CN117430673A - Antibacterial peptide composition for inhibiting and killing gram-negative bacteria and application thereof - Google Patents

Antibacterial peptide composition for inhibiting and killing gram-negative bacteria and application thereof Download PDF

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CN117430673A
CN117430673A CN202210868364.6A CN202210868364A CN117430673A CN 117430673 A CN117430673 A CN 117430673A CN 202210868364 A CN202210868364 A CN 202210868364A CN 117430673 A CN117430673 A CN 117430673A
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antibacterial
antibacterial peptide
thanatin
peptide composition
peptide
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李彬
夏学峰
张雪寒
周俊明
汪伟
李澧
范宝超
周金柱
朱雪蛟
赵永祥
李基棕
李运川
胡屹屹
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Jiangsu Academy of Agricultural Sciences
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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
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    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • A—HUMAN NECESSITIES
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    • A61P31/04—Antibacterial agents
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    • C07K7/00—Peptides having 5 to 20 amino acids in a fully defined sequence; Derivatives thereof
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    • Y02A—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE
    • Y02A50/00—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE in human health protection, e.g. against extreme weather
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Abstract

The invention discloses an antibacterial peptide composition for inhibiting and killing gram-negative bacteria and application thereof. An antibacterial peptide composition, wherein the antibacterial peptide composition is composed of two antibacterial peptides, namely Thanatin and BF-15a3; the sequence of the antimicrobial peptide Thanatin is shown in SEQ ID NO. 1; the sequence of the antibacterial peptide BF-15a3 is shown as SEQ ID NO. 2. The antibacterial peptide composition not only has the function of synergistically killing common pathogenic microorganisms, but also has very excellent antibacterial activity on clinically separated multi-drug resistant pathogenic bacteria and does not have hemolytic activity. The antibacterial peptide used in the antibacterial peptide composition is synthesized artificially, has the advantages of small molecular weight, convenient artificial synthesis, strong bactericidal effect, broad antibacterial spectrum, low hemolytic activity and the like, and has wide application prospect.

Description

Antibacterial peptide composition for inhibiting and killing gram-negative bacteria and application thereof
Technical Field
The invention belongs to the technical field of biological medicines, and particularly relates to an antibacterial peptide composition for inhibiting and killing gram-negative bacteria and application thereof.
Background
Coli O157H 7 is a common food-borne pathogenic bacterium and forms a serious threat to international public health safety. Coli O157H 7 is one of the most common sources of human infection, and may cause a variety of syndromes such as mild diarrhea, severe bloody diarrhea, hemorrhagic colitis or renal failure due to Hemolytic Uremic Syndrome (HUS) due to its low infection dose and strong acid resistance [3] . Coli O157: H7 has become one of the most deadly food-borne pathogens because of its strong hazard and pathogenicity.
Antibiotics, one of the biggest medical findings in the 20 th century, play a vital role in the treatment of microbial pathogen infections. The use of antibiotics makes the treatment of many diseases more efficient, but bacteria gradually evolve over the long term use of antibiotics, which makes them highly resistant. In recent years, infections caused by drug-resistant bacteria have frequently exploded around the world due to abuse or misuse of antibiotics. According to the world health organization report, the level of bacterial resistance has peaked in the last decade, severely threatening human health and safety. Thus, new antimicrobial agents are urgently needed to alleviate this problem.
The antibacterial peptide has broad-spectrum antibacterial activity, a unique multi-target action mechanism can effectively inhibit multi-drug resistant bacteria, and the bacteria are not easy to generate resistance, however, the popularization and application of the antibacterial peptide in the fields of food and medicine are limited due to the problems of cost, toxicity, stability, drug resistance and the like.
Disclosure of Invention
The object of the present invention is to provide an antimicrobial peptide composition for inhibiting and killing gram-negative bacteria.
It is another object of the present invention to provide the use of the antimicrobial peptide composition.
