Antibacterial peptide and application thereof

Abstract

The present application relates to a kind of bovine milk endogenous antibacterial peptide and its application, it is from endogenous enzyme hydrolysis casein of antibacterial polypeptide compound PKTKVIPYVRYL of milk, its amino acid sequence is Pro-Lys-Thr-Lys-Val-Ile-Pro-Tyr-Val-Arg-Tyr-Leu.Gram-positive bacteria and gram-negative bacteria have extensive inhibitory activity respectively, including gingival porphyromonas, escherichia coli, staphylococcus aureus etc..The antibacterial peptide can be used as the health care product and / or medical equipment or drug, food bacteriostatic agent, new functional food, pet food and animal feed additive and daily chemical in preventing and / or reducing infection disease, with good application prospect.

Description

Technical Field

[0001] This invention relates to a bovine milk endogenous antimicrobial peptide PKTKVIPYVRYL and its applications. Background Technology

[0002] Antibiotics are a class of drugs that kill or inhibit microorganisms such as bacteria. They kill or inhibit microbial growth by targeting specific biological processes of microorganisms, such as cell wall synthesis, protein synthesis, nucleic acid synthesis, and metabolism. However, the overuse of antibiotics has led to increasingly prominent problems, including bacterial resistance, side effects, disruption of normal flora resulting in weakened immunity, poor therapeutic efficacy of existing antibiotics, and a narrowing of the antibacterial spectrum. Therefore, finding substances that can replace or improve the effectiveness of antibiotics has always been a research hotspot in this field. Antimicrobial peptides, as a novel treatment for bacterial infections, have attracted widespread attention due to their natural origin, broad-spectrum antibacterial activity, safety, high efficacy, and lack of drug resistance, and show promising research and application prospects.

[0003] Antimicrobial peptides are a class of small-molecule polypeptide compounds that can inhibit bacterial growth and reproduction, and are widely found in nature. Antimicrobial peptides can bind to the bacterial cell membrane surface, disrupting the structure and function of the cell membrane, leading to loosening or destruction of the cell membrane, thereby affecting biological reactions on the cell membrane and even causing cell death. In addition, some antimicrobial peptides also exhibit inhibitory activity against certain viruses and have shown effects such as enhancing immunity and promoting wound healing. Their broad biological activities demonstrate their promising application prospects in antibacterial applications. Developing safe, stable, and readily available antimicrobial peptides is of great significance.

[0004] Milk is rich in nutrients and is the primary source of exogenous protein for humans, containing abundant proteins and endogenous peptides. Due to the strong hydrolytic effect of endogenous enzymes in milk on proteins, a large number of stable endogenous peptides exist in milk, some of which possess biological activity. A simple and rapid method, using heat treatment followed by direct ultrafiltration, is employed to obtain endogenous peptides from milk. Therefore, starting from endogenous peptides in milk, multi-omics technologies are used to identify endogenous peptides with antibacterial activity, providing new solutions for clinical anti-infective treatments and offering new products and directions for the development of simple and safe microbial inhibitors and natural foods. Summary of the Invention

[0005] This invention relates to a bovine milk endogenous antimicrobial peptide PKTKVIPYVRYL and its applications. This antimicrobial peptide is used in the preparation of products such as microbial growth inhibitors, anti-infective drugs, antioxidants, food preservatives, pet food, animal feed, and cosmetics. It can be added to one of the acceptable carriers or excipients in pharmaceuticals, medical devices, food, pet food, animal feed, oral care products, and cosmetics. It is also used in the preparation of drugs for preventing and / or reducing diseases caused by Escherichia coli, Staphylococcus aureus, Porphyromonas gingivalis, and Streptococcus mutans, either individually or collectively, such as intestinal infections, gingivitis, or periodontitis, or in novel infant formula.

[0006] To achieve the above objectives, the present invention uses the polypeptide PKTKVIPYVRYL as an effective component for inhibiting the growth activity of pathogenic microorganisms.

[0007] It has the amino acid sequence in SEQ ID NO:1 in the sequence listing; the polypeptide PKTKVIPYVRYL is an endogenous active ingredient in bovine milk, which may or may not contain pharmaceutically or medically acceptable carriers or excipients.

[0008] The amino acid sequence of the antibacterial polypeptide compound PKTKVIPYVRYL is Pro-Lys-Thr-Lys-Val-Ile-Pro-Tyr-Val-Arg-Tyr-Leu. It has a molecular weight of 1476.78 Da, is a white powder, soluble in water, and exhibits strong inhibitory effects on the growth and activity of various pathogenic bacteria.

[0009] This invention discloses endogenous antimicrobial peptides from bovine milk that exhibit broad inhibitory activity against both Gram-positive and Gram-negative bacteria, including *Porphyromonas gingivalis*, *Escherichia coli*, and *Staphylococcus aureus*. These antimicrobial peptides show promising applications as health supplements and / or medical devices or pharmaceuticals for the prevention and / or reduction of infectious diseases, as well as as antimicrobial agents in food, novel functional foods, pet food and animal feed additives, and daily chemical products.

[0010] Compared with the prior art, the present invention has the following beneficial effects:

[0011] This invention utilizes heat-processed cow's milk to obtain and determine the structure and physicochemical properties of a polypeptide. It is the first to verify the broad-spectrum antibacterial activity of this polypeptide. Furthermore, cow's milk is readily available and inexpensive, and the extraction process for its endogenous peptides is simple and easy to operate, requiring no additional chemical reagents. The extracted endogenous peptides are safe, stable, and readily available. Therefore, it has promising applications in the preparation and prevention of infectious diseases caused by *E. coli*, including drugs and / or health products or medical devices, daily chemical products, novel functional foods, pet food, and animal feed. Attached Figure Description

[0012] Figure 1 Figure 1 shows the antibacterial effect of the antimicrobial peptide PKTKVIPYVRYL. Figure 2a shows the inhibition zone experiment results of PKTKVIPYVRYL inhibiting Staphylococcus aureus. The positive control is chlorhexidine, and the negative control is sterile water. Note: + represents chlorhexidine, - represents sterile water, and a represents the antimicrobial peptide PKTKVIPYVRYL. Detailed Implementation

[0013] 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.

[0014] Example 1

[0015] Preparation and identification of endogenous peptides from bovine milk

[0016] (1) Sample preparation

[0017] Take 200 μL of raw milk (common name: cow's milk) and dilute it with sterile water to 10 times the volume of the raw milk. Heat it in a water bath at 95°C for 5 min to denature it. Use an ultrafiltration tube (Millipore) with a molecular weight cutoff of 10 kDa to ultrafilter at 14000×g and 20°C for 20 min. Collect the clear liquid that has passed through the ultrafiltration membrane (i.e., ultrafiltrate) to obtain the milk endogenous peptides.

[0018] (2) HPLC-MS / MS analysis

[0019] The milk endogenous peptide sample was desalted using a C18 column (Waters Oasis HLB SPE column, 1cc / 10mg, 31.7μm) as follows: The commercially available C18 column was activated with 1.5mL of methanol and then equilibrated with 1.5mL of 0.1% (V / V) TFA (trifluoroacetic acid)-H2O solution. The sample was added to the C18 column, and the salt was eluted with 1.5mL of 0.1% (V / V) TFA (trifluoroacetic acid)-H2O solution. Then, the sample was 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℃ to obtain the specified endogenous peptide sample. The endogenous peptide samples obtained from the above steps were redissolved in 0.1% (V / V) FA (formic acid)-H2O solution, with a sample concentration of 0.5 mg / mL (endogenous peptide sample / solution). A loading volume of 2 μL was used 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. Samples were loaded onto a C18 pretreated column (capillary column: Polymicro, inner diameter * length: 75 μm id * 3 cm; packing material: 3 μm). (Sepucbd) and C18 (capillary column: Polymicro, inner diameter * length: 75μm id * 20cm; packing material: 2.4μm, Mass spectrometry analysis was performed on a Reprosil analytical column. Tandem mass spectrometry was acquired 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. The top 20 strong precursor ions with charge states 2–7 were analyzed using high-energy collisional dissociation (HCD) mode at a mass resolution of 35,000. Dynamic exclusion was enabled with a 60-second exclusion time.

[0020] Thermo EASY-nLC was used to transport the mobile phase. Reverse-phase gradient separation was performed using mobile phase A (H₂O / 0.1% FA (formic acid, V / V) solution) and mobile phase B (80% ACN / 0.1% FA (formic acid, V / V) aqueous solution). The program settings were as follows: 0–2 min, 0–12% B (V / V); 2–72 min, 12–50% B; 72–75 min, 50–90% B; 75–90 min, 90% B; flow rate 250 nL / min. Gradient elution with mobile phase B was performed for 75 min at a flow rate of 500 nL / min. Finally, the system was flushed with phase B for 15 min to equilibrate.

[0021] (3) Data retrieval

[0022] The *RAW format files acquired by mass spectrometry were searched using MaxQuant (Version 2.0.3.0) software. The protein database was established by the laboratory based on information from the bovine protein database (6035 proteins) on the Uniprot website (http: / / www.uniprot.org / ). 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 mode was selected. The mass tolerance for precursor ions was 20 ppm, and the free ion density (FDR) was <1%.

[0023] (4) Obtain target peptide information by LC-MS / MS

[0024] 1047 peptides were identified in endogenous bovine milk peptide samples, with molecular weights ranging from 1.6 kDa to 9.1 kDa, of which approximately 66% were derived from casein. The endogenous bovine milk peptide PKTKVIPYVRYL is derived from the protein precursor Alpha-S2-casein, with an isoelectric point of 10.01, a molecular weight of 1476.78, and 12 amino acids. Biological information obtained from the amino acid sequence of SEQ ID NO: 1 using online tools indicates that this peptide has an average hydrophobicity (GRAVY) of -0.175, an instability coefficient of -6.59, and an Aliphatic index of 113.33.

[0025] Information of SEQ ID No. 1 (a) Sequence characteristics

[0026] *Length: 12 amino acids

[0027] *Type: Amino acid

[0028] * Chain type: branched single chain (b) Molecular type: protein

[0029] Sequence description: PKTKVIPYVRYL

[0030] Example 2

[0031] Antibacterial activity of peptides

[0032] The polypeptide PKTKVIPYVRYL with a purity of 98.58% was synthesized by Nanjing Jietai Biotechnology Co., Ltd. using a solid-phase method. The antibacterial activity of the polypeptide PKTKVIPYVRYL was investigated using Staphylococcus aureus, a Gram-positive bacterium, as the target bacterial species.

[0033] (1) Strains and resuscitation

[0034] Staphylococcus aureus (CICC10384) was preserved at -80℃ using the glycerol preservation method. Before the experiment, Staphylococcus aureus was inoculated into TSB broth (Beijing Solarbio Science & Technology Co., Ltd.), with the target strain inoculated at a volume of 1%. The strain was then anaerobically cultured at 37℃ for 12 hours to allow for resuscitation. The bacterial suspension was then re-inoculated into fresh TSB broth at a volume of 1% for further revival.

[0035] Repeat the inoculation and resuscitation process twice to restore the viability of the strain.

[0036] (2) Culture medium

[0037] The antibacterial experiment used 90 mm diameter microbial culture dishes (with 20 mL of bacterial culture medium). SA (Staphylococcus aureus) was prepared with 1.5% agar (m / v, g / ml) at a bacterial concentration of 1 × 10⁻⁶. 6 TSB broth medium at CFU / mL was purchased from Sangon Biotech (Shanghai) Co., Ltd. The medium was sealed and stored at 4℃ for later use.

[0038] (3) Antibacterial test

[0039] The antimicrobial activity of the antimicrobial peptide PKTKVIPYVRYL against Staphylococcus aureus was determined using a perforation method. Seven wells were punched on a plate using a perforator, with a spacing of approximately 4 cm between each well and a diameter of 3.8 ± 0.2 mm. The synthetic standard peptide was aliquoted at 5 mg / tube, and 40 μL of sterile water was added to each aliquot, vortexing until completely dissolved. 20 μL of the standard peptide was added to each well, resulting in an actual addition of 2.5 mg per well. The positive control chlorhexidine (2 MIC, 9.435 μg / ml) and the negative control sterile water were added using the same method and volume. After addition, the petri dishes were incubated at 37°C for 18 h, and the appearance and size of inhibition zones were observed and recorded.

[0040] (4) Experimental Results

[0041] The antibacterial results and inhibition zone diameter of the milk antimicrobial peptide PKTKVIPYVRYL against Staphylococcus aureus are shown in the figure. Figure 1 See Table 1. The results show that the polypeptide has good inhibitory activity against Staphylococcus aureus and exhibits stable and efficient antibacterial properties.

[0042] Table 1. Antibacterial results of peptide PKTKVIPYVRYL

[0043]

[0044] Note: The numbers represent the diameter of the inhibition zone.

[0045] Example 3

[0046] Antibacterial activity of peptides

[0047] The polypeptide PKTKVIPYVRYL was synthesized by Nanjing Jietai Biotechnology Co., Ltd. using a solid-phase method, with a purity of 98.58%. The antibacterial activity of the polypeptide PKTKVIPYVRYL was investigated using Gram-negative bacteria *Escherichia coli*, *Porphyromonas gingivalis*, and Gram-positive bacteria *Streptococcus mutans* as target bacteria.

[0048] (1) Strains and resuscitation

[0049] Escherichia coli (CICC10899), Porphyromonas gingivalis (ATCC33277), and Streptococcus mutans (ATCC25175) were preserved at -80℃ using the glycerol preservation method. Before the experiment, Escherichia coli was inoculated into LB broth (Sangon Biotech (Shanghai) Co., Ltd.), while Porphyromonas gingivalis and Streptococcus mutans were inoculated into BHI broth (Beijing Sobolai Technology Co., Ltd.) at a 1% volume. The cultures were incubated at 37℃ for 12 hours to revive the bacteria. The bacterial suspension was then re-inoculated at a 1% volume into fresh media of the corresponding media (LB broth for Escherichia coli, and BHI broth for Porphyromonas gingivalis and Streptococcus mutans, respectively) for further revival. This inoculation and revival process was repeated twice to restore the viability of the strains.

[0050] (2) Antibacterial test

[0051] Take 1 mL of bacterial culture, wash twice with PBS buffer, and then dilute to 10⁻⁶. 7 CFU / mL bacterial suspensions were used to obtain bacterial suspensions of Escherichia coli, Porphyromonas gingivalis, and Streptococcus mutans, respectively.

[0052] The antimicrobial peptide VAVALAPKHPIK samples were dissolved in the corresponding culture media for the above-mentioned bacteria (LB broth for Escherichia coli, BHI broth for Porphyromonas gingivalis and Streptococcus mutans, respectively) to prepare 100 mg / mL antimicrobial peptide solutions. The solutions were then sterilized by passing the solutions through a 0.22 μm membrane for later use, thus obtaining peptide solutions for different bacteria.

[0053] 100 μL of the bacterial suspension at the above concentration was mixed with 100 μL of the corresponding culture medium (LB broth for Escherichia coli, BHI broth for Porphyromonas gingivalis and Streptococcus mutans) to form the control group; 100 μL of the bacterial suspension at the above concentration was mixed with 100 μL of the corresponding peptide solution to form the experimental group. These were added sequentially to different wells of a 96-well cell culture plate. Escherichia coli was cultured aerobically at 37°C, while Porphyromonas gingivalis and Streptococcus mutans were cultured anaerobically at 37°C. After culturing for 12 hours, the absorbance (OD value) was measured at 600 nm. The inhibition rate of the samples for each bacterium was calculated using the following formula:

[0054]

[0055] OD1 control group: 100 μL bacterial suspension + 100 μL culture medium

[0056] OD2 experimental group: 100 μL bacterial culture + 100 μL peptide solution

[0057] (4) Experimental Results

[0058] The antimicrobial effects of the antimicrobial peptide PKTKVIPYVRYL against *Escherichia coli*, *Porphyromonas gingivalis*, and *Streptococcus mutans* are shown in Table 2. The table lists the antimicrobial effects of PKTKVIPYVRYL against these bacteria. The results show that this peptide PKTKVIPYVRYL has good inhibitory activity against both Gram-negative and Gram-positive bacteria, exhibiting broad-spectrum and highly effective antimicrobial properties.

[0059] Table 2. Antibacterial results of peptide PKTKVIPYVRYL

[0060]

Claims

1. Use of an antibacterial peptide in the preparation of a microbial growth inhibitor or an anti-infective drug, wherein the microbe is one or more than two of Escherichia coli, Staphylococcus aureus, Porphyromonas gingivalis and Streptococcus mutans; the amino acid sequence of the antibacterial peptide is shown as SEQ ID NO: 1; and the infection is caused by one or more than two of Escherichia coli, Staphylococcus aureus, Porphyromonas gingivalis and Streptococcus mutans.

2. Use according to claim 1, characterized in that: The microbial growth inhibitor or the anti-infective drug is an active ingredient of the antibacterial peptide, with or without the addition of a pharmaceutically acceptable carrier or adjuvant.

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