Synthetic antibacterial peptide and application thereof

Abstract

The application discloses an artificially synthesized antibacterial peptide and application thereof. The amino acid sequence of the antibacterial peptide is FLFRVASKVFKALIGKFKKK, the molecular weight is 2355.98 Da, the isoelectric point is 11.43, and the net charge number is 7. Compared with the original antibacterial peptide D51, the antibacterial peptide significantly improves the antibacterial activity on Bacillus cereus, more efficiently inhibits the spore germination and biofilm formation of Bacillus cereus, and can effectively control the pollution of Bacillus cereus in food. The antibacterial peptide has the advantages of good stability, low hemolysis, difficulty in causing drug resistance, small synthesis difficulty and low production cost.

Description

TECHNICAL FIELD

[0001] The present application belongs to the field of biotechnology, and particularly relates to an artificially synthesized antibacterial peptide and application thereof. BACKGROUND

[0002] Bacillus cereus is a spore-forming, aerobic or facultative anaerobic, gram-positive bacillus that is widely distributed. Bacillus cereus is also an important foodborne opportunistic pathogen. Vegetative cells and spores can easily enter the food chain through crops and contaminate various foods and packaging materials. The emetic and enterotoxins produced by Bacillus cereus can cause food poisoning, including vomiting and diarrhea syndrome, and can also cause local tissue and systemic infections, and even death (Bacillus cereus food intoxication and toxicoinfection. Compr Rev Food Sci Food Saf. 2021, 20:3719-3761.). The ability of spores and biofilm formation makes this special bacterium have strong resistance to heat, chemical treatment and mechanical conditions, leading to deterioration of dairy products, meat and vegetables, which poses a continuous safety risk to consumers and the food industry. According to the China Public Health Emergency Reporting Management Information System, among bacterial food poisoning in China, food poisoning caused by Bacillus cereus ranks in the top three in terms of the number of occurrences and the number of cases (Chu FJ, et al. Epidemiological analysis of food poisoning in China from 2008 to 2010 based on the national network reporting system for public health emergencies [J]. Chinese Journal of Food Hygiene, 2012, 24(4): 387-390.).

[0003] The overuse of antibiotics and chemical preservatives has led to the emergence of multiple drug-resistant strains of Bacillus cereus, and there is a very urgent need for new antibacterial agents. Antimicrobial peptides (AMP) are a class of small molecule polypeptides widely existing in organisms in nature, which have broad-spectrum antibacterial properties, low bactericidal concentration, good stability, and are not easy to produce drug resistance to target strains, etc. They have completely different targets and mechanisms of action from antibiotics, and have good bacteriostatic effect on antibiotic-resistant bacteria (Development of bactericidal peptides against multidrug-resistant Acinetobacter baumannii with enhanced stability and low toxicity. Int J Mol Sci, 2022, 23, 2191.), and thus have a wide application prospect.

[0004] Pre-preliminary study found that the synthetic antibacterial peptide D51 has a low inhibitory concentration on Bacillus cereus (Alinguistic model for the rational design of antimicrobial peptides. Nature, 2006, 443(7113): 867-9.). However, people still hope to obtain antibacterial peptides with stronger antibacterial effect, so as to reduce the dosage and cost. SUMMARY

[0005] The primary purpose of the present application is to overcome the shortcomings and deficiencies of the prior art, and provide a synthetic antibacterial peptide.

[0006] Another purpose of the present application is also to provide the application of the above-mentioned synthetic antibacterial peptide.

[0007] The purpose of the present application is achieved by the following technical scheme: a synthetic antibacterial peptide, named antibacterial peptide D51-P11K, has an amino acid sequence of Phe-Leu-Phe-Arg-Val-Ala-Ser-Lys-Val-Phe-Lys-Ala-Leu-Ile-Gly-Lys-Phe-Lys-Lys-Lys, abbreviated as FLFRVASKVFKALIGKFKKK.

[0008] The application of the above-mentioned antibacterial peptide D51-P11K in inhibiting the activity of Bacillus cereus, inhibiting the spore germination of Bacillus cereus and / or inhibiting the biofilm formation of Bacillus cereus.

[0009] The application of the above-mentioned antibacterial peptide D51-P11K in preparing a microbial inhibitor.

[0010] The microorganism is preferably Bacillus cereus.

[0011] The microbial inhibitor includes food preservatives and drugs for preventing and treating bacterial infections.