The aim of the invention can be achieved by the following technical scheme:
an antibacterial peptide composition comprises antibacterial peptide Thanatin and antibacterial peptide BF-15a3. The method for obtaining the antibacterial peptide is chemical synthesis, and the amino acid sequence is Thanatin: GSKKPVPIIYCNRRTGKCQRM (SEQ ID NO. 1); BF-15a3: VKRWKKWKRKWKKWV (SEQ ID NO. 2).
As a preferred aspect of the invention, the composition has a mass ratio of Thanatin to BF-15a3 of from 1:1.5 to 3, preferably from 1:1.8 to 2, more preferably from 1:2.
The application of the antibacterial peptide composition in preparing a product is characterized in that the product is selected from any one of the following:
1) An antibacterial agent;
2) A medicament for preventing and/or treating a disease caused by a bacterial infection;
3) Food preservative.
As a preferred aspect of the present invention, the bacterium is a gram-negative bacterium.
As a preferred embodiment of the present invention, the bacterium is E.coli, preferably E.coli resistant to carbapenems or not resistant to carbapenems.
An antibacterial agent comprising the antibacterial peptide composition of the present invention.
As a preferred aspect of the invention, the agent is a solution in which the concentration of Thanatin is 0.4-2.4. Mu.g/mL.
As a preferred embodiment of the present invention, the concentration of Thanatin in the reagent is 1. Mu.g/mL and the concentration of BF-15a3 is 2. Mu.g/mL.
The antibacterial peptide composition and the antibacterial agent are applied to food preservation.
The antibacterial peptide composition not only has the function of synergistically killing common pathogenic microorganisms, but also has very excellent antibacterial activity on clinically separated multi-drug resistant pathogenic bacteria, and does not have hemolytic activity. The antibacterial peptide used in the antibacterial peptide composition is synthesized artificially, has the advantages of small molecular weight, convenient artificial synthesis, strong bactericidal effect, broad antibacterial spectrum, low hemolytic activity and the like, and has wide application prospect.
The antibacterial peptide composition for inhibiting and killing gram-negative bacteria provided by the invention has the following beneficial effects:
1) The composition has good synergistic bactericidal effect when used for inhibiting and killing gram-negative bacteria, can greatly reduce the use amount of the antibacterial peptide, keeps the original bactericidal effect, and has better biological safety.
2) The food, health care product, medicine and other products prepared by the composition can inhibit and kill more than 99 percent of bacteria, thereby reducing the possibility of diseases caused by the infection of escherichia coli.
3) Products such as foods, health-care products, medicines and the like prepared by the composition can inhibit and kill escherichia coli including drug-resistant escherichia coli with high efficiency, and meanwhile, the bacteria cannot generate resistance.
Drawings
FIG. 1 is a graph showing the bactericidal kinetics of an antimicrobial peptide combination against E.coli WX1002, ATCC 43895.
FIG. 2 is a graph showing a hemolytic test of the antibacterial peptide composition.
T+B refers to thanatin+BF-15a3. As used herein, MIC refers to that of E.coli O157: H7WX1002, that of Thanatin is 4. Mu.g/mL, and that of BF-15a3 is 8. Mu.g/mL.
FIG. 3 cytotoxicity T+B of thaumatin, BF-15a3 and combinations thereof on HEK-293T cells and IPEC-J2 cells refers to thaatin+BF-15 a3. As used herein, MIC refers to that of E.coli O157: H7WX1002, that of Thanatin is 4. Mu.g/mL, and that of BF-15a3 is 8. Mu.g/mL. NS, not sign, no significant difference, indicating no statistical significance of the difference between the means (p > 0.05).
FIG. 4 induction of resistance to E.coli O157:H7WX1002 by thanatin and BF-15a3.
FIG. 5 MIC (μg/mL) of thanatin and BF-15a3 for E.coli O157:H7WX1002 after 100℃treatment.
FIG. 6 bacteriostatic activity of thanatin and BF-15a3 on E.coli O157: H7WX1002 after treatment with different pH.
FIG. 7 sterilizing effect of thanatin, BF-15a3 and combinations thereof on E.coli O157: H7WX1002 on the surfaces of lettuce (A) and cucumber (B) during storage at 4 ℃.
