Bovine milk endogenous antibacterial peptide and application thereof
By developing the bovine milk endogenous antimicrobial peptide VAVALAPKHPIK, the problem of antibiotic side effects has been solved, achieving broad-spectrum inhibition against Gram-positive and Gram-negative bacteria, and its applications are in pharmaceuticals, health products, and food.
Patent Information
- Authority / Receiving Office
- CN · China
- Patent Type
- Patents(China)
- Current Assignee / Owner
- Filing Date
- 2023-07-06
- Publication Date
- 2026-03-17
AI Technical Summary
Existing antibiotics have side effects such as allergic reactions, gut microbiota dysbiosis, and multidrug resistance in the treatment of non-viral infections, necessitating the search for safe and effective alternatives.
A bovine milk endogenous antimicrobial peptide, VAVALAPKHPIK, was developed. It has broad-spectrum antimicrobial activity and inhibits bacterial growth and reproduction by binding to the cell membrane of pathogenic bacteria, disrupting their structure.
This antimicrobial peptide has broad inhibitory activity against both Gram-positive and Gram-negative bacteria and can be used to prepare drugs, health products, food, cosmetics, etc., to prevent and reduce infectious diseases. It is safe, stable, and readily available.
Smart Images
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Abstract
Description
Technical Field
[0001] This invention relates to a bovine milk endogenous antimicrobial peptide, VAVALAPKHPIK, and its applications. Background Technology
[0002] Antibiotics are a class of drugs used to treat non-viral infections by killing or inhibiting the growth and reproduction of certain microorganisms. However, the clinical use of antibiotics has encountered numerous problems, such as allergies, gut microbiota dysbiosis, superinfections, and multidrug resistance. Therefore, finding alternatives to antibiotics has been a hot research topic 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, showing promising prospects for research and application.
[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 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. Therefore, starting from endogenous peptides in milk, using multi-omics technologies to identify endogenous peptides with antibacterial activity can provide new solutions for clinical anti-infective treatments, and offer 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, VAVALARPKHPIK, 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 pharmaceutical, medical device, food, pet food, animal feed, oral care products, and cosmetics products using an acceptable carrier or excipient. 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 VAVALAPKHPIK 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 VAVALAPKHPIK is an active ingredient against Escherichia coli, Staphylococcus aureus, Porphyromonas gingivalis and Streptococcus mutans, with or without pharmaceutically or medically acceptable carriers or excipients.
[0008] The antibacterial polypeptide compound VAVALARPKHPIK has the amino acid sequence Val-Ala-Val-Ala-Leu-Ala-Arg-Pro-Lys-His-Pro-Ile-Lys. It has a molecular weight of 1399.68 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 was derived from heat-processed milk, and the structure and physicochemical properties of the polypeptide were 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. Attached Figure Description
[0012] Figure 1 This image shows the antibacterial effect of the antimicrobial peptide VAVALAPKHPIK. Figure 'a' represents the inhibition zone experiment results of VAVALAPKHPIK against *Escherichia coli*. The positive control is chlorhexidine, and the negative control is sterile water. Note: + represents chlorhexidine, - represents sterile water, and 'a' represents the antimicrobial peptide VAVALAPKHPIK. 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 (ultrafiltrate) that has passed through the ultrafiltration membrane to obtain the milk endogenous peptide.
[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). The loading volume was 2 μL, and HPLC-MS / MS analysis was performed 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, V / V) and mobile phase B (80% ACN / 0.1% FA, 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 bovine milk endogenous peptide VAVALARPKHPIK has an isoelectric point of 11.17, a molecular weight of 1399.68, and contains 13 amino acids. Biological information obtained from the amino acid sequence of SEQ ID NO: 1 using online tools indicates that this peptide has a GRAVY value of 0.262, an instability coefficient of 15.30, and an Aliphatic index of 127.69.
[0025] Information of SEQ ID No. 1 (a) Sequence characteristics
[0026] *Length: 13 amino acids
[0027] *Type: Amino acid
[0028] * Chain type: branched single chain (b) Molecular type: protein
[0029] Sequence description: VAVALAPKHPIK
[0030] Example 2
[0031] Antibacterial activity of peptides
[0032] The polypeptide VAVALARPKHPIK was synthesized by Nanjing Jietai Biotechnology Co., Ltd. using a solid-phase method, with a purity of 97.91%. The antibacterial activity of the polypeptide VAVALARPKHPIK was investigated using Gram-negative Escherichia coli as the target bacterium.
