conducive to controlled release and enhanced antimicrobial activity.

[0067] The method is applicable to a variety of antimicrobial peptides, including nisin, polylysine, caecal peptide, defensin, bacitracin, lactobacillus or their salts. This diversity is conducive to the preparation of specific antimicrobial properties to address specific pathogens or applications.

[0068] The structure-locking component is necessary to maintain the native conformation of the peptide and can be selected from a variety of materials, including cyclodextrin, polylysine, zein, silk fibroin, sericin or their salts, providing adaptability to different environmental conditions and applications.

[0069] The encapsulation material for achieving controlled release can be a polymer or lipid component, which can be selected from polyvinyl pyrrolidone, solid lipids, natural waxes or their salts. This encapsulation strategy ensures the long-term release of the antimicrobial peptide and enhances its efficacy.

[0070] In order to enhance membrane permeability and further optimize antimicrobial activity, a membrane permeation enhancer is included in the preparation process, which can be selected from citric acid, phytic acid, myristic acid, fumaric acid, mandelic acid, succinic acid, lauric acid, magnolol, poly-L-lysine or its salts to enhance the ability of the peptide to fight pathogens.

[0071] The solvent used to carry out the synthesis process is selected from water, isopropanol or ethanol to ensure that all components can be properly mixed to form an acid- and alkali-resistant and heat-stable antimicrobial peptide.

[0072] According to a third aspect of the present invention, there is provided use of an antimicrobial peptide as a preservative.

[0073] The use of the acid- and alkali-resistant and heat-stable antimicrobial peptides as described above to prepare preservatives provides a safe and effective solution for extending the shelf life of various products, including but not limited to food.

[0074] The core of the efficacy of this preservative is the addition of an acid-resistant, alkali-resistant and heat-stable antimicrobial peptide. As mentioned earlier, the antimicrobial peptide can maintain its natural conformation even under adverse conditions such as extreme pH values ​​and temperature changes. This stability is a crucial property when used in food preservation.

[0075] The resulting acid- and alkali-resistant, heat-stable, non-toxic preservatives offer many advantages to the food industry. They effectively inhibit the growth of spoilage microorganisms and pathogenic bacteria, prolonging the freshness and safety of food, and do not require the introduction of harmful or toxic ingredients, making them a safe consumer choice.

[0076] The versatility of this preservative allows it to be incorporated into a wide range of foods. It can be used in a variety of forms, including liquid, powder, coating or encapsulated, depending on the specific application and the desired release characteristics. The controlled release properties of the preservative, derived from the encapsulation of the antimicrobial peptide, ensure that the preservative has a sustained antimicrobial effect over time, further enhancing its effectiveness.

[0077] Furthermore, the non-toxicity of preservatives makes them a preferred choice for clean label and health-conscious foods, meeting the growing consumer demand for natural and safe food additives while addressing the critical issues of food spoilage and safety.

[0078] According to a third aspect of the invention, there is provided the use of an antimicrobial peptide as a food additive. Similarly, the acid and alkali resistant, heat stable, non-toxic preservative may also be used as a food additive to improve the shelf life, safety and overall quality of food, and therefore, the addition of the preservative to a food formulation enables manufacturers to meet consumer expectations for safe, fresh and minimally processed products.

[0079] In summary, the acid- and alkali-resistant, heat-stable, non-toxic preservatives prepared by the antimicrobial peptides of the present invention provide a groundbreaking solution to the problems of food preservation and quality improvement. Their versatility, stability and safety have the potential to make them a valuable asset in the food industry, providing opportunities for clean label products and solving key issues related to food safety and waste reduction.

[0080] Example

[0081] In the following examples, nisin (E number E234 approved by the U.S. Food and Drug Administration), one of the commercially available antimicrobial peptides, is used as an exemplary embodiment. Due to its limited antimicrobial spectrum and the weakening of its antimicrobial effect under the conditions of pH, temperature changes and enzyme reactions, it has not been widely used as a preservative in the food field.

[0082] Example 1: Method for combining a structural locking component with nisin

[0083] The peptide chain structure of nisin is sensitive to changes in pH and temperature, which seriously affects its high antibacterial activity; therefore, a structure-locking component is introduced into nisin to bind to the molecules on its peptide chain and lock the peptide in the desired conformation, thereby limiting the secondary and tertiary structural changes that may occur in the peptide when the pH and temperature change. Figure 1 The display molecule is bound to the peptide chain molecule via the structure-locking component. In this embodiment, cyclodextrin is used as the structure-locking component.

[0084] 1 g of nisin and 1 g of cyclodextrin were dissolved in 98 ml of deionized water, and the resulting mixture was stirred for different durations, namely 2 hours, 4 hours, 6 hours, 8 hours and overnight, wherein the cyclodextrin used was β-cyclodextrin, to produce a slightly acidic solution with a pH of about 5. Different ratios of nisin and cyclodextrin mixtures (2:1, 1:1 and 1:2) were prepared.

[0085] According to Table 1, the particle size increases with the extension of the stirring time, and the particle size fluctuates between 44 nm and 895 nm. The polydispersity index (PDI) value exceeds 0.5 when the stirring time is in the range of 0 h to 8 h. However, after stirring overnight (24 h), the particle size stabilizes at 694 nm and the polydispersity index drops below 0.5. Similar results were observed in samples with different ratios of nisin to cyclodextrin. Therefore, in order to obtain a stable uniform solution, all samples were stirred overnight unless otherwise specified in the steps.

[0086] Table 1. Nisin and β-cyclodextrin at different mixing times and ratios

[0087] Figure 3 Shows the appearance of different ratios of nisin:β-cyclodextrin solutions. The results show that similar particle sizes can be produced when nisin is mixed with β-cyclodextrin at a ratio of 2:1, 1:1, and 1:2. However, when the ratio of nisin to cyclodextrin solution is 1:2, insoluble excess β-cyclodextrin is visible at the bottom of its container. Therefore, the 1:2 ratio solution was not selected for subsequent experiments. In the following experiments, the ratio of nisin to cyclodextrin is 1:1 for uniformity and convenience.

[0088] Thin layer chromatography (TLC) was performed to evaluate the binding between cyclodextrin and nisin. TLC is an affinity-based chromatographic technique used to separate and identify compounds in a solution mixture based on their different affinities for a stationary phase and a liquid mobile phase, which affects the migration rate of individual compounds and thus separates the compounds in the mixture between the two phases. Therefore, each compound has a unique retention factor (Rf) value. TLC is characterized by versatility, simplicity, high sensitivity, relatively low cost, rapid development time and reproducibility.

[0089] Briefly, approximately 1-5 μL of sample is spotted on a designated line on a thin layer chromatography plate coated with silica, and the plate is immersed in a liquid mobile phase containing a solvent system (preferably 1:9 dichloromethane: methanol) in a thin layer chromatography reaction chamber for reaction until the solvent front reaches the top of the thin layer chromatography plate and is marked. Subsequently, the thin layer chromatography plate is inspected under long-wave ultraviolet (UVA) and short-wave ultraviolet (UVC) light to detect fluorescent compounds. To further confirm the activity of compound spots under UV light, the thin layer chromatography plate is stained with iodine and vanillin. Finally, the retention factor value of each compound is calculated by dividing the distance traveled by the distance traveled by the solvent. Figure 4A and 4BShown are the TLC setup and the TLC plate stained with iodine and vanillin, respectively.

[0090] The retention factor values ​​of nisin, cyclodextrin and the mixture were calculated and listed in Table 2. The results showed that the retention factor values ​​of the nisin-cyclodextrin mixture were different from those of nisin and cyclodextrin alone, indicating successful binding between cyclodextrin and nisin.

[0091] Table 2. Retention factor values ​​of nisin, various cyclodextrins, and nisin-cyclodextrin mixtures

[0092] Example 2: Protective effect of cyclodextrin on nisin

[0093] The criteria for evaluating the effect of pH on the physical properties of nisin included measuring the particle size of nisin solutions at different pH values ​​(pH 4, 7 and 10). The particle sizes were measured at 185 nm, 711 nm and 450 nm at pH 4, 7 and 10, respectively. These size changes indicate that the degree of protonation of the amino residues on the peptide chain leads to changes in the twisting angle of the peptide chain in acidic (pH 4), neutral (pH 7) and alkaline (pH 10) environments. Comparison of the percentage change in particle size of nisin-cyclodextrin mixtures at pH 4 and 7 indicates the effective protection and / or confinement of cyclodextrin (samples with a primary particle size of more than 1000 nm were excluded at this stage when subjected to different pH levels).

[0094] The steps of preparing nisin-cyclodextrin solutions in different pH environments are as follows: 1 g nisin and 1 g cyclodextrin are dissolved in 98 mL deionized water, and the mixture is stirred overnight; the obtained sample is then freeze-dried to obtain a powder. The powder is dissolved in different pH solutions (pH 4, 7, and 10), and the particle size of the sample is measured using a Zeta particle sizer.

[0095] Table 3 shows that under different pH conditions, the main (average) particle sizes of α-cyclodextrin, γ-cyclodextrin and methyl-β-cyclodextrin are all less than 1000 nanometers, so subsequent experiments can be carried out.

[0096] Table 3. Particle size of nisin-cyclodextrin mixture at pH 4, 7 and 10

[0097] Example 3. Antimicrobial properties of structure-locked antimicrobial peptides

[0098] The antimicrobial properties of the formulations were screened and evaluated using the zone of inhibition test method, and the antimicrobial efficacy was determined by the petri plate count method.

[0099] For the inhibition zone test, 0.2 g of freeze-dried nisin:cyclodextrin powder was dissolved in 9.8 g of the solutions at pH 4, 7 and 10, respectively, and stirred overnight. The particle sizes of the resulting solutions are listed in Table 3.

[0100] In the inhibition test area, the formulations treated with different pH values ​​were inoculated on nutrient agar plates cultured with Staphylococcus aureus and Escherichia coli. Briefly, a volume of 100 μL of Staphylococcus aureus or Escherichia coli solution was added to the agar plate and evenly spread using an applicator; subsequently, 20 μL of the formulation solution was loaded onto a 6 mm sterile disc and the disc was placed on the surface of the agar plate inoculated with Staphylococcus aureus or Escherichia coli, and then these agar plates were incubated at 37°C overnight, and the resulting clear inhibition zone was recorded. The results are shown in Fig.10 shown.

[0101] For the Petri dish count method, prepare solutions of Staphylococcus aureus and Escherichia coli and dilute to 10 7 cfu / mL, and then 0.2mL of Staphylococcus aureus and Escherichia coli solutions were added to 1.8mL of nisin: cyclodextrin formula solutions treated with different pH values, and the bacteria were evenly distributed in the solution by vortexing, and the mixture was finally incubated at room temperature for 30 minutes. After incubation, 100μL of each mixture was collected and serially diluted from 10 to 100,000 times, including a blank control group; then, 100μL of all diluted samples were evenly spread on agar plates and incubated at 37°C overnight; finally, the colonies formed on the plates were counted and recorded, and the results are summarized in Table 4.

[0102] Table 4. Antibacterial effect of nisin:cyclodextrin formulation on Staphylococcus aureus

[0103] At the same concentration (1%) and the same contact time (30 minutes), the antibacterial results of the nisin:cyclodextrin formula against Staphylococcus aureus showed that the combination of nisin and various cyclodextrins increased its antibacterial effect compared to unformulated nisin. This phenomenon is attributed to the improved and increased stability, solubility and permeability of nisin when interacting with cyclodextrins.

[0104] However, due to the poor adhesion and permeability between nisin and the bacterial outer membrane, Gram-negative bacteria (such as Escherichia coli) show resistance to nisin. Previous studies have shown that metal chelators such as citric acid and phytic acid (G. Zhao et al., Food Control, 2023) enhance the antibacterial effect of nisin against Gram-negative bacteria (IJ G Márquez et al., Can J Microbiol, 2020). It has also been shown that honokiol has similar functions to these metal chelators and is able to chelate iron (II) ions (S. Kantham et al., Neurosci, 2017). Similarly, polylysine, as a peptide, also has the ability to chelate metal ions. Therefore, the addition of honokiol and poly-L-lysine to the formula can enhance the adhesion between the bacterial membranes of nisin and promote penetration to facilitate subsequent cytoplasmic leakage, thereby achieving maximum antibacterial effect. The present invention further evaluates the antibacterial effect of incorporating honokiol and poly-L-lysine on Escherichia coli, and the results are summarized in Table 5. The inhibition zone test image is shown in Fig.11 middle.

[0105] Table 5. Antibacterial effects of formulations containing honokiol and poly-L-lysine on Staphylococcus aureus and Escherichia coli

[0106] Example 4: Encapsulation of structure-locked antimicrobial peptides

[0107] Encapsulation is the process of encapsulating and / or trapping one substance in another to form particles or capsules. Solid lipids, polyvinyl pyrrolidone (PVP) and natural waxes are commonly used encapsulation materials, among which solid lipids are a class of lipids with a high melting point, which are solid at room temperature and are often used as matrix materials for encapsulating bioactive compounds and / or drugs to improve their stability, bioavailability and controlled release properties; polyvinyl pyrrolidone is a water-soluble polymer, which is commonly used as a coating agent and / or sealant in various applications. Polyvinyl pyrrolidone has a high molecular weight, is non-toxic and biocompatible, and is also suitable for biomedical and pharmaceutical applications; natural waxes are complex mixtures of lipids extracted from plants, animals or microorganisms, have a high melting point, and are solid at room temperature, therefore, they are suitable for use as encapsulation materials. Figure 2 Encapsulation of a structure-locked antimicrobial peptide is shown in FIG.

[0108] One of the suitable methods for preparing polyvinyl pyrrolidone nanoparticles is as follows. Briefly, 1 g of nisin and 1 g of polyvinyl pyrrolidone were dissolved in 98 mL of deionized water and stirred overnight to obtain a homogenous solution. Then, 1 mL of the resulting homogenous solution was added to a 1.5 mL microcentrifuge tube for subsequent antibacterial testing. 10 mg / mL of protease was added to the homogenous solution, and the mixture was incubated at 37 ° C overnight to enzymatically digest the nisin in the solution. After incubation, 20 μL of the treated solution was transferred to a 6 mm sterile disc and placed on an agar plate inoculated with Staphylococcus aureus together with the sample that was not treated with the enzyme, and finally the inoculated agar plate containing the sample was incubated at 37 ° C overnight.

[0109] For solid lipids and natural waxes, 1 g of solid lipid or wax was added to 49 g of ethanol and heated at 70-80 °C with stirring at 400 rpm until all solid lipid or paraffin was dissolved. Then 1 g of nisin was dissolved in 49 g of deionized water. The lipid or wax ethanol solution was added dropwise to the nisin solution under vigorous stirring and stirred for 30 minutes. The resulting mixture was milky white. After that, 1 mL of the sample was transferred to a 1.5 mL microcentrifuge tube for subsequent antimicrobial testing. 10 mg / mL of protease was added to the solution and incubated overnight at 37 °C to enzymatically digest the nisin in the solution. After incubation, the sample was heat treated to release the nisin embedded in the nanoparticles. Subsequently, 20 μL of the treated solution was added to a 6 mm sterile disc for antimicrobial testing. The sterile disc was placed on an agar plate inoculated with Staphylococcus aureus together with the untreated sample, and the plate was incubated overnight at 37 °C.

[0110] The inhibition zone of each sample was measured and recorded, and when the inhibition zone was 1 mm larger than the sterile disc (6 mm), it indicated antimicrobial activity. The results of the inhibition zone test are summarized in Table 6, and the inhibition zone test image is shown in Figure 5.

[0111] Table 6. Protective effects of solid lipids, natural waxes and polymers

[0112] Among the 17 antimicrobial tests performed, the combination of nisin with myristic acid, monoacylglycerides, capric acid, or propolis still showed antimicrobial effects after protease digestion; in contrast, the other combinations showed no antimicrobial effects after protease digestion. These results show that the combination of nisin with myristic acid, monoacylglycerides, capric acid, or propolis formulations is able to partially and / or completely encapsulate and / or entrap nisin. Although myristic acid and capric acid can cause precipitation in the resulting solution (e.g. Figure 6), but this issue can be addressed, optimized and further developed through subsequent formulation and processing. On the other hand, the combination of nisin and propolis did not lead to precipitation and showed only a slight reduction in the inhibition zone after protease digestion.

[0113] Example 5. Physical properties of encapsulated structure-locked antimicrobial peptides

[0114] To prepare the 10% propolis ethanol extract, 1 g of propolis was dissolved in 9 g of ethanol and the mixture was sonicated for at least 60 minutes to ensure complete dissolution. The suspension was then filtered with qualitative filter paper to remove large debris. Subsequently, the filtrate was further filtered using a 0.45 μm nylon or PTFE syringe filter. Finally, the resulting filtrate was collected as the 10% propolis ethanol extract and stored at room temperature for subsequent experiments.

[0115] In another experiment, 1 g of nisin, 1 g of cyclodextrin and 1 g of poly-L-lysine were dissolved in 96 g of deionized water to obtain a light yellow uniform solution, and the solution was stirred overnight to ensure sufficient mixing. Finally, the particle size of nisin / poly-L-lysine with different cyclodextrins was measured with a particle size analyzer. The results are shown in Table 7.

[0116] Table 7. Effects of different cyclodextrins on nisin / poly-L-lysine particle size

[0117] The results showed that the sample containing only 1% nisin: α-cyclodextrin: poly-L-lysine (1:1:1) had a polydispersity index of less than 0.5. Therefore, this formula was selected to incorporate propolis extract to maximize its antibacterial effect. Briefly, 1 g of 10% propolis ethanol extract was added dropwise to a solution consisting of 99 g nisin / cyclodextrin / poly-L-lysine under vigorous stirring. In addition, propolis extract was added to 1% nisin: methyl-β-cyclodextrin and 1% nisin: β-cyclodextrin / epichlorohydrin copolymer, respectively, to form nanoparticles, and the particle size was measured with a particle size analyzer. The results are shown in Table 7.

[0118] To examine the morphology of the nanoparticles, a scanning electron microscope (SEM) was used for observation. The nisin / cyclodextrin / poly-L-lysine / propolis solution was freeze-dried to a powder form, which was then transferred to a silicon wafer and adhered to the surface of a copper support using carbon conductive tape. A thin layer of gold was deposited on the surface of the powder to enhance the conductivity of the sample. A high-resolution image was captured using a scanning electron microscope, and the captured image is shown in FIG7 , which shows nanoparticles with a size of approximately 600 nanometers. In addition, the scanning electron microscope image shows that there is a thin layer around the nanoparticles, which may be unencapsulated nisin / cyclodextrin / poly-L-lysine. These unencapsulated materials may act as antimicrobial agents before the encapsulated antimicrobial agents are released from the propolis-encapsulated nanoparticles.

[0119] Example 6: Preparation of an acid- and alkali-resistant and heat-stable antimicrobial peptide sample AMP_NACDPLLHKP_05-05-1

[0120] The method for preparing AMP_NAMECDPLLHKP_05-05-1 includes the following steps: 1 g of nisin, 1 g of α-cyclodextrin, 0.5 g of poly-L-lysine and 0.5 g of honokiol are dissolved in 96 g of deionized water. The mixture is stirred overnight to obtain a light yellow uniform solution; then, 1 g of 10% propolis ethanol extract is added dropwise to 99 g of nisin / cyclodextrin / poly-L-lysine solution, and the solution is vigorously stirred to obtain a mixed solution with a milky appearance. The details of this formula are summarized in Table 8. A portion of the mixed solution is diluted ten times and transferred to a cuvette, and the particle size is measured using a particle size analyzer. The results are listed in Table 9, and the particle distribution curve is as shown in Table 9. Figure 8 The remaining part of the solution was freeze-dried to obtain a powder form for subsequent scanning electron microscopy observation, where the nanostructure of AMP_NAMECDPLLHKP_05-05-1 is as shown Fig. 9 shown.

[0121] Table 8. Formula of AMP_NAMECDPLLHKP_05-05-1 raw material Percentage (solution) Percentage (powder) Nisin 1％ 37.7％ α-Cyclodextrin 1％ 37.7％ Poly-L-lysine 0.5％ 18.9％ Propolis Extract 0.1％ 3.8％ Honokiol 0.05％ 1.9％ Ethanol 1％ - Deionized water 96.35％ -

[0122] Table 9. Granularity of AMP_NAMECDPLLHKP_05-05-1 Main size (nanometer) Polydispersity index Sample-1 648.9 0.425 Sample-2 630.3 0.328 Sample-3 655.0 0.388 average: 644.7 0.380

[0123] The results showed that the particle size of AMP_NAMECDPLLHKP_05-05-1 was always below 1000 nm and the polydispersity remained below 0.5.

[0124] To further evaluate the stability of the sample, the freeze-dried AMP_NAMECDPLLHKP_05-05-1 solution was placed in an environmental chamber at 40°C and 75% relative humidity (RH) for 3 months. This test was designed to simulate the 2-year shelf life of the product. The particle size and antimicrobial effect of the sample AMP_NAMECDPLLHKP_05-05-1 were measured at 0, 1, and 2 months (Table 10). In addition, the particle size at the 3-month time point was also measured, and the antimicrobial effect was evaluated using the Petri dish counting method.

[0125] Table 10. Granularity of AMP_NAMECDPLLHKP_05-05-1 under accelerated conditions

[0126] Example 7: Antibacterial effect of AMP_NACDPLLHKP_05-05-1 in an acidic environment (pH 4) (refer to the Technical Standards for Disinfectants in 2002)

[0127] According to the method of "Technical Standard for Disinfectants 2002", antibacterial tests were performed using Staphylococcus aureus and Escherichia coli. 2 mL of AMP_NAMECDPLLHKP_05-05-1 solution (equivalent to 1% nisin activity) and 2 mL of 1% nisin were transferred to 15 mL sterile centrifuge tubes and freeze-dried for three days to obtain freeze-dried powder. 1.8 mL of pH 4 solution was added to the freeze-dried powder and mixed evenly overnight to obtain pH 4-treated AMP_NAMECDPLLHKP_05-05-1 solution and nisin solution. Prepare Staphylococcus aureus and Escherichia coli solutions and dilute to 10 7 cfu / mL, 0.2mL of Staphylococcus aureus or Escherichia coli solution was added to 1.8mL of AMP_NAMECDPLLHKP_05-05-1 solution and nisin solution treated with pH 4, vortexed to evenly distribute the bacteria in the solution, and then the mixture was stirred and cultured at room temperature for 30 minutes. After 30 minutes of culture, 100μL of the bacterial solution was collected and serially diluted 10 to 100000 times, and the blank sample was subjected to the same steps. Finally, 100μL of the diluted sample was evenly spread on the inoculated agar culture dish and cultured overnight at 37℃, and then the colonies formed on the culture dish were counted and recorded. The results are shown in Table 11.

[0128] Table 11. Antibacterial effect of AMP_NAMECDPLLHKP_05-05-1 on Escherichia coli and Staphylococcus aureus at pH 4 according to the test method of "2002 Technical Standard for Disinfectants"

[0129] These results showed that nisin had good antibacterial effect against S. aureus; however, no significant antibacterial effect of nisin against E. coli was detected, and AMP_NAMECDPLLHKP_05-05-1 was observed to have better antibacterial performance against both S. aureus and E. coli. By comparing the log reduction values ​​against S. aureus and E. coli, AMP_NAMECDPLLHKP_05-05-1 was at least better than unformulated nisin, with log difference values ​​at least higher by 5.11 logs (E. coli) and 3.97 logs (S. aureus).

[0130] Example 8: Antibacterial effect of AMP_NACDPLLHKP_05-05-1 in neutral (pH 7) and alkaline (pH 10) environments

[0131] The antibacterial test was carried out in accordance with the method of "Technical Standard for Disinfectants 2002", including tests on Staphylococcus aureus and Escherichia coli. 2 mL of AMP_NAMECDPLLHKP_05-05-1 solution (equivalent to 1% nisin activity) and 2 mL of 1% nisin were transferred to 15 mL sterile centrifuge tubes and freeze-dried for three days to obtain lyophilized powder. Subsequently, 1.8 mL of pH 7 or pH 10 solution was added to the lyophilized powder, and the mixture was evenly mixed overnight to obtain pH 7 or pH 10 treated solutions of AMP_NAMECDPLLHKP_05-05-1 and nisin.

[0132] Prepare Staphylococcus aureus and Escherichia coli solutions and dilute to approximately 10 7 Then, 0.2 mL of the Staphylococcus aureus and Escherichia coli solutions were added to 1.8 mL of the pH-treated AMP_NAMECDPLLHKP_05-05-1 solution and nisin solution, and the mixture was vortexed to ensure that the bacteria were evenly distributed in the solution, and then the mixture was stirred and incubated at room temperature for 30 minutes.

[0133] After 30 minutes of incubation, 100 μL of the bacterial solution was collected and serially diluted 10 to 100,000 times, and the blank sample was subjected to the same steps. Finally, 100 μL of the diluted sample was evenly spread on the inoculated agar culture dish and incubated overnight at 37°C, and then the colonies formed on the culture dish were counted and recorded. The results of the test are summarized in Table 12 (results of pH 7 treatment) and Table 13 (results of pH 10 treatment).

[0134] Table 12. Antibacterial effect of AMP_NAMECDPLLHKP_05-05-1 on Escherichia coli and Staphylococcus aureus at pH 7 according to the test method of "2002 Technical Standard for Disinfectants"

[0135] Table 13. Antibacterial effect of AMP_NAMECDPLLHKP_05-05-1 on Escherichia coli and Staphylococcus aureus at pH 10 according to the test method of "2002 Technical Standard for Disinfectants"

[0136] These results showed that nisin had a significant antibacterial effect against S. aureus, while no significant antibacterial effect was observed against E. coli. In contrast, AMP_NAMECDPLLHKP_05-05-1 exhibited enhanced antibacterial properties against both S. aureus and E. coli at both pH 7 and pH 10.

[0137] When comparing the difference in log reduction against S. aureus and E. coli after treatment at pH 7 or pH 10 for 30 minutes, AMP_NAMECDPLLHKP_05-05-1 was at least 5.33 logs (E. coli) and 2.91 logs (S. aureus) higher than unformulated nisin at pH 7. Similarly, at pH 10, AMP_NAMECDPLLHKP_05-05-1 showed a difference of 5.61 logs (E. coli) and 2.84 logs (S. aureus) compared to unformulated nisin, indicating that the antibacterial effect of AMP_NAMECDPLLHKP_05-05-1 (based on remaining bacterial cfu / mL) was at least 50% higher.

[0138] Example 9: According to the 2002 Technical Standards for Disinfectants, the antibacterial effect of AMP_NACDPLLHKP_05-05-1 on Escherichia coli and Staphylococcus aureus after high temperature treatment at 90°C for 15 minutes

[0139] The antibacterial test was carried out according to the method of Technical Standard for Disinfectants 2002, using Staphylococcus aureus and Escherichia coli as test organisms. First, 2 mL of AMP_NAMECDPLLHKP_05-05-1 solution (equivalent to 1% nisin activity) was transferred to a 15 mL sterile centrifuge tube and freeze-dried for three days to obtain a lyophilized powder. Subsequently, 1.8 mL of PBS solution was added to the freeze-dried powder and mixed thoroughly overnight to produce a uniform AMP_NAMECDPLLHKP_05-05-1 solution.

[0140] Before the antibacterial test, half of the AMP_NAMECDPLLHKP_05-05-1 solution sample was heat treated at 90°C for at least 15 minutes. Then, the Staphylococcus aureus and Escherichia coli solutions were prepared and diluted to about 10 7 cfu / mL, and then 0.2 mL of Staphylococcus aureus or Escherichia coli solution was added to each AMP_NAMECDPLLHKP_05-05-1 sample solution (heat-treated and untreated), and finally the mixture was vortexed to ensure uniform distribution of bacteria in the solution, followed by stirring and incubation at room temperature for 30 minutes.

[0141] After 30 minutes of incubation, 100 μL of the bacterial solution was collected and serially diluted 10 to 100,000 times, and the blank sample was subjected to the same steps. Finally, 100 μL of the diluted sample was evenly spread on the inoculated agar plate and incubated at 37°C overnight, and then the colonies formed on the plate were counted and recorded, and the results are summarized in Table 14.

[0142] Table 14. Antibacterial effect of AMP_NAMECDPLLHKP_05-05-1 on Escherichia coli and Staphylococcus aureus before and after heat treatment

[0143] The antibacterial results showed that the antibacterial effect of AMP_NAMECDPLLHKP_05-05-1 against Staphylococcus aureus and Escherichia coli did not show significant changes in antibacterial effect after heat treatment at 90 °C for 15 min ( Table 14 ), and the change values ​​were only 0.53 (Escherichia coli) and 0 (Staphylococcus aureus), both of which were less than log 1.