Detailed Description
Material reagent
12 antibacterial peptides (sequence information is shown in Table 1) were synthesized by solid phase method from Nanjing Jinsri biotechnology Co., ltd, and purity was verified by high performance liquid chromatography-mass spectrometry>90% >; rabbit red blood cells, purchased from nanjingsen multiple gamma biotechnology limited; PCR Mix was purchased from Nanjinopran biosciences Inc.; DNA markers were purchased from beijing qingke biotechnology limited; gelred nucleic acid dyes were purchased from Shanghai Biotechnology; restriction endonuclease XbaI was purchased from Dalianbao bioengineering (Takara) Inc.; sensittre TM NARMS gram negativeSex bacteria drug sensitive plate, purchased from us Thermo Fisher Scientific company.
Experimental strains
Standard strain E.coli O157H 7 ATCC43895; e.coli O157: H7WX1002 was isolated from slaughterhouses, O157: H7 JL1801 split-line farms, and E.coli O157: H7WX1002, JL1801 strains harbored the NDM-5 resistance gene.
The following examples facilitate a better understanding of the present invention, but are not intended to limit the same. The experimental methods in the following examples are conventional methods unless otherwise specified. The experimental materials used in the examples described below, unless otherwise specified, were purchased from conventional biochemical reagent stores.
Example 1 determination of minimum inhibitory concentration of antibacterial peptide
The minimum inhibitory concentration (Minimum inhibitory concentration, MIC) of the 12 antimicrobial peptides (table 1) was measured using the method adopted by the national clinical laboratory standards committee (NCCLS) with some modifications. 2mg of the antibacterial peptide was dissolved in 1mL of ddH 2 In O, 0.22 μm filter is used for filtering, and 2mg/mL of antibacterial peptide stock solution is prepared and stored in a refrigerator at-20 ℃. Using ddH 2 O2-fold serial dilutions of the antibacterial peptide solution were performed to obtain final test concentrations of 1-1024. Mu.g/mL. A bacterial suspension was prepared by adding single colonies on LB plates to deionized water to adjust to 0.5 McFarland, mcF. Next, the bacterial suspension was diluted 100-fold with Mueller-Hinton Broth (MHB) medium to give a final bacterial concentration of 1X 10 6 CFU/mL. Subsequently, 50. Mu.L of the peptide solution of different concentrations and 50. Mu.L of the bacterial solution were added to a 96-well plate, incubated at 37℃for 18 hours, and absorbance at 600nm was measured using a microplate reader, with the lowest concentration at which OD value did not significantly change being used as MIC of the antibacterial peptide. For comparison, MIC of meropenem was also determined.
MIC values of them for 3 strains of E.coli O157: H7 strain (standard strain E.coli O157: H7 ATCC43895, E.coli O157: H7WX1002 carrying blaNDM-5 gene and E.coli O157: H7 JL 1801) were determined, and MIC values of E.coli ATCC25922 were determined as quality control.
As shown in Table 2, the anti-bacterial peptides were differentThe bacterial activities are very different. The MIC values of each antibacterial peptide for the escherichia coli O157:H27 and the carbapenem-resistant escherichia coli O157:H27 are not obviously different, so that the activity of the antibacterial peptide is not influenced by the drug resistance phenotype or the drug resistance mechanism of bacteria, and the antibacterial peptide has a sterilization mode which is distinct from that of antibiotics. Wherein thanatin and BF-15a3 have lower MIC values (GM) for E.coli O157:H7 O157 4, 10. Mu.g/mL respectively), followed by BF-15a4, chensinin-1b, indolicidin, cLf-BF-15a3 (GM) O157 16, 25, 25, 32, 32 μg/ml in this order).
TABLE 1.12 sequence information of antibacterial peptides
TABLE 2 minimum inhibitory concentration (MIC, μg/mL) of 12 antibacterial peptides against E.coli
Note that: GM (GM) O157 Geometric mean of MIC of the antibacterial peptide for E.coli O157: H7. GM was calculated with 1024. Mu.g/mL when no antibacterial activity was detected at 512. Mu.g/mL O157 . The minimum hemolysis concentration (Minimum hemolytic concentration, MHC) refers to the lowest concentration of peptide that results in a 5% hemolysis rate of rabbit red blood cells. Selection Index (SI) is the MHC and GM of peptides O157 Is a ratio of (2). If not detected at 128. Mu.g/mL>A therapeutic index was calculated with 256. Mu.g/mL, with a hemolysis rate of 5%.
