Antimicrobial peptide against bacillus cereus, preparation method therefor and use thereof
Patent Information
- Authority / Receiving Office
- WO · WO
- Patent Type
- Applications
- Current Assignee / Owner
- Filing Date
- 2024-08-30
- Publication Date
- 2026-04-09
AI Technical Summary
Existing antimicrobial peptides have shortcomings in terms of antibacterial activity, stability, and drug resistance, making it difficult to effectively inhibit Bacillus cereus contamination, especially in food where the risk of food poisoning is high and difficult to detect.
A novel class of antimicrobial peptides was designed and synthesized with the amino acid sequence [KR]GG[KR]XC[FWY]C[KR]X[KR][FWY]C[LIAV]CXG[KR][KR]. Prepared by the Fmoc solid-phase synthesis method, it has specific molecular weight, isoelectric point and net charge number, and is suitable for the preparation of antimicrobial drugs, feed additives and food preservatives.
This antimicrobial peptide has a strong inhibitory effect on Bacillus cereus, good stability, is not prone to inducing drug resistance, has high synthesis efficiency, and low cost. It can effectively inhibit the growth and germination of Bacillus cereus in food and is suitable for food preservation and disease prevention.
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Abstract
Description
Antibacterial peptide against bacillus cereus and preparation method and application thereof TECHNICAL FIELD
[0001] The present application belongs to the field of biotechnology, and particularly relates to characterization, preparation and application of an artificial synthetic antibacterial peptide against bacillus cereus. BACKGROUND
[0002] Bacillus cereus is a gram-positive bacteria with facultative anaerobic spore production, and is a common foodborne pathogen. It can produce enterotoxins, leading to diarrhea and vomiting food poisoning, and even death in severe cases. It widely exists in soil, dust and water, and can grow and reproduce under relatively lenient conditions, and can produce defensive spores under harsh conditions. Therefore, bacillus cereus contamination is found in various pre-packaged foods, such as rice products, dairy products and meat products. Studies have found that when the number of bacillus cereus in the ingested food is >10 2 CFU / g (ml), there is a risk of foodborne illness, but it does not cause sensory changes, making it difficult for people to detect, so it is easy to eat. The eating habits in China mainly include rice, noodles and other starch products, and there is a habit of eating leftover food and overnight food, which makes food poisoning caused by bacillus cereus occur frequently. According to reports, among bacterial food poisoning in China, food poisoning caused by bacillus cereus ranks third in terms of the number of occurrences and the number of people affected.
[0003] Antimicrobial peptides (AMPs) are a class of polypeptides widely existing in nature, and have broad-spectrum antibacterial activity, and can quickly kill various bacteria, fungi, viruses and parasites, etc. The extensive use of antibiotics has led to the emergence of drug-resistant bacteria, which has prompted people to develop new antibacterial agents, and AMP is an excellent choice due to its unique biological activity and special action mechanism different from traditional antibiotics, and has good bacteriostatic effect on antibiotic-resistant bacteria. Therefore, AMP has become one of the most potential antibiotic substitutes, and its application in food, medicine, skin care and cosmetic preservatives is also becoming more and more widespread, and has good development prospects.
[0004] Although antimicrobial peptides have universal advantages, there are also some obvious shortcomings, such as low antibacterial activity, poor stability, high toxicity, etc. of some antimicrobial peptides. The antimicrobial peptides obtained by modification or new synthesis of existing antimicrobial peptides can greatly improve some of the above shortcomings to meet different application requirements, achieve the effect of reducing the amount and reducing the cost. The present application discloses a synthetic antibacterial peptide obtained by modification of host defense peptides, which has strong antibacterial effect and low antibacterial concentration for most bacteria. SUMMARY
[0005] In order to overcome the shortcomings and deficiencies existing in the prior art, the purpose of the present application is to provide a kind of synthetic antibacterial peptide, solve the problems in the above background art.
[0006] The purpose of the present application is achieved by the following technical solutions:
[0007] A kind of synthetic antibacterial peptide, its amino acid sequence general formula is [KR]GG[KR]XC[FWY]C[KR]X[KR][FWY]C[LIAV]CXG[KR][KR].
[0008] Expression [KR] in the bracket indicates that, in the first, fourth, ninth, eleventh, eighteenth and nineteenth positions in the syntax, lysine, arginine is also acceptable; [FWY] indicates that, in the seventh, twelfth position in the syntax, phenylalanine, tryptophan, tyrosine is also acceptable; [LIAV] indicates that, in the fourteenth position in the syntax, leucine, isoleucine, alanine, valine is also acceptable. Further, the amino acid sequence of the antibacillus cereus antibacterial peptide is as shown in SEQ ID NO. 1-SEQ ID NO. 50.
[0009] Table 1
[0010]
[0011] .
[0012] Further, the anti-Bacillus cereus antibacterial peptide has a molecular weight ranging from 1907.36 to 2583.08 Da, an isoelectric point ranging from 9.62 to 11.31, and a net charge number ranging from 6 to 8.
[0013] Further, the synthetic antibacterial peptide XWC26 has an amino acid sequence of KGGKLCWCKNKWCICLGKR, a molecular weight of 2224.79 Da, an isoelectric point of 9.70, and a net charge number of 6.
[0014] The application provides an application of a synthetic antibacterial peptide in preparation of antibacterial drugs, feed additives or food preservatives.
[0015] In the application, the anti-Bacillus cereus antibacterial peptide is applied to preparation of microbial inhibitors.
[0016] The application further provides a preparation method of the synthetic antibacterial peptide.
[0017] The application provides a synthetic method of an anti-Bacillus cereus antibacterial peptide.
[0018] Compared with the prior art, the application has the following beneficial effects:
[0019] The application finds a polypeptide with a new amino acid sequence through bioinformatics technology design and screening. BRIEF DESCRIPTION OF DRAWINGS
[0020] Fig. 1 is a result graph of cell morphology of Bacillus cereus after the antibacterial peptide XWC26 acts on the Bacillus cereus.
[0021] Fig. 2 is a result graph of the inhibitory effect of the antibacterial peptide XWC26 on spore germination of Bacillus cereus.
[0022] Fig. 3 is a result graph of detection of hemolytic activity of the antibacterial peptide XWC1.
[0023] Fig. 4 is a result graph of sterilization of the antibacterial peptide XWC26 in food samples. Embodiments of the present application
[0024] The present application is further illustrated by the following examples, which do not limit the present application. It should be noted that, if the following processes are not particularly detailed, they can be implemented or understood by those skilled in the art according to the prior art. If the reagents or instruments used are not marked with the manufacturer, they are considered to be conventional products that can be purchased on the market.
[0025] Example 1: Design and screening of antibacterial peptides
[0026] The present application aims to solve the above technical problems by modifying the sequence and spatial structure of host defense peptides with certain conservation, designing and screening an antibacterial peptide library with strong antibacterial activity and effective inhibition of drug-resistant bacteria, and verifying the antibacterial activity.
