Antibacterial peptide and antibacterial application thereof

Abstract

The invention discloses an antibacterial peptide and antibacterial application thereof. The amino acid sequence of the antibacterial peptide is as shown in SEQ ID No.1, and the C end of the antibacterial peptide is amidated. The antibacterial peptide provided by the invention has the activity of resisting staphylococcus aureus and escherichia coli, and plays a role in inhibiting bacteria by destroying cell membranes. The antibacterial peptide is excellent in antibacterial activity, good in stability and not prone to generating drug resistance, can be used for synthesizing and preparing antibacterial compositions, and has great potential in application in the fields of food and medicine.

Description

Technical Field

[0001] The present invention relates to the technical field of peptide inhibitors, in particular to an antimicrobial peptide and its antimicrobial application. Background Art

[0002] A century ago, the advent of insulin ushered in the era of peptide therapeutics. Currently, more than 80 peptide drugs have been approved by regulatory agencies, and 550-750 peptides are in the clinical or preclinical stage. Even so, the growing emergence of antibiotic resistance remains a global problem that endangers healthcare systems and human health. The emergence and rapid spread of drug-resistant pathogens have led to an increasing number of difficult-to-treat infections. Staphylococcus aureus and Escherichia coli, as typical pathogens, not only cause food and drug spoilage, damage the market economy, and cause public health problems, but also cause foodborne diseases, posing great safety risks to human health. In this environment, humans are eager to seek new antibacterial drugs, and antimicrobial peptides (AMPs) are considered to be potential candidates for treating infections caused by microbial resistance that are otherwise unpredictable. Unlike traditional antimicrobial agents that interfere with bacterial nucleic acid or protein synthesis, most AMPs inhibit bacteria by destroying bacterial cell membranes. This mechanism of action makes it less likely that bacteria will develop resistance to AMPs. Therefore, the development of antimicrobial drugs with broad-spectrum and efficient bactericidal effects and a lower tendency to develop resistance is a technical problem that needs to be solved urgently. Summary of the invention

[0003] The purpose of the present invention is to provide an antimicrobial peptide having a broad-spectrum, highly efficient bactericidal effect and a lower tendency to develop drug resistance and its antimicrobial application.

[0004] To achieve the above purpose, the technical solution specifically adopted by the present invention is as follows:

[0005] In a first aspect, the present invention provides an antimicrobial peptide, the amino acid sequence of which is shown in SEQ ID No. 1, and the C-terminus of the antimicrobial peptide has an amidation modification.

[0006] In a second aspect, the present invention provides use of the antimicrobial peptide as described in the first aspect in the preparation of an antimicrobial product.

[0007] As a preferred embodiment of the second aspect, the antibacterial target of the antibacterial product is Gram-positive bacteria or Gram-negative bacteria.

[0008] As a preferred embodiment of the second aspect, the Gram-positive bacteria is Micrococcus luteus, Staphylococcus carnosus, Staphylococcus aegypti or Bacillus subtilis.

[0009] As a preference in the above second aspect, the Gram-negative bacteria is Escherichia coli.

[0010] As a preferred embodiment of the second aspect, when the antibacterial product is used to inhibit Staphylococcus aureus, the minimum inhibitory concentration is 4 μM.

[0011] As a preferred embodiment of the second aspect, when the antibacterial product is used to inhibit Escherichia coli, the minimum inhibitory concentration is 7 μM.

[0012] As a preferred embodiment of the second aspect, the antibacterial product is an antibacterial drug, a food preservative, or a feed additive.

[0013] As a preferred embodiment of the second aspect, the antibacterial action mode of the antibacterial product is cell membrane damage.

[0014] In a third aspect, the present invention provides an antimicrobial drug, a food preservative or a feed additive, the active ingredient of which includes the antimicrobial peptide described in the first aspect.

[0015] Compared with the prior art, the present invention has the following beneficial effects:

[0016] 1. The antimicrobial peptide provided by the present invention has an inhibitory effect on both Gram-positive bacteria and Gram-negative bacteria, especially has a more prominent antibacterial activity against Staphylococcus aureus and Escherichia coli, and has good thermal stability and pH tolerance.