Example 2 synergistic antibacterial action of the antimicrobial peptide Thanatin and BF-15a3 composition
After the Minimum Inhibitory Concentrations (MIC) of the antimicrobial peptides thanatin and BF-15a3 were measured, the synergistic effect of the two antimicrobial peptides was detected by a checkerboard assay, which was performed on E.coli WX1002, JL1801 (containing the drug resistance gene blaNDM-5) and E.coli ATCC43895, as follows:
1) Diluting the test strain with MHB broth culture medium to obtain strain concentration of 1×10 6 CFU/mL of bacterial suspension.
2) The antimicrobial peptides Thanatin and BF-15a3 were diluted separately so that the concentrations of the respective antimicrobial peptides were 8 XMIC, 4 XMIC, 2 XMIC, 1/2 XMIC, 1/4 XMIC, and 1/8 XMIC in this order.
3) mu.L of the antimicrobial peptide Thanatin dilution, 25. Mu.L of the antimicrobial peptide BF-15a3 dilution and 50. Mu.L of the bacterial solution were each added to 96-well plates (8 XMIC dilution of the antimicrobial peptide A in row 1, 8 XMIC dilution of the antimicrobial peptide B in column 1, 4 XMIC dilution of the antimicrobial peptide A in row 2, 4 XMIC dilution of the antimicrobial peptide B in column 2, and so on) in a 7X 7 checkerboard pattern, and the final concentrations of each of the antimicrobial peptides were 2 XMIC, 1/2 XMIC, 1/4 XMIC, 1/8 XMIC, 1/16 XMIC, 1/32 XMIC. The antibacterial peptide alone was used as a control.
4) The 96-well plate is placed in a 37 ℃ incubator for incubation for 18 hours, and an enzyme label instrument reads OD600nm to judge MIC in combined bacteria inhibition. The corresponding fractional inhibitory concentration index (Fraction inhibition concentration index, FICI) was calculated as follows.
Fici= (MIC of antibacterial peptide a when used in combination/MIC of antibacterial peptide a when used alone) + (MIC of antibacterial peptide B when used in combination/MIC of antibacterial peptide B when used alone). FICI is less than or equal to 0.5 and is synergistic; FICI is less than or equal to 0.5 and less than or equal to 1, and the addition effect is achieved; FICI 1 is less than or equal to 4 and is irrelevant; FICI >4 is antagonistic.
The experimental results are shown in Table 3. The results show that for three gram-negative bacteria, the combined inhibitory concentration index (FICI) of the antimicrobial peptides Thanatin and BF-15a3 is lower than 0.5, so that Thanatin and BF-15a3 have good synergistic effects on gram-negative bacteria including drug-resistant bacteria.
TABLE 3 synergistic effects of the antibacterial peptides Thanatin and BF-15a3
Example 3 antimicrobial peptide composition kinetics of Sterilization of multidrug resistant bacteria
The test strain is escherichia coli O157:H27 ATCC43895 and WX1002, wherein the WX1002 drug resistance spectrum is amoxicillin/clavulanic acid-ceftriaxone-ceftiofur-cefoxitin-ampicillin-sulfaisoxazole-meropenem.
1) Regulating concentration of bacterial liquid of Escherichia coli ATCC43895 and WX1002 to 1×10 with MHB culture medium 6 CFU/mL。
2) The bacterial solution in the step 1) and the equivalent antibacterial peptide solution or the antibacterial peptide combination solution are mixed in a test tube and incubated at 37 ℃.
3) Taking out proper amount of the mixed solution at 0, 10min, 20min, 30min, 40min, 50min, 60min and 120min respectively, and performing plate coating counting after 10-time gradient dilution.
