Cow milk endogenous antibacterial peptide and application thereof

Abstract

The invention relates to a cow's milk endogenous antibacterial peptide and application thereof, the cow's milk endogenous antibacterial peptide is an antibacterial peptide compound VAVALARPKHPIK from endogenous enzyme hydrolyzed cow cheese protein, and the amino acid sequence of the antibacterial peptide compound is Val-Ala-Val-Ala-Leu-Ala-Arg-Pro-Lys-His-Pro-Ile-Lys. The compound has wide inhibitory activity on gram-positive bacteria and gram-negative bacteria, including porphyromonas gingivalis, escherichia coli, staphylococcus aureus and the like, respectively. The antibacterial peptide can be used as a health-care product and / or a medical instrument or a medicine for preventing and / or reducing infectious diseases, a food bacteriostatic agent, novel functional food, pet food, animal feed additives and daily chemicals, and has a good application prospect.

Description

Technical Field

[0001] The invention relates to an endogenous antimicrobial peptide VAVALARPKHPIK from bovine milk and application thereof. Background Art

[0002] Antibiotics are a class of drugs used to treat non-viral infections that can kill or inhibit the growth and reproduction of certain microorganisms. Currently, many problems have arisen in the clinical use of antibiotics, such as allergies, intestinal flora disorders, superinfections, multidrug resistance and other side effects. Therefore, the search for alternatives to antibiotics has always been a hot topic in this field. As a new type of substance for the treatment of bacterial infections, antimicrobial peptides have attracted widespread attention due to their natural origin, broad-spectrum antibacterial properties, safety and high efficiency, and lack of drug resistance. They have good prospects for scientific research and application.

[0003] Antimicrobial peptides are a class of small molecule polypeptide compounds that can inhibit bacterial growth and reproduction, and are widely present in nature. Antimicrobial peptides can disrupt the structure and function of the bacterial cell membrane by binding to the surface of the bacterial cell membrane, causing the cell membrane to loosen or destroy, thereby affecting the biological response on the cell membrane and even causing cell death. In addition, some antimicrobial peptides also have inhibitory activity against some viruses, and show effects such as improving immunity and promoting wound healing. Its wide range of biological activities shows that it has good application prospects in antibacterial applications. It is of great significance to develop safe, stable and easily available antimicrobial peptides.

[0004] Cow's milk is the main source of exogenous protein for humans, and contains rich proteins and endogenous peptides. Since the endogenous enzymes in cow's milk have a strong hydrolysis effect on proteins, there are a large number of stable endogenous peptides in cow's milk, some of which have biological activity. Therefore, starting from the endogenous peptides in cow's milk, the use of multi-omics technology to find endogenous peptides with antibacterial activity provides a new solution for clinical anti-infection treatment, and provides new products and new directions for the development of simple and safe microbial inhibitors and natural foods. Summary of the invention

[0005] The present invention relates to an endogenous antimicrobial peptide VAVALARPKHPIK of bovine milk and its application, and the application of the antimicrobial peptide in the preparation of microbial growth inhibitors, anti-infective drugs, antioxidants, food preservation and antiseptics, pet food, animal feed, cosmetics and other products, wherein one of the acceptable carriers or excipients in pharmacology, medical devices, food, pet food, animal feed, oral products and cosmetics can be added. The antimicrobial peptide is used in the preparation of drugs or new infant formula foods for preventing and or reducing diseases caused by Escherichia coli, Staphylococcus aureus, Porphyromonas gingivalis and Streptococcus mutans together or by a single bacteria, such as intestinal infection, gingivitis or periodontitis.

[0006] To achieve the above purpose, the present invention uses the polypeptide VAVALARPKHPIK as an effective ingredient for inhibiting the growth of pathogenic microorganisms.

[0007] It has the amino acid sequence of SEQ ID NO: 1 in the sequence table; the polypeptide VAVALARPKHPIK is an active ingredient against Escherichia coli, Staphylococcus aureus, Porphyromonas gingivalis and Streptococcus mutans, and acceptable carriers or excipients in pharmacology, medical devices and daily necessities may or may not be added.

[0008] The amino acid sequence of the peptide compound VAVALARPKHPIK with antibacterial activity is Val-Ala-Val-Ala-Leu-Ala-Arg-Pro-Lys-His-Pro-Ile-Lys. The molecular weight is 1399.68Da, it is a white powder, soluble in water, and has a strong inhibitory effect on the growth and activity of various pathogenic bacteria.

