Cicada slough oligopeptide with antibacterial activity and application thereof

Abstract

The invention provides cicada slough oligopeptide with antibacterial activity, and the amino acid sequence of the cicada slough oligopeptide is shown as SEQ ID No.1. The cicada slough oligopeptide amino acid sequence is subjected to codon optimization, a target gene is synthesized, a recombinant expression vector is constructed, and the constructed recombinant expression vector is transformed into engineering bacteria to obtain recombinant engineering bacteria; and carrying out amplification culture on the recombinant engineering bacteria, inducing short peptide expression, and separating and purifying an expression product to obtain the purified cicada slough short peptide with antibacterial activity. The invention provides application of the cicada slough oligopeptide with the antibacterial activity. The cicada slough oligopeptide is used for preparing a broad-spectrum antibacterial preparation, a wound dressing, a preparation for protecting the activity of probiotics, a food preservative and a feed additive. The cicada slough oligopeptide is low in production cost, can effectively inhibit the activity of pathogenic bacteria, has the characteristics of high temperature resistance and acid resistance, is high in environmental adaptability, and provides a key material basis for research and development of novel antibacterial products.

Description

Technical Field

[0001] This invention belongs to the field of biotechnology, specifically relating to a short peptide of cicada molting with antibacterial activity and its applications. Background Technology

[0002] Currently, antibiotic overuse leading to bacterial resistance has become a major challenge in global public health, making the development of novel antibacterial active substances with unique mechanisms of action and low risk of resistance an urgent priority. Bioactive peptides, due to their advantages such as strong targeting, high safety, and low likelihood of inducing resistance, have become a research focus in the antibacterial field. Cicada molting, a traditional Chinese medicine, contains abundant active ingredients, but the antibacterial potential of its short peptides has not been fully explored. Existing technologies, such as traditional extraction methods for obtaining cicada molting active peptides, suffer from low efficiency and unstable activity. Chemical synthesis methods face limitations such as high cost and difficulties in large-scale production. Furthermore, most antibacterial substances are prone to inadvertently harming probiotics during their action or becoming inactive under high temperature or acidic environments, limiting their application scenarios. Summary of the Invention

[0003] The technical problem to be solved by this invention is to address the shortcomings of the prior art by providing a cicada molting short peptide with antibacterial activity and its application. The cicada molting short peptide prepared by this method not only solves the drawbacks of traditional preparation methods, but also has the characteristics of no inhibition of probiotics, high temperature resistance, and acid resistance. Moreover, it has low production cost and can be mass-produced, providing a key material basis for the research and development of new antibacterial products.

[0004] To solve the above-mentioned technical problems, the technical solution adopted by the present invention is: a short peptide of cicada molting with antibacterial activity, wherein the amino acid sequence of the short peptide of cicada molting with antibacterial activity is shown in SEQ ID No.1.

[0005] This invention provides applications of cicada molting short peptides with antibacterial activity, wherein the applications include one or more of the following: The cicada molting short peptide is used to prepare a broad-spectrum antibacterial agent; The cicada molting short peptide is used to prepare wound dressings; The cicada molting short peptide is used to prepare a formulation that protects the activity of probiotics; The cicada molting short peptide is used to prepare a food preservative. The cicada molting short peptide is used to prepare feed additives.

[0006] Preferably, when the cicada molting short peptide is used to prepare an antibacterial agent, the antibacterial agent has an antibacterial range that includes pathogenic bacteria and pathogenic fungi.

[0007] Preferably, when the cicada molting short peptide is used to prepare an antibacterial preparation, the pathogenic bacteria include one or more of Salmonella enteritidis, Streptococcus mutans, drug-resistant Staphylococcus aureus, multidrug-resistant Escherichia coli, and Vibrio. The pathogenic fungi include one or more of the following: *Pyrophyllus*, *Alternaria*, *Fusarium graminearum*, *Botrytis cinerea*, and *Phyllostachys rubrum*.

[0008] Preferably, when the cicada molting short peptide is used to prepare a formulation that protects the activity of probiotics, the probiotics include one or more of Bifidobacterium, Lactobacillus, Bacillus subtilis, and Enterococcus faecalis.

[0009] Preferably, when the cicada molting short peptide is used to prepare feed additives, the feed additives include livestock and poultry feed additives and aquatic feed additives.

