Antibacterial peptide, application and product thereof

Abstract

The present application relates to a kind of lactoferrin coexisting component antibacterial peptide and its application, it is from endogenous enzyme hydrolysis cheese protein antibacterial polypeptide compound YQKFALPQYLKTVYQHQK, its amino acid sequence is Tyr-Gln-Lys-Phe-Ala-Leu-Pro-Gln-Tyr-Leu-Lys-Thr-Val-Tyr-Gln-His-Gln-Lys.Gram-positive bacteria and gram-negative bacteria have extensive inhibitory activity respectively, including escherichia coli, staphylococcus aureus, klebsiella pneumoniae, acinetobacter baumannii etc..The antibacterial peptide can be used as the health care product and / or medical equipment or drug, food bacteriostatic agent, green biological pesticide, new functional food, daily chemical in preventing and / or reducing infection disease, it has good application prospect.

Description

Technical Field

[0001] This invention relates to an antimicrobial peptide YQKFALPQYLKTVYQH QK coexisting component of lactoferrin and its application. Background Technology

[0002] Antibiotics are first-line drugs for clinical anti-infective treatment, but their widespread and even overuse has led to a series of serious problems, such as allergies, gut microbiota dysbiosis, superinfections, and multidrug resistance. In particular, the emergence and spread of multidrug-resistant bacteria have severely weakened the efficacy of antibiotics and threatened global public health security. Therefore, exploring and developing highly effective and safe alternative antibiotics has always been a research hotspot in this field.

[0003] Antimicrobial peptides (AMPs) are among the most promising follow-up agents. AMPs are endogenous small polypeptides widely found in various organisms in nature and are important effector molecules of the innate immune system. Unlike traditional antibiotics that act on specific intracellular targets, AMPs mainly bind to negatively charged bacterial cell membranes through electrostatic interactions, thereby disrupting membrane integrity through "carpet-like" or "bucket-like" models, leading to leakage of contents and cell death. This predominantly physical bactericidal mechanism makes it difficult for pathogens to develop resistance through single gene mutations. In addition, AMPs exhibit multiple biological functions, including immunomodulation, promoting wound healing, and neutralizing endotoxins, giving them unique therapeutic advantages beyond simple bactericidal action. Nevertheless, the large-scale preparation, in vivo stability, and potential cytotoxicity of natural AMPs remain major obstacles to their clinical translation. Therefore, the continuous discovery and identification of novel AMPs with well-defined activities and readily available sources has significant scientific and applied value. Milk is an important dietary source of exogenous proteins and bioactive components for humans. It is rich in protein composition and contains a variety of bioactive substances. Lactoferrin (LF) is an important multifunctional natural bioactive protein in milk, attracting much attention due to its important roles in iron metabolism, immune regulation, and broad-spectrum antibacterial activity. In the natural state of lactoferrin and during its isolation and purification, small peptides often coexist with it in the same system or are non-covalently bound to it. These peptides coexisting with lactoferrin are partly derived from the limited hydrolysis of lactoferrin itself and partly from the co-degradation of other milk proteins in the system, forming a complex and diverse natural bioactive peptide library. Peptides isolated from lactoferrin coexisting compounds possess various biological potentials, including antioxidant, immunomodulatory, and antibacterial activities. Based on this, this study used lactoferrin coexisting compounds as the research object, employing liquid chromatography-tandem mass spectrometry (LC-MS / MS) and bioinformatics techniques to systematically analyze the endogenous peptide group within them, aiming to screen and identify new sequences with broad-spectrum antibacterial activity. This study not only provides a new molecular basis for elucidating the synergistic antibacterial mechanism of lactoferrin, but also holds promise for providing important candidate molecules and strategic directions for the development of novel natural food preservatives, functional ingredients, and clinical anti-infective lead compounds. Summary of the Invention

[0004] This invention relates to an antimicrobial peptide YQKFALPQYLKTVYQHQ K in a lactoferrin coexistence compound and its applications. This antimicrobial peptide can be used in the preparation of products such as microbial growth inhibitors, anti-infective drugs, antioxidants, food preservatives, biopesticides, animal feed, and cosmetics. It can be added to one of the acceptable carriers or excipients in pharmaceuticals, medical devices, food, biopesticides, animal feed, oral care products, and cosmetics. It can also be used in the preparation of drugs for preventing and / or reducing diseases caused by common or single bacteria such as Escherichia coli, Staphylococcus aureus, Klebsiella pneumoniae, and Acinetobacter baumannii, such as intestinal infections, lung infections, and urinary tract infections, or in novel infant formula foods.