[0033] (1) Strains and resuscitation
[0034] Escherichia coli (CICC10899) was preserved at -80℃ using the glycerol preservation method. Before the experiment, E. coli was inoculated into LB broth medium (Sangon Biotech (Shanghai) Co., Ltd.), with the target strain inoculated at a volume of 1%. The culture was incubated at 37℃ for 12 hours to allow the strain to recover. The bacterial culture was then inoculated again into fresh LB broth medium at a volume of 1% for further recovery. This inoculation and recovery process was repeated twice to restore the viability of the strain.
[0035] (2) Culture medium
[0036] The antibacterial experiment used 90 mm diameter microbial culture dishes (containing 20 mL of bacterial culture medium). E. coli was prepared with 1.5% agar (m / v, g / ml) at a bacterial concentration of 1 × 10⁻⁶. 6LB broth medium at CFU / mL was purchased from Sangon Biotech (Shanghai) Co., Ltd. The medium was sealed and stored at 4℃ for later use.
[0037] (3) Antibacterial test
[0038] The antimicrobial activity of antimicrobial peptides against *Escherichia coli* was determined using a perforation method. Seven wells were punched in 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 synthesized standard peptide was dispensed at 5 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 2.5 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 loading, the plates were incubated at 37°C for 18 h, and the presence and size of inhibition zones were observed and recorded.
[0039] (4) Experimental Results
[0040] The antibacterial results and inhibition zone diameters of the milk antimicrobial peptides VAVALAPK and HPIK 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.
[0041] Table 1. Antibacterial results of peptides VAVALAPK and HPIK
[0042]
[0043] Note: The numbers represent the diameter of the inhibition zone.
[0044] Example 3
[0045] Antibacterial activity of peptides
[0046] The peptide VAVALARPKHPIK was synthesized by Nanjing Jietai Biotechnology Co., Ltd. using a solid-phase method, with a purity of 97.91%. The antibacterial activity of the peptide VAVALARPKHPIK was investigated using Gram-negative Porphyromonas gingivalis and Gram-positive Staphylococcus aureus and Streptococcus mutans as target bacteria.
[0047] (1) Strains and resuscitation
[0048] Staphylococcus aureus (CICC10384), Porphyromonas gingivalis (ATCC33277), and Streptococcus mutans (ATCC25175) were preserved at -80℃ using the glycerol preservation method. Before the experiment, Staphylococcus aureus was inoculated into TSB broth (Beijing Solarbio Science & Technology Co., Ltd.), while Porphyromonas gingivalis and Streptococcus mutans were inoculated into BHI broth (Beijing Solarbio Science & 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 (TSB broth for Staphylococcus aureus, 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.
[0049] (2) Antibacterial test
[0050] 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 Staphylococcus aureus, Porphyromonas gingivalis, and Streptococcus mutans, respectively. The antimicrobial peptide VAVALARPKHPIK samples were dissolved in the corresponding culture media for each of the above bacteria (TSB broth for Staphylococcus aureus, BHI broth for Porphyromonas gingivalis and Streptococcus mutans, respectively) to prepare 100 mg / mL antimicrobial peptide solutions. These solutions were then sterilized by passing the solution through a 0.22 μm membrane for later use, yielding peptide solutions for different bacteria. 100 μL of the bacterial suspension at the above concentration was mixed with 100 μL of the corresponding culture medium (TSB broth for Staphylococcus aureus, BHI broth for Porphyromonas gingivalis and Streptococcus mutans, respectively) 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. Staphylococcus aureus 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:
[0051] OD1 control group: 100 μL bacterial suspension + 100 μL culture medium
[0052] OD2 experimental group: 100 μL bacterial culture + 100 μL peptide solution
[0053] (4) Experimental Results
[0054] The antimicrobial effects of the antimicrobial peptide VAVALARPKHPIK against Staphylococcus aureus, Porphyromonas gingivalis, and Streptococcus mutans are shown in Table 2. The results indicate that VAVALARPKHPIK exhibits good inhibitory activity against both Gram-negative and Gram-positive bacteria, demonstrating broad-spectrum and highly effective antimicrobial properties.
[0055] Table 2 Antibacterial results of peptides VAVALAPK and HPIK
[0056]
Claims
1. An antibacterial peptide, characterized in that: The amino acid sequence of the antibacterial peptide is shown in SEQ ID NO:
1.
2. Use of the antibacterial peptide of claim 1 for the preparation of a microbial growth inhibitor or an anti-infective drug. The microorganism is one or two or more of Escherichia coli, Staphylococcus aureus, Porphyromonas gingivalis and Streptococcus mutans, and the infection is a disease caused by one or two or more of Escherichia coli, Staphylococcus aureus, Porphyromonas gingivalis and Streptococcus mutans together or a single bacterium.
3. Use according to claim 2, characterized in that: The microorganism growth inhibitor and anti-infective drug is an active ingredient of the antibacterial peptide, with or without a pharmaceutically acceptable carrier or adjuvant.