[0010] This invention also provides a method for preparing the above-mentioned antibacterial cicada molting short peptide, the method being: S1. After codon optimization of the amino acid sequence of the cicada molting short peptide, gene synthesis was performed to construct a recombinant expression vector; S2. The recombinant expression vector constructed in S1 is transformed into engineered bacteria to obtain recombinant engineered bacteria; S3. The recombinant engineered bacteria obtained in S2 were inoculated into LB liquid medium for expansion culture, and then isopropyl-β-D-thiogalactoside was added to induce expression. The bacterial cells were collected by centrifugation, resuspended, broken, and the supernatant was collected by centrifugation to obtain a crude extract of cicada molting short peptide with antibacterial activity. S4. The crude extract of cicada molting short peptide with antibacterial activity obtained in S3 is purified, and after freeze-drying, the purified cicada molting short peptide with antibacterial activity is obtained.

[0011] Preferably, the conditions for expanding the culture in S3 are: culture at a temperature of 37℃ and a rotation speed of 200 r / min until OD600 = 0.6 to 0.8; the conditions for inducing expression are: inducing expression at a temperature of 28℃ for 8 h.

[0012] Preferably, the conditions for centrifugation and re-centrifugation in S3 are: centrifugation for 10 minutes at a temperature of 4°C and a rotation speed of 8000 r / min; the condition for crushing is: crushing for 30 minutes at a power of 300W.

[0013] Preferably, the purification method described in S4 is Ni 2+ -NTA affinity chromatography; the freeze-drying conditions are: drying at -50℃ for 4 hours.

[0014] Compared with the prior art, the present invention has the following advantages: 1. This invention uses engineered bacteria expression technology, which has a lower production cost compared to chemical synthesis and traditional extraction methods. The purified cicada molting short peptide has a purity of ≥96%, meeting the requirements of industrial applications.

[0015] 2. The cicada molting short peptide of the present invention has targeted antibacterial activity. Experimental verification shows that the MIC of the cicada molting short peptide against pathogenic bacteria is 1.8±0.05μg / mL~11.3±1.1μg / mL; the MIC against probiotics is greater than 200μg / mL. At the conventional application concentration, it does not inhibit the growth of probiotics at all, thus avoiding disruption of the microecological balance of humans and animals.

[0016] 3. The antibacterial cicada molting short peptide of the present invention differs from the targeted inhibition mechanism of traditional antibiotics, and is less likely to induce drug resistance gene mutations in bacteria. No increase in drug resistance was observed after 10 generations of continuous use of pathogenic bacteria. It still maintains good inhibitory activity against common drug-resistant strains in livestock and poultry farming (Streptococcus mutans, Staphylococcus aureus), with MIC maintained at 5.7±0.6μg / mL~11.3±1.1μg / mL.

[0017] 4. The antibacterial cicada molting short peptide of the present invention has strong environmental adaptability, and its high temperature resistance and acid resistance make it suitable for acidic environments and high temperature processing applications. After treatment with gastric and trypsin for 4 hours, the activity residual rate is still more than 85%, which solves the problem of most antibacterial peptides being inactivated in acidic environments.

[0018] The present invention will now be described in further detail with reference to the accompanying drawings and embodiments. Attached Figure Description

[0019] Figure 1 Figure 1 shows the predicted physicochemical properties and structure of the cicada molting short peptide in Example 1 of the present invention; wherein, Figure A is the predicted physicochemical properties of the amino acid sequence of the cicada molting short peptide; Figure B is the predicted hydrophilic and hydrophobic regions of the amino acid sequence of the cicada molting short peptide; Figure C is the predicted transmembrane region of the amino acid sequence of the cicada molting short peptide; and Figure D is the predicted α-helix conformation of the cicada molting short peptide.

[0020] Figure 2 This is a verification diagram of double enzyme digestion of the recombinant vector plasmid in Example 1 of the present invention.

[0021] Figure 3 This is a high-performance liquid chromatography (HPLC) chromatogram of the purified cicada molting short peptide from Example 1 of the present invention.

[0022] Figure 4 This is a graph showing the antibacterial effect of cicada molting short peptides against bacterial pathogens in Example 2 of the present invention.

[0023] Figure 5 This is a graph showing the antibacterial effect of cicada molting short peptides against fungal pathogens in Example 2 of the present invention.