[0027] The present application is designed based on the following general formula of amino acid sequence: [KR]GG[KR]XC[FWY]C[KR]X[KR][FWY]C[LIAV]CXG[KR][KR]
[0028] (The expression [KR] in the parentheses means that, in the syntax, lysine and arginine are also acceptable at the first position)
[0029] Table 2 Sequence list of antibacterial peptides XCW1-XCW50
[0030] Sequence list of antibacterial peptides Sequence list of antibacterial peptides Sequence list of antibacterial peptides Sequence list of antibacterial peptides Sequence list of antibacterial peptides Sequence 1 XCW1 Sequence 11 XCW1 Sequence 21 XCW21 Sequence 31 XCW31 Sequence 41 XCW41 Sequence 2 XCW2 Sequence 12 XCW2 Sequence 22 XCW22 Sequence 32 XCW32 Sequence 42 XCW42 Sequence 3 XCW3 Sequence 13 XCW3 Sequence 23 XCW23 Sequence 33 XCW33 Sequence 43 XCW43 Sequence 4 XCW4 Sequence 14 XCW4 Sequence 24 XCW24 Sequence 34 XCW34 Sequence 44 XCW44 Sequence 5 XCW5 Sequence 15 XCW5 Sequence 25 XCW25 Sequence 35 XCW35 Sequence 45 XCW45 Sequence 6 XCW6 Sequence 16 XCW6 Sequence 26 XCW26 Sequence 36 XCW36 Sequence 46 XCW46 Sequence 7 XCW7 Sequence 17 XCW7 Sequence 27 XCW27 Sequence 37 XCW37 Sequence 47 XCW47 Sequence 8 XCW8 Sequence 18 XCW8 Sequence 28 XCW28 Sequence 38 XCW38 Sequence 48 XCW48 Sequence 9 XCW9 Sequence 19 XCW9 Sequence 29 XCW29 Sequence 39 XCW39 Sequence 49 XCW49 Sequence 10 XCW10 Sequence 20 XCW10 Sequence 30 XCW30 Sequence 40 XCW40 Sequence 50 XCW50
[0031] Example 2: Synthesis of antibacterial peptide
[0032] The antibacterial peptide of the present application is synthesized by conventional polypeptide solid phase synthesis. Taking antibacterial peptide XWC2 as an example, its sequence is: KGGKLCWCKWKYCICLGKR.
[0033] Sequence characteristics: the sequence type is amino acid sequence, contains 19 amino acid residues, the molecular weight size is 2273.89 Da, and the net charge number is 6.
[0034] The specific synthesis steps are as follows: 1) resin swelling: take the degree of substitution of 0.1 mmol 2-Chlorotrityl Chloride Resin resin, put the resin into the reaction tube, add DCM (15 ml / g) solvent, and shake for 40 min. 2) Connect the first amino acid: filter out the DCM solvent through the sand core, add 3 times the amount of Fmoc-Arg(Pbf)-OH amino acid, then add 2,4,6-trimethylpyridine, and finally add DCM for dissolution, shake for 12 h, and then wash with DMF for 3 times. 3) Deprotection: add 10 ml of 20% piperidine DMF solution (15 ml / g), shake for 6 min, then remove the piperidine DMF solution, add another 10 ml of 20% piperidine DMF solution (15 ml / g), and shake for 20 min. 4) Detection: remove the piperidine DMF solution, take a dozen resins, wash with ethanol three times, add one drop of ninhydrin and phenol solution, heat at 105℃-110℃ for 3 min, and turn dark blue for positive reaction. 5) First washing: wash with DMF (10 ml / g) for 3 times. 6) Blocking: add methanol (10 ml / g) and 3 times the amount of DIEA, shake for 20 min, and then wash with DMF for 3 times. 7) Condensation (generate the second amino acid Lysine in the sequence): add 3 times the amount of Fmoc-Lys(boc)-OH amino acid, HCTU, and DIEA to the reaction tube, dissolve with as little DMF as possible, and react for 1 h. 8) Second washing: wash with DMF (10 ml / g) once, and then wash with DMF (10 ml / g) twice. 9) Repeat the above operation to connect the amino acids in the sequence from left to right. 10) After connecting the last amino acid, deprotection, and wash the resin according to the following method: DMF (10 ml / g) twice, DCM (10 ml / g) twice, and dry for 8 min. 11) Cut the polypeptide from the resin: the ratio of resin and cleavage solution is 10 ml / g, constant temperature shaking for 150 min. (The cleavage solution is prepared according to the volume percentage, which can be TFA 95%, water 2.5%, and TIS 2.5%). 12) Blow dry and wash: blow the cleavage solution as much as possible with nitrogen, chromatograph with ether, wash with ether six times, and then dry at room temperature to obtain the crude peptide. 13) Analyze and identify the polypeptide by HPLC-MS: (1) Prepare a solution of the crude peptide with a concentration of 1 mg / ml. (2) Filter the solution with a 0.45 μm filter membrane. (3) Analysis: take 10 μl for HPLC-MS analysis. The mobile phase is water and acetonitrile, and the time is 25 min, isocratic elution, first equilibrate the HPLC with isocratic gradient for 5 min, then inject the sample, gradient water 40%, acetonitrile 60%; get the purity and MS identification. 13) Freeze-dry the purified solution to obtain the finished product. 14) Seal the white powder-like polypeptide and store it at -20 degrees.
