CN117164669A - Litopenaeus vannamei extension factor antibacterial peptide BCE3 and application thereof - Google Patents
Litopenaeus vannamei extension factor antibacterial peptide BCE3 and application thereof Download PDFInfo
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Abstract
The invention discloses a litopenaeus vannamei extension factor antibacterial peptide BCE3, the amino acid sequence of which is GSFRYAWVLDKLK, and the molecular weight of which is 1582.9 daltons. The antibacterial peptide BCE3 can have a strong inhibition effect on bacillus cereus, can be used for preparing medicines and feed additives for treating or preventing diseases caused by bacillus cereus, and can also be used for preparing food preservatives.
Description
Technical Field
The invention relates to the technical field of biology, in particular to a litopenaeus vannamei extension factor antibacterial peptide BCE3 and application thereof.
Background
Bacillus cereus @Bacillus cereus) Is a pathogenic microorganism which can cause food-borne diseases, and can be widely distributed in the environment and food because of forming spores which can resist high temperature and low humidity. Bacillus cereus is capable of producing both types of toxins that cause vomiting and diarrhea. Among them, food poisoning associated with starch-based foods such as rice and grain is usually caused by vomit-type toxins. This is because these foods are rich in carbohydrates, which provides the most desirable environment for the production of vomitoxin. In addition, improper storage of cooked rice, cereal-based foods after cooking can also cause bacillus cereus contamination, which leads to frequent occurrence of bacillus cereus food poisoning worldwide.
At present, aiming at the situation that the food-borne pathogenic bacteria are increasingly polluted, the addition of chemical preservatives, such as potassium sorbate, benzoic acid and the like, is the most effective method. However, because of the safety of chemical preservatives, consumers often exhibit untrustworthy chemical additives because they are considered carcinogenic, teratogenic, and residual toxicity. The search for chemical drug alternatives is therefore also a major hotspot in current research. Antibacterial peptides (AMPs) are a class of naturally occurring, small molecule bioactive peptides with antibacterial or fungal action, also known as host defenses, produced by organisms such as bacteria, plants, vertebrates and invertebrates. The compound has the advantages of rapid sterilization, low drug resistance risk and small side effect, is considered as a potential substitute of chemical preservative, and has good application prospect.
In recent years, shrimp have become an important component of crustacean consumption. However, during processing of shrimp, the utilization of shrimp meat is mainly focused, while the heads, shells and tails of shrimp are generally considered as processing byproducts, which contain a large amount of underutilized quality protein. Bacillus subtilis is a common gram-positive bacterium, and proteases produced during its growth are capable of degrading proteins in shrimp processing byproducts to produce active peptides. According to research reports, some active peptides show antibacterial activity. Thus, shrimp processing byproducts can be converted to valuable products enriched in active peptides by appropriate techniques and bioconversion processes.
Therefore, the research discovers a novel antibacterial peptide BCE3 through fermenting shrimp heads by bacillus subtilis, shows strong antibacterial activity on bacillus cereus, further discusses the antibacterial mechanism of the bacillus cereus, and provides a theoretical basis for preventing and treating food-borne pathogenic microorganisms.
Disclosure of Invention
The invention aims to overcome the defects of the prior art, and provides a litopenaeus vannamei extension factor antibacterial peptide BCE3 and application thereof, which solve the problems in the prior art.
One of the technical schemes adopted for solving the technical problems is as follows: provides a litopenaeus vannamei extension factor antibacterial peptide BCE3. The amino acid sequence is GSFRYAWVLDKLK, such as SEQ ID NO:1.
The molecular weight of the antibacterial peptide is 1582.9 daltons, the positive charge is +2, and the total hydrophobicity ratio is 46%.
The second technical scheme adopted by the invention for solving the technical problems is as follows: provides an application of Litopenaeus vannamei extension factor antibacterial peptide BCE3 in preparing antibacterial drugs for inhibiting and/or killing bacillus cereus.
The third technical scheme adopted by the invention for solving the technical problems is as follows: an antibacterial drug is provided, the active ingredients of which comprise Litopenaeus vannamei extension factor antibacterial peptide BCE3, the amino acid sequence of the antibacterial peptide BCE3 is SEQ ID NO:1.
in a preferred embodiment of the present invention, the active ingredient of the antibacterial agent is the antibacterial peptide BCE3 of the elongation factor of litopenaeus vannamei, and the amino acid sequence of the antibacterial peptide BCE3 is SEQ ID NO:1.
in a preferred embodiment of the present invention, the antimicrobial agent is used to inhibit and/or kill bacillus cereus.