[0024] Figure 6 This is a graph showing the antibacterial effect of cicada molting short peptides on probiotics in Example 2 of the present invention.

[0025] Figure 7This is a graph showing the effect of cicada molting short peptides on egg preservation in Example 2 of the present invention. Detailed Implementation

[0026] Example 1 This embodiment describes a method for preparing cicada molting short peptides with antibacterial activity. The method is as follows: S1. Based on the amino acid sequence of the cicada molting short peptide predicted by the whole genome and transcriptome, the amino acid sequence of the cicada molting short peptide is codon optimized (adapted to E. coli preferred codons) and then the gene is synthesized. The amino acid sequence of the cicada molting short peptide is MVVMSTTMMIVVLVMLVMMLIVVVVVVMMMAE (SEQ ID No. 1); For example... Figure 1 As shown in Figure A, its sequence length is 32 amino acid residues, theoretical isoelectric point is 4.00, and molecular weight is 3574.81 Da (Daltons). Structural analysis of the cicada molting short peptide yielded the following results: Figure 1 As shown in B and C, it has an amphiphilic α-helical spatial conformation, with the N-terminus being a hydrophilic region and the C-terminus being a hydrophobic transmembrane region, as... Figure 1 As shown in D, the α-helical conformation of the two parents enables the peptide chain to form a stable helical shape in space, which promotes binding to the cell membrane of pathogenic bacteria (such as Escherichia coli) and has a weaker binding to the cell membrane of probiotics (such as lactic acid bacteria). S2. The vector pET-28a was double-digested with restriction endonucleases EcoR Ⅰ (R0101S, NEB) and Xho Ⅰ (R0146S, NEB) to obtain the linearized vector pET-28a. The system for the double enzyme digestion reaction was as follows: 10×Buffer 5 μL, pET-28a vector (1 μg / μL) 2 μL, EcoRI (10 U / μL) 1 μL, XhoRI (10 U / μL) 1 μL, ddH2O 41 μL; the conditions for the double enzyme digestion reaction were: 37℃ water bath reaction for 0.5 h, followed by inactivation at 65℃ for 10 min to terminate the reaction; S3. Using T4 DNase (EL0014, Thermo Fisher Scientific), the gene synthesized in S1 was ligated with the linearized vector pET-28a obtained in S2, and then transformed into *E. coli* DH5α competent cells. Positive clones were screened and sequenced to verify the correctness of the target gene sequence. Plasmids were then extracted using a plasmid extraction kit (DP103, Tiangen), and double enzyme digestion was performed for verification. Figure 2 As shown, the large fragment of the vector and the small fragment encoding the cicada molting short peptide were successfully digested with enzymes to obtain the recombinant expression vector; The ligation reaction system was as follows: 2 μL of 10×T4 DNA Ligase Buffer, approximately 50 ng of linearized vector pET-28a, approximately 100 ng of target gene fragment, 1 μL of T4 DNA Ligase (5 U / μL), and ddH2O to a final volume of 20 μL. The ligation reaction conditions were: 14 h in a 16°C water bath, followed by inactivation at 65°C for 10 min after ligation to terminate the reaction. S4. Transform the recombinant expression vector obtained in S3 into Escherichia coli BL21 competent cells to obtain recombinant engineered bacteria; S5. The recombinant engineered bacteria obtained in S4 were inoculated into LB liquid medium (containing 50 μg / mL kanamycin) and cultured at 37℃ and 200 r / min until OD. 600 =0.8 (OD) 600 =0.6~0.8), then add isopropyl-β-D-thiogalactoside (IPTG) to a final concentration of 0.5 mmol / L and induce expression at 28℃ for 8 h. Collect the bacterial cells by centrifugation at 4℃ and 8000 r / min for 10 min, resuspend in phosphate buffered saline (PBS), and sonicate at 300W for 30 min with a 5 s interval between every 3 s of sonication. Then centrifuge at 4℃ and 8000 r / min for 10 min and collect the supernatant to obtain a crude extract of cicada molting short peptides with antibacterial activity. S6, using Ni 2+ -NTA affinity chromatography was used to purify the crude extract of cicada molting peptides with antibacterial activity obtained from S5 (the cicada molting peptides contain a 6×His tag). The column was equilibrated with PBS buffer containing 20 mmol / L imidazole. After loading the sample, the column was eluted with a gradient of PBS buffers containing 50 mmol / L, 100 mmol / L, and 250 mmol / L imidazole. The elution peak at 250 mmol / L imidazole was collected. The eluent was desalted by ultrafiltration using an ultrafiltration membrane with a molecular weight cutoff of 3 kDa, and dried at -50°C for 4 h to obtain the purified cicada molting peptides. Figure 3 As shown, the purity of the purified cicada molting peptide, as determined by high performance liquid chromatography (HPLC), was 96.1136%.