[0144] Example 10: Antifungal Effect of AMP_NACDPLLHKP_05-05-1

[0145] According to the international standard EN 1650:2019, antifungal tests were performed in an accredited third-party laboratory (BUREAU VERITAS HONGKONG LIMITED, BV), using Candida albicans and Aspergillus brasiliensis as test organisms. To prepare the sample, 1 g of nisin was dissolved in 99 mL of a pH 4 solution and stirred overnight to obtain a uniform 1% nisin solution treated with pH 4. Subsequently, 100 mL of AMP_NAMECDPLLHKP_05-05-1 solution (equivalent to 1% nisin activity) was transferred to a 100 mL sterile container and freeze-dried for three days, and the resulting lyophilized powder was added to 100 mL of a pH 4 solution and stirred overnight to obtain a pH 4-treated AMP_NAMECDPLLHKP_05-05-1 solution. Both samples with a pH value of 4 were tested for antifungal effects, and the results are shown in Tables 15 and 16.

[0146] Table 15. Antifungal test results of 1% nisin solution treated with pH 4

[0147] Table 16. Antifungal test results of AMP_NAMECDPLLHKP_05-05-1 solution treated with pH 4

[0148] The antifungal results showed that unformulated nisin exhibited only slight antifungal activity against Candida albicans and Aspergillus brasiliensis. In contrast, the formulated nisin, AMP_NAMECDPLLHKP_05-05-1, showed significantly increased antifungal activity against Candida albicans and slightly increased antifungal activity against Aspergillus brasiliensis. Specifically, the antifungal results of AMP_NAMECDPLLHKP_05-05-1 showed a significant increase in antifungal activity, with a difference of 5.11 logs (Candida albicans) and 0.11 logs (A. brasiliensis) compared to unformulated nisin.

[0149] Example 11, Safety Assessment of AMP_NACDPLLHKP_05-05-1

[0150] The MTT assay was performed using a human cell line (HaCat) to evaluate the cytotoxicity of AMP_NACDPLLHKP_05-05-1 and compared with the results of nisin. The quantitative evaluation was calculated using the following formula: Survival rate (%) = [absorbance value (wavelength 570-650) of test group / positive control group / negative control group] / [absorbance value (wavelength 570-650) of blank control group]*100; The results are shown in Tables 17-19.

[0151] Table 17. Results of AMP_NAMECDPLLHKP_05-05-1 (mean ± SD)

[0152] Table 18. Results of Nisin (mean ± SD) Group (V / V) Absorbance Qualitative grading of cell morphology Survival rate (%) Blank control group 0.23±0.02 0 100.00±8.99 Negative control group 0.20±0.01 0 86.11±6.21 Positive control group 0.06±0.01 4 23.52±4.63 10％ 0.18±0.01 2 75.09±4.43 1％ 0.19±0.01 1 78.78±2.95 0.1％ 0.26±0.03 0 108.96±12.73 0.01％ 0.23±0.01 0 96.99±3.74 0.001％ 0.24±0.04 0 100.55±15.86 0.0001％ 0.25±0.01 0 106.76±5.39

[0153] Table 19. Qualitative morphological grading of cytotoxicity

[0154] According to the above results, it can be seen that the cytotoxicity of AMP_NAMECDPLLHKP_05-05-1 is similar to that of nisin, indicating that the sample has no cytotoxicity.

[0155] In addition, samples were sent to an accredited third-party laboratory to evaluate their chemical and biological safety, and the results are shown in Table 20.

[0156] Table 20. Safety Assessment *ROHS and SVHC testing is performed by Bureau Veritas **Skin irritation, acute oral toxicity and acute skin toxicity were tested by SGS Standards Technical Services

[0157] From the results, it can be seen that the sample passed all safety assessments.

[0158] Example 12: Application of AMP_NACDPLLHKP_05-05-1 as a preservative and verification of its effect

[0159] AMP_NAMECDPLLHKP_05-05-1 powder can be used with a variety of excipients to suit different usage scenarios. For example, freeze-dried AMP_NAMECDPLLHKP_05-05-1 powder was dissolved in deionized water and stirred overnight to obtain a 1% nisin active spray for food (such as meat and fruit); in addition, freeze-dried AMP_NAMECDPLLHKP_05-05-1 was incorporated into a cream base to form a mixed and uniform cream without liquefaction and / or phase separation.

[0160] To test the preservative effectiveness of AMP_NAMECDPLLHKP_05-05-1, samples were commissioned to an accredited third-party laboratory to evaluate its preservative effectiveness according to the standards listed in BS EN ISO 11930:2019 and United States Pharmacopoeia USP43-NF38(2020) General Chapter 51, and the results are summarized in Table 21.

