Polypeptide with efficacy of preventing and controlling bacterial disease of black seabream and application thereof

Abstract

The application provides a polypeptide with the efficacy of preventing and controlling bacterial diseases of black seabream and application thereof, the polypeptide is prepared by adding an extraction buffer to tissue material of the black seabream after the tissue material is cut and crushed, homogenizing, ultrafiltration grading, gel filtration chromatography and reverse phase high performance liquid chromatography separation and purification of the homogenate. The polypeptide has good antibacterial property, has excellent bacteriostatic effect on main pathogenic bacteria of the black seabream, i.e. Vibrio alginolyticus, Vibrio harveyi, Vibrio vulnificus and Aeromonas hydrophila, the feed additive prepared by the application can be used for preventing and treating bacterial diseases of the black seabream, applied to black seabream culture, can significantly enhance the non-specific immunity of the black seabream, improve the survival rate after challenge, has no negative effect on the normal growth of the black seabream, and has good economic and application prospect.

Description

Technical Field

[0001] This invention belongs to the field of functional polypeptide screening and application technology for aquaculture diseases, specifically involving a polypeptide with the effect of preventing and controlling bacterial diseases in black sea bream and its application. Background Technology

[0002] Black sea bream (Acanthopagrus schlegelii) belongs to the family Sea Breamidae and is one of the important marine aquaculture fish in coastal areas of my country, widely distributed in the Yellow Sea, East China Sea, and South China Sea. Black sea bream has advantages such as rapid growth, strong adaptability, tender flesh, and high nutritional value. In recent years, it has experienced rapid development in marine cage culture and recirculating aquaculture systems, with its farming scale continuously expanding, and has become an important part of my country's marine aquaculture industry.

[0003] With the increasing stocking density and the emergence of problems such as water quality fluctuations, uneaten feed accumulation, and increased organic pollution in the aquaculture environment, black sea bream are susceptible to various pathogenic microorganisms during the aquaculture process, especially bacterial diseases, which are on the rise and seriously restrict the healthy development of the industry. Vibrio spp.-induced vibriosis often manifests as surface bleeding, ulcers, and visceral necrosis; hemorrhagic septicemia caused by Aeromonas spp. can lead to widespread bleeding, ascites, and increased mortality; in addition, Edwardsiella tarda infection often causes enteritis and liver lesions, while streptococcal infection can cause neurological symptoms and exophthalmos. These bacteria are mostly opportunistic pathogens, which can easily break out rapidly under high-density aquaculture and environmental stress conditions, causing large-scale mortality and serious economic losses.

[0004] Currently, the prevention and control of bacterial diseases in black sea bream mainly relies on antibiotics and chemical drugs, such as florfenicol and enrofloxacin, which are controlled through mixing with feed or spraying into the water. While these methods can inhibit the growth of pathogens in the short term, long-term or improper use can easily lead to increased drug resistance in pathogens, reducing drug sensitivity and treatment effectiveness. At the same time, antibiotic residues in fish and aquaculture water are becoming increasingly prominent, not only affecting the quality and safety of aquatic products but also posing potential risks to the ecological environment, thus becoming a significant factor restricting the sustainable development of aquaculture.

[0005] Antimicrobial peptides (AMPs) are a class of naturally occurring small-molecule bioactive polypeptides widely found in plants, animals, and microorganisms, and are an important component of the body's innate immune system. They primarily kill pathogens by interacting with bacterial cell membranes, disrupting membrane structure, and exhibiting broad-spectrum antibacterial activity, low susceptibility to inducing drug resistance, and some immunomodulatory functions. Therefore, antimicrobial peptides are considered ideal candidates to replace traditional antibiotics. However, the screening of specific antimicrobial peptides targeting the main pathogens of black sea bream and their stable application in feed remain relatively insufficient, and safe, efficient, and suitable antimicrobial peptide products for black sea bream farming systems are still lacking.

[0006] Therefore, developing an antimicrobial peptide that targets the main bacterial pathogens of black sea bream, has good stability and application potential, