An antimicrobial peptide that inhibits foodborne pathogens, its acquisition method and application

Abstract

This invention provides an antimicrobial peptide that inhibits foodborne pathogens, its acquisition method, and its applications, relating to the fields of bioactive peptides and biomedical engineering technology. The amino acid sequence of the antimicrobial peptide is: Gly-Leu-Thr-Leu-Lys-His-Leu-Lys-Lys-Leu-Ile-Phe. This antimicrobial peptide can effectively inhibit the growth of foodborne pathogens such as Staphylococcus aureus, Salmonella, Escherichia coli, Pseudomonas aeruginosa, and Listeria monocytogenes. This invention overcomes the shortcomings of existing technologies by using a screening method combining gastrointestinal digestive enzyme-peptidomics technology and experiments to discover and identify antimicrobial peptides with antimicrobial activity, providing new natural antimicrobial agents for the food, pharmaceutical, and other related fields.

Description

Technical Field

[0001] This invention relates to the field of bioactive peptides and biomedical engineering technology, specifically to an antimicrobial peptide that inhibits foodborne pathogens, its acquisition method, and its application. Background Technology

[0002] The gut microbiota is a crucial component of host health, playing an indispensable role in maintaining well-being by influencing nutrient metabolism, immune responses, and disease-fighting mechanisms. With in-depth research into the interaction between the microbiota and the host, scientists have gradually recognized that naturally occurring antimicrobial peptides (AMPs) released from dietary proteins during gastrointestinal digestion can not only reshape the structure of the gut microbiota but also possess the potential to regulate immune function, exert antimicrobial and antipathogenic effects. These antimicrobial peptides, generated from the breakdown of food-derived proteins, have significant biological importance in promoting host health, particularly in combating gut pathogens.

[0003] Mealworms (Tenebrio molitor), as a food source rich in high-quality protein, have been approved as a novel food by the European Union. Recent studies have shown that incorporating mealworm-based feeds into poultry diets is a strategy to enhance gastrointestinal function and gut health. Consuming mealworms can improve the composition of the mammalian gut microbiota and enhance animal resistance to pathogens and immune efficacy, indicating that mealworm protein and its gastrointestinal digests are rich in antimicrobial peptides. However, related research is limited, restricting the development of the mealworm antimicrobial peptide industry.

[0004] Gastrointestinal digestive enzyme-peptidomics technology has become a widely used method in recent years for exploring potential active peptides in foodborne protein hydrolysates. This method helps to elucidate the potential antimicrobial peptide fragments of yellow mealworm proteins in gastrointestinal digests. Therefore, combining gastrointestinal digestive enzyme-peptidomics technology with solid-phase synthesis technology can help to discover antimicrobial peptides from yellow mealworms. Summary of the Invention

[0005] To address the shortcomings of existing technologies, this invention provides an antimicrobial peptide that inhibits foodborne pathogens, along with its acquisition method and applications. An antimicrobial peptide with antimicrobial activity is discovered and identified through a screening method combining gastrointestinal digestive enzyme-peptidomics technology and experiments, providing new natural antimicrobial agents for the food, pharmaceutical, and other related fields.

[0006] To achieve the above objectives, the present invention provides the following technical solution:

[0007] An antimicrobial peptide that inhibits foodborne pathogens, the amino acid sequence of which is: Gly-Leu-Thr-Leu-Lys-His-Leu-Lys-Lys-Leu-Ile-Phe (abbreviated as GLTLKHLKKLIF).

[0008] Preferably, the method for obtaining the antimicrobial peptide is any one of enzymatic hydrolysis and purification, microbial expression, or solid-phase chemical synthesis.

[0009] The antimicrobial peptides are used to inhibit Staphylococcus aureus, Salmonella, Escherichia coli, Pseudomonas aeruginosa, and Listeria monocytogenes.

[0010] Preferably, the MIC of the antimicrobial peptide in inhibiting Staphylococcus aureus, Salmonella, Escherichia coli, Pseudomonas aeruginosa, and Listeria monocytogenes is 4-256 μg / mL.

[0011] An antimicrobial composition comprising at least one of an antimicrobial peptide having the amino acid sequence Gly-Leu-Thr-Leu-Lys-His-Leu-Lys-Lys-Leu-Ile-Phe, or a pharmaceutically acceptable salt thereof, an isotopic label thereof, or a conjugate thereof with a carrier.

[0012] Preferably, the antibacterial composition is used in the preparation of preservatives, antibacterial preservatives, feed additives, oral care preparations, skin care preparations, disinfectants, or medical dressings.

[0013] This invention provides an antimicrobial peptide that inhibits foodborne pathogens, a method for obtaining the peptide, and its application. Compared with existing technologies, its advantages are as follows:

[0014] This invention utilizes a screening method combining gastrointestinal digestive enzyme-peptidomics technology and experiments to identify multiple antimicrobial peptides. Among them, Gly-Leu-Thr-Leu-Lys-His-Leu-Lys-Lys-Leu-Ile-Phe significantly outperforms the commercial antimicrobial agent NisinZ in its antibacterial activity against foodborne pathogens such as Staphylococcus aureus, Escherichia coli, Salmonella, and Pseudomonas aeruginosa. Furthermore, this antimicrobial peptide exhibits low hemolytic activity. Attached Figure Description

[0015] Figure 1 This is a schematic diagram showing the number of polypeptides produced by digestion with pepsin, trypsin, and chymotrypsin in Example 1 of the present invention.

[0016] Figure 2 The images show the ESI-MS (left) and RP-HPLC (right) spectra of peptide AP1 in this embodiment of the invention.

[0017] Figure 3 The images show the ESI-MS (left) and RP-HPLC (right) spectra of peptide AP2 in this embodiment of the invention.

[0018] Figure 4 The images show the ESI-MS (left) and RP-HPLC (right) spectra of peptide AP3 in this embodiment of the invention.

[0019] Figure 5 The images show the ESI-MS (left) and RP-HPLC (right) spectra of peptide AP4 in this embodiment of the invention.

[0020] Figure 6 The images show the ESI-MS (left) and RP-HPLC (right) spectra of peptide AP5 in this embodiment of the invention.

[0021] Figure 7 The images show the ESI-MS (left) and RP-HPLC (right) spectra of peptide AP6 in this embodiment of the invention.

[0022] Figure 8 The images show the ESI-MS (left) and RP-HPLC (right) spectra of peptide AP7 in this embodiment of the invention.

[0023] Figure 9 This is a schematic diagram showing the antibacterial rate of different concentrations of polypeptide AP2 and nisin Z against S. aureusATCC25923 in the embodiments of the present invention;

[0024] Figure 10 This is a schematic diagram showing the antibacterial rate of different concentrations of polypeptide AP2 and nisin Z against S. typhimurium ATCC14028 in the embodiments of the present invention.

[0025] Figure 11 This is a schematic diagram illustrating the antibacterial rates of different concentrations of polypeptide AP2 and nisin Z against E. coli ATCC25922 in embodiments of the present invention.

[0026] Figure 12 This is a schematic diagram showing the antibacterial rate of different concentrations of polypeptide AP2 and nisin Z against P. aeruginosa ATCC9027 in the embodiments of the present invention.

[0027] Figure 13 This is a schematic diagram showing the inhibition rate of different concentrations of polypeptide AP2 and nisin Z against L. monocytogenes ATCC19115 in the embodiments of the present invention.

[0028] Figure 14 This is a schematic diagram illustrating the effects of different concentrations of the antimicrobial peptide AP2 on the cell membrane of E. coli ATCC25922 in embodiments of the present invention. Detailed Implementation

[0029] To make the objectives, technical solutions, and advantages of the embodiments of the present invention clearer, the technical solutions of the embodiments of the present invention will be clearly and completely described below in conjunction with the embodiments of the present invention. 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.

[0030] Example 1:

[0031] Obtaining antimicrobial peptides:

[0032] 1. Protein database construction

[0033] First, complete protein sequences of the mealworm (Tenebrio molitor) were obtained from the UniProt database. To ensure data comprehensiveness, all annotated and experimentally validated protein sequences were selected. The downloaded protein sequences underwent normalization, including removing low-quality sequences, redundancy (such as different versions of the same protein), and low-complexity regions (such as sequences rich in repetitive amino acids). These preprocessing steps help improve the accuracy of subsequent virtual digestion and screening. All protein sequences were formatted into FASTA format files for input into the virtual digestion tool for analysis. Ultimately, the mealworm proteome retrieved using UniProtKB contained approximately 49,132 protein sequences.

[0034] 2. Gastrointestinal digestive enzymes

[0035] Enzyme digestion rules were set: Based on the digestive enzyme characteristics of mealworms, common proteolytic enzymes were selected for virtual enzyme digestion.

[0036] Trypsin: The cleavage site is after "Lys" and "Arg". Pepsin: The cleavage site is after "Phe" and "Tyr".

[0037] Chymotrypsin: The cleavage sites are after "Phe" and "Leu". The PeptideCutter program was used to digest the protein with selected pepsin, trypsin, and chymotrypsin to obtain polypeptide fragments.

[0038] 3. Peptide screening

[0039] Considering the structural characteristics of antimicrobial peptides (stable secondary structure) and the feasibility of solid-phase synthesis (peptides longer than 20 amino acids are typically costly and difficult to purify), only peptides with lengths of 6-25 amino acid residues were retained, resulting in a total of 3,782,689 candidate peptides. Specifically, pepsin, trypsin, and chymotrypsin generated 1,286,027, 906,333, and 1,590,329 peptides, respectively. Figure 1 Further peptide length distribution analysis showed that short to medium peptides (6–10 residues) accounted for more than 50% of the total peptide library, while long peptides (20–24 residues) accounted for less than 10%.

[0040] 4. Screening based on physicochemical properties

[0041] The net charge, secondary structure composition, and hydrophobic amino acid content of antimicrobial peptides significantly affect their antimicrobial activity. Screening criteria were set as follows: charge number 2–3, helix content 50–60%, fold content 30–40%, and hydrophobic amino acid ratio 30–50%. The BioPython tool was used to calculate the charge number, helix content, and fold content of the peptides, and filtering was performed according to the above criteria, resulting in 7 peptide sequences, as shown in Table 1 below.

[0042] Table 1

[0043]

[0044] 5. Hemolysis test:

[0045] Fresh mouse-derived mammalian erythrocytes were co-incubated with different concentrations of peptides. If the peptides exhibited hemolytic activity, they would disrupt the erythrocyte membrane, leading to the release of hemoglobin into the supernatant. After centrifugation to remove cell debris, the absorbance of the supernatant was measured at a specific wavelength (typically 540 nm or 414 nm), with absorbance values ​​directly proportional to the degree of hemolysis. The complete hemolysis group was defined as 100% hemolysis, and the control group as 0% hemolysis; the relative hemolysis rate at each concentration was calculated.

[0046] Peptide solution: Dissolve the lyophilized peptides in PBS buffer to prepare a high-concentration stock solution (e.g., 1-10 mM), filter sterilize using a 0.22 μm filter membrane, aliquot and store at -80°C.

[0047] Negative control:

[0048] 0% hemolysis control: PBS buffer + red blood cell suspension (buffer treatment only, representing no hemolysis).

[0049] Positive control:

[0050] 100% hemolysis control: 0.1% Triton X-100 (v / v) + red blood cell suspension (complete destruction of red blood cells);

[0051] The results are shown in Table 2 below, HC 50 This refers to the concentration of the peptide that causes 50% hemolysis of red blood cells. A higher value indicates lower hemolytic toxicity and better selectivity of the peptide. Table 2 shows the HC values ​​of these peptides. 50 Values ​​between 119.8 and 186.8 μM indicate low hemolytic activity.

[0052] Table 2

[0053]

[0054] 6. Preparation and characterization of the above-mentioned polypeptides AP1-AP7.

[0055] Solid-phase peptide synthesis (SPPS) was employed. SPPS is a highly efficient synthetic method suitable for synthesizing medium-length peptides, allowing for the stepwise addition of amino acid residues to a solid support. Rink Amide MBHA resin was selected, as it is suitable for synthesizing peptides with C-terminal amides. All amino acids used in the synthesis were Fmoc-protected, with the side-chain protecting groups selected according to the type of amino acid to ensure no unnecessary side reactions occurred during the synthesis process. Dimethyl thionamide (DMF) was used as a solvent to dissolve the amino acids, and the resin surface was cleaned with dichloromethane (DCM) to maintain resin activity.

[0056] Synthesis steps: Removal of amino acid protecting groups: First, remove the Fmoc protecting groups on the resin by removing the Fmoc groups with a 20% piperidine / DMF solution, in preparation for the next step of adding amino acids.

[0057] Amino acid addition: For each amino acid added, N,N'-diisothiocyanate (DIC) was used as the coupling agent, and HOBt (1-hydroxybenzotriazole) was used as the catalyst. The coupling reaction was carried out at room temperature. After each reaction step, the resin was washed with DMF, and the binding of the amino acid residues was detected by ultraviolet spectrophotometry.

[0058] After peptide chain synthesis, the synthesized peptide fragments are removed from the resin using hydrogen fluoride (HF) to obtain crude peptides. Residual hydrogen fluoride remains after removal, therefore, the peptides need to be thoroughly washed in dichloromethane and dried to remove all residual solvent.

[0059] To ensure the purity of the synthesized peptides, reversed-phase high-performance liquid chromatography (RP-HPLC) was used for peptide purification. Column selection: A C18 reversed-phase column (250 mm × 4.6 mm, 5 μm particle size) was used, suitable for separating hydrophobic peptides. Solvent system: The mobile phase consisted of two solutions: Solution A was water (0.1% TFA, trifluoroacetic acid), and Solution B was acetonitrile (0.1% TFA). Under gradient elution, the proportion of acetonitrile was gradually increased (0% to 80%) to separate peptides of different polarities. Purification conditions: The flow rate and solvent gradient were finely adjusted during elution to ensure efficient and accurate separation of the target peptides. Elution was monitored using a UV detector at a wavelength of 220 nm. The purified target peptides were collected based on elution time and UV absorbance.

[0060] The purity of the purified peptides was confirmed by HPLC analysis. The purity of the target peptide was required to exceed 95% to ensure the reliability of subsequent experiments.

[0061] The purified peptides were analyzed by mass spectrometry (ESI-MS) to confirm their molecular weight and structure. The purified peptides were dissolved in a suitable solvent, commonly water / acetonitrile (1:1 ratio), with an appropriate amount of TFA added to facilitate mass spectrometry measurements. An Agilent 6125B mass spectrometer equipped with an electrospray ionization (ESI) source was used. The molecular ion peak (m / z value) of the peptide was measured by mass spectrometry to confirm its mass. Based on the mass spectrometry analysis results, the molecular weight of the synthesized peptides was compared with the theoretical molecular weight to ensure consistency with expectations. The ESI-MS and RP-HPLC spectra of AP1-AP7 are shown below. Figure 2-8 As shown.

[0062] 7. In vitro antibacterial test

[0063] The activity of the peptide was verified by in vitro antibacterial experiments. The specific steps are as follows:

[0064] Experimental bacterial strains were selected to comprehensively evaluate the antimicrobial activity of the antimicrobial peptide AP1-AP7. Five common foodborne pathogens were chosen: * Staphylococcus aureus* (ATCC25923): a Gram-positive bacterium widely found in food and medical environments, often causing food poisoning and skin infections. * Escherichia coli* (ATCC25922): a common Gram-negative bacterium, usually a foodborne pathogen, easily causing intestinal infections. * Salmonella typhimurium* (ATCC14028): a Gram-negative bacterium, a common foodborne pathogen that can cause serious gastrointestinal diseases.

[0065] *Pseudomonas aeruginosa* ATCC 9027: A Gram-negative bacterium with widespread drug resistance, it can cause hospital-acquired infections. *Listeria monocytogenes* ATCC 19115: A Gram-positive bacterium, it is the pathogen causing listeriosis, can survive at low temperatures, and is an important pathogen in food safety.

[0066] Culture medium selection: All strains were cultured in Luria-Bertani (LB) medium, which is suitable for the growth of most common bacteria and can maintain their activity. Inoculation conditions: After inoculation, the strains were cultured at 37°C and 100% humidity for 24 hours under normal growth conditions.

[0067] MIC determination:

[0068] Preparation of antimicrobial peptide solutions: The synthesized peptides were dissolved in sterile deionized water to prepare solutions of different concentrations. To ensure complete dissolution, gentle shaking was used and the solution was kept at room temperature. Concentration gradient range preparation: Different concentrations of peptide solutions (1000 μg / mL, 500 μg / mL, 250 μg / mL, 125 μg / mL, 64 μg / mL) were prepared according to experimental requirements. The minimum inhibitory concentration (MIC) of the antimicrobial peptides was determined using the two-fold dilution broth microdilution method. This method is a standard antimicrobial activity assay that can determine the antimicrobial effect of antimicrobial agents at different concentrations. A 96-well plate was prepared, with 100 μL of ILB medium added to each well. 50 μL of peptide solution of different concentrations (from high to low) was added to each well. For example, 1000 μg / mL was added to the first column, 500 μg / mL to the second column, and so on. Next, 50 μL of bacterial suspension was added to each well, resulting in a final bacterial concentration of 10^5 CFU / mL. Each experimental strain used an independent well as a positive control (containing a known concentration of the antibiotic nisin Z) and a negative control (containing only bacterial culture medium and bacterial suspension). Cultivation and observation: The 96-well plate was incubated at 37°C for 24 hours. After cultivation, the bacterial growth in each well was observed. The bacterial growth was determined by comparing the optical density (OD600 nm) values ​​before and after cultivation. Whether bacteria grew at each concentration was recorded, and the lowest peptide concentration that could inhibit bacterial growth was calculated, which is the MIC. MIC calculation: Based on the observation results of each well, the MIC is the lowest peptide concentration that can significantly inhibit bacterial growth. If the concentration is too high, there will be no obvious bacterial growth in the well; conversely, if the concentration is too low, it indicates that the concentration cannot inhibit bacterial growth. The MIC value of the peptide was determined through the experimental results.

[0069] The MIC results are shown in Table 3. Peptide AP2 (GLTLKHLKKLIF) showed significant antimicrobial activity against multiple bacteria among the seven synthesized sequences. Its MIC values ​​against Staphylococcus aureus were 32 μg / mL, against Escherichia coli, Salmonella, Pseudomonas aeruginosa, and Listeria monocytogenes were 64 μg / mL and 32 μg / mL, respectively. Therefore, peptide AP2 is confirmed as an antimicrobial peptide, exhibiting strong antibacterial activity against Staphylococcus aureus and Listeria monocytogenes with low MIC values; while its MIC values ​​against Escherichia coli, Salmonella, and Pseudomonas aeruginosa were higher, but it still effectively inhibited bacterial growth (Table 3 and 3). Figure 9-13 ).

[0070] Table 3

[0071]

[0072] 8. Zeta potential and particle size distribution

[0073] Zeta potential assay: The synthesized antimicrobial peptide was dissolved in pure water at a concentration of 1 mg / mL. The zeta potential of the antimicrobial peptide was measured using a Malvern Zetasizer Nano ZS instrument, which is an important parameter for assessing the stability of the peptide solution. In neutral aqueous solution, the zeta potential of the antimicrobial peptide was +25 mV, indicating that it has strong charge stability and low solubility risk.

[0074] Particle size distribution analysis: Particle size distribution was measured using a Malvern Zetasizer Nano ZS. Dynamic light scattering (DLS) was performed at 25 °C to measure the particle size distribution of the peptide in aqueous solution. The particle size distribution results showed that the antimicrobial peptide GLTLKHLKKLIF had a particle size of approximately 189 nm, indicating good dispersibility of the peptide particles in the solution.

[0075] Table 4. Zeta potential and particle size distribution of antimicrobial peptide AP2

[0076]

[0077] 9. Cell membrane disruption

[0078] This experiment selected *Escherichia coli* (E. coli ATCC25922) as the model strain due to its standardized culture characteristics and common use in antimicrobial activity testing. Bacterial culture: *E. coli* was cultured in Luria-Bertani (LB) medium at 37°C until it reached the logarithmic growth phase. Antimicrobial peptide treatment: The synthesized antimicrobial peptide AP2 was dissolved in sterile physiological saline to prepare antimicrobial peptide solutions of different concentrations (1×MIC and 4×MIC). The antimicrobial peptide solutions were added to the *E. coli* suspension to treat different concentrations. The treatment time was set to 1 hour to ensure sufficient contact between the peptide and the bacteria.

[0079] Scanning electron microscopy (SEM) observation: Bacterial samples treated with antimicrobial peptides were washed three times with PBS to remove unbound antimicrobial peptides and culture medium components. Bacteria were fixed overnight at 4°C using 2.5% glutaraldehyde (in PBS). After fixation, dehydration was performed using a gradient of ethanol (30%, 50%, 70%, 80%, 90%, n=100%), with each step lasting 15 minutes. Samples were dried using a Leica EM CPD300 critical point desiccator to avoid morphological changes due to moisture evaporation. The dried samples were then placed in a Leica EM ACE200 coating system for metal coating, typically gold, to enhance conductivity and facilitate electron microscopy observation.

[0080] The treated bacteria were observed using a Hitachi SU8010 scanning electron microscope. Before treatment, *E. coli* exhibited a typical rod-shaped morphology with an intact and smooth cell membrane. After treatment with the antimicrobial peptide, significant morphological changes were observed on the bacterial surface. At a concentration of 1×MIC, the bacterial membrane showed shrinkage and slight perforation; at a concentration of 4×MIC, the bacterial cell membrane ruptured extensively, showing obvious indentations and perforations, indicating that the antimicrobial peptide inhibited bacterial growth by disrupting the integrity of the cell membrane. Figure 14 ).

[0081] Example 2:

[0082] Skin care products were prepared using the antimicrobial peptide AP2 obtained above as raw material, and the formula of the skin care products was as follows (mass fraction): white petrolatum 5.2%, glyceryl stearate 2.4%, isostearyl neopentyl ester 3.4%, sodium polyacrylate 7.5%, glyceryl stearate GSM 4.3%, cyclomethyl silicone oil 4.6%, tremella polysaccharide 4.0%, white oil 1.8%, and an appropriate amount of antimicrobial peptide AP2, with the remainder being water;

[0083] Referring to Table 5 below, different experimental groups of skincare products were set up according to the amount of antimicrobial peptide AP2 added:

[0084] Table 5

[0085]

[0086] The specific preparation method for skincare products is as follows:

[0087] Raw material preparation: Weigh out the oil phase components such as white petrolatum, isostearyl neopentyl ester, glyceryl stearate GSM, glyceryl stearate, cyclomethyl silicone oil and white oil, and place them in an oil phase container for later use; weigh out purified water (reserve a small amount for later use) in another container and add sodium polyacrylate.

[0088] Aqueous phase preparation: First, heat and stir the purified water in the aqueous phase container to allow the sodium polyacrylate to fully swell and form a uniform aqueous phase viscosity system; then, dissolve the antimicrobial peptide AP2 and tremella polysaccharide in sequence, maintain the system temperature at 70-75 ℃, and stir until all components are completely and uniformly dispersed.

[0089] Oil phase preparation: White petrolatum, isostearyl neopentyl ester, glyceryl stearate GSM, glyceryl stearate, cyclomethyl silicone oil and white oil placed in an oil phase container are heated to 70-75 ℃ and stirred to melt and dissolve the components and form a homogeneous oil phase.

[0090] Emulsification: Under the condition that both the oil phase and the aqueous phase are maintained at 70-75 ℃, the oil phase is added to the aqueous phase dropwise or continuously at a slow constant rate, while homogenizing is performed using a high-speed homogenizer; the homogenization conditions are 12000 rpm for 5 min, until a uniform, fine and stable water-in-oil emulsion is formed.

[0091] Cooling and addition of active ingredients: After emulsification, switch to low-speed stirring or intermittent stirring to allow the emulsion to cool naturally to below 40 ℃. When the system temperature drops to ≤40 ℃, slowly add the antimicrobial peptide AP2 under low temperature and low shear conditions;

[0092] Continue stirring at low speed to disperse the peptides evenly. When the emulsion temperature drops to room temperature (about 20-25 ℃) and the appearance, viscosity and dispersibility are stable, defoaming is performed. Finally, the product is filled, sealed and labeled to obtain the finished antibacterial skin care product.

[0093] The antibacterial activity of the finished antibacterial skincare products prepared in the blank control group and experimental groups 1-4 was determined. The specific testing process is as follows:

[0094] Experimental strains were selected from five common foodborne pathogens: Staphylococcus aureus (S. aureus ATCC25923), Escherichia coli (E. coli ATCC25922), Salmonella typhimurium (S. typhimurium ATCC14028), Pseudomonas aeruginosa (P. aeruginosa ATCC9027), and Listeria monocytogenes (L. monocytogenes ATCC19115).