The fourth technical scheme adopted for solving the technical problems is as follows: the effective components of the feed additive comprise litopenaeus vannamei extension factor antibacterial peptide BCE3, wherein the amino acid sequence of the antibacterial peptide BCE3 is SEQ ID NO:1.
in a preferred embodiment of the present invention, the effective component of the feed additive is a litopenaeus vannamei elongation factor antibacterial peptide BCE3, and the amino acid sequence of the antibacterial peptide BCE3 is SEQ ID NO:1.
in a preferred embodiment of the invention, the feed additive is used for inhibiting and/or killing bacillus cereus.
The fifth technical scheme adopted by the invention for solving the technical problems is as follows: the food preservative comprises the following active ingredients of Litopenaeus vannamei elongation factor antibacterial peptide BCE3, wherein the amino acid sequence of the antibacterial peptide BCE3 is SEQ ID NO:1.
in a preferred embodiment of the invention, the food preservative is used to inhibit and/or kill bacillus cereus.
According to the invention, the antibacterial activity of the antibacterial peptide BCE3 on bacillus cereus is researched through MBC and Time-kill; and observing the influence of the antibacterial peptide BCE3 on the bacterial cell wall by detecting the change of the alkaline phosphatase activity, and observing the damage degree of the antibacterial peptide BCE3 on the bacterial cell membrane by propidium iodide staining; meanwhile, genome DNA of bacillus cereus is extracted respectively, and the influence of the genome DNA on bacterial DNA is verified. Experimental results show that the peptide has obvious inhibition effect on bacillus cereus. The antibacterial peptide disclosed by the invention is a cationic antibacterial peptide, and can be used for inhibiting the synthesis of bacterial DNA by destroying the bacterial cell wall, changing the permeability of a cell membrane, penetrating the cell membrane and combining with the bacterial genomic DNA, so that the antibacterial effect is exerted.
The antibacterial peptide BCE3 will destroy bacteria from the following actions: the antibacterial peptide BCE3 can damage the bacterial cell wall, change the permeability of bacterial cell membrane, penetrate the cell membrane, combine with bacterial genome DNA and inhibit the synthesis of bacterial DNA, thereby leading to bacterial death.
The antimicrobial peptides of the invention can be synthesized using methods known to those skilled in the art, such as solid phase synthesis, and purified using methods known to those skilled in the art, such as high performance liquid chromatography.
The implementation of the invention has the following beneficial effects:
according to the invention, the litopenaeus vannamei extension factor is taken as a research object, and an antibacterial peptide BCE3 with a brand new amino acid sequence is discovered through screening. Antibacterial activity of antibacterial peptide BCE3 on bacillus cereus was studied. Experimental results show that the peptide has a strong inhibition effect on bacillus cereus, and the antibacterial mechanism is that bacterial cell walls are firstly destroyed, the permeability of cell membranes is changed, and then the peptide is combined with bacterial genome DNA to inhibit the synthesis of bacterial DNA, so that bacterial death is caused. The antibacterial peptide BCE3 can be used for preparing medicines, feed additives or food preservatives for preventing or inhibiting bacillus cereus infection.
Drawings
FIG. 1 is a view showing the structure of a predictive model of the antibacterial peptide BCE3 of the present invention.
FIG. 2 is a graph showing the measurement of MBC at the minimum bactericidal concentration of the antibacterial peptide BCE3 of the present invention against Bacillus cereus.
Wherein, the A-F antibacterial peptide BCE3 is respectively as follows: 0. mu.g/mL, 500. Mu.g/mL, 250. Mu.g/mL, 125. Mu.g/mL, 62.5. Mu.g/mL, 31.3. Mu.g/mL.
FIG. 3 is a graph showing time-killing of the antibacterial peptide BCE3 of the present invention against Bacillus cereus, wherein 1/2 XMBC, 1 XMBC, 2 XMBC are 62.5. Mu.g/mL, 125. Mu.g/mL, 250. Mu.g/mL, respectively.
FIG. 4 is a graph showing the effect of the antibacterial peptide BCE3 of the present invention on the cell wall of Bacillus cereus (alkaline phosphatase leakage).
FIG. 5 is a graph showing the effect of the antibacterial peptide BCE3 on the cell membrane permeability of Bacillus cereus by propidium iodide staining.