[0012] The present application has the following advantages and effects relative to the prior art:

[0013] The antibacterial peptide D51-P11K provided by the present application is obtained by rational molecular design on the basis of antibacterial peptide D51. The antibacterial peptide D51-P11K significantly improves the antibacterial activity on Bacillus cereus compared with the original antibacterial peptide D51, more efficiently inhibits the spore germination and biofilm formation of Bacillus cereus, and can effectively control the pollution of Bacillus cereus in food. The antibacterial peptide D51-P11K also has the advantages of good stability, low hemolyticity, low drug resistance, small synthesis difficulty and low production cost. BRIEF DESCRIPTION OF DRAWINGS

[0014] Figure 1Figure 1 is a bactericidal kinetics curve of antibacterial peptide D51 and D51-P11K against Bacillus cereus.

[0015] Figure 2 Figure 2 is a result graph of morphological influence of antibacterial peptide D51-P11K on Bacillus cereus cells.

[0016] Figure 3 Figure 3 is a result graph of inhibitory effect of antibacterial peptide D51 and D51-P11K on spore germination of Bacillus cereus.

[0017] Figure 4 Figure 4 is a result graph of inhibitory effect of antibacterial peptide D51 and D51-P11K on biofilm formation of Bacillus cereus.

[0018] Figure 5 Figure 5 is a result graph of thermal stability detection of antibacterial peptide D51-P11K.

[0019] Figure 6 Figure 6 is a result graph of detection of hemolytic activity of antibacterial peptide D51-P11K.

[0020] Figure 7 Figure 7 is a bactericidal effect graph of antibacterial peptide D51 and D51-P11K in food samples. DETAILED DESCRIPTION

[0021] The application will be further described in detail below with reference to the examples and the accompanying drawings, but the embodiments of the application are not limited thereto.

[0022] Example 1 Design of D51 mutant

[0023] Based on the amino acid sequence Phe-Leu-Phe-Arg-Val-Ala-Ser-Lys-Val-Phe-Pro-Ala-Leu-Ile-Gly-Lys-Phe-Lys-Lys-Lys (abbreviated as FLFRVASKVFPALIGKFKKK) of antibacterial peptide D51, four mutant peptides were obtained by amino acid mutation to improve positive charge and change hydrophobicity, and the physicochemical parameter analysis of the mutant peptides was performed by using an online tool as shown in Table 1. The antibacterial peptide D51 and the mutant peptides were synthesized by Nanjing Kingsrui Biological Technology Co., Ltd.

[0024] Table 1 Sequence and physicochemical properties of antibacterial peptide D51 and mutant peptides thereof

[0025]

[0026] The D51 amino acid substitution peptides in Table 1 are D51-P11K (P at position 11 is changed to K), D51-P11G / A12K (PA at positions 11 and 12 is changed to GK), D51-P11K / A12K (PA at positions 11 and 12 is changed to K), and D51-F1K / A12K (FA at positions 1 and 12 is changed to K).

[0027] Example 2 Minimum inhibitory concentration (MIC) determination

[0028] In the experiment, the model strain Bacillus cereus ATCC14579 (purchased from the American Type Culture Collection) and the emetic Bacillus cereus NCTC11143 (purchased from the British National Culture Collection) were selected as the test strains. The Bacillus cereus was streaked on LB solid medium and incubated at 37°C for 16 hours. A single colony was inoculated in 5 mL of LB liquid medium and incubated overnight. The next morning, the culture was transferred to LB liquid medium at a ratio of 1:100 (bacterial solution: medium) and incubated until the logarithmic growth phase. The OD 600 value of the bacterial solution was measured by a spectrophotometer, and the solution was diluted to 1×10 6 CFU / mL. 50 μL of the diluted bacterial solution was added to a 96-well plate, and 50 μL of the antibacterial peptide was added to the 96-well plate containing the bacterial suspension according to the two-fold dilution method. The final concentrations were 128 μg / mL, 64 μg / mL, 32 μg / mL, 16 μg / mL, 8 μg / mL, 4 μg / mL, 2 μg / mL, 1 μg / mL, 0.5 μg / mL, 0.25 μg / mL, respectively. Ampicillin was used as a positive control. The 96-well plate was incubated at 37°C for 16 hours, and the OD 600 of each well was measured. The minimum inhibitory concentration was the lowest antibacterial peptide concentration at which no bacterial growth was observed. The experiment was repeated three times, with three replicates each time.