[0035] Example 3: Minimum inhibitory concentration (MIC) and minimum bactericidal concentration (MBC) determination
[0036] The 50 antibacterial peptides of general formula predicted and synthesized in Examples 1 and 2 were subjected to minimum inhibitory concentration (MIC) determination to screen the peptides with the smallest MIC value and the strongest antibacterial activity.
[0037] In this experiment, the model strain ATCC14579 of Bacillus cereus was selected as the test strain. The Bacillus cereus was streaked on the anti-MH solid medium and incubated at 37°C for 16h. 3-5 colonies were selected and inoculated in 3mL of MH liquid medium for overnight culture. The next morning, the OD 600 value of the bacterial solution was measured by spectrophotometer, and it was diluted to 5×10 5 CFU / mL. 90μL of the diluted bacterial solution was added to the 96-well plate, and the antibacterial peptides were diluted according to the double dilution method. 10μL of each was added to the 96-well plate containing the bacterial suspension, with the final concentration being 128μg / mL, 64μg / mL, 32μg / mL, 16μg / mL, 8μg / mL, 4μg / mL, 2μg / mL, 1μg / mL, 0.5μg / mL, 0.25μg / mL, respectively. Ampicillin was used as a control. The 96-well plate was incubated at 37°C for 16h, 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. To determine the minimum bactericidal concentration (MBC), 10μL of the suspension was taken from each well and spotted on agar plates. The lowest peptide concentration that resulted in no bacterial colony growth was recorded as the MBC. This experiment was repeated 3 times with 3 replicates each.
[0038] The results are shown in Table 3. The MIC value of XWC26 against Bacillus cereus was 2μg / mL, and the antibacterial ability was the most significant. The MIC values of XWC1, XWC7, XWC35 and XWC46 against Bacillus cereus were all 4μg / mL, and the antibacterial ability was better than that of the other antibacterial peptides.
[0039] Further minimum bactericidal concentration (MBC) determination was performed on the antibacterial peptides with MIC≤4μg / mL (XWC1, XWC7, XWC26, XWC35, XWC46). The results are shown in Table 4. The MBC value of antibacterial peptide XWC7 was twice that of the MIC value, and the MBC values of the other 4 peptides were consistent with their MIC values. The experiment showed that the above antibacterial peptides had strong antibacterial activity against Bacillus cereus.
[0040] Table 3 MIC of XWC1-AMP50 against Bacillus cereus (μg / mL)
[0041] Peptides MIC Peptides MIC Peptides MIC Peptides MIC Peptides MIC XCW14 XCW118 XCW2164 XCW3116 XCW4132 XCW28 XCW1232 XCW2232 XCW3216 XCW428 XCW38 XCW138 XCW238 XCW3316 XCW438 XCW416 XCW148 XCW248 XCW3464 XCW4432 XCW58 XCW1516 XCW258 XCW354 XCW4516 XCW616 XCW168 XCW262 XCW3616 XCW464 XCW74 XCW178 XCW278 XCW3716 XCW4764 XCW816 XCW18128 XCW288 XCW388 XCW4816 XCW964 XCW1964 XCW2916 XCW3916 XCW4932 XCW1032 XCW208 XCW30>128 XCW408 XCW5032
[0042] Table 4 MBC of antibacterial peptides XWC1, etc. against Bacillus cereus (μg / mL)
[0043] Peptides MBC XCW14 XCW78 XCW262 XCW354 XCW464
[0044] Example 4: Determination of antibacterial spectrum
[0045] The antibacterial peptides (XWC1, XWC7, XWC26, XWC35, XWC46) with MIC≤4 μg / mL in Example 3 were subjected to antibacterial activity determination, and the antibacterial activity against Gram-positive bacteria (Staphylococcus aureus, etc.) and Gram-negative bacteria (Escherichia coli, etc.) was evaluated.