[0027] Example 2 This embodiment illustrates the application of the antibacterial cicada molting short peptide prepared in Example 1.

[0028] (i) Short peptides from cicada molting with antibacterial activity are used to prepare broad-spectrum antibacterial agents, wound dressings, and preparations that protect the activity of probiotics: 1. Experimental materials: Pathogenic bacterial strains: *Salmonella enteritidis* (ATCC13076), *Streptococcus mutans* (ATCC25175), *Staphylococcus aureus* (MRSA, clinical isolate), multidrug-resistant *Escherichia coli* (ETEC, swine diarrhea isolate), and *Vibrio* (shrimp enteritis isolate); Pathogenic fungal strains: *Botryosphaeria berengeriana* (NW311), *Alternaria* (ATCC336535), *Fusarium graminearum* (wheat disease isolate), *Botrytis cinerea* (grape mold isolate), and *Valsa* (apple rot isolate); Probiotic strains: *Bifidobacterium* The bacteria tested included ATCC15697, Lactic acid bacteria (ATCC4356), Bacillus subtilis (ATCC6633), and Enterococcus faecalis (ATCC19433); the short peptide sample was the purified cicada molting short peptide with antibacterial activity prepared in Example 1, denoted as cicada molting short peptide.

[0029] 2. Experimental methods: The Oxford cup method was used, with ddH2O as the negative control, cicada molting peptide at a mass concentration of 1 μg / mL as the experimental group, and oxytetracycline at a mass concentration of 1 μg / mL and 50% ethanol as the positive control groups, to detect the antibacterial effect of cicada molting peptide against pathogenic bacteria. The same method was also used to detect the antibacterial effect of cicada molting peptide against pathogenic fungi, and finally, to detect the antibacterial effect of cicada molting peptide against probiotics.

[0030] Given the close association between pathogenic bacteria and probiotics and the risk of infection and health in humans and animals, a micro-broth dilution method was further employed to dilute the cicada molting peptides into 10 concentration gradients: 0.125 μg / mL, 0.25 μg / mL, 0.5 μg / mL, 1 μg / mL, 2 μg / mL, 5 μg / mL, 10 μg / mL, 20 μg / mL, 100 μg / mL, and 200 μg / mL. These concentrations were then mixed with pathogenic bacterial suspensions (1×10⁻⁶). 6CFU / mL), probiotic suspension (1×10) 6 CFU / mL were mixed at a 1:1 volume ratio and incubated at 37℃ in a 5% CO2 incubator for 24 h. The bacterial survival rate at each concentration was measured. At the same time, high temperature group (80℃~121℃ for 30 min), acidic group (pH 2.0~4.0 for 4 h), and protease treatment group (1 mg / mL pepsin and 1 mg / mL trypsin for 4 h each) were set up to treat cicada molting short peptides. The above experiments were repeated to detect the residual antibacterial rate of cicada molting short peptides against pathogenic bacteria after treatment and to verify environmental tolerance. Each experiment was repeated 3 times.

[0031] 3. Experimental Results: like Figure 4 As shown, the antibacterial effect of cicada molting peptides against pathogenic bacteria was tested. The results showed that a concentration of 1 μg / mL of cicada molting peptides had an antibacterial effect comparable to that of oxytetracycline at the same concentration, and even better than oxytetracycline against some pathogenic bacteria. Figure 5 As shown, the results of the test on the antibacterial effect of cicada molting peptides against fungal pathogens showed that cicada molting peptides at a mass concentration of 1 μg / mL had a significant antibacterial effect on all tested fungal pathogens. Figure 6 As shown, the results of the test on the antibacterial effect of cicada molting peptide on probiotics showed that cicada molting peptide with a mass concentration of 1 μg / mL had no significant antibacterial effect on probiotics.