[0029] The results are shown in Table 2. The MIC value of D51 for the two Bacillus cereus strains was 16 μg / mL, while the MIC value of the mutant peptide D51-P11K for the two Bacillus cereus strains was 8 μg / mL, which was significantly stronger than D51. The MIC values of the mutant peptides D51-P11G / A12K, D51-P11K / A12K, and D51-F1K / A12K for the two Bacillus cereus strains were in the range of 32-64 μg / mL, and the antibacterial activity was reduced by 2-4 times compared to the original peptide D51. It is confirmed that increasing the number of positive charges in a certain range can increase the antibacterial activity, but when the number of positive charges exceeds a certain critical value, the electrostatic repulsion between antibacterial peptide molecules is stronger than the electrostatic attraction between the antibacterial peptide molecules and the cell membrane, which hinders the aggregation of antibacterial peptide molecules on the cell membrane and the formation of membrane pores, resulting in a decrease in antibacterial activity.

[0030] Table 2 MIC of D51 and mutant peptides against two B. cereus strains

[0031]

[0032] Example 3 Time-kill curve of antibacterial peptides against B. cereus

[0033] The method for determining the bactericidal kinetics of antibacterial peptide D51-P11K was based on the traditional bactericidal-time method. The B. cereus NCTC 11143 in the logarithmic growth phase was adjusted to 1 x 10 6 CFU / mL, 100 μL bacterial suspension was added to each well of a 96-well plate, and 100 μL of antibacterial peptide D51 and D51-P11K dilutions were added to each well, respectively, to achieve a final concentration of 2 x MIC, and the final concentration of ampicillin was 2 x MIC. After mixing, 20 μL was taken for dilution and plating to calculate the initial number of bacteria, and then sampling and dilution plating were performed every 20 min. The plates were incubated at 37°C for 16 h, and then plate counting was performed.

[0034] Figure 1 The results show that antibacterial peptides D51 and D51-P11K have bactericidal ability against B. cereus, D51-P11K kills all bacteria within 60 min, while D51 needs 80 min to kill all bacteria. However, when ampicillin at a concentration of 2 x MIC is used, viable bacteria can still be detected at the end of the experiment (200 min). These results show that antibacterial peptide D51-P11K kills B. cereus more quickly than D51, and the bactericidal speed of both is significantly stronger than that of ampicillin.

[0035] Example 4 Morphological effect of antibacterial peptide D51-P11K on B. cereus cells

[0036] The B. cereus NCTC 11143 in the logarithmic growth phase was adjusted to an OD 600 of 0.1, and antibacterial peptide D51-P11K was added to achieve a final concentration of 2 x MIC, with PBS as a negative control. Incubation was performed at 37°C for 1 h, followed by centrifugation at 5000 rpm for 15 min, and the supernatant was discarded. The cells were washed twice with water, and then 3% (v / v) glutaraldehyde was added for fixation at 4°C overnight. The cells were centrifuged at 5000 rpm for 15 min, and then washed twice with deionized water. The cells were dehydrated with 70%, 80%, and 95% ethanol solutions, respectively, for 10 min each time, and then with 100% ethanol twice. Then, the cells were subjected to critical point drying, and then sputtered with gold using an ion sputtering instrument. The cell morphology was observed under a scanning electron microscope.

[0037] The results of the scanning electron microscope are shown in Figure 4. Figure 2As shown, the control group bacterial cell membranes were intact, plump, and smooth. However, after treatment with the antimicrobial peptide D51-P11K, the bacterial membrane surface underwent significant changes, becoming wrinkled, rough, and even showing obvious lysis leading to the outflow of intracellular substances. This indicates that the antimicrobial peptide D51-P11K has a destructive effect on the cell membrane of Bacillus cereus NCTC11143.

[0038] Example 5: Experiment on the inhibition of Bacillus cereus spore germination by antimicrobial peptides

[0039] Bacillus cereus NCTC11143 was cultured for 48 h, washed twice with PBS, and 100 μL was spread onto spore medium and cultured for 5 days. Cells were then scraped from the culture plate and washed three times with PBS. The cells were centrifuged at 3000 rpm for 20 min and resuspended in PBS. The spore suspension was incubated at 80°C for 10 min to kill vegetative cells, and then washed three times with PBS to remove vegetative cells. The spore suspension was diluted with an appropriate amount of LB medium. 100 μL of the spore suspension and 100 μL of peptide solutions of different concentrations were added to 96-well plates and incubated for 16 h. A 20 μL sample was then diluted and spread. After 16 h of incubation, plate counts were performed. Plates without antimicrobial peptides served as controls. This experiment was repeated three times, with three replicates per well.