FIG. 6 is a graph showing the effect of the antibacterial peptide BCE3 of the present invention on cell membrane permeability of Bacillus cereus (nucleic acid leakage).
FIG. 7 is a graph showing the effect of the antibacterial peptide BCE3 of the present invention on cell membrane permeability of Bacillus cereus (protein leakage).
FIG. 8 is a graph showing the effect of the antibacterial peptide BCE3 on the cell membrane permeability of Bacillus cereus by fluorescence microscopy. Wherein a is the bright field diagram of the control group, b is the fluorescent diagram of the PI channel of the control group, c is the combined diagram of a and b, d is the bright field diagram of the antibacterial peptide BCE3 treatment, e is the fluorescent diagram of the PI channel of the antibacterial peptide BCE3 treatment, and f is the combined diagram of d and e.
FIG. 9 is a graph showing the effect of the antibacterial peptide BCE3 on apoptosis of Bacillus cereus by flow cytometry analysis.
FIG. 10 is a diagram showing the electrophoresis of the combination of the antibacterial peptide BCE3 of the present invention with the genomic DNA of Bacillus cereus.
Wherein, strip 7: blank control;
band 6-1: the mass ratio of the antibacterial peptide BCE3 to the DNA is 10/10, 10/8, 10/6, 10/4, 10/2 and 10/1 respectively.
FIG. 11 is a fluorescence spectrum of the antibacterial peptide BCE3 of the present invention competitively binding to EB genomic DNA of Bacillus cereus.
FIG. 12 is a graph showing the concentration of the antibacterial peptide BCE3 of the present invention in Bacillus cereus during rice storage. Wherein 1 XMBC and 2 XMBC are concentrations of the added antibacterial peptide BCE3 of 125 mug/mL and 250 mug/mL respectively, and the blank control group is to replace the antibacterial peptide BCE3 with the same amount of sterile PBS.
Detailed Description
For a better understanding of the present invention, reference will now be made in detail to the following examples and accompanying drawings, which are included to provide a further understanding of the invention, and it is to be understood by those skilled in the art that the following examples are not intended to limit the scope of the invention.
The experimental methods used in the following examples are conventional methods unless otherwise specified.
Materials, reagents and the like used in the examples described below are commercially available unless otherwise specified.
Example 1: screening of Litopenaeus vannamei elongation factor antibacterial peptide BCE3
Firstly, fermenting litopenaeus vannamei by using bacillus subtilis, homogenizing the shrimp heads, and preparing samples. The bacillus subtilis is used as a starter, and fresh penaeus vannamei boone heads are homogenized at 37 ℃ and subjected to liquid fermentation at 160 r/min. After fermentation, the supernatant was centrifuged at 3000 r/min for 5 min and filtered through a 3000 Da filter. Stored at-20℃for further analysis.
Chromatographic conditions: sample injection amount: 5.0 Mu L (mu L)
Chromatographic column: c18 analytical chromatographic column, length 25 cm, inner diameter 75 μm.
Mobile phase: a:0.1% aqueous methanol solution B: acetonitrile
In combination with the search software: maxquantv1.6.5.0, database: the uniprot shramp shrimp protein library is used for distinguishing and identifying the obtained peptide fragment mass spectrogram, then an APD3 online server is used for screening the sequence of the peptide fragment, a Swiss-model server is used for predicting the peptide fragment structure, and finally antibacterial peptide with strong antibacterial effect on bacillus cereus is obtained through screening, the sequence GSFRYAWVLDKLK is named BCE3, the molecular weight is 1582.9 Da, the chemical synthesis (synthesized by Beijing midbody matt biotechnology Co., ltd.) is performed, and antibacterial activity verification is performed.
Example 2: 3D structure prediction of antibacterial peptide BCE3
The structure of the antibacterial peptide BCE3 was predicted using an online structure prediction server Swiss-model, and edited and modified using Pymol software to obtain the secondary structure of the antibacterial peptide BCE3, as shown in FIG. 1.
Example 3: minimum Bactericidal Concentration (MBC) assay for the antibacterial peptide BCE3
Culturing Bacillus cereus at 37deg.C to logarithmic phase, and diluting to 10 in 0.01M pH 7.2 phosphate buffer 5 CFU/mL. Antibacterial peptide BCE3 was dissolved in phosphate buffer, mixed with bacteria at equal volumes at 37 ℃ and incubated 2 h. The Minimum Bactericidal Concentration (MBC) refers to the concentration of antimicrobial peptides that are capable of killing bacteria after incubation at 37 ℃. As shown in FIG. 2, the Minimum Bactericidal Concentration (MBC) of the antibacterial peptide BCE3 against Bacillus cereus was 125. Mu.g/mL.