[0005] To achieve the above objectives, the present invention uses the polypeptide YQKFALPQYLKTVYQHQK as an effective component for inhibiting the growth activity of pathogenic microorganisms.

[0006] It has the amino acid sequence in SEQ ID NO: 1 in the sequence listing; the polypeptide YQKFALPQYLKTVYQHQK is an effective ingredient for inhibiting the growth of pathogenic microorganisms, and may or may not contain pharmaceutically or medically acceptable carriers or excipients.

[0007] The antibacterial polypeptide compound YQKFALPQYLKTVYQHQK has the following amino acid sequence: Tyr-Gln-Lys-Phe-Ala-Leu-Pro-Gln-Tyr-Leu-Lys-Thr-Val-Tyr-Gln-His-Gln-Lys. It has a molecular weight of 2283.66 Da, is a white powder, soluble in water, and exhibits strong inhibitory effects on the growth and activity of various pathogenic bacteria.

[0008] Compared with the prior art, the present invention has the following beneficial effects: This invention is derived from the coexisting components of lactoferrin. The structure and physicochemical properties of the polypeptide were obtained and determined. The broad-spectrum antibacterial activity of the polypeptide was verified for the first time. Therefore, it has good application prospects in the preparation of drugs and / or health products or medical devices, daily chemical products, and novel functional foods for the prevention and / or reduction of infectious diseases caused by Escherichia coli, Staphylococcus aureus, Klebsiella pneumoniae, Acinetobacter baumannii, etc. Attached Figure Description

[0009] Figure 1 The image shows the antibacterial effect of the antimicrobial peptide YQKFALPQYLKTVYQHQK. Figure a shows the inhibition zone experiment results of the peptide YQKFALPQYLKTVYQHQK against Escherichia coli. The positive control was chlorhexidine, and the negative control was sterile water. Note: + represents chlorhexidine, - represents sterile water. Detailed Implementation

[0010] The present invention will now be described in detail with reference to specific embodiments. These examples will help those skilled in the art and researchers to further understand the present invention, but do not constitute any limitation thereof. Any modifications made by any person within the scope of the claims of this invention are still within the protection scope of the claims of this invention.

[0011] Example 1 Isolation and identification of lactoferrin coexisting components (1) Sample preparation Take 200 mg of lactoferrin, dilute it with sterile water to a concentration of 200 mg / mL (w / v), heat it in a water bath at 95 °C for 5 min to denature it, and use an ultrafiltration tube (Millipore) with a molecular weight cutoff of 30 kDa to ultrafilter at 14000 × g and 20 °C for 20 min. Collect the ultrafiltrate to obtain the lactoferrin coexisting component.

[0012] (2) HPLC-MS / MS analysis The lactoferrin coexisting component sample was desalted using a C18 column (1cc / 10mg, Waters Oasis HLB SPE column) as follows: The commercially available C18 column was activated with 1.5mL methanol for 15min, then equilibrated with 1.5mL of 0.1% (V / V) TFA (trifluoroacetic acid)-H2O solution for 30min. The sample was then added to the C18 column, and the salt was eluted with 1.5mL of 0.1% (V / V) TFA (trifluoroacetic acid)-H2O solution. The eluent was then eluted with 1.5mL of 80% (V / V) ACN / 0.1% (V / V) TFA (trifluoroacetic acid)-H2O solution. The eluent was collected, aliquoted, lyophilized, and stored at -80℃. The endogenous peptide samples obtained from the above steps were reconstituted with 0.1% (V / V) FA (formic acid)-H2O, with a sample concentration of 0.5 mg / mL. All samples were loaded into the same 2 μL volume for HPLC-MS / MS analysis, with an injection volume of 1 μg. Nano LC-MS / MS analysis was performed using an Orbitrap Fusion Lumos Tribrid mass spectrometer (Thermo Fisher, San Jose, CA) in information-dependent acquisition (DDA) mode. Pre-treated columns were C18 (10 mm × 0.3 mm, Waters) and C18 (3 μm, 120 Å, Dr Masch GmbH, Georgia) packed with 20 cm × 200 μm d. Tandem mass spectrometry was performed in positive ion data-dependent mode, with the mass spectrum scan set to a full scan range of ions with a charge-to-mass ratio (m / z) of 375–1650. The ion transport capillary temperature was 250 °C, and the spray voltage was 2 kV. Mass spectrometry analysis of the top 20 strong precursor ions with charge states 2-7 was performed using high-energy collisional dissociation (HCD) mode at a mass resolution of 35,000. Dynamic exclusion was enabled with an exclusion time of 60 seconds.