[0032] As shown in Table 1, Generation 0 and Generation 10 refer to strains that have not been treated with cicada molting peptides and strains that have been treated with cicada molting peptides for 10 consecutive generations, respectively. The MICs of pathogenic bacteria are: Salmonella enteritidis 1.8 μg / mL, Staphylococcus aureus resistant to drugs 2.3 μg / mL, Streptococcus mutans 5.7 μg / mL, and multidrug-resistant Escherichia coli 11.3 μg / mL. The MICs of all probiotics are >200 μg / mL, indicating that the bacteria strongly inhibit pathogenic bacteria, have no effect on probiotics, and no obvious increase in drug resistance was found.

[0033] Table 1. Minimum inhibitory concentrations (MICs) of cicada molting short peptides against pathogenic bacteria and probiotics. As shown in Table 2, after high temperature and acid treatment, the residual inhibition rate of cicada molting short peptide against pathogenic bacteria was still above 95%, proving that it has high temperature and acid resistance. After being treated with gastric and trypsin for 4 hours, the residual inhibition rate of cicada molting short peptide against pathogenic bacteria was still above 85%, proving that it has high gastrointestinal environment stability.

[0034] Table 2. Effects of temperature, pH, and storage conditions on cicada molt short peptides In summary, cicada molting peptides possess excellent broad-spectrum antibacterial activity, exhibiting significant inhibitory effects on common bacterial pathogens (including drug-resistant strains) and fungal pathogens commonly found in clinical, animal husbandry, and crop cultivation fields, without significant adverse effects on beneficial bacteria in the human and animal gut. Furthermore, they possess good heat and acid resistance and gastrointestinal stability, making them suitable for preparing broad-spectrum antibacterial agents and wound dressings to promote wound healing and reduce infection risks. They can also be used to prepare formulations that protect the activity of beneficial bacteria, demonstrating broad application prospects and significant practical value in fields such as pharmaceuticals, food preservation, livestock and aquaculture, and crop disease control.

[0035] (ii) Cicada molting short peptides with antibacterial activity are used to prepare food preservatives: 1. Preparation of preservatives: The antibacterial cicada molting short peptide prepared in Example 1 was mixed with sodium alginate (Solebo) at a mass ratio of 1:8, dissolved in purified water, and a 1.5% preservative solution was prepared. The solution was then autoclaved at 121°C for 30 minutes for later use.

[0036] 2. Experimental methods: Fresh eggs (with intact shells and no damage) were selected and divided into two groups of 30 eggs each. The experimental group was immersed in the above-mentioned preservative solution for 1 minute, drained, and then placed in a room temperature environment of 25°C. The control group was immersed in a sodium alginate solution containing the same mass as the above-mentioned preservative solution for 1 minute, drained, and then placed in a room temperature environment of the same size. The eggs were observed regularly for spoilage (spreading of yolk, cloudy egg white, and off-odors indicate spoilage), and the shelf life was determined.

[0037] 3. Experimental Results: As shown in Table 3, eggs in the control group began to spoil at room temperature on day 15, with a spoilage rate of 41.52±3.61% on day 25; eggs in the experimental group began to show slight spoilage at room temperature on day 35, with a spoilage rate of 38.44±2.89% on day 50, extending the shelf life by more than double. Figure 7 As shown in the figure, the shell breakage test showed that the egg yolks and egg whites of the experimental group (fresh eggs + cicada exuviae short peptide preservative) were in good condition and free from bacterial contamination, while the spoiled eggs of the control group (fresh eggs) were found to be contaminated with bacteria.

[0038] Table 3. Application of cicada molting short peptides in egg preservation. Note: Different lowercase letters in the same line indicate significant differences (P<0.05), while the same letter indicates no significant differences (P>0.05).

[0039] (III) Cicada molting short peptides with antibacterial activity are used to prepare livestock and poultry feed additives: 1. Preparation of feed additives: The antibacterial cicada molting short peptide prepared in Example 1 was mixed with maltodextrin (Xiwang) and silicon dioxide (Weifang Sanjia Chemical Co., Ltd.) at a mass ratio of 1:8:1 to prepare a powdered feed additive.

