CN114149484B - Antibacterial peptide MAMP-03 and application thereof - Google Patents

Abstract

The invention relates to a polypeptide compound MAMP-03 with microbial inhibitory activity from hydrolyzed milk protein, and the amino acid sequence of the polypeptide compound MAMP-03 is Leu-Asp-Thr-Asp-Tyr-Lys-Lys-Tyr (LDTDYKKY). The polypeptide MAMP-03 has wide inhibitory activity on gram-positive bacteria and gram-negative bacteria, including escherichia coli, porphyromonas gingivalis, streptococcus mutans and the like. Has the advantages of natural and safe raw materials and low production cost, and can be used as an active ingredient for inhibiting the growth of microorganisms to be applied to medicines, foods and daily necessities.

Description

Antibacterial peptide MAMP-03 and application thereof

Technical Field

The invention relates to an antibacterial peptide MAMP-03 (LDTDYKKY) from hydrolyzed milk protein and application thereof.

Background

Antibiotics are drugs capable of resisting pathogenic microorganisms, and are the largest type of antibacterial and anti-inflammatory drugs. Antibiotics are substances produced by bacteria, fungi or other microorganisms in the course of life, and have the effect of inhibiting or killing pathogenic microorganisms such as bacteria, fungi, etc., so they are widely used as anti-infective agents for various infectious diseases. The remarkable effect of antibiotics in the treatment of infections also leads to bacterial resistance, making the treatment of part of the infectious diseases increasingly difficult. The antibacterial peptide is used as a novel antibiotic, has broad-spectrum antibacterial activity, can not easily cause the microorganism to generate drug resistance, and has wide application prospect.

Antibacterial peptides are widely distributed in nature, and as a novel antibiotic which is widely concerned, more and more antibacterial peptides are found in microorganisms, animals and plants. The structure-activity relationship of the antibacterial peptide analyzed at present has two clearer characteristics, namely positive charged cations and amphiphilic sequence structures. However, antimicrobial peptides still face a number of problems to be solved as antimicrobial drugs: some cationic antimicrobial peptides can exert a greater toxic effect on mammalian cells; the amphiphilic structure requires a certain amount of basic amino acids in the amino acid sequence, and the characteristic that the basic amino acids are easy to hydrolyze by protease makes the amphiphilic structure have a greatly limited effect after being used as a medicine entering a human body. Therefore, it is of great importance to develop safe, stable, low-cytotoxicity antimicrobial peptides.

Milk is taken as high-quality natural food, is rich in protein and polypeptide resources, and combines the advantages of the antibacterial peptide and the characteristics of high-quality and safe protein sources. The milk has rich protein content, forms stable antibacterial peptide under the hydrolysis of protease, and has good potential as a microorganism growth inhibitor. Therefore, further searching for peptides with bacteriostatic activity from hydrolyzed milk protein peptides would provide a new direction for the preparation of healthy and safe microbial growth inhibitors.

Disclosure of Invention

The invention aims to provide an antibacterial peptide MAMP-03 derived from hydrolyzed milk protein, and application thereof in preparing medicines, daily necessities or microorganism growth inhibitors for preventing and/or reducing intestinal tract infection diseases, oral diseases and skin diseases caused by microorganisms.

In order to achieve the above purpose, the invention uses the polypeptide MAMP-03 as an active ingredient for inhibiting the growth activity of microorganisms.

It has the sequence table SEQ ID NO:1, an amino acid sequence in seq id no; the polypeptide MAMP-03 is an active ingredient of medicines, foods and daily necessities for resisting colibacillus, porphyromonas gingivalis and streptococcus mutans, and can be added with carriers or auxiliary materials acceptable in pharmacy, foods and daily necessities.

The amino acid sequence of the polypeptide compound MAMP-03 with the inhibitory activity on escherichia coli, porphyromonas gingivalis and streptococcus mutans is LDTDYKKY, leu-Asp-Thr-Asp-Tyr-Lys-Lys-Tyr. The molecular weight is 1045.16Da, white powder is easy to dissolve in water, and has strong inhibition effect on the growth of escherichia coli, porphyromonas gingivalis and streptococcus mutans.