[0009] The endogenous antimicrobial peptides of bovine milk of the present invention have a wide range of inhibitory activities against Gram-positive bacteria and Gram-negative bacteria, including Porphyromonas gingivalis, Escherichia coli, Staphylococcus aureus, etc. The antimicrobial peptides can be used as health products and / or medical devices or drugs for preventing and / or reducing infectious diseases, food antibacterial agents, new functional foods, pet foods and animal feed additives, and daily chemicals, and have good application prospects.

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

[0011] The present invention obtains and determines the structure and physicochemical properties of the polypeptide derived from heat-processed cow's milk, and verifies for the first time the broad-spectrum antibacterial activity of the polypeptide. Therefore, it has good application prospects in the preparation and prevention and / or reduction of infectious diseases caused by Escherichia coli, and / or health products or medical devices, daily chemicals, and new functional foods. BRIEF DESCRIPTION OF THE DRAWINGS

[0012] Figure 1 Figure 1 is the antibacterial effect diagram of the antimicrobial peptide VAVALARPKHPIK. a is the antibacterial zone test result of the peptide VAVALARPKHPIK inhibiting 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 VAVALARPKHPIK. DETAILED DESCRIPTION

[0013] The present invention is described in detail below in conjunction with specific embodiments. The following examples will help the technical personnel and researchers in the field to further understand the present invention, but do not constitute any limitation of the present invention. Any modification made by anyone within the scope of the claims of the present invention in any form is still within the protection scope of the claims of the present invention.

[0014] Example 1

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

[0016] (1) Sample preparation

[0017] Take 200 μL of cow's milk (common name: milk) stock solution, dilute it with sterile water to 10 times the volume of the cow's milk stock solution, heat it in a water bath at 95°C for 5 minutes to denature it, use an ultrafiltration tube (Millipore) with a molecular weight cutoff of 10K Da to ultrafilter at 14000×g and 20°C for 20 minutes, collect the clarified liquid (ultrafiltrate) that passes through the ultrafiltration tube filter membrane, and obtain the cow's milk endogenous peptides.

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

[0019] The bovine milk endogenous peptide sample was desalted using a C18 column (Waters Oasis HLB SPE column, 1cc / 10mg, 31.7μm)) according to the following steps: the commercial 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, and then eluted with 1.5mL of 80% (V / V) ACN / 0.1% (V / V) TFA (trifluoroacetic acid)-H2O solution. The eluate was collected, packaged, lyophilized, and stored at -80°C to obtain a standard endogenous peptide sample. The endogenous peptide samples obtained by the above steps were re-dissolved in 0.1% (V / V) FA (formic acid)-H2O solution, the sample concentration was 0.5 mg / mL (endogenous peptide sample / solution), the sample 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. The sample was loaded onto a C18 pretreatment column (capillary column: Polymicro, inner diameter * length: 75 μm id * 3 cm; filler: 3 μm, (Sepucbd)) and C18 (capillary column: Polymicro, inner diameter*length: 75μm id*20cm; filler: 2.4μm, (Reprosil)) analytical column. Tandem mass spectra were acquired in positive ion data-dependent mode, with a full scan of ions in the mass-to-charge ratio (m / z) range of 375-1650, an ion transport capillary temperature of 250 °C, and a spray voltage of 2 kV. High energy collision dissociation (HCD) mode was used to analyze the top 20 strong precursor ions with charge states of 2-7, with a mass resolution of 35,000. Dynamic exclusion was enabled with an exclusion time of 60 seconds.

[0020] Thermo EASY-nLC was used to transport the mobile phase. Mobile phase A was H2O / 0.1% FA (formic acid, V / V) solution, and mobile phase B was 80% ACN / 0.1% FA (formic acid, V / V) aqueous solution for reversed-phase gradient separation. 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, mobile phase B gradient elution 75 min, flow rate 500 nL / min. Finally, the system was flushed with phase B for 15 min to balance.

[0021] (3) Data retrieval

[0022] MaxQuant (Version 2.0.3.0) software was used to perform database retrieval on the *RAW format files collected by mass spectrometry. The protein database was established by the laboratory based on the information of the bovine protein database (bovine, the number of proteins is 6035) on the Uniprot website (http: / / www.uniprot.org / ). During the retrieval process, the search parameters were as follows: non-specific enzyme cleavage, methionine oxidation (+15.9949Da), lysine acetylation (+42.011Da), and phosphorylation (+79.966Da) were set as variable modifications, and there was no fixed modification. The standardless quantitative method of MaxQuant was selected. The mass tolerance deviation of the parent ion was 20ppm, and the FDR was <1%.