[0046] Each test strain was streak-inoculated on an anti-MH solid culture medium, and incubated in a 37°C incubator for 16 h, and 3-5 colonies were selected and inoculated in 3 mL of MH liquid culture medium for overnight culture. The next morning, the OD value of the bacterial solution was measured by spectrophotometer, and it was diluted to 5×10 600 5 CFU / mL. 90 μL of the diluted bacteria solution was added to a 96-well plate, and 10 μL of the diluted antibacterial peptide was added to the 96-well plate containing the bacterial suspension in accordance with 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 control. The 96-well plate was incubated in a 37°C incubator for 16 h, 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.
[0047] The results are shown in Table 5. The tested gram-positive bacteria (e.g., Staphylococcus aureus, Bacillus subtilis, etc.) showed stronger antibacterial activity than the gram-negative bacteria (Escherichia coli, Pseudomonas aeruginosa, etc.). The experiment showed that the above antibacterial peptide had broad antibacterial activity, and the effect on gram-positive bacteria was stronger.
[0048] Table 5. Antibacterial spectrum determination of antibacterial peptide XWC1
[0049]
[0050] Example 5: Observation of the cell morphology of Bacillus cereus before and after the action of antibacterial peptide XWC26
[0051] Bacillus cereus ATCC14579 was inoculated in an antibiotic-free LB liquid medium and incubated overnight. The next morning, the OD 600 value of the bacterial solution was measured by a spectrophotometer, and the solution was diluted to 1 x 10 8 CFU / mL. Antibacterial peptide XWC26 was added to make the final concentration 2 x MIC, and PBS was used as a negative control. The solution was incubated at 37°C for 1 h, then centrifuged at 8000 rpm for 3 min, and the supernatant was discarded. The bacterial cells were washed with PBS buffer for 3 times, and then 3% (v / v) glutaraldehyde was added for overnight fixation at 4°C. After sufficient fixation, the bacterial cells were centrifuged at 8000 rpm for 5 min, and washed with PBS buffer for 3 times. The bacterial cells were then dehydrated with ethanol solutions with a volume fraction of 30%, 50%, 70%, 80%, and 90% for 15 min each, and 100% ethanol for 2 times. Then, the bacterial cells were subjected to critical point drying, sprayed with gold by an ion sputtering instrument, and observed under a scanning electron microscope.
[0052] The scanning electron microscope results are shown in Figure 1. The cell morphology of the Bacillus cereus cells without peptide treatment is normal, the cell membrane is complete, and the surface is smooth. However, after treatment with the antibacterial peptide XWC26, the membrane surface of the bacteria changes significantly, becoming wrinkled and rough, with obvious membrane rupture and cytoplasmic components exuding. The experiment shows that the antibacterial peptide XWC26 can destroy the cell membrane of Bacillus cereus, change the permeability of the cell membrane, and ultimately lead to the death of the bacteria.
[0053] Example 6: Inhibition of Bacillus cereus spore germination by antibacterial peptides
[0054] Bacillus cereus ATCC14579 was inoculated in an antibiotic-free LB liquid medium, and after 48 h of culture, 100 μL of the diluted bacterial solution was spread on a spore culture medium for 5 days. Then the cells were scraped from the culture plate and washed 3 times with PBS buffer, centrifuged at 4000 rpm for 20 min and resuspended in PBS buffer. The spore suspension was killed by incubating in a 80°C water bath for 10 minutes, and then washed 3 times with PBS buffer to remove the vegetative cells. The spore suspension was diluted with an appropriate amount of LB medium. 200 μL of the spore suspension and 200 μL of the peptide solution at different concentrations were added to a 96-well plate, and after overnight incubation, 10 μL of the sample was diluted and spread. After 16 h of incubation in an incubator, plate counting was performed, and the antibacterial peptide was not added as a control. This experiment was repeated 3 times, with 3 replicates each time.
[0055] As shown in Figure 2, compared with the untreated control, the spore germination rate of Bacillus cereus treated with antibacterial peptide XWC26 at a concentration of 2 μg / mL was 75%, and as the concentration increased to 8 μg / mL, the spore germination rate decreased to 15%, and when the concentration increased to 16 μg / mL, the spore germination rate was less than 10%. The experiment shows that antibacterial peptide XWC26 at high concentration can effectively inhibit the germination of Bacillus cereus spores.
[0056] Example 7: Stability determination of antibacterial peptides
[0057] Thermal stability determination: antibacterial peptides (XWC1, XWC7, XWC26, XWC35, XWC46) were placed at 25, 50, 75 and 100°C for 1 h, then restored to room temperature, and the MIC against Bacillus cereus was determined. The untreated antibacterial peptide (4°C) was used as a control. This test was repeated 3 times, with 3 replicates each time.
[0058] Acid-base stability assay: Buffer solutions were prepared at pH 2 (50 mM, glycine-HCl buffer), 6 (50 mM, sodium phosphate buffer) or 10 (50 mM, glycine-NaOH buffer). The above-mentioned antibacterial peptides were dissolved in the buffer solutions and incubated at 37 °C for 1 h, and the MICs against B. cereus were determined. The antibacterial peptides dissolved in PBS buffer were used as a control. The experiment was repeated 3 times with 3 replicates each.
[0059] Protease stability assay: Pepsin, trypsin and proteinase-K were dissolved in PBS buffer at a final concentration of 20 μg / mL. The above-mentioned antibacterial peptides were mixed with the above-mentioned proteases at a volume ratio of 1:1 at 37 °C for 1 h. Then, the peptide-protease mixtures were heated at 60 °C for 15 min to inactivate the protease activity. The MICs against B. cereus were determined. The antibacterial peptides mixed in PBS buffer were used as a control. The experiment was repeated 3 times with 3 replicates each.
[0060] Table 6 is the temperature tolerance result. It can be found from the table that the activity of the above-mentioned 5 antibacterial peptides remains unchanged after being treated at a temperature of up to 100 °C, indicating that the above antibacterial peptides have good thermal stability and can tolerate heat treatment during food processing.
[0061] Table 7 is the acid-base tolerance result. The antibacterial activity of XWC7 is not affected by pH changes; XWC1, XWC26, XWC35 and XWC46 maintain antibacterial activity at pH 2-6 and have a slight decrease in activity at pH 10, indicating that the antibacterial peptides have good stability under acidic and neutral conditions. In alkaline conditions, precipitation occurs due to reaching the isoelectric point of the protein, which affects the antibacterial activity to a certain extent.
[0062] Table 8 is the protease tolerance result. XWC1, XWC26, XWC35 and XWC46 maintain activity under the influence of pepsin, and XWC7 has a slight decrease in activity. However, the activity of the above antibacterial peptides decreases by 8-16 times under the influence of trypsin or proteinase-K, indicating that the above antibacterial peptides maintain good stability in the presence of pepsin and poor stability in the presence of trypsin or proteinase-K.
[0063] Table 6 Temperature tolerance assay of antibacterial peptide XWC1 and the like
[0064]
[0065] Table 7 Acid-base tolerance assay of antibacterial peptide XWC1 and the like
[0066]
[0067] Table 8 Protease tolerance assay of antibacterial peptide XWC1 and the like
[0068]
[0069] Example 8: Hemolytic activity assay of antibacterial peptides
[0070] Take 100 μL of 6% (v / v) mouse red blood cell suspension (Shanghai Yuan Ye) and add to a 96-well plate, then add 100 μL of different concentrations of antibacterial peptides (XWC1, XWC7, XWC26, XWC35, XWC46) to a final concentration of 1-128 μg / mL, and take the same amount of PBS and 1% (v / v) Triton X-100 as negative and positive control groups, respectively. Incubate the 96-well plate at 37°C for 1 hour, centrifuge at 1000 r / min for 3 min, take the supernatant to a new 96-well plate, measure the OD 540 nm value of each well, and calculate the hemolysis rate, the formula is as follows: hemolysis rate (%) = (sample OD 540 nm-negative control OD 540 nm) / (positive control OD 540 nm-negative control OD 540 nm) x 100
[0071] Figure 3 shows the hemolytic activity of the above antibacterial peptides with varying concentrations. As can be seen from the figure, the antibacterial peptides have almost no hemolytic activity below a concentration of 2 μg / mL, begin to show hemolytic activity when the concentration is increased to 4 μg / mL, and show obvious hemolytic activity when the concentration is increased to 8 μg / mL, with a hemolysis rate higher than 10%. This shows that the above antibacterial peptides have relatively stable biological safety at their minimum inhibitory concentration (MIC ≤ 4 μg / mL), but still need to be improved.
[0072] Example 9: Application example of the antibacterial peptides in food
[0073] Take cooked rice as the experimental object, and study the preservative effect of antibacterial peptide XWC26 in food. The rice sample is taken from a student cafeteria and heated in a microwave oven at high heat for 3 min to kill microorganisms. Dilute 10 g of cooked rice with 40 mL of distilled water and shake for 10 min. Then, artificially contaminate the rice sample with 5 x 10 5 CFU / mL of Bacillus cereus, and place at 25°C for 1 h. Add the antibacterial peptide to the sample to a final concentration of 8 μg / mL, and incubate at 25°C for 3 h, without adding the antibacterial peptide as a control. Take samples every 1 h, dilute and plate. Incubate the plates at 37°C for 16 h, and count the average number of colonies. The experiment is performed 3 times.
[0074] The results are shown in Figure 4, which shows that the sample treated with antibacterial peptide XWC26 decreases by about 2 orders of magnitude within 2 hours, indicating that it can inhibit and kill the bacteria in the rice sample in a short time, and can be used for food preservation.
[0075] The above embodiments are only preferred embodiments of the present application, which are used for explaining the present application, but not limit the present application. Any changes, replacements, modifications, etc. made by those skilled in the art without departing from the spirit and essence of the present application shall belong to the protection scope of the present application.
Claims
1. An anti-Bacillus cereus antibacterial peptide, characterized in that: The amino acid sequence of the anti-Bacillus cereus antibacterial peptide is [KR]GG[KR]XC[FWY]C[KR]X[KR][FWY]C[LIAV]CXG[KR][KR].
2. The anti-Bacillus cereus peptide of claim 1, wherein: The expression [KR] in the brackets means that, in the syntax, lysine and arginine are also acceptable at the first, fourth, ninth, eleventh, eighteenth and nineteenth positions; [FWY] means that, in the syntax, phenylalanine, tryptophan and tyrosine are also acceptable at the seventh and twelfth positions; and [LIAV] means that, in the syntax, leucine, isoleucine, alanine and valine are also acceptable at the fourteenth position.
3. The Bacillus licheniformis antibacterial peptide of claim 1, wherein: The amino acid sequence of the anti-Bacillus cereus antibacterial peptide is as shown in SEQ ID NO. 1-SEQ ID NO.
50.
4. The Bacillus licheniformis antibacterial peptide of claim 1, wherein: The molecular weight of the anti-Bacillus cereus antibacterial peptide ranges from 1907.36-2583.08 Da, the isoelectric point ranges from 9.62-11.31, and the net charge number ranges from 6-8.
5. The anti-Bacillus cereus peptide of claim 1, wherein, One of the synthesized antibacterial peptides, XWC26, has an amino acid sequence of KGGKLCWCKNKWCICLGKR, a molecular weight of 2224.79 Da, an isoelectric point of 9.70, and a net charge number of 6.
6. The synthesis method of the anti-Bacillus cereus antibacterial peptide according to any one of claims 1-5, which uses CTC resin as a solid-phase carrier and Fmoc solid-phase synthesis method to synthesize a peptide with the amino acid sequence of the target product.
7. The use of the anti-Bacillus cereus antibacterial peptide according to any one of claims 1-5 in inhibiting the activity of Bacillus cereus and inhibiting the germination of Bacillus cereus spores.
8. The use of the anti-Bacillus cereus antibacterial peptide according to claim 1 in preparing a microbial inhibitor.
9. Use according to claim 8, characterized in that: The microbial inhibitor is a food preservative, a cosmetic preservative or a drug for preventing and treating bacterial infection.
10. Use according to claim 8, characterized in that: The microorganism is Bacillus cereus, as well as other gram-positive bacteria and gram-negative bacteria.