[0040] 2. Experimental methods: One hundred and twenty 21-day-old weaned piglets were randomly divided into three groups of 40 piglets each: a blank control group, a pathogenic bacteria infection group, and a cicada molting peptide supplementation group. The blank control group was fed a basal diet without any treatment. The pathogenic bacteria infection group was fed a basal diet and, on the third day, was given pathogenic Escherichia coli (ETEC, 1×10⁻⁶) via oral gavage. 8 CFU / head); the cicada molting short peptide group was given 0.2% of the above powdered feed additive in the basal diet, and on the 3rd day, the same dose of pathogenic Escherichia coli was administered by gavage; the experimental period was 14 days, and the diarrhea rate and average daily weight gain of piglets were recorded. After the experiment, the number of pathogenic bacteria (pathogenic Escherichia coli) and probiotics (lactic acid bacteria) in the intestines of piglets were detected.

[0041] 3. Experimental Results: As shown in Table 4, the diarrhea rate was 5.28±0.38% in the blank control group, 75.85±2.32% in the pathogenic bacteria infection group, and 12.5±0.89% in the cicada molting short peptide supplementation group, which was 84.6% lower than that in the pathogenic bacteria infection group. The average daily weight gain was 415.53±6.87g in the blank control group, 226.72±4.19g in the pathogenic bacteria infection group, and 405.36±5.54g in the cicada molting short peptide supplementation group, which was 78.8% higher than that in the pathogenic bacteria infection group. The intestinal flora showed a significant decrease in the number of pathogenic Escherichia coli in the cicada molting short peptide supplementation group compared to the pathogenic bacteria infection group, while the number of lactic acid bacteria significantly increased, approaching the level of the blank control group. This demonstrates that the cicada molting short peptide-based livestock and poultry feed additive can effectively inhibit pathogenic Escherichia coli, protect intestinal probiotics, and prevent swine bronchitis diarrhea.

[0042] Table 4 shows the use of cicada molting short peptides with antibacterial activity as pig feed additives. Note: Different lowercase letters in the same line indicate significant differences (P<0.05), while the same letter indicates no significant differences (P>0.05).

[0043] (iv) Cicada molting short peptide additives with antibacterial activity are used in the preparation of aquatic feed additives: 1. Preparation of feed additives: The antibacterial cicada molting short peptide prepared in Example 1 was mixed with maltodextrin at a mass ratio of 1:10 and spray-dried to prepare a powdered additive.

[0044] 2. Experimental methods: 120 whiteleg shrimp weighing approximately 50g were randomly divided into two groups of 60 shrimp each from shrimp ponds with vibrio disease. The experimental group was fed with 0.3% of the feed additive prepared above, while the control group was not fed with the additive. Both groups were cultured under the same water quality and temperature conditions, fed twice daily, for a culture period of 30 days. The number of pathogenic bacteria (Vibrio) and probiotics (Lactobacillus) in the shrimp intestines was tested regularly, and the incidence of disease was recorded.

[0045] 3. Experimental Results: As shown in Table 5, after the aquaculture period, the number of Vibrio bacteria in the intestines of white shrimp in the experimental group was significantly lower than that in the control group, while the number of lactic acid bacteria was significantly higher. The incidence rate of Vibrio bacteria in the intestines of white shrimp in the control group was 21.67±0.62%, while that in the experimental group was 5.13±2.32%, a significant reduction of 76.33%. This demonstrates that the aquatic feed additive prepared from cicada molting short peptides with antibacterial activity can effectively inhibit intestinal pathogens, promote the growth of probiotics, and improve the disease resistance of aquatic animals.

[0046] Table 5 shows cicada molting short peptides with antibacterial activity used as feed additives for white shrimp. Note: Different lowercase letters in the same line indicate significant differences (P<0.05), while the same letter indicates no significant differences (P>0.05).

[0047] In summary, when this cicada molting peptide is used in the preparation of broad-spectrum antibacterial agents, wound dressings, and probiotic preparations that protect the gut, it exhibits good antibacterial activity, high temperature resistance, acid resistance, and can protect gut probiotics, promoting wound healing, reducing infection risk, adapting to the acidic environment of the stomach, and inhibiting pathogenic bacteria in the gut. When used in the preparation of food preservatives, it can effectively extend the shelf life of food and has high safety as a natural preservative. Furthermore, this cicada molting peptide can be used in the preparation of feed additives. When used in the preparation of livestock and poultry feed additives, it can significantly increase the daily weight of piglets, reduce the number of Escherichia coli in their bodies, and increase the number of lactic acid bacteria, thereby reducing their diarrhea rate. When used in the preparation of aquatic feed additives, it can significantly reduce the number of Vibrio in white shrimp and significantly increase the number of lactic acid bacteria in their bodies, thereby significantly reducing the incidence of Vibrio in white shrimp.