[0095] Culture medium selection: All strains were cultured in Luria-Bertani (LB) medium, which is suitable for the growth of most common bacteria and can maintain their activity. Inoculation conditions: After inoculation, the strains were cultured at 37°C and 100% humidity for 24 hours under normal growth conditions.

[0096] MIC determination:

[0097] Preparation of blank control group and experimental groups 1-4 skin care product solutions: Dissolve the blank control group and experimental groups 1-4 skin care products in sterile deionized water to prepare solutions of different concentrations. To ensure complete dissolution, use gentle shaking and maintain at room temperature. Concentration gradient range preparation: Prepare blank control group and experimental groups 1-4 skin care product solutions of different concentrations (100 mg / mL, 50 mg / mL, 25 mg / mL, 12.5 mg / mL, 6.25 mg / mL, and 3.125 mg / mL) according to experimental requirements. The minimum inhibitory concentration (MIC) of the antimicrobial peptide was determined using the two-fold dilution broth microdilution method. Prepare 96-well plates, adding 100 μL of LB medium to each well. Add 50 μL of different concentrations of blank control group and experimental groups 1-4 skin care product solutions (from high to low concentration) to each well. Then, add 50 μL of bacterial suspension to each well, resulting in a final bacterial concentration of 10^5 CFU / mL. Incubate the 96-well plates at 37°C for 24 hours. After culturing, the growth of bacteria in each well was observed. The bacterial growth was determined by comparing the optical density (OD600 nm) values ​​before and after culturing. Whether bacteria grew at each concentration was recorded, and the minimum concentration of the antimicrobial peptide that could inhibit bacterial growth was calculated; this is the MIC. Based on the observations of each well, the MIC is the lowest concentration of antimicrobial peptide that can significantly inhibit bacterial growth. If the concentration is too high, there will be no obvious bacterial growth in the well; conversely, if the concentration is too low, it indicates that the concentration cannot inhibit bacterial growth. The MIC value of the antimicrobial peptide was determined through the experimental results.

[0098] The MIC results are shown in Table 6. The results show that experimental groups 1-4 of this invention have good inhibitory effects on the five common pathogenic bacteria selected, with MICs below 25 mg / mL.

[0099] Table 6

[0100]

[0101] The above embodiments are only used to illustrate the technical solutions of the present invention, and are not intended to limit it. Although the present invention has been described in detail with reference to the foregoing embodiments, those skilled in the art should understand that modifications can still be made to the technical solutions described in the foregoing embodiments, or equivalent substitutions can be made to some of the technical features. Such modifications or substitutions do not cause the essence of the corresponding technical solutions to deviate from the spirit and scope of the technical solutions of the embodiments of the present invention.

Claims

1. An antibacterial peptide that inhibits foodborne pathogens, characterized in that, The amino acid sequence of the antibacterial peptide is Gly-Leu-Thr-Leu-Lys-His-Leu-Lys-Lys-Leu-Ile-Phe.

2. The antimicrobial peptide according to claim 1, characterized in that: The antibacterial peptide is obtained by any one of enzymatic separation and purification, microbial expression, and solid-phase chemical synthesis.

3. Use of the antibacterial peptide of claim 1 for inhibiting Staphylococcus aureus, Salmonella, Escherichia coli, Pseudomonas aeruginosa, and Listeria monocytogenes, excluding the use for therapeutic purposes.

4. Use according to claim 3, characterized in that: The MIC of the antibacterial peptide for inhibiting Staphylococcus aureus, Salmonella, Escherichia coli, Pseudomonas aeruginosa, and Listeria monocytogenes is 4-256 μg / mL.

5. An antimicrobial composition, characterized by: The antibacterial composition comprises at least one of the antibacterial peptide with the amino acid sequence of Gly-Leu-Thr-Leu-Lys-His-Leu-Lys-Lys-Leu-Ile-Phe, or a pharmaceutically acceptable salt, an isotopically labeled compound thereof, or a combination thereof with a carrier.

6. The antimicrobial composition according to claim 5, wherein: The antibacterial composition is applied to the preparation of antiseptics, bacteriostatic preservatives, feed additives, oral care preparations, skin care preparations, disinfectants, or medical dressings.

Images