[0040] like Figure 3 As shown, compared with the untreated control, the spore germination rate of Bacillus cereus spores treated with antimicrobial peptide D51-P11K at a concentration of 8 μg / mL was 68%, while the spore germination rate of the D51 group was close to that of the control group. With increasing concentrations of antimicrobial peptides D51 and D51-P11K, the spore germination rates were 73% and 40.6%, respectively. When the concentration of antimicrobial peptides increased to 32 μg / mL, D51-P11K exhibited perfect germination inhibitory activity, with a spore germination rate of only 1%, while the spore germination rate after D51 treatment was still 40% (*p<0.05, **p<0.01). These results indicate that antimicrobial peptide D51-P11K can effectively inhibit the germination of Bacillus cereus spores, and its inhibitory effect is higher than that of D51.

[0041] Example 6: Experiment on the inhibition of biofilm formation by antimicrobial peptides in Bacillus cereus

[0042] The effect of the antimicrobial peptide D51-P11K on biofilm formation in Bacillus cereus was investigated using a conventional crystal violet staining assay. Logarithmic-phase Bacillus cereus NCTC11143 was diluted to a final concentration of 1.0 × 10⁻⁶ in TSB medium. 7CFU / mL, D51 and D51-P11K were added in the final concentration of 4, 8, 16, 32 μg / mL, PBS as control group, incubated in 37℃ incubator for 48 h, carefully removed the supernatant and washed with PBS for 3 times, remove the planktonic bacteria, stained with 0.1% crystal violet solution for 15 min, washed with PBS to remove excess dye, air dry at room temperature, add 100 μL of absolute ethanol to each well, avoid light for 30 min at room temperature, use microplate reader to measure the A value of each well at 595 nm. The experiment was repeated 3 times, each time 3 parallel wells.

[0043] Figure 4 The results of the inhibition rate of antibacterial peptide D51 and D51-P11K on B. cereus biofilm formation showed that the biofilm formation ability was negatively correlated with the concentration of antibacterial peptide. When the concentration of antibacterial peptide D51 and D51-P11K was 4 μg / mL, there was no significant effect on the formation of bacterial biofilm; when the concentration of antibacterial peptide was 8 μg / mL, only the D51-P11K treatment group showed slight inhibition; further increasing the concentration of antibacterial peptide to 16 μg / mL, the inhibition rate of D51 on B. cereus biofilm was 24%, while the inhibition rate of D51-P11K treatment group was more than 50%; when the concentration of antibacterial peptide was 32 μg / mL, D51-P11K could significantly inhibit the formation of B. cereus biofilm, with an inhibition rate of more than 90%, while the inhibition rate of D51 treatment group was less than 65% (*p<0.05, **p<0.01). It is shown that antibacterial peptide D51-P11K can efficiently inhibit the formation of B. cereus biofilm, and the inhibition effect is significantly higher than that of D51. Biofilm formation provides protection for bacteria in harsh environments, which is considered to be a major health risk point in the food industry, therefore, the inhibition of B. cereus biofilm formation by antibacterial peptide D51-P11K is a prominent feature of the peptide, indicating that it can be applied to the preservation and preservation of B. cereus food-related and prevention of B. cereus biofilm infection in clinic.

[0044] Example 7 Determination of the thermal stability of antibacterial peptide D51-P11K

[0045] The antibacterial peptide D51-P11K was placed at 20, 40, 60, 80 and 100℃ for 1 h, then restored to room temperature, and the MIC of B. cereus NCTC11143 was determined. The untreated antibacterial peptide (4℃) was used as a control. The experiment was repeated 3 times, each time with 3 parallel samples.

[0046] Figure 5 The results of temperature tolerance can be found in the table that after 80℃ treatment, the activity of antibacterial peptide D51-P11K remained unchanged, and after 100℃ treatment, the activity decreased slightly, indicating that antibacterial peptide D51-P11K has good thermal stability and can tolerate heat treatment during food processing.

[0047] Example 8 Hemolytic activity assay of antibacterial peptides

[0048] Fresh C57 mice (purchased from Guangdong Medical Laboratory Animal Center) were used to prepare red blood cells. The blood was centrifuged at 1000 r / min for 10 min, and the supernatant was discarded. The red blood cells were washed repeatedly with PBS until the supernatant was clear. The red blood cells were resuspended with PBS to prepare a 4% (v / v) red blood cell suspension. 100 μL of the cell suspension was added to a 96-well plate, and different concentrations of antibacterial peptide D51-P11K were added to a final concentration of 4-128 μg / mL. The same amount of PBS and 1% (v / v) Triton X-100 were used as negative and positive controls, respectively. The 96-well plate was incubated at 37°C for 30 min, centrifuged at 1000 r / min for 10 min, and the supernatant was transferred to a new 96-well plate. The OD 540 nm value of each well was measured, and the hemolysis rate was calculated. The formula for calculating the hemolysis rate is as follows:

[0049] Hemolysis rate (%) = (sample OD 540 nm - negative control OD 540 nm) / (positive control OD 540 nm - negative control OD 540nm ) x 100%

[0050] The hemolytic activity of antibacterial peptides varied with the concentration of antibacterial peptide D51-P11K, as shown in Figure 6 From the figure, it can be seen that antibacterial D51-P11K had almost no hemolytic activity at a concentration of 4-32 μg / mL. When the concentration was increased to 64 μg / mL, the hemolysis rate was still less than 10%, and only when the concentration of antibacterial peptide D51-P11K was increased to 128 μg / mL did it show obvious hemolytic activity. This indicates that antibacterial peptide D51-P11K has good biological safety.

[0051] Example 9 Application of antibacterial peptide D51-P11K in food

[0052] Since rice is one of the foods most severely infected by Bacillus cereus, cooked rice was chosen as the experimental object to study the preservative effect of antibacterial peptide D51-P11K in food. The rice sample was taken from a student cafeteria and heated in a microwave oven at high heat for 5 min to kill all microorganisms. The cooked rice (10 g) was diluted with 40 mL of distilled water and shaken for 10 min. Then, 1.5 x 10 5Rice samples artificially contaminated with Bacillus cereus NCTC11143 (CFU / mL) were incubated at 25°C for 1 hour. Different concentrations of the antimicrobial peptides D51 and D51-P11K were added to the samples to a final concentration of 32 μg / mL, and incubated at 25°C for 4 hours. Samples were taken every hour, diluted with PBS, and plated. The plates were incubated at 37°C for 16 hours, and the average colony count was counted. This experiment was performed three times.

[0053] The results are as follows Figure 7 As shown, after treatment with antimicrobial peptide D51-P11K, the bacterial count decreased by approximately 2.5 orders of magnitude within 2 hours, and within 3 hours, the bacterial count dropped to undetectable levels, indicating that D51-P11K could kill all bacteria in the rice sample within 3 hours. However, viable bacteria could still be detected after 4 hours of treatment with antimicrobial peptide D51 (*p<0.05, **p<0.01). This indicates that antimicrobial peptide D51-P11K can also exert a bactericidal effect in food matrices, and its bactericidal effect is stronger than that of D51. Therefore, antimicrobial peptide D51-P11K can be used for food preservation.

[0054] The above embodiments are preferred embodiments of the present invention, but the embodiments of the present invention are not limited to the above embodiments. Any changes, modifications, substitutions, combinations, or simplifications made without departing from the spirit and principle of the present invention shall be considered equivalent substitutions and shall be included within the protection scope of the present invention.

Claims

1. A synthetic antibacterial peptide, characterized in that: The amino acid sequence of the artificial synthetic antibacterial peptide is FLFRVASKVFKALIGKFKKK.

2. Use of the artificial synthetic antibacterial peptide of claim 1 for inhibiting the activity of Bacillus cereus, inhibiting the spore germination of Bacillus cereus and / or inhibiting the biofilm formation of Bacillus cereus for non-diagnostic and therapeutic purposes.

3. Use of the artificial synthetic antibacterial peptide according to claim 1 for the preparation of a microbial inhibitor, characterized in that: The microorganism is Bacillus cereus.

4. Use according to claim 3, characterized in that: The microbial inhibitor is a food preservative or a drug for preventing and treating bacterial infection.

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