Example 4: time-kill curve (Time-kill) determination of the antibacterial peptide BCE3
The time-sterilization profile of the antimicrobial peptide BCE3 was determined using plate colony counting. Culturing Bacillus cereus at 37deg.C to logarithmic phase, and diluting to 10 in 0.01M pH 7.2 phosphate buffer 5 CFU/mL. Peptides with concentrations of 1/2 xMBC, 1 xMBC and 2 xMBC were mixed with the same volume of the bacterial liquid, and incubated in a biochemical incubator at a constant temperature of 37 ℃. At various time points (i.e., 0.5, 1, 1.5, 2, 2.5, and 3 h), 0.02 mL bacterial suspension was obtained and after 24 h incubation on nutrient broth plates at 37℃the bacteria were countedAnd (5) falling. The results are shown in FIG. 3. 1 XMBC of the antibacterial peptide BCE3 can kill Bacillus cereus within 3 h.
Example 5: effect of the antibacterial peptide BCE3 on bacterial cell walls
Culturing Bacillus cereus at 37deg.C to logarithmic phase, collecting a certain amount of bacterial liquid, washing the collected bacterial liquid with 0.01M phosphate buffer solution with pH of 7.2 for three times, and adjusting bacterial liquid concentration to 10 8 CFU/mL. Mixing with antibacterial peptide BCE3 in equal volume until final concentration is 1/2 xMBC, 1 xMBC, and 2 xMBC respectively, mixing phosphate buffer solution and bacterial suspension with equal amount as blank control, culturing in biochemical incubator at 37deg.C constant temperature for 2 h, taking out bacterial suspension, centrifuging at 3500 rpm for 10 min, and obtaining supernatant. According to the alkaline phosphatase AKP kit, at OD 510 Under the condition, the activity of AKP enzyme in each supernatant is measured in a microplate reader by using a 96-well plate, and finally the influence of the polypeptide on the bacterial cell wall is reflected according to the change of the activity of AKP enzyme. The results are shown in FIG. 4. Alkaline phosphatase (AKP enzyme) is an intracellular enzyme located between the cell wall and the cell membrane and leaks out of the bacteria into the extracellular environment when the cell wall is damaged. Therefore, the damage condition of the antibacterial peptide to the cell wall of the thallus can be judged by detecting the activity of AKP enzyme. The AKP enzyme activity of the bacillus cereus treated by the antibacterial peptide BCE3 is increased, and the AKP enzyme activity is increased in a dose-dependent manner.
Example 6: effect of the antibacterial peptide BCE3 on bacterial cell membrane permeability
Propidium Iodide (PI) staining analysis of the effect of the antibacterial peptide BCE3 on bacterial cell membrane permeability. Bacillus cereus was inoculated into LB broth, cultured at 37℃and 200 r/min to grow to logarithmic phase. Centrifuging a certain amount of bacterial liquid in logarithmic phase, removing supernatant, cleaning the collected bacterial cells with 0.01M pH 7.2 phosphate buffer solution for three times, and regulating bacterial liquid concentration to 10 5-6 CFU/mL. Peptides were dissolved in phosphate buffer to a concentration of 1/2 XMBC, 1 XMBC, 2 XMBC, and the blank was replaced with phosphate buffer and incubated with bacteria at 37℃in equal volumes for 2 h. After the incubation, 100. Mu.L of the supernatant was removed by centrifugation, 100. Mu.L of PI dye (30. Mu.M) was added, and the mixture was vortexed and mixed at 25℃at 4℃Incubate in the dark for 15 min. After staining was completed, washed 2 times with 0.1M PBS and finally resuspended to 100 μl. The fluorescence spectrum of the sample at excitation wavelength 535 nm and emission wavelength 570-750 nm was measured using a multifunctional microplate reader. PI is a nucleic acid dye that has the strongest fluorescence absorbance at excitation wavelength 535 nm and emission wavelength 615 nm. It cannot cross normal cell membranes, but PI can enter the cell membrane to bind DNA and display red fluorescence when the cell membrane is damaged or ruptured. The degree of damage to the cells can be determined based on the magnitude of the fluorescence intensity. The binding of PI to bacterial genomic DNA treated with different concentrations of antimicrobial peptide is shown in fig. 5. The fluorescence intensity of bacillus cereus treated by the antibacterial peptide BCE3 with different concentrations is higher than that of a blank control group, which indicates that the antibacterial peptide BCE3 is used for damaging bacterial cell membranes and increasing the permeability of the cell membranes. The fluorescence intensity of the 2 XMBC antimicrobial peptide BCE3 treated samples was greater than that of the 1/2 XMBC antimicrobial peptide treated samples, indicating that high doses of the antimicrobial peptide BCE3 increased the permeability of the bacterial cell membrane resulting in more PI entering the intracellular binding DNA to fluoresce.
