Antibacterial polypeptides and uses and bacterial growth inhibitors, anti-infective drugs

Abstract

The present application relates to a kind of polypeptides with inhibitory activity to gram-negative bacteria and gram-positive bacteria represented by escherichia coli and staphylococcus aureus derived from Daqu.The amino acid sequence is Tyr-Pro-Leu-Gly-Gln-Gly-Ser-Phe-Arg-Pro.Polypeptide YPLGQGSFRP has good inhibitory activity to gram-negative bacteria and gram-positive bacteria, as natural safe antibacterial agent, it has good application prospect in preparing medicine and / or health care product for preventing and / or reducing intestinal infectious disease caused by escherichia coli and staphylococcus aureus.

Description

Technical Field

[0001] This invention relates to an antibacterial polypeptide YPLGQGSFRP derived from Daqu (a type of Chinese koji) and its application in the preparation of bacterial growth inhibitors and anti-infective drugs. Background Technology

[0002] Antibiotics are special secondary metabolites produced by microorganisms. Even at low concentrations, they can inhibit bacterial growth and metabolism by hindering nucleic acid formation, suppressing protein synthesis, altering cell membrane permeability, and interfering with cell wall synthesis. Antibiotics are widely used due to their significant antibacterial effects; however, overuse has led to bacterial resistance, making the treatment of bacterial infections increasingly difficult.

[0003] Antimicrobial peptides (AMPs), as a novel type of antibiotic, are widely available, possessing 10-50 amino acids, strong cationicity, amphiphilicity, and broad-spectrum antibacterial activity. Compared to traditional antibiotics, the greatest advantage of antimicrobial peptides is that they primarily bind to bacterial cell membranes through electrostatic attraction and, through hydrophobic interactions, bind to phospholipids and insert into the cytoplasmic membrane, thus disrupting the bacterial cell membrane structure. This external force action generally does not induce drug resistance in microorganisms. However, the application of antimicrobial peptides currently faces two major challenges: firstly, the antimicrobial activity of natural antimicrobial peptides is relatively low; and secondly, the stability and cytotoxicity of antimicrobial peptides are difficult to control. Therefore, the development of safe, stable, and low-cytotoxic antimicrobial peptides is of great significance.

[0004] Daqu (a type of starter culture) is produced by microbial fermentation of grains at high temperatures. The antimicrobial peptides derived from grain protein in Daqu are food-derived, naturally safe, and possess good potential as antimicrobial drugs. Therefore, this invention screens an antimicrobial peptide derived from wheat protein, a Daqu substrate, which can be studied as a drug for the prevention and / or reduction of intestinal infections caused by Escherichia coli and Staphylococcus aureus. Summary of the Invention

[0005] The purpose of this invention is to provide the application of an antimicrobial polypeptide YPLGQGSFRP derived from wheat protein, a substrate of Daqu (a type of starter culture), in the preparation of medicaments for the prevention and / or reduction of intestinal infections caused by Escherichia coli and Staphylococcus aureus.

[0006] To achieve the above objectives, the present invention uses the polypeptide YPLGQGSFRP as an effective component for inhibiting microbial growth activity.

[0007] It has the amino acid sequence in SEQ ID NO: 1, and the polypeptide YPLGQGSFRP is the active ingredient of drugs against Escherichia coli and Staphylococcus aureus, wherein pharmaceutically acceptable carriers or excipients may be added.

[0008] YPLGQGSFRP, a polypeptide compound with the amino acid sequence Tyr-Pro-Leu-Gly-Gln-Gly-Ser-Phe-Arg-Pro, exhibits inhibitory activity against Escherichia coli and Staphylococcus aureus. It has a molecular weight of 1121.24 Da, is a white powder, readily soluble in water, and shows strong inhibitory effects on the growth of Escherichia coli and Staphylococcus aureus.

[0009] Compared with the prior art, the present invention has the following beneficial effects: This invention obtained and determined the structure of the above-mentioned polypeptide from Daqu (a type of starter culture), and for the first time verified that the above-mentioned polypeptide has good antibacterial activity, and has good application prospects in the preparation of drugs for preventing and / or reducing intestinal infectious diseases caused by Escherichia coli and Staphylococcus aureus. Attached Figure Description

[0010] Figure 1 This is a graph showing the antibacterial results of the polypeptide YPLGQGSFRP. Figure 1 (A) is a diagram showing the experimental results of the peptide YPLGQGSFRP inhibiting Escherichia coli; Figure 1 (B) is a diagram showing the experimental results of the polypeptide YPLGQGSFRP inhibiting Staphylococcus aureus. Detailed Implementation

[0011] Example 1: Preparation and Identification of Peptide YPLGQGSFRP A combined LC-MS / MS and bottom-up proteomics approach was employed. Using commercially available high-temperature koji (RH-02, Luzhou Ruihua Bio-koji Making Co., Ltd.) as raw material, the koji was extracted with water and ethanol using a gradient process, followed by centrifugation, ultrafiltration, and LC-MS / MS analysis. Based on structure-activity relationship characteristics, peptides exhibiting inhibitory effects against *Escherichia coli* and *Staphylococcus aureus* were screened.