[0013] The mobile phase was transported using a Thermo EASY-nLC system. Reverse-phase gradient separation was performed using mobile phase A (H₂O / 0.1% FA (formic acid) solution) and mobile phase B (80% ACN / 0.1% FA (formic acid) solution). The program was set to linear gradient elution of 12%–90% mobile phase B for 75 min at a flow rate of 500 nL / min. Finally, the system was equilibrated by washing with phase A for 15 min.

[0014] (3) Data retrieval Mass spectrometry data was acquired using MaxQuant (Version 2.0.3.0) software. Database searches were performed using RAW format files. The protein database was established by the laboratory based on information from its self-built bovine endogenous peptide database (5319 endogenous peptides). During the search, the following parameters were used: non-specific enzyme digestion; methionine oxidation (+15.9949 Da), lysine acetylation (+42.011 Da), and phosphorylation (+79.966 Da) were set as variable modifications, with no fixed modifications. MaxQuant's standard-free quantification method was selected. The tolerance for precursor ion mass was 20 ppm, and the free dose ratio (FDR) was <1%.

[0015] (4) Obtain target peptide information by LC-MS / MS More than 500 (517) peptides were identified in the lactoferrin coexisting component sample, of which approximately 45% originated from casein. The polypeptide YQKFALPQYLKTVYQHQK derived from the lactoferrin coexisting component is an Alpha-S1-casein protein precursor with an isoelectric point of 9.70, a molecular weight of 2283.66, and 18 amino acids. Biological information obtained from the amino acid sequence shown in SEQ ID NO: 1 using online tools indicates that the peptide has an average hydrophobicity GRAVY of -1.039, an instability coefficient of 17.75, and an Aliphatic index of 65.00.

[0016] Information of SEQ ID No. 1 (a) Sequence characteristics Length: 18 amino acids Type: Amino acid Chain type: branched single chain (b) Molecular type: Protein YQKFALPQYLKTVYQHQK Sequence description: SEQ ID No. 1 Example 2 Antibacterial activity of peptides The peptide VAVALARPKHPIK was synthesized by Nanjing Jietai Biotechnology Co., Ltd. using a solid-phase method, with a purity greater than 95%. The antibacterial activity of the peptide YQKFALPQYLKTVYQHQK was investigated using Gram-negative Escherichia coli as the target bacterium.

[0017] (1) Strains and resuscitation Escherichia coli (CICC10899) was preserved at -80℃ using the glycerol preservation method. Before the experiment, the bacteria were inoculated into liquid culture medium and cultured at 37℃ for 12 hours to revive the strain and restore its activity.

[0018] (2) Culture medium The antibacterial experiment used a 90mm microbial culture dish. E. coli It contains 1.5% agar (m / v, g / mL) and has a bacterial concentration of 1×10⁻⁶. 6 LB broth medium at CFU / mL was purchased from Sangon Biotech (Shanghai) Co., Ltd. The medium was sealed and stored at 4℃ for later use.

[0019] (3) Antibacterial test The antimicrobial activity of antimicrobial peptides against *Escherichia coli* was determined using a perforation method. A perforator was used to punch holes in agar plates of culture medium in petri dishes. The holes were 3.8 ± 0.2 mm in diameter and spaced approximately 1.5 cm apart. The synthesized standard peptide was dispensed at 4 mg / tube, with 40 μL of sterile water added to each tube, and vortexed until completely dissolved. 20 μL of the standard peptide was added to each well, resulting in an actual dosage of 2.0 mg per well. The positive control chlorhexidine (2 MIC) and the negative control sterile water were added using the same method and volume. After sample addition, the petri dishes were incubated at 37°C for 18 hours. The presence and size of inhibition zones were observed and recorded.

[0020] (4) Experimental Results The antibacterial results and inhibition zone diameters of the milk antimicrobial peptides YQKFALPQYLKTVYQHQK against Escherichia coli are shown in the figures below. Figure 1 See Table 1. The results show that the polypeptide has good inhibitory activity against Escherichia coli and exhibits stable and efficient antibacterial properties.

[0021]

[0022] Table 1. Antibacterial results of peptides YQKFALPQYLKTVYQHQK Note: The numbers represent the diameter of the inhibition zone. Example 3 Antibacterial activity of peptides The polypeptide YQKFALPQYLKTVYQHQK with a purity of 97.86% was synthesized by Nanjing Jietai Biotechnology Co., Ltd. using a solid-phase method. The antibacterial activity of the polypeptide YQKFALPQYLKTVYQHQK was investigated using Gram-negative Klebsiella pneumoniae, Acinetobacter baumannii, and Gram-positive Staphylococcus aureus as target bacteria.