Example 7: effect of antibacterial peptide BCE3 on bacterial cell membrane permeability (nucleic acid, protein leakage)
The integrity of the bacterial cell membrane is judged by measuring the leakage of proteins within the bacterial cell. Bacillus cereus was inoculated into LB broth, cultured at 37℃and 200 r/min to the log phase of growth. Centrifuging a certain amount of bacterial liquid in logarithmic phase, removing supernatant, cleaning the collected bacterial cells with 0.01M pH 7.2 phosphate buffer solution for three times, and adjusting bacterial liquid concentration to 10 8 CFU/mL. The peptides were dissolved in phosphate buffer to a final concentration of 1/2 XMBC, 1 XMBC, 2 XMBC, the blank was replaced with phosphate buffer, the equivalent volume was mixed with bacteria, and the positive control was replaced with nisin (900 IU/mg; microphone, shanghai, china). Mixing, incubating at 37deg.C for a certain time, sampling at 0 min, 60 min, 120 min, and 180 min, centrifuging at 10000 r/min for 2 min, collecting supernatant, measuring optical density at 260 nm and 280 nm, and respectively detecting leakage of intracellular acids and proteins. The results are shown in FIGS. 6-7. Nucleic acids and proteins in culture under normal conditionsSubstantially stabilized at a lower level. And after the antibacterial peptide BCE3 is treated for 180 min, the leakage amount of nucleic acid and protein in the bacterial liquid is increased, and after the antibacterial peptide BCE3 is treated by 1 XMBC (125 mug/mL), the leakage amount of nucleic acid protein in the bacterial liquid is equivalent to that of nisin treated by 500 mug/mL.
Example 8: analysis of the Effect of the antibacterial peptide BCE3 on cell permeability by fluorescence microscopy
Taking a certain amount of bacterial liquid cultured to logarithmic phase, centrifuging, discarding supernatant, cleaning the collected bacterial cells with sterile 0.01M pH 7.2 phosphate buffer solution for three times, and regulating bacterial liquid concentration to 1×10 6 CFU/mL. Antibacterial peptide BCE3 was added to the bacterial solution to a final concentration of 1×mbc and incubated at 37 ℃ for 2 h. The blank was replaced with an equal amount of PBS. After the incubation, 100. Mu.L of the mixture was added with an equal amount of PI dye, mixed by vortexing at 25℃and incubated at 4℃for 15 min in the absence of light. After staining was completed, washed 2 times with sterile PBS and finally resuspended to 100 μl. After panning and using a fluorescence microscope, PI channels were selected to capture images. As shown in fig. 8, in the PBS-treated control group, most cells did not fluoresce red, and only a small amount of weak red fluorescence, presumably caused by normal death of bacterial cells. The cell membrane of bacillus cereus treated by the antibacterial peptide BCE3 with the concentration of 1 XMBC is damaged, and most cells emit red fluorescence after being stained by PI. This result shows that 1 XMBC concentration of the antibacterial peptide BCE3 has a destructive effect on the integrity of bacterial cell membranes, which is a proof of the numerical results of PI fluorescence intensity.