[0012] The specific method is as follows: (1) Sample preparation 1g of Daqu sample was mixed with 20 mL of ultrapure water, shaken at 37℃ and 200 rpm for 2 h, and then at 4℃ and 15000× g Centrifuge at 15 min at 37°C, collect the supernatant (denoted as supernatant A), add 20 mL of 50% ethanol-water solution to the precipitate (denoted as precipitate A), and incubate at 37°C, 200 rpm for 2 h with shaking. Then incubate at 4°C, 15000 × 10⁻⁶ rpm for 2 h with shaking. gCentrifuge at 15 min at 37°C, collect the supernatant (referred to as supernatant B), add 20 mL of ethanol to the precipitate (referred to as precipitate B), and incubate at 37°C and 200 rpm for 2 h with shaking. Then incubate at 4°C and 15000 × 10⁻⁶ rpm. g Centrifuge at a certain speed for 15 min, and collect the supernatant (denoted as supernatant C). Supernatants A, B, and C are ultrafiltered using ultrafiltration membranes with a molecular weight cutoff of 10,000 Da, and the corresponding filtrates A, B, and C are desalted using a C18-SPE column (Waters Oasis HLB SPE). After desalting, the samples are lyophilized and stored at -80℃ to obtain the corresponding lyophilized samples A, B, and C.

[0013] (2) LC-MS / MS analysis Samples A, B, and C, which were lyophilized, were reconstituted in 0.1% (v / v) FA-H2O solution. Mass spectrometry analysis was performed using LTQ-OrbitrapVelos (a high-resolution mass spectrometer combining a dual-partial-pressure linear trap and an electrostatic field orbital trap). The sample loading was 2 μg. Mobile phase A in the liquid chromatography was an aqueous solution (v / v) containing 0.1% formic acid, and mobile phase B was acetonitrile (v / v) containing 0.1% formic acid. The gradient elution program was as follows: 0-80 min, 7%-27% B (v / v); 80-95 min, 27%-40% B; 95-97 min, 40%-90% B; 97-107 min, 90% B; 107-109 min, 90%-0% B; 109-126 min, 0% B, flow rate 60 μL / min The mass spectrometer used an ESI ion source with an ion transport temperature of 250℃, a spray voltage of 2.2 kV, a normalized collision energy of 35%, a first-order spectral resolution of 60,000, a scan range of 400-2000 m / z, and a data acquisition mode of data-dependent (DDA). The 20 strongest precursor ions from the first-order spectrum were selected for second-order fragmentation. Dynamic exclusion was set as follows: repeat count, 2; repeat time, 30 s; exclusion time, 60 s.

[0014] (3) Data retrieval RAW files use MaxQuant TMThe software (v.1.5.3.30) searched the wheat (379 proteins) database (http: / / www.uniprot.org / ). Search parameters were as follows: no enzyme digestion, maximum missed digestion count, and fixed modifications were set; the variable modification was set to methionine oxidation (+15.9949 Da). The mass tolerance for precursor ions was 20 ppm, and for fragment ions, it was 0.5 Da. Peptides with a PSM false positive rate (FDR) <1% were considered valid data for analysis. A total of 475 peptides from 39 proteins were identified in the experiment. Since antimicrobial peptides typically possess characteristics such as containing hydrophobic or basic amino acids, strong cationicity, and amphiphilicity, the structure-activity relationship of antimicrobial peptides was used to screen some potential antimicrobial peptides from the identification results. The results are shown in Table 1. Table 1. Identification results of some potential antimicrobial peptides

[0015] (4) Property analysis of polypeptide YPLGQGSFRP The polypeptide YPLGQGSFRP is derived from α / β gliadin (P02863, f224-233), a major storage protein in wheat seeds. The peptide has an isoelectric point of 8.75 and a molecular weight of 1121.24 Da. Biological information obtained using the online tool ExPASy indicates that the polypeptide is positively charged, has an instability coefficient of 40.60, an aliphatic amino acid index of 39.00, and a hydrophilicity of -0.750.

[0016] Information of SEQ ID No. 1 (a) Sequence characteristics * Length: 10 amino acids * Type: Amino acid * Chain type: Linear single chain (b) Molecular type: protein Sequence description: SEQ ID No.1 YPLGQGSFRP Example 2: Detection of antibacterial activity of peptide YPLGQGSFRP Three potential antimicrobial peptides, YPLGQGSFRP, IFWGIPALLK, and AAFSPPVSLHSALSLLAAGAGS, were selected and synthesized by Nanjing Jietai Biotechnology Co., Ltd. using a solid-phase synthesis method, achieving purities of 95.46%, 99.73%, and 95.31%, respectively. The antimicrobial activity of these three potential antimicrobial peptides was investigated using Gram-positive Escherichia coli and Gram-negative Staphylococcus aureus as target bacteria.