4) Sterilization curves were drawn with sterile water treatment as a negative control.
The experimental results are shown in FIG. 1. The result shows that the antibacterial peptide combination of 1 mug/mL Thanatin plus 2 mug/mL BF-15a3 can be rapidly sterilized for more than 99% in 2 hours, and the effect equivalent to that of high-concentration 4 mug/mL Thanatin or 8 mug/mL BF-15a3 can be achieved.
Example 4 hemolysis experiment of antibacterial peptide composition
The PBS buffer was pH7.4, 0.01M PBS buffer.
The preparation method of the 8% erythrocyte suspension is as follows: (1) 10mL of 40% rabbit red blood cell suspension was taken, centrifuged at 3000g for 5min, and after discarding the supernatant, the pellet was resuspended to 50mL by washing twice with PBS buffer, yielding 8% red blood cell suspension.
The concentration of the antimicrobial peptide thanatin is adjusted to 8ug/ml and 32ug/ml; the concentration of the antibacterial peptide BF-15a3 is adjusted to 16 mug/mL and 64 mug/mL; the concentration of the composition was 8. Mu.g/mL of thanatin+16. Mu.g/mL of BF-15a3+16. Mu.g/mL of Thanatin+64. Mu.g/mL of BF-15a3.
1) Taking a 96-well plate, adding 100ul of antibacterial peptide and antibacterial peptide composition with each concentration, respectively adding 100ul of 8% erythrocyte suspension,
2) The 96-well plate was incubated at 37℃for 1h, centrifuged at 3000g for 5min, and 100. Mu.L of supernatant was aspirated.
3) The OD570nm value of the supernatant obtained in step 2, i.e. the OD570nm value of the antimicrobial peptide-treated group, was measured.
4) Step 2 was replaced by step a, and the other steps were unchanged, giving a negative control OD570nm value. The step a is as follows: the 96-well plate was taken and 100 μl PBS buffer was added to each well.
5) And (3) replacing the step 2 with the step b, wherein other steps are unchanged, and obtaining the positive control OD570nm value. The step b is as follows: the 96-well plate was taken and 100. Mu.L Triton X-100 was added to each well.
The antimicrobial peptides exert antimicrobial activity through non-receptor mediated membrane permeation mechanisms, which may reduce the biocompatibility of the antimicrobial peptides with normal cells. In view of the limited use of antimicrobial peptides in the food field due to their possible haemolytic and cytotoxic properties, this example evaluates the haemolytic activity of antimicrobial peptides and antimicrobial peptide compositions on rabbit red blood cells. The results show (FIG. 2) that the hemolytic activity of thanatin and BF15-a3 was negligible at the concentrations tested. Furthermore, there was no significant difference in OD570nm (p > 0.05) between thanatin or BF-15a3 alone and in combination, meaning that their combination did not lead to synergistic hemolysis, which clearly facilitates the synergistic application of the two antimicrobial peptides.
Example 5 cytotoxicity assay of antibacterial peptide compositions
Cytotoxicity was determined using human embryonic kidney cells (HEK-293T) and porcine small intestine cells (IPEC-J2). The experiments were performed according to the methods reported previously, with appropriate modifications. Briefly, HEK-293T cells (5X 10) 4 cells/mL) and IPEC-J2 cells (5X 10) 4 cells/mL) were incubated with DMEM medium containing 10% (v/v) fetal bovine serum (Fetal bovine serum, FBS) and DMEM/F12 medium containing 10% (v/v) FBS, respectively, at 37deg.C, 5% CO 2 Culturing in 96-well culture plate under humidified environment for 24 hr (cell growth over 90%). The cells were washed twice with the corresponding serum-free medium and then treated with a concentration of peptide (diluted with serum-free medium). After 18h incubation with the antimicrobial peptide, 10 μl of CCK-8 reagent was added to each well and incubated in a cell incubator for 2h, and absorbance at 450nm was measured. Cell viability was calculated according to the CCK-8 instructions. The formula is as follows:
OD sample : cell + peptide + CCK-8
OD negative : cell +CCK-8
OD blank : medium+CCK-8
As shown in FIG. 3, thanatin and BF-15a3 were less toxic to HEK-293T at 4 XMIC, with cell viability of 95.1% and 97.2%, respectively. Their toxicity to IPEC-J2 was similar to HEK-293T, with cell viability at 4 XMIC of 93.2% and 96.9%, respectively. Importantly, the combined use of thanatin and BF-15a3 did not result in an increase in cytotoxicity, and there was no significant difference (p > 0.05) in cytotoxicity from the peptide alone (4×mic thanatin or BF-15a 3).