Example 9: effect of the antibacterial peptide BCE3 on apoptosis
The effect of the antibacterial peptide BCE3 on apoptosis of bacillus cereus was analyzed by flow cytometry. The bacterial liquid activated to logarithmic phase is regulated to 1X 10 concentration 5-6 CFU/mL. 1 h, 2 h and 3 h were treated with 1 XMBC, 2 XMBC antimicrobial peptide BCE3 at 37℃respectively, mixed with equal amounts of sterile 0.01M PBS (pH 7.2) and bacterial suspension as negative controls, and mixed with equal amounts of nisin and bacterial suspension as positive controls. After incubation, treatment was performed according to Annexin V-FITC/PI apoptosis kit instructions and analyzed using a flow cytometer equipped with CytExpert software. The results are shown in FIG. 9. Wherein the cells of the LL region are normal living cells. After 2 XMBC of the antibacterial peptide BCE3 acted on Bacillus cereus 3 h, the viable cell rate was 6.99%, which is lower than that of the positive control group (67.04%). When the bacillus cereus is treated by the antibacterial peptide BCE3 with different concentrations, the normal living cell rate of bacteria is also reduced along with the increase of the concentration of the antibacterial peptide and the extension of the action time, and the living cell rate after the treatment of the antibacterial peptide BCE3 is lower than that of a positive control. These results all show that the antibacterial peptide BCE3 has a strong antibacterial effect on bacillus cereus, and the antibacterial effect has time-concentration dependence and is superior to nisin.
Example 10: interaction of the antibacterial peptide BCE3 with bacterial DNA
The interaction of the antibacterial peptide BCE3 with the genomic DNA of Bacillus cereus was studied using a DNA gel blocking method. Bacteria were cultured in 50 mL Nutrient Broth (NB) at 37 ℃ for 12 h and bacterial genomic DNA was extracted using a bacterial genomic DNA extraction kit. The optical density ratio at 260 nm and 280 nm (OD 1.70. Ltoreq.OD 260 /OD 280 Less than or equal to 1.90) evaluating the purity of the extracted genomic DNA. Next, bacillus cereus DNA (161 ng/. Mu.L) was mixed with a continuous amount of the antibacterial peptide BCE3 at 25℃for 90 min, and the mixture was subjected to electrophoresis on a 1.5% agarose gel. Gel blocking was observed under ultraviolet radiation using a gel imaging system (QuickGel 6200, monad, su zhou, china) as shown in fig. 10. Agarose gel blocking experiments the binding of the antimicrobial peptides to the bacterial DNA can be analyzed by analyzing the electrophoretic mobility of the samples. If the antibacterial peptide BCE3 binds to the DNA of Bacillus cereus, the mobility of the antibacterial peptide BCE3 in electrophoresis is affected, namely, the antibacterial peptide BCE has a certain hysteresis compared with the migration distance of the DNA alone. In band 6-1, blocking occurred at a mass ratio of antibacterial peptide BCE3 to DNA of 10/10, and as the concentration of antibacterial peptide increased, it was observed that all DNA was blocked near the spotting wells. At the same time, the band of DNA is darkened significantly, probably because the antibacterial peptide BCE3 binds to DNA, competing for the binding site of the nucleic acid dye to DNA, darkening the band of DNA.
Example 11: fluorescent spectroscopic experiment of competitive binding of antibacterial peptide BCE3 and EB to DNA
Fluorescent spectroscopic experiments of competitive binding of the antibacterial peptide BCE3 to Ethidium Bromide (EB) to DNA the mode of action of the antibacterial peptide BCE3 with bacterial genomic DNA: bacillus cereus genomic DNA was diluted to 50. Mu.g/mL with TE buffer. The reaction was performed in 96-well plates, first 5. Mu.L of DNA solution and 10. Mu.L of EB solution with a concentration of 100. Mu.g/mL were added to each well, mixed well, and incubated in a biochemical incubator at 37℃for 10 min in the absence of light. Then 50. Mu.L of peptide solution with different concentrations is added, the blank group is replaced by 50. Mu.L of sterile distilled water, and the mixture is placed in a biochemical incubator for incubation for 30 min at 37 ℃ in a dark place. After the incubation, the fluorescence spectrum of the sample at excitation wavelength 535 nm and emission wavelength 550-750 nm is measured with a multifunctional microplate reader. In aqueous solution, EB fluorescence is weak, but its fluorescence intensity is greatly enhanced when it is bound to double-stranded DNA by way of intercalation with higher affinity. If the EB-DNA complex system is in competition with the EB bound to the DNA by the presence of a substance that acts similarly to the DNA, the fluorescence intensity of the system will decrease, indicating that the competition binds to the DNA in the same intercalating mode as the EB. Therefore, by measuring the change in fluorescence spectrum of the action of the DNA-EB complex system and the competitor, it was determined whether the competitor also binds to DNA by intercalation as in EB. As is clear from FIG. 11, the fluorescence intensity of the EB-DNA complex decreased with increasing concentration of the antibacterial peptide BCE3, which indicates that the antibacterial peptide BCE3 was insert-bonded to the Bacillus cereus DNA, and that EB previously bonded to the DNA base pair was competed for the substitution of EB to be insert-bonded to the DNA, thereby decreasing the fluorescence intensity of the whole system.