Compared with the prior art, the invention has the following beneficial effects:

the invention is obtained from hydrolyzed milk protein and determines the structure of the polypeptide, and the polypeptide has better antibacterial activity, so that the polypeptide has good application prospect as a medicament and/or health-care product and/or daily necessities or microorganism growth inhibitor for preventing and/or reducing infectious diseases caused by escherichia coli, porphyromonas gingivalis and streptococcus mutans.

Drawings

FIG. 1 shows the results of bacteriostasis of the polypeptides of the invention against E.coli, porphyromonas gingivalis and Streptococcus mutans, wherein: FIG. 1A shows the experimental results of the inhibition of E.coli by the polypeptide MAMP-03; FIG. 1B is an experimental result of the polypeptide MAMP-03 inhibiting Porphyromonas gingivalis; FIG. 1C shows the results of an experiment for inhibiting S.mutans by the polypeptide MAMP-03.

Detailed Description

Example 1

Preparation and identification of hydrolyzed milk protein peptide.

(1) Sample preparation

Dissolving 1g of milk casein in 10ml of water, adding 20mg of trypsin, reacting for 4 hours at 37 ℃, centrifuging for 20 minutes at 14000 Xg and 4 ℃ after the enzymolysis process is finished, and obtaining a supernatant which is a hydrolyzed milk protein peptide sample.

(2) LC-MS/MS analysis

Hydrolyzed milk protein peptide solution samples were desalted by a C18 column (Waters Oasis HLB SPE column, 1cc/30mg,30 um) as follows: the C18 column was equilibrated with 1.5mL of 0.1% (V/V) TFA (trifluoroacetic acid) -H2O solution, 1mL of 0.1% (V/V) TFA-H2O solution was added to the C18 column after the sample was reconstituted, and 1.5mL of 80% (V/V) ACN (acetonitrile)/0.1% (V/V) TFA-H2O solution was used for elution, and the eluate was collected and lyophilized at-80℃for storage. The desalted sample was dissolved in 0.1% (V/V) FA (formic acid) -H2O to prepare a solution having a concentration of 0.2g/L, and the solution was loaded in an amount of 8. Mu.L and subjected to mass spectrometry on a 15cm capillary analytical column (inner diameter: 180 μm). The LC-MS/MS system consisted of an Agilent 1100HPLC system and an LTQ-Orbitrap Velos mass spectrometer. Mobile phase a was an aqueous solution containing 0.1% (V/V) formic acid and mobile phase B was an aqueous solution containing 0.1% formic acid (V/V) and 98% acetonitrile (V/V). The gradient elution procedure was as follows: 0-80 min, 5-25% B (V/V, the same applies below); 80-95 min, 25-35% B; 95-97 min, 35-90% B; 97-107 min,90% B;107 to 109min,90 to 0 percent of B; 109-126 min,0% B; the flow rate was 70. Mu.L/min. Complete mass scans were obtained in an Orbitrap mass analyzer at 60000 resolution (m/z 400-2000). Ion collision dissociation (CID) is carried out on 15 ions which are strongest, and then MS/MS scanning is carried out, wherein the scanning range is between 400 and 2000 m/z. The dynamic exclusion function is set as follows: repeating 2; duration 30s; the exclusion duration was 60s. System control and data collection was performed by Xcalibur software. Three mass spectrometric analyses were performed for each sample.

(3) Data retrieval

RAW files were retrieved with MaxQuant software in a protein database (laboratory at http:// www.uniprot.org/download protein information, build milk protein database, number of proteins 7). The search parameters are as follows: the variable modification was set to methionine oxidation (+ 15.9949 Da), acetylation (+ 42.011Da, phosphorylation (+ 79.966 Da) and mass tolerance deviation of parent ion 20ppm, fragment ion 0.8Da. Control false positive rate (FDR) <1% when deriving peptide fragment, and analysis was performed on peptide fragment of derived result Score >20 as valid data.