Example 6 antibacterial peptide induced resistance detection
The overnight E.coli O157: H7WX1002 was added to the MHB medium containing 0.5 XMIC antimicrobial peptide at a ratio of 1:100, and after shaking culture at 37℃and 200rpm for 12 hours, passaging was continued in this step, and the MIC was determined after each passage. The concentration of the antibacterial peptide is gradually increased along with the increase of MIC of the indicator bacteria, so that the concentration of the antibacterial peptide is always 0.5 xMIC, and the continuous passage is carried out for 20 times.
To evaluate the effect of the antimicrobial peptide on the development of bacterial resistance, we continued subculture of E.coli O157:H2 1002 with the antimicrobial peptide at sub-inhibitory concentrations, as shown in FIG. 4, with an increase of at most 2-fold in MIC of thanatin and BF-15a3 used alone or in combination in consecutive 20-generation cultures, while the MIC of control meropenem continued to increase by 8-fold.
Example 7 stability of antibacterial peptide composition
7.1 thermal stability
According to the previous report, the heat resistance of the antibacterial peptide was determined by measuring its MIC for E.coli O157:H7WX1002 after incubation at 100℃for various times. Briefly, peptides were dissolved in sterile deionized water and incubated at 100℃for 0, 10, 30 and 60min. Thereafter, their MIC was detected. The results show that: after 1h incubation at 100 ℃, the MIC values of thanatin and BF-15a3 remained unchanged, maintaining their original antimicrobial activity (fig. 5), indicating that they have excellent residual antimicrobial activity after heat treatment of the food.
7.2 stability in different pH environments
To test the resistance of the antimicrobial peptides to different pH values, experiments were performed using previously reported methods, with some modifications [153] . ddH with different pH values (2, 4, 6, 8 and 10, adjusted with 1M HCl and 1M NaOH) 2 The peptide was diluted with O and incubated at 37℃for 2H, then incubated with an equal volume of E.coli O157:H7WX1002 solution (5X 10) 5 CFU/mL) was mixed and incubated at 37 ℃ for 2h. A gradient dilution of the mixture was spread on LB plates and incubated at 37℃for 18h. Colony forming units were counted and the inhibition of the peptide (final concentration 2×mic) was calculated.
As shown in FIG. 6, the antimicrobial activity of thanatin and BF-15a3 was hardly affected when the pH value was between 4 and 10. The antibacterial peptide can meet the sterilization requirement of most of foods under the pH condition.
Example 8 sterilizing Effect of antibacterial peptide combination on fresh-cut vegetables
Preparation of bacterial cultures: inoculating single colony of Escherichia coli O157H 7WX1002 into LB culture medium, and culturing at 37deg.C for about 6 hr to OD 600nm With a value of 1, bacterial cultures were incubated at ddH 2 Diluting to 1×10 in O 6 CFU/mL for inoculation.
Inoculating escherichia coli O157 to H7: fresh lettuce and cucumber are purchased from local markets in south-Beijing, china and washed with deionized water to remove dust and soil particles.
Lettuce leaves and cucumber epidermis were cut into small pieces (2 cm. Times.2 cm) and irradiated with ultraviolet light for 30 minutes. Each sample was inoculated with 20 μl of the prepared bacterial suspension and allowed to stand at room temperature for 30 minutes to allow bacteria to attach. Next, the sample surface was coated with 40. Mu.L of the antibacterial peptide solution and stored at 4℃and 25℃respectively. With ddH 2 O and 100mg/L NaClO were used as negative and positive controls to compare, respectively, instead of the antimicrobial peptide.