[0161] Table 21. Effectiveness of preservatives

[0162] The foregoing description has been presented for the purpose of illustrating and describing the present invention, but is not intended to be exhaustive or to limit the invention to the precise form disclosed, as many modifications and variations will be apparent to those skilled in the art.

[0163] The embodiment was chosen and described in order to best explain the principles of the invention and its practical application, thereby enabling others skilled in the art to understand the invention for various embodiments and with various modifications as are suited to the particular use contemplated.

Claims

1. An acid-base resistant and heat-stable antimicrobial peptide, characterized in that: The acid-base-resistant and heat-stable antimicrobial peptide is non-covalently bonded to a structure-locking component to keep the antimicrobial peptide in a natural conformation and is encapsulated by a controlled-release encapsulation material.

2. The acid- and alkali-resistant and thermostable antimicrobial peptide according to claim 1, wherein the non-covalent bond is selected from a hydrogen bond, a van der Waals bond, a hydrophobic interaction or an electrostatic interaction.

3. The acid- and alkali-resistant and thermostable antimicrobial peptide according to claim 1, wherein the structure-locking component is bonded to the hydrophobic residue of the antimicrobial peptide.

4. The acid-base resistant and heat-stable antimicrobial peptide according to claim 1, wherein the antimicrobial peptide is selected from nisin, polylysine, caecal peptide, defensin, bacitracin, lactic acid bacteria, their salts or any combination thereof.

5. The acid-base resistant and heat-stable antimicrobial peptide according to claim 1, wherein the structure-locking component is selected from cyclodextrin, polylysine, zein, silk fibroin, silk sericin, salts thereof or any combination thereof.

6. The acid- and alkali-resistant and thermostable antimicrobial peptide according to claim 1, wherein the encapsulating material is a polymer or a lipid component comprising polyvinyl pyrrolidone, solid lipids, natural waxes and salts thereof.

7. The acid-base resistant and heat-stable antimicrobial peptide according to claim 1, wherein the acid-base resistant and heat-stable antimicrobial peptide has at least 99% antibacterial effect on Escherichia coli, Staphylococcus aureus and Candida albicans.

8. The acid- and alkali-resistant and thermostable antimicrobial peptide according to claim 1, wherein the acid- and alkali-resistant and thermostable antimicrobial peptide is stable and can function normally at a pH value of 4-10.

9. The acid-base resistant and heat-stable antimicrobial peptide according to claim 1, wherein the acid-base resistant and heat-stable antimicrobial peptide is stable and can function normally at a temperature of 0-120°C.

10. A method for preparing an acid- and alkali-resistant and heat-stable antimicrobial peptide, characterized in that: include: capping and / or grafting a structure-locked component onto the hydrophobic residues of the antimicrobial peptide to obtain a structure-locked peptide; as well as The structure-locked peptide is homogenized in a solvent with an encapsulating material and a membrane permeation enhancer to form an acid- and alkali-resistant and heat-stable antimicrobial peptide.

11. method according to claim 10, wherein the weight percentages of the antimicrobial peptide, the structure-locking component, the encapsulating material, the membrane permeation enhancer and the solvent are 0.5-38wt%, 0.5-38wt%, 0.01-5wt%, 0.01-47wt% and 0-98.98%.

12. The method according to claim 11, wherein the antimicrobial peptide is 0.5-1wt%, the structure-locking component is 0.5-1wt.%, the encapsulating material is 0.01-0.1wt%, the membrane permeation enhancer is 0.01-0.55wt%, and the solvent is 97.35-98.98wt%.

13. The method according to claim 11, wherein the antimicrobial peptide is 25-38 wt%, the structure-locking component is 25-38 wt.%, the encapsulating material is 3-5 wt%, and the membrane permeation enhancer is 19-47 wt%.

14. The method of claim 10, wherein the size of the structure-locked peptide is in the range of 100-1000 nanometers.

15. The method of claim 10, wherein the antimicrobial peptide is selected from nisin, polylysine, caecal peptide, defensin, bacitracin, lactic acid bacteria, their salts or any combination thereof.

16. The method of claim 10, wherein the structure-locking component is selected from cyclodextrin, polylysine, zein, silk fibroin, silk sericin, salts thereof, or any combination thereof.

17. The method of claim 10, wherein the encapsulating material is a polymer or lipid component including polyvinyl pyrrolidone, solid lipids, natural waxes and salts thereof.

18. The method of claim 10, wherein the membrane permeation enhancer is selected from citric acid, phytic acid, myristic acid, fumaric acid, mandelic acid, succinic acid, lauric acid, honokiol, poly-L-lysine or a salt thereof.

19. The method of claim 10, wherein the solvent is selected from water, isopropanol or ethanol.

20. An acid-base resistant, heat-stable and non-toxic preservative, characterized in that: It comprises the acid-base-resistant and heat-stable antimicrobial peptide as claimed in claim 1.

21. An acid-base resistant, heat-stable and non-toxic food additive, characterized in that: It comprises the acid-base-resistant and heat-stable antimicrobial peptide as claimed in claim 1.

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