(4) LC-MS/MS (liquid Crystal-mass spectrometry/mass spectrometry) for obtaining target peptide fragment information

More than five hundred pieces of peptide fragment information were identified in hydrolyzed milk protein peptide samples, of which more than about 10% were derived from beta-lactoglobulin. The polypeptide LDTDYKKKY protein precursor is bovine beta-lactoglobulin (f 111-118), the isoelectric point of peptide fragment is 5.84, the molecular mass is 1045.16Da, the polypeptide LDTDYKKKY protein precursor has 8 amino acids, a shorter sequence and a lower relative molecular mass, and the combination of mass spectrum identification results shows that the sequence can stably exist and is not easy to be continuously hydrolyzed by protease. Obtaining the SEQ ID NO by an online tool: 1, the instability coefficient of the peptide segment is-10.04, the aliphatic amino acid index and the hydrophilicity are 48.75 and-1.788 respectively, the peptide segment is hydrophobic, and the peptide segment has the characteristic of antibacterial peptide.

Information of SEQ ID No.1

(a) Sequence characterization

* Length: 8 amino acids

* Type (2): amino acids

* Chain type: single strand

(b) Molecular type: proteins

Sequence description:

SEQ ID No.1

LDTDYKKY

example 2

Antimicrobial activity of the polypeptide.

The chemically synthesized polypeptide MAMP-03 was custom made by Shanghai Jie peptide technology Co., ltd and had a purity of 97.6%. Taking escherichia coli and porphyromonas gingivalis in gram-negative bacteria and staphylococcus aureus and streptococcus mutans in gram-positive bacteria as target strains, and examining the antibacterial activity of the polypeptide MAMP-03.

(1) Strain and resuscitation

Coli (Escherichia coli K), staphylococcus aureus (Staphylococcus aureus), porphyromonas gingivalis (Porphyromonas gingivalis, p.g., ATCC 33277) and streptococcus mutans (Streptococcus mutans, m.s., ATCC 25175) were all purchased from the chinese strain resource pool collection and stored at-80 ℃ by glycerol storage. Coli was inoculated into LB broth (beijing soleba technology limited) before the experiment, staphylococcus aureus was inoculated into TSB broth (beijing soleba technology limited), porphyromonas gingivalis and streptococcus mutans were respectively inoculated into BHI broth (beijing soleba technology limited), and the target strains were all inoculated in 1% volume. Culturing at 37 ℃ for 12 hours, and inoculating the bacterial liquid into fresh culture medium again in an amount of 1% by volume for culturing and reviving. The inoculation recovery operation was repeated 2 times to restore the activity of the strain.

(2) Culture medium

The experiment used square dishes with sides of 90 mm. Coli bacteriumAnd Staphylococcus aureus bacteria inhibition experiment using double-layer culture medium, wherein the lower layer is supported by 20mL of 2% (m/V, g/mL) agar aqueous solution, and the upper layer is respectively added with 8mL of agar containing 0.7% (m/V, g/mL) and bacteria concentration of 1×10 ^{^5} CFU/mL LB medium and TSB medium were prepared as LB plates and TSB plates, respectively. Sealing the two prepared plate culture mediums and then preserving at 4 ℃. The bacteriostasis experiment of Porphyromonas gingivalis uses Columbia agar medium (Nanjing Quanlong biotechnology Co., ltd.) 15mL of the medium to prepare a plate, 200. Mu.L of bacterial liquid with bacterial concentration of 1X 10≡5CFU/mL is added on the plate, and the plate is uniformly coated by using balls. The bacteria inhibition experiment of mutans streptococcus uses BHI culture medium containing 1.5% (m/V, g/mL) agar and bacterial concentration of 1X 10≡5CFU/mL, and 15mL of bacteria-containing culture medium is added into each culture dish to prepare a flat plate.

(3) Antibacterial experiments

Holes with the inner diameter of 2mm are manufactured on the upper layers of an LB plate and a TSB plate, holes with the inner diameter of 8mm are manufactured in a BHI plate, and oxford cups with the inner diameter of 8mm are added into a Columbia agar plate and are used for an antibacterial experiment respectively.

The antibacterial peptide sample was dissolved in sterile water to prepare a 100mg/ml solution, which was added to 20. Mu.L of each of the agar plate wells and oxford cups. Coli and staphylococcus aureus were treated with sterile water as negative controls and 0.3mg/mL chlorhexidine as positive controls for porphyromonas gingivalis and streptococcus mutans. The loading volumes of the negative control and the positive control are the same as the antibacterial peptide sample solution.

Standing the plate after sample addition in a refrigerator at 4 ℃ for 3 hours, taking out the plate after the sample liquid in the hole is completely absorbed and diffused, inversely culturing escherichia coli and staphylococcus aureus in a constant temperature incubator at 37 ℃ for 16 hours, and inversely culturing porphyromonas gingivalis and streptococcus mutans in an anaerobic incubator at 37 ℃ for 16 hours under the conditions of 80% nitrogen (V/V), 10% hydrogen (V/V) and 10% carbon dioxide (V/V). After the cultivation is finished, whether a bacteriostasis ring appears or not is observed, and the size of the bacteriostasis ring is measured and recorded.

(4) Experimental results

The results of the bacteriostasis on the four bacteria are shown in tables 1 and 2. The result shows that the polypeptide MAMP-03 has growth inhibition activity on escherichia coli, porphyromonas gingivalis and streptococcus mutans.

Table 1 polypeptide MAMP-03 against E.coli and Staphylococcus aureus (zone of inhibition, mm)

1, the number represents the diameter (mm) of the inhibition zone, and 4.5 is 4.5 (mm) of the inhibition zone;

2. -indicating no bacteriostatic activity.

Table 2 results of bacteriostasis of polypeptide MAMP-03 on Porphyromonas gingivalis and Streptococcus mutans (zone of bacteriostasis, mm)

1, the number represents the diameter (mm) of the inhibition zone, and 13.0 x 13.0 represents the diameter (mm) of the inhibition zone.

Claims (4)

1. An application of antibacterial peptide in preparing microorganism growth inhibitor, wherein one or more than two pharmaceutically acceptable carriers or auxiliary materials can be added, and the application is characterized in that: the antibacterial peptide is polypeptide LDTDYKKY, and the amino acid sequence of the polypeptide is specifically as follows: leu-Asp-Thr-Asp-Tyr-Lys-Lys-Tyr; the microorganism growth inhibitor can inhibit the growth of one or more than two of Escherichia coli, porphyromonas gingivalis and Streptococcus mutans.

2. An application of antibacterial peptide in preparing anti-infective medicament, wherein one or more than two pharmaceutically acceptable carriers or auxiliary materials can be added, and the antibacterial peptide is characterized in that: the antibacterial peptide is polypeptide LDTDYKKY, and the amino acid sequence of the polypeptide is specifically as follows: leu-Asp-Thr-Asp-Tyr-Lys-Lys-Tyr; the infection is infection or bacterial imbalance caused by one or more than two of colibacillus, porphyromonas gingivalis and streptococcus mutans.

3. An application of antibacterial peptide in preparing oral products, wherein one or more than two acceptable carriers or auxiliary materials in the oral products can be added, and the antibacterial peptide is characterized in that: the antibacterial peptide is polypeptide LDTDYKKY, and the amino acid sequence of the polypeptide is specifically as follows: leu-Asp-Thr-Asp-Tyr-Lys-Lys-Tyr; the oral product is used for preventing and/or reducing or treating oral diseases caused by one or more than two of Escherichia coli, porphyromonas gingivalis and Streptococcus mutans.

4. The application of the antibacterial peptide in preparing cosmetics, wherein one or more than two acceptable carriers or auxiliary materials in the cosmetics can be added, is characterized in that: the antibacterial peptide is polypeptide LDTDYKKY, and the amino acid sequence of the polypeptide is specifically as follows: leu-Asp-Thr-Asp-Tyr-Lys-Lys-Tyr; the cosmetic is used for preventing and/or reducing or treating skin diseases caused by one or more than two of Escherichia coli, porphyromonas gingivalis and Streptococcus mutans.

Images