[0048] The above description is merely a preferred embodiment of the present invention and is not intended to limit the invention in any way. Any simple modifications, alterations, and equivalent changes made to the above embodiments based on the inventive essence shall still fall within the protection scope of the present invention.

Claims

1. A short peptide derived from cicada molting with antibacterial activity, characterized in that, The amino acid sequence of the cicada molting short peptide with antibacterial activity is shown in SEQ ID No.

1.

2. The application of the cicada molting short peptide with antibacterial activity as described in claim 1, characterized in that, The application includes one or more of the following: The cicada molting short peptide is used to prepare a broad-spectrum antibacterial agent; The cicada molting short peptide is used to prepare wound dressings; The cicada molting short peptide is used to prepare a formulation that protects the activity of probiotics; The cicada molting short peptide is used to prepare a food preservative. The cicada molting short peptide is used to prepare feed additives.

3. The application of the antibacterial cicada molting short peptide according to claim 2, characterized in that, When the cicada molting short peptide is used to prepare a broad-spectrum antibacterial agent, the antibacterial range of the antibacterial agent includes pathogenic bacteria and pathogenic fungi.

4. The application of the antibacterial cicada molting short peptide according to claim 3, characterized in that, When the cicada molting short peptide is used to prepare a broad-spectrum antibacterial agent, the pathogenic bacteria include one or more of Salmonella enteritidis, Streptococcus mutans, drug-resistant Staphylococcus aureus, multidrug-resistant Escherichia coli, and Vibrio. The pathogenic fungi include one or more of the following: *Pyrophyllus*, *Alternaria*, *Fusarium graminearum*, *Botrytis cinerea*, and *Phyllostachys rubrum*.

5. The application of the antibacterial cicada molting short peptide according to claim 2, characterized in that, When the cicada molting short peptide is used to prepare a formulation that protects the activity of probiotics, the probiotics include one or more of Bifidobacterium, lactic acid bacteria, Bacillus subtilis, and Enterococcus faecalis.

6. The application of the antibacterial cicada molting short peptide according to claim 2, characterized in that, When the cicada molting short peptide is used to prepare feed additives, the feed additives include livestock and poultry feed additives and aquatic feed additives.

7. A method for preparing the cicada molting short peptide with antibacterial activity as described in claim 1, characterized in that, The method is as follows: S1. After codon optimization of the amino acid sequence of the cicada molting short peptide, gene synthesis was performed to construct a recombinant expression vector; S2. The recombinant expression vector constructed in S1 is transformed into engineered bacteria to obtain recombinant engineered bacteria; S3. The recombinant engineered bacteria obtained in S2 were inoculated into LB liquid medium for expansion culture, and then isopropyl-β-D-thiogalactoside was added to induce expression. The bacterial cells were collected by centrifugation, resuspended, broken, and the supernatant was collected by centrifugation to obtain a crude extract of cicada molting short peptide with antibacterial activity. S4. The crude extract of cicada molting short peptide with antibacterial activity obtained in S3 is purified, and after freeze-drying, the purified cicada molting short peptide with antibacterial activity is obtained.

8. The method according to claim 7, characterized in that, The conditions for expanding the culture described in S3 are: culture at a temperature of 37℃ and a rotation speed of 200 r / min until OD600 = 0.6 to 0.8; the conditions for inducing expression are: inducing expression at a temperature of 28℃ for 8 h.

9. The method according to claim 7, characterized in that, The conditions for centrifugation and re-centrifugation in S3 are: centrifugation for 10 minutes at a temperature of 4℃ and a rotation speed of 8000 r / min; the condition for crushing is: crushing for 30 minutes at a power of 300W.

10. The method according to claim 7, characterized in that, The purification method described in S4 is Ni 2+ -NTA affinity chromatography; the freeze-drying conditions are: drying at -50℃ for 4 hours.

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