Colony count was determined: after storage for 0, 1, 12, 24 and 48 hours, the samples were placed in 10ml of 0.2% tween-80 PBS solution and rolled slowly up and down 30 times. Dilutions (100 μl) were plated onto LB plates and incubated overnight at 37 ℃. CFU is then counted.
As shown in FIG. 7A, treatment with thanatin, BF-15a3 and combinations thereof inhibited bacterial growth during storage compared to the control group. The combination of thanatin and BF-15a3 reduced the E.coli O157: H7 number more rapidly than the treatment with the two antimicrobial peptides alone. After 2d of storage, the number of colonies after the combined treatment with 0.5×MIC (thanatin+BF-15 a 3) was significantly lower (p < 0.05) than the number of colonies treated with a single antimicrobial peptide (1×MIC thanatin, BF-15a 3). In addition to the NaClO treatment group, 2 XMIC (thanatin+BF-15 a 3) treatment was most effective against E.coli O157:H27, and after 12 hours the colony count of E.coli O157:H27 was reduced from 4.5log CFU/piece to below the detection limit of the plate count method.
The antibacterial peptide was similar to lettuce in terms of sterilization on cucumber surfaces, and the colony count was significantly lower in the 0.5,1,2×MIC (thanatin+BF-15 a 3) treated group than in the control group (p < 0.05) after 2d storage at 4℃as shown in FIG. 7B. The results show that the combination of the thanatin and the BF-15a3 not only can inactivate the escherichia coli O157:H7 in vitro, but also can effectively kill the escherichia coli O157:H7 on the surfaces of lettuce and cucumbers during storage, and has better sterilization effect than single use.
The result shows that the antibacterial peptide combination Thanatin+BF-15a3 can quickly kill drug-resistant escherichia coli on the surfaces of lettuce and cucumbers, and the effect similar to 100mg/L NaClO can be achieved when the antibacterial peptide combination Thanatin+16 mu g/mL BF-15a3 is 8 mu g/mL.
The above-described embodiments are only preferred embodiments of the invention, and not all embodiments of the invention are possible. Any obvious modifications thereof, which would be apparent to those skilled in the art without departing from the principles and spirit of the present invention, should be considered to be included within the scope of the appended claims.

Claims (9)

1. An antimicrobial peptide composition characterized by: the antibacterial peptide is composed of two antibacterial peptides, namely Thanatin and BF-15a3; the sequence of the antimicrobial peptide Thanatin is shown in SEQ ID NO. 1; the sequence of the antibacterial peptide BF-15a3 is shown as SEQ ID NO. 2.
2. The antimicrobial peptide composition of claim 1, wherein: the mass ratio of the Thanatin to the BF-15a3 in the composition is 1:1.5-3, preferably 1:1.8-2, and more preferably 1:2.
3. Use of an antimicrobial peptide composition according to claim 1 or 2 for the preparation of a product, characterized in that said product is selected from any one of the following:
1) An antibacterial agent;
2) A medicament for preventing and/or treating a disease caused by a bacterial infection;
3) Food preservative.
4. Use according to claim 3, characterized in that the bacteria are gram-negative bacteria.
5. Use according to claim 3, characterized in that the bacteria are escherichia coli, preferably carbapenem-resistant or carbapenem-intolerant escherichia coli.
6. An antibacterial agent comprising the antibacterial peptide composition according to claim 1 or 2.
7. The antibacterial agent according to claim 6, wherein the agent is a solution in which the concentration of Thanatin is 0.4-2.4 μg/mL.
8. The antibacterial agent according to claim 6, wherein the concentration of Thanatin in the agent is 1 μg/mL and the concentration of BF-15a3 is 2 μg/mL.
9. Use of the antibacterial peptide composition according to claim 1 or 2, the antibacterial agent according to any one of claims 6 to 8 for preserving foods.
CN202210868364.6A 2022-07-22 2022-07-22 Antibacterial peptide composition for inhibiting and killing gram-negative bacteria and application thereof Pending CN117430673A (en)

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