[0023] (1) Strains and resuscitation Staphylococcus aureus ( Staphylococcus aureus CICC10384), Klebsiella pneumoniae ( Klebsiella pneumoniaeAcinetobacter baumannii (ATCC13883) and Acinetobacter baumannii (ATCC19606) were preserved at -80℃ using the glycerol preservation method. Before the experiment, the bacteria were inoculated into the corresponding liquid culture medium (TSB broth for Staphylococcus aureus, and CAMHB broth for Klebsiella pneumoniae) and cultured at 37℃ for 12 h to revive the bacteria and restore their viability.

[0024] (2) Antibacterial test Take 1 mL of bacterial culture, wash twice with PBS buffer, and then dilute to 10⁻⁶. 7 Bacterial suspensions were obtained for Staphylococcus aureus, Klebsiella pneumoniae, and Acinetobacter baumannii. Antimicrobial peptide samples YQKFALPQYLKTVYQHQK were dissolved in the corresponding culture media for each of the bacteria (TSB broth for Staphylococcus aureus, CAMHB broth for Klebsiella pneumoniae) to prepare 100 mg / mL solutions. These solutions were then sterilized by passing the solutions through a 0.22 μm membrane. 100 μL of the corresponding antimicrobial peptide solution or the corresponding culture medium was mixed with 100 μL of the corresponding bacterial suspension (TSB broth for Staphylococcus aureus or the suspension obtained after dissolving the antimicrobial peptide sample; CAMHB broth for Klebsiella pneumoniae and the suspension obtained after dissolving the sample). These mixtures were then added sequentially to different wells of a 96-well cell culture plate. Staphylococcus aureus, Klebsiella pneumoniae, and Klebsiella pneumoniae were cultured at 37°C under aerobic conditions. The absorbance of the above bacterial cultures was measured at 600 nm after culturing for 12 hours. The inhibition rate of each sample for different bacteria was calculated using the following formula:

[0025] OD1 control group: 100 μL bacterial suspension + 100 μL culture medium OD2 experimental group: 100 μL bacterial culture + 100 μL antimicrobial peptide solution (4) Experimental Results The antimicrobial peptide YQKFALPQYLKTVYQHQK's inhibitory effects against Staphylococcus aureus, Klebsiella pneumoniae, and Acinetobacter baumannii are shown in Table 2. The table lists the inhibitory results of the antimicrobial peptide YQKFALPQYLKTVYQHQK. The results indicate that this peptide YQKFALPQYLKTVYQHQK exhibits good inhibitory activity against both Gram-negative and Gram-positive bacteria, demonstrating broad-spectrum and highly effective antimicrobial properties.

[0026] Table 2. Antibacterial results of peptides VAVALAPKHPIK

Claims

1. An antimicrobial peptide, characterized in that: The amino acid sequence of the antimicrobial peptide is shown in SEQ ID NO:

1.

2. The antimicrobial peptide according to claim 1, characterized in that: The antimicrobial peptide is YQKFALPQYLKTVYQHQK, and its amino acid sequence is Tyr-Gln-Lys-Phe-Ala-Leu-Pro-Gln-Tyr-Leu-Lys-Thr-Val-Tyr-Gln-His-Gln-Lys.

3. The use of the antimicrobial peptide according to claim 1 or 2 in the preparation of products such as microbial growth inhibitors, anti-infective drugs, antioxidants, food preservatives, cosmetics, health products, biological pesticides, or animal feed.

4. An antimicrobial peptide product, characterized in that: The antimicrobial peptide product is a microbial growth inhibitor or anti-infective drug, health product, biopesticide, animal feed or medical device. The microbial growth inhibitor or anti-infective drug, health product, biopesticide, animal feed or medical device uses the antimicrobial peptide YQKFALPQYLKTV YQHQK as the active ingredient, with or without pharmaceutically or medically acceptable carriers or excipients.

5. The application according to claim 3 or the antimicrobial peptide product according to claim 4, characterized in that: The microorganisms are one or more of Escherichia coli, Staphylococcus aureus, Klebsiella pneumoniae, and Acinetobacter baumannii.

6. The application according to claim 3 or the antimicrobial peptide product according to claim 4, characterized in that: The infection is caused by a combination or a single bacterium, such as Escherichia coli, Staphylococcus aureus, Klebsiella pneumoniae, or Acinetobacter baumannii, including intestinal infections, lung infections, or urinary tract infections.

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