Example 12: application of antibacterial peptide BCE3 in rice
Bacillus cereus was cultured in LB medium at 37℃to logarithmic phase and washed three times with PBS and resuspended to 1X 10 3-4 CFU/mL, adding 1 mL bacteria solution into sterilized cooked rice for artificial contamination, adding equal amount of antibacterial peptide BCE3 (1 XMBC, 2 XMBC) for treating sample, and placing the blank control group with equal amount of sterile PBS instead of antibacterial peptide BCE3 at 25deg.C, sampling at 2 hr, 4 hr, 6 hr, 8 hr, and collecting sample according to national standard GB4789.2-2022, by a method of determining the total number of food microbiologically examined colonies. As a result, as shown in FIG. 12, the initial bacterial concentration was 2.5 log CFU/mL, the number of Bacillus cereus in the blank group was increased to 4.8 log CFU/mL during the experiment, and after the antibacterial peptide BCE3 treatment at 1 XMBC and 2 XMBC, the number of Bacillus cereus was reduced to 2.0 log CFU/mL and 1.1 log CFU/mL, respectively, indicating that the antibacterial peptide BCE3 can effectively inhibit the growth of Bacillus cereus in rice.
In conclusion, the invention provides a brand new antibacterial peptide BCE3 with a Minimum Bactericidal Concentration (MBC) of 125 mug/mL for bacillus cereus, which shows that the antibacterial peptide BCE3 has a stronger inhibition effect on the bacillus cereus. The antibacterial peptide BCE3 disclosed by the invention firstly breaks the cell wall of bacteria, penetrates the cell membrane of the bacteria, changes the permeability of the cell membrane, and then is combined with bacterial genome DNA to inhibit the synthesis of the bacterial DNA, so that the bacteria die.
While specific embodiments of the invention have been described above, it will be appreciated by those skilled in the art that the specific embodiments described are illustrative only and not intended to limit the scope of the invention, and that equivalent modifications and variations of the invention in light of the spirit of the invention will be covered by the claims of the present invention.
Claims (10)
1. An antibacterial peptide BCE3 of the litopenaeus vannamei extension factor has an amino acid sequence shown in SEQ ID NO:1.
2. The use of the litopenaeus vannamei elongation factor antibacterial peptide BCE3 as claimed in claim 1 for the preparation of antibacterial drugs, characterized in that: the antibacterial drug is used for inhibiting and/or killing bacillus cereus.
3. An antibacterial agent characterized in that: the effective components of the anti-bacterial peptide comprise the anti-bacterial peptide BCE3 of the Litopenaeus vannamei extension factor, wherein the amino acid sequence of the anti-bacterial peptide BCE3 is SEQ ID NO:1.
4. an antimicrobial agent as claimed in claim 3 wherein: the effective component of the antibacterial peptide is Litopenaeus vannamei elongation factor antibacterial peptide BCE3, and the amino acid sequence of the antibacterial peptide BCE3 is SEQ ID NO:1.
5. an antibacterial agent according to any one of claims 3 or 4 wherein: the antibacterial drug is used for inhibiting and/or killing bacillus cereus.
6. A feed additive, characterized in that: the effective components of the anti-bacterial peptide comprise the anti-bacterial peptide BCE3 of the Litopenaeus vannamei extension factor, wherein the amino acid sequence of the anti-bacterial peptide BCE3 is SEQ ID NO:1.
7. the feed additive of claim 6, wherein: the effective component of the antibacterial peptide is Litopenaeus vannamei elongation factor antibacterial peptide BCE3, and the amino acid sequence of the antibacterial peptide BCE3 is SEQ ID NO:1.
8. a feed additive according to any one of claims 6 or 7, wherein: the feed additive is used for inhibiting and/or killing bacillus cereus.
9. A food preservative characterized by: the effective component of the antibacterial peptide is Litopenaeus vannamei elongation factor antibacterial peptide BCE3, and the amino acid sequence of the antibacterial peptide BCE3 is SEQ ID NO:1.
10. a food preservative as claimed in claim 9, wherein: the food preservative is used for inhibiting and/or killing bacillus cereus.
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