[0017] 2. The antimicrobial peptide provided by the present invention exerts its antibacterial effect by destroying the cell membrane of the indicator bacteria. Its antibacterial mechanism is based on the membrane targeting mechanism, so it will make it less likely for pathogens to develop drug resistance. BRIEF DESCRIPTION OF THE DRAWINGS

[0018] Figure 1 The high performance liquid chromatogram and HPLC component analysis results of the antimicrobial peptide CRRI15 (9);

[0019] Figure 2 This is the mass spectrometry molecular weight identification result of the antimicrobial peptide CRRI15 (9);

[0020] Figure 3 The results of the pH stability test of the antimicrobial peptide CRRI15 (9) are shown;

[0021] Figure 4 The results of the thermal stability test of the antimicrobial peptide CRRI15 (9) are shown;

[0022] Figure 5 The change of cell membrane conductivity of Escherichia coli DH5α as indicator bacteria;

[0023] Figure 6 The change of cell membrane conductivity of Staphylococcus aureus D48 as indicator bacteria;

[0024] Figure 7The SEM images of the Escherichia coli DH5α cell morphology in the control group and the antimicrobial peptide CRRI15(9)-treated group;

[0025] Figure 8 The SEM images of the cell morphology of Staphylococcus aureus D48 in the control group and the group treated with antimicrobial peptide CRRI15(9);

[0026] Fig. 9 TEM images of the cell morphology of Escherichia coli DH5α in the control group and the group treated with antimicrobial peptide CRRI15(9);

[0027] Fig.10 TEM electron micrographs of the cell morphology of Staphylococcus aureus D48 in the control group and the group treated with antimicrobial peptide CRRI15(9). DETAILED DESCRIPTION

[0028] The technical solution of the present invention is further described below in conjunction with the accompanying drawings and specific embodiments. Unless otherwise defined, the technical terms or scientific terms used in the present invention should have the common meanings understood by people with ordinary skills in the field to which the present invention belongs.

[0029] The present invention provides an antimicrobial peptide, whose amino acid sequence is shown in SEQ ID No. 1, and whose C-terminus has an amidation modification. For the convenience of description, the present invention names the antimicrobial peptide as antimicrobial peptide CRRI15 (9), and its complete amino acid sequence can be expressed as VLLKILRWL-NH2.

[0030] The antimicrobial peptide CRRI15(9) provided by the present invention has antimicrobial activity against Staphylococcus aureus and Escherichia coli, and has an antibacterial effect by destroying the cell membrane. CRRI15(9) has excellent antimicrobial activity, good stability, and is not prone to drug resistance. It can be used to synthesize and prepare antimicrobial compositions, and has great potential for application in the fields of food and medicine.

[0031] The present invention also provides the use of the antimicrobial peptide in the preparation of antimicrobial products.

[0032] The antibacterial product has a broad spectrum of activity against Gram-positive or Gram-negative bacteria and is not sensitive to temperature and acid-base environments. Among them, the Gram-positive bacteria that can be effectively inhibited are Micrococcus luteus, Staphylococcus carnosus, Staphylococcus aureus or Bacillus subtilis, and the Gram-negative bacteria that can be effectively inhibited are Escherichia coli. When the antibacterial product is used to inhibit Staphylococcus aureus, the minimum inhibitory concentration is 4μM, and when it is used to inhibit Escherichia coli, the minimum inhibitory concentration is 7μM.

[0033] The specific form of the antibacterial product of the present invention can be adjusted according to actual needs, including but not limited to antibacterial drugs, food preservatives or feed additives.

[0034] The following is a series of examples to demonstrate the synthesis and property test results of the antimicrobial peptide CRRI15 (9).

[0035] Example 1 Synthesis and Antibacterial Properties of Antimicrobial Peptide CRRI15 (9)

[0036] CRRI15(9) was synthesized by solid phase chemical synthesis, purified and analyzed by high performance liquid chromatography, and its molecular weight was identified by mass spectrometry. Its minimum inhibitory concentration, thermal stability, and pH stability were also determined to comprehensively evaluate the antibacterial activity of CRRI15(9) as a novel antimicrobial peptide.

[0037] 1. Solid phase synthesis, purification and identification of CRRI15 (9)

[0038] CRRI15(9) was synthesized by solid phase chemical synthesis. The amino acids were connected to the resin from the C-terminus to the N-terminus in sequence using the Fmoc solid phase synthesis method. After the entire target sequence was connected, the peptide chain was eluted from the resin with TFA to obtain a crude product. The crude product of the synthesized antimicrobial peptide CRRI15(9) was purified and tested by high performance liquid chromatography. Water and acetonitrile were selected as the mobile phase system. 0.1% trifluoroacetic acid was used as the ion. The Waters high performance liquid chromatography system C18 reverse phase chromatography column was used for purification, preparation and analysis. The molecular weight of the sample was identified using the Shimadzu LC-MS detection system.

[0039] The amino acid sequence of the synthesized antimicrobial peptide CRRI15 (9) is VLLKILRWL-NH2. The HPLC chromatogram and HPLC component analysis report are as follows: Figure 1 As shown in the figure, the shape, height and area of ​​the peaks indicate that there are fewer impurities, the sample concentration and content are higher, and the product purity calculated by the peak area normalization method reaches 95%, which meets the requirements for further experiments. The mass spectrometry molecular weight identification results are shown in the figure. Figure 2 As shown, according to the mass-to-charge ratio (m / z) of the molecular ion peak of CRRI15 (9), its molecular weight can be inferred to be 1152.70 Da, which is consistent with the theoretical molecular weight of 1153.52 Da, so the synthetic sample can be used for the next test.

[0040] 2. Determination of minimum inhibitory concentration (MIC)

[0041] First, the antimicrobial peptide CRRI15 (9) was dissolved in sterile water and serially diluted twofold. 100 μL of the dilutions of different concentrations were added to a 96-well plate and cultured with 100 μL of the culture medium at a ratio of 1:1 (v / v) until the logarithmic phase. The OD was then adjusted. 600The indicator bacteria with a value of about 0.2 were mixed so that the final concentration of the antimicrobial peptide was 1-464 μM (the concentration decreased from the first well to the twelfth well). For the negative control, the indicator strain was mixed with sterile water, LB medium was used as a blank control, and then the 96-well plate was incubated at 37°C for 16 hours. The absorbance at 600nm was measured using an ELISA instrument. The concentration corresponding to the well where the OD value no longer increased was the minimum inhibitory concentration (MIC) of the antimicrobial peptide against the indicator bacteria.

[0042] The minimum inhibitory concentration can specifically illustrate the antibacterial ability of antimicrobial peptides and is an important indicator of antibacterial activity. The MIC test results of CRRI15 (9) for various indicator bacteria are shown in Table 1:

[0043] Table 1 MIC of CRRI15(9) against 10 indicator bacteria

[0044]

[0045] Six Gram-positive bacteria (G + ), 4 strains of Gram-negative bacteria (G - ) was used as the indicator bacteria. The test results showed that CRRI15 (9) had certain antibacterial activity against both Gram-positive and Gram-negative bacteria, especially against Gram-positive bacteria (MIC≤15μM). Among them, the best inhibitory effect was on Staphylococcus aureus D48, with an MIC of 4μM. It also showed excellent antibacterial effects on Micrococcus luteus 10209, Staphylococcus carnosus pet20, Staphylococcus aureus PCM2106, and Bacillus subtilis BAS2, with an MIC of 7μM. The inhibitory effect on Gram-negative bacteria was poor, and it only had a strong inhibitory effect on Escherichia coli DH5α, with an MIC of 7μM. It had poor antibacterial effects on the other three strains of Salmonella.

[0046] 3. Stability test

[0047] Thermal stability: Dissolve the antimicrobial peptide in sterile water to a concentration of 0.4 mg / mL. Heat in a water bath at 50°C, 60°C, 70°C, 80°C, 90°C, and 100°C for 1 hour. After cooling to room temperature, use Staphylococcus aureus as an indicator bacteria to detect the remaining antibacterial activity, and the antimicrobial peptide that has not been heat-treated is used as a blank control. The remaining antibacterial activity is the size of the inhibition zone compared to the control that has not been heat-treated, expressed as a percentage.

[0048] pH tolerance: The pH of the antimicrobial peptides was adjusted to 2.0, 3.0, 4.0, 5.0, 6.0, 7.0, 8.0, 9.0, and 10.0 with 1M HCl and NaOH, and the residual antibacterial activity of the antimicrobial peptides was detected with Staphylococcus aureus as the indicator bacteria. The antimicrobial peptides without acid or alkali treatment were used as blank controls, and the residual antibacterial activity was expressed as a percentage of the size of the inhibition zone compared with the control without acid or alkali treatment to detect its tolerance to acid or alkali environments.

[0049] The results of pH stability and thermal stability tests are shown in Figure 3 and Figure 4 As shown, the results indicate that CRRI15 (9) has good thermal stability and pH stability. The loss of antibacterial activity of CRRI15 (9) is minimal in the temperature range of 37°C-90°C. Even after being treated at 100°C for 1 hour, it can still maintain 85.4% of its antibacterial activity. When pH ≤ 8, it can maintain about 90% of its antibacterial activity. In an alkaline environment with a higher pH value, it does not lose its activity and can retain more than 60% of its antibacterial activity. Considering the good antibacterial ability and stability of CRRI15 (9), it has a good foundation for application in the fields of food and biomedicine.

[0050] Example 2 Study on the antibacterial mechanism of antimicrobial peptide CRRI15 (9)

[0051] Indicator bacteria with better antibacterial effect of CRRI15(9) (G+: Staphylococcus aureus and G-: Escherichia coli) were selected, and their cell membrane conductivity before and after the addition of CRRI15(9) was detected. Combined with scanning electron microscopy and transmission electron microscopy observations, their antibacterial action mode was comprehensively explored.

[0052] 1. Changes in cell membrane conductivity

[0053] Fresh bacterial suspension of Escherichia coli and Staphylococcus aureus was inoculated into 10 mL LB liquid medium at a 1% inoculum volume, and cultured overnight at 37°C, 180 rpm until the exponential growth phase. Centrifuge at 4°C, 10,000 rpm for 5 min, discard the supernatant, wash the precipitate with 5 mM HEPES buffer and centrifuge twice, and resuspend it with the same buffer until the OD600 value is about 0.2. Add the antimicrobial peptide CRRI15 (9) to a final concentration of 1×MIC, and measure its conductivity with a conductivity meter. Detect once every 5 min, read and record when the value is stable, monitor the changes for 60 min, and repeat three times. Use an equal volume of deionized water as a negative control and 1% TritonX-100 as a positive control.

[0054] When the cell membrane is damaged, electrolytes flow out of the cell, and the conductivity of the solution increases. Therefore, it is possible to infer whether the cell membrane is damaged by measuring the change in the conductivity of the extracellular solution. The experimental results of observing the effect of CRRI15 (9) on cell membrane conductivity using Escherichia coli DH5α and Staphylococcus aureus D48 as indicator bacteria are shown below. Figure 5 and Figure 6 As shown, it is not difficult to see that in the two groups of experiments, the negative control group Ctrl using deionized water had almost no fluctuation. After CRRI15(9) at a concentration of 1×MIC was added to the system, the conductivity increased to varying degrees. For Staphylococcus aureus D48, it rose rapidly in the first 10 minutes, and gradually remained stable after reaching the peak; while for Escherichia coli DH5α, the rising trend of conductivity was relatively gentle, and it did not reach the highest value until 35 minutes, and there was a small range of fluctuations. In the two groups of experiments, CRRI15(9) showed similar trends and effects to TritonX-100. It can be seen that the antimicrobial peptide CRRI15(9) can destroy the bacterial cell membrane, increase its permeability, and cause electrolyte outflow.

[0055] 2. Scanning electron microscopy observation

[0056] Single colonies of Escherichia coli DH5α and Staphylococcus aureus D48 were picked and cultured in 10 mL LB liquid medium at 37°C, 180 rpm overnight, and then inoculated into new LB liquid medium at a 1% inoculum, and cultured at 37°C until the logarithmic growth phase. The cells were collected after centrifugation (6000 rpm, 15 min, 4°C) three times with sterile PBS buffer, and the cells were resuspended to an OD600 of 0.2. The antimicrobial peptide CRRI15 (9) was added to the bacterial suspension to a final concentration of 1×MIC, and incubated at 37°C for 1 hour. The static bacterial solution was washed three times with PBS buffer at 4°C, 5000×g, 5 min, the supernatant was discarded, and the bacterial precipitate was collected. 1 mL of 2.5% glutaraldehyde solution was added to the washed bacteria, and fixed at 4°C for 16 hours. The fixed sample was washed three times with 0.1M PBS buffer, each for 15 minutes. The cell samples were dehydrated (15 min) using ethanol solutions with a concentration gradient ranging from 30% to 100%. The samples were dried and the cell surfaces were plated with a 10 nm gold film. The morphology and ultrastructural changes of Escherichia coli and Staphylococcus aureus cells were then observed using a scanning electron microscope.

[0057] In order to further study the antibacterial mechanism of antimicrobial peptide CRRI15(9), the morphological changes of Escherichia coli DH5α and Staphylococcus aureus D48 cells were observed by scanning electron microscopy (SEM) after treatment with antimicrobial peptide CRRI15(9) for 1 h. Figure 7 and Figure 8As shown in the SEM observations of the two bacteria, A and B are the results of the control group, and C and D are the results of the treatment group. Figure 7 and Figure 8 As can be seen from A and B, the bacteria that were not treated with CRRI15 (9) had a smooth and intact bacterial surface, with clear and intact spherical and rod-shaped structures, and clear boundaries between cells. Figure 8 As can be seen from the arrows C and D in the figure, after CRRI15(9) treatment of Staphylococcus aureus D48, the bacteria no longer maintained their normal morphology. The cell surface was obviously wrinkled and sunken, the bacteria were significantly deformed, and tended to dissolve. At the same time, holes were formed and the contents flowed out. Figure 7 As can be seen from the arrows C and D, the bacteria were also damaged to varying degrees, with uneven cell surfaces that appeared abnormally rough, accompanied by adhesion and leakage of cell contents. This shows that CRRI15 (9) can damage the bacterial cell membrane, causing it to no longer maintain its normal structural morphology.

[0058] 3. Transmission electron microscopy observation

[0059] The pretreatment, preparation and fixation of bacteria are consistent with the above-mentioned scanning electron microscope observation steps. The samples fixed overnight are washed twice with 0.1M PBS buffer, embedded in 50°C agar droplets, taken out after embedding and cut into small pieces, and then fixed with 1% osmic acid solution for 2h, and then washed twice with the same buffer solution, each time for 15min. The cell samples are dehydrated using ethanol solutions (50%-90%) and acetone solutions (100%) with different concentration gradients, each for 10min. The embedding agent and acetone are added in different proportions and mixed for infiltration overnight. The fully infiltrated Escherichia coli and Staphylococcus aureus samples are sliced ​​and stained, and the morphology and ultrastructural changes of the cells are observed by transmission electron microscopy.

[0060] Transmission electron microscopy (TEM) can visually observe the internal structure and integrity of bacteria. Similarly, Escherichia coli DH5α and Staphylococcus aureus D48 are used as indicator bacteria, and their transmission electron microscopy images are as follows: Fig. 9 and Fig.10 As shown in the transmission electron microscopy observations of the two bacteria, A and B are the results of the control group, and C and D are the results of the treatment group. Fig.10 As can be seen from A and B, the Staphylococcus aureus D48 cells that were not treated with CRRI15 (9) were intact, had typical cell wall and cell membrane structural characteristics, and the cytoplasm was full, uniform, and dense. Fig. 9 It can also be seen from A and B that the results of the control group of E. coli DH5α are the same. Fig. 9 C, D and Fig.10As can be seen from C and D, after being treated with CRRI15 (9), the contents of Staphylococcus aureus D48 cells are no longer uniform, and gaps appear ( Fig.10 The orange arrow in C shows that the membrane surface is discontinuous and the cell wall is missing ( Fig.10 The orange and blue arrows in D); the interior of the E. coli DH5α cells also underwent obvious changes, with the cytoplasm scattered and disordered ( Fig. 9 The orange arrows in C and D) are accompanied by cell membrane damage and leakage of contents ( Fig. 9 The experimental results show that CRRI15 (9) has a strong destructive effect on the external structure of bacterial cells, causing changes in the internal morphology of the cells, the outflow of contents and death.

[0061] The above-described embodiments are only some preferred implementations of the present invention, but are not intended to limit the present invention. A person skilled in the relevant technical field may make various changes and modifications without departing from the spirit and scope of the present invention. Therefore, any technical solution obtained by equivalent replacement or equivalent transformation falls within the protection scope of the present invention.

Claims

1. An antimicrobial peptide, the amino acid sequence of which is shown in SEQ ID No. 1, and the C-terminus of which has an amidation modification.

2. Use of the antimicrobial peptide as claimed in claim 1 in the preparation of antimicrobial products.

3. The use according to claim 2, characterized in that The antibacterial product is effective against Gram-positive bacteria or Gram-negative bacteria.

4. The use according to claim 2, characterized in that The Gram-positive bacteria are Micrococcus luteus, Staphylococcus carnosus, Staphylococcus aegypti or Bacillus subtilis.

5. The use according to claim 2, characterized in that The Gram-negative bacteria is Escherichia coli.

6. The use according to claim 2, characterized in that When the antibacterial product is used to inhibit Staphylococcus aureus, the minimum inhibitory concentration is 4 μM.

7. The use according to claim 2, characterized in that When the antibacterial product is used to inhibit Escherichia coli, the minimum inhibitory concentration is 7 μM.

8. The use according to claim 2, characterized in that The antibacterial products are antibacterial drugs, food preservatives and feed additives.

9. The use according to claim 2, characterized in that The antibacterial action mode of the antibacterial product is damage to the cell membrane.

10. An antimicrobial drug, food preservative or feed additive, the active ingredient of which comprises the antimicrobial peptide according to claim 1.

Images