[0023] (4) LC-MS / MS to obtain target peptide information

[0024] 1047 peptides were identified in the endogenous peptide samples of bovine milk, with a molecular weight range of 1.6KDa to 9.1KDa, of which about 66% were derived from casein. The protein precursor of the endogenous peptide VAVALARPKHPIK from bovine milk is Alpha-S1-casein, with an isoelectric point of 11.17, a molecular mass of 1399.68, and 13 amino acids. The biological information of the amino acid sequence shown in SEQ ID NO: 1 was obtained through online tools, and the average hydrophobicity GRAVY of the peptide was 0.262, the instability coefficient was 15.30, and the Aliphatic index was 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: VAVALARPKHPIK

[0030] Example 2

[0031] Antimicrobial activity of peptides

[0032] Nanjing Jiepeptide Biotechnology Co., Ltd. was commissioned to synthesize the peptide VAVALARPKHPIK by solid phase method with a purity of 97.91%. The antibacterial activity of the peptide VAVALARPKHPIK was investigated using Gram-negative bacteria Escherichia coli as the target bacteria.

[0033] (1) Strains and recovery

[0034] Escherichia coli (CICC10899) was stored at -80°C by glycerol preservation. Before the experiment, Escherichia coli was inoculated into LB broth medium (Shanghai Biotechnology Co., Ltd.), and the target strain was inoculated at 1% volume. The target strain was cultured at 37°C for 12 hours to recover the strain, and the bacterial solution was inoculated into fresh LB broth medium at 1% volume to recover the strain. The inoculation recovery operation was repeated twice to restore the strain to its vitality.

[0035] (2) Culture medium

[0036] The antibacterial experiment used a 90 mm diameter microbial culture dish (loaded with 20 mL of bacterial culture medium). E. coli (Escherichia coli) contained 1.5% agar (m / v, g / ml) and a bacterial concentration of 1×10 6CFU / mL LB broth culture medium was purchased from Sangon Biotech (Shanghai) Co., Ltd. The culture medium was sealed and stored at 4°C for future use.

[0037] (3) Antibacterial test

[0038] The punching method was used to determine the antibacterial ability of antimicrobial peptides against Escherichia coli. Use a puncher to punch 7 holes on the plate, with a spacing of about 4 cm between each hole and a hole diameter of 3.8±0.2 mm. The synthetic standard peptide was dispensed at 5 mg / tube, and 40 μL of sterile water was added to each dispense tube and vortexed until completely dissolved. The sample was added at a loading volume of 20 μL / well, and the actual amount of standard peptide added to each well was 2.5 mg. The positive control chlorhexidine (2MIC, 9.435 μg / ml) and the negative control sterile water were added in the same method and volume. After adding the sample, the culture dish was placed in a 37°C incubator for 18 hours to observe whether an inhibition zone appeared, and the size of the inhibition zone was measured and recorded.

[0039] (4) Experimental results

[0040] The antibacterial results and antibacterial zone diameters of bovine milk antimicrobial peptide VAVALARPKHPIK against Escherichia coli are shown in Figure 1 and Table 1. The results showed that the polypeptide had good inhibitory activity against Escherichia coli and had stable and efficient antibacterial properties.

[0041] Table 1 Antibacterial results of peptide VAVALARPKHPIK

[0042]

[0043] Note: The numbers represent the diameter of the inhibition zone

[0044] Example 3

[0045] Antimicrobial activity of peptides

[0046] Nanjing Jiepeptide Biotechnology Co., Ltd. was commissioned to synthesize the peptide VAVALARPKHPIK by solid phase method with a purity of 97.91%. The antibacterial activity of the peptide VAVALARPKHPIK was investigated with Gram-negative bacteria Porphyromonas gingivalis and Gram-positive Staphylococcus aureus and Streptococcus mutans as target bacteria.

[0047] (1) Strains and recovery

[0048] Staphylococcus aureus (CICC10384), Porphyromonas gingivalis (ATCC33277) and Streptococcus mutans (ATCC25175) were stored at -80°C by glycerol preservation. Before the experiment, Staphylococcus aureus was inoculated into TSB broth medium (Beijing Suolaibao Technology Co., Ltd.), and Porphyromonas gingivalis and Streptococcus mutans were inoculated into BHI broth medium (Beijing Suobaolai Technology Co., Ltd.) The target strains were inoculated at 1% volume. The strains were cultured at 37°C for 12 hours to recover, and the bacterial liquid was inoculated again at 1% volume into the fresh corresponding culture medium (TSB broth medium for Staphylococcus aureus, BHI broth medium for Porphyromonas gingivalis and Streptococcus mutans) for culture recovery. The inoculation recovery operation was repeated twice to restore the strains. The strains were restored to vitality.

[0049] (2) Antibacterial test

[0050] Take 1 mL of bacterial solution and wash twice with PBS buffer and then dilute to 10 7 CFU / mL bacterial liquid, Staphylococcus aureus, Porphyromonas gingivalis and Streptococcus mutans were obtained. The antimicrobial peptide VAVALARPKHPIK samples were dissolved in the culture medium corresponding to the above bacterial culture (TSB broth medium for Staphylococcus aureus, BHI broth medium for Porphyromonas gingivalis and Streptococcus mutans), prepared into 100 mg / mL antimicrobial peptide solution, sterilized by 0.22 μm membrane for standby use, and peptide solutions corresponding to different bacteria were obtained. 100 μL of the above concentration of bacterial solution was mixed with 100 μL of the corresponding culture medium (TSB broth medium for Staphylococcus aureus, BHI broth medium for Porphyromonas gingivalis and Streptococcus mutans). This was the control group; 100 μL of the above concentration of bacterial solution was mixed with 100 μL of the above corresponding peptide solution. This was the experimental group, and added to different wells of the 96-well cell culture plate in turn. Staphylococcus aureus was cultured under aerobic conditions at 37°C, and Porphyromonas gingivalis and Streptococcus mutans were cultured under anaerobic conditions at 37°C. The absorbance (OD value) of the above bacterial solutions was detected at 600 nm after culturing for 12 hours. The antibacterial rate of the samples corresponding to different bacteria was calculated according to the following formula:

[0051] OD1 control group: 100 μL bacterial solution + 100 μL culture medium

[0052] OD2 experimental group: 100 μL bacterial solution + 100 μL peptide solution

[0053] (4) Experimental results

[0054] The antibacterial results of the antimicrobial peptide VAVALARPKHPIK against Staphylococcus aureus, Porphyromonas gingivalis and Streptococcus mutans are shown in Table 2, which lists the antibacterial results of the antimicrobial peptide VAVALARPKHPIK. The results show that the peptide VAVALARPKHPIK has good inhibitory activity against both Gram-negative and Gram-positive bacteria, and has broad-spectrum and high-efficiency antibacterial properties.

[0055] Table 2 Antibacterial results of peptide VAVALARPKHPIK

[0056]

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 an endogenous antimicrobial peptide of bovine milk, and is an antimicrobial polypeptide compound derived from endogenous enzymatic hydrolysis of bovine milk casein, and has a sequence of VAVALARPKHPIK, and an amino acid sequence of: Val-Ala-Val-Ala-Leu-Ala- Arg-Pro-Lys-His-Pro-Ile-Lys.

3. 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 preservation and antiseptics, cosmetics, health products, pet food or animal feed or medical devices.

4. The use according to claim 3, characterized in that: The microbial growth inhibitor, anti-infective drug, antioxidant, food preservation, cosmetics, health care product, pet food, animal feed or medical device uses the antimicrobial peptide as an active ingredient, with or without carriers or excipients acceptable to pharmacology, medical devices or daily necessities.

5. The use according to claim 3 or 4, characterized in that: The microorganism is one or more than one of Gram-positive bacteria or Gram-negative bacteria; the infection is a disease caused by one or more than two of Gram-positive bacteria or Gram-negative bacteria.

6. The use according to claim 3 or 4, characterized in that: The microorganism is one or two or more of Escherichia coli, Staphylococcus aureus, Porphyromonas gingivalis and Streptococcus mutans.

7. The use according to claim 3 or 4, characterized in that: The infection is a disease caused by one or more of Escherichia coli, Staphylococcus aureus, Porphyromonas gingivalis and Streptococcus mutans, or a single bacterium, such as one or more of intestinal infection, gingivitis or periodontitis.

Images