[0017] (1) Strains and resuscitation Escherichia coli ( Escherichia coli K12) and Staphylococcus aureus ( Staphylococcus aureus All samples were preserved at -80℃ using the glycerol preservation method. Before the experiment, one loopful of Escherichia coli was inoculated into 5 mL of liquid LB medium and one loopful of Staphylococcus aureus was inoculated into 5 mL of liquid TSB medium. The samples were then incubated at 37℃ and 200 rpm for 12 h to allow the strains to recover.

[0018] After 12 hours, 100 μL of *E. coli* culture was transferred to 5 mL of liquid LB medium, and 100 μL of *Staphylococcus aureus* culture was transferred to 5 mL of liquid TSB medium. Both cultures were then incubated again at 37°C and 200 rpm for 12 hours for resuscitation. This resuscitation process was repeated 2-3 times to restore the viability of the strain.

[0019] (2) Culture medium The antibacterial experiment used round petri dishes with a diameter of 90 mm. The culture media were LB solid medium and TSB solid medium, each containing 1.5% agar by mass. The final concentration of LB solid medium was 1×10⁻⁶. 5 CFU / mL E. coli, TSB solid medium mixed to a final concentration of 1×10⁻⁶ 5 Staphylococcus aureus at CFU / mL. The culture medium containing the bacterial suspension was poured into six different round petri dishes (Escherichia coli and Staphylococcus aureus were each poured into three petri dishes), with the culture medium filling height in the petri dishes to 4 mm. After sealing, the dishes were stored at 4°C to obtain six plates.

[0020] (3) Antibacterial test Three 3 mm wells were prepared on six agar plates, spaced 3 cm apart. The six plates were divided into three groups: one plate containing *E. coli* and one plate containing *Staphylococcus aureus* were grouped together. In each group, 1.5 mg of the same standard peptide dissolved in 50 μL of sterile water was added to two wells of two plates, (three standard peptides, three experimental groups). Each group was tested in duplicate. A blank control, 50 μL of sterile water, was added to the third well. After adding the samples, the plates were incubated at 4°C for 4 h until the sample solution was completely absorbed and diffused. Then, they were incubated upside down at 37°C for 12 h, and the size of the inhibition zone was observed and recorded.

[0021] (4) Experimental results The inhibition zone diameters of the three peptides against *Escherichia coli* and *Staphylococcus aureus* are shown in Table 2. Peptides IFWGIPALLK and AAFSPPVSLHSALSLLAAGAGS showed no inhibitory effect on *Escherichia coli* and only a weak inhibitory effect on *Staphylococcus aureus*; while YPLGQGSFRP showed inhibitory effects on both *Escherichia coli* and *Staphylococcus aureus*, with a slightly better inhibitory effect on *Escherichia coli* than on *Staphylococcus aureus*. These results indicate that peptide YPLGQGSFRP has inhibitory activity against both Gram-negative and Gram-positive bacteria, with higher inhibitory activity against Gram-negative bacteria.

[0022] Figure 1 The study demonstrated the inhibitory effect of the peptide YPLGQGSFRP on Escherichia coli and Staphylococcus aureus.

[0023] Table 2. Antibacterial effect of peptide YPLGQGSFRP sequence list <110> Dalian Institute of Chemical Physics, Chinese Academy of Sciences <120> Antimicrobial peptides and their applications, bacterial growth inhibitors, and anti-infective drugs <160> 1 <170> SIPOSequenceListing 1.0 <210> 1 <211> 10 <212> PRT <213> Artificial Sequence <400> 1 Tyr Pro Leu Gly Gln Gly Ser Phe Arg Pro 1 5 10

Claims

1. An antimicrobial polypeptide, characterized in that: The polypeptide is YPLGQGSFRP, which has the amino acid sequence in SEQ ID NO: 1; specifically, the amino acid sequence of the polypeptide is Tyr-Pro-Leu-Gly-Gln-Gly-Ser-Phe-Arg-Pro.

2. The use of the polypeptide of claim 1 in the preparation of an inhibitor for the growth of Escherichia coli and / or Staphylococcus aureus.

3. The use of the polypeptide of claim 1 in the preparation of a medicament for the prevention and / or treatment of intestinal bacterial infections caused by Escherichia coli and / or Staphylococcus aureus.

4. The application according to claim 2 or 3, characterized in that: It uses the polypeptide YPLGQGSFRP as the active ingredient, with the addition of a pharmaceutically acceptable carrier.

5. A growth inhibitor for Escherichia coli and / or Staphylococcus aureus, characterized in that: It uses the polypeptide described in claim 1 as its active ingredient.

6. The Escherichia coli and / or Staphylococcus aureus growth inhibitor according to claim 5, characterized in that: It uses the polypeptide of claim 1 as the active ingredient, wherein a pharmaceutically acceptable carrier is added.

7. A drug for the prevention and / or treatment of intestinal bacterial infections caused by *Escherichia coli* and / or *Staphylococcus aureus*, characterized in that: It uses the polypeptide described in claim 1 as its active ingredient.

8. The medicament according to claim 7, characterized in that: It uses the polypeptide of claim 1 as the active ingredient, wherein a pharmaceutically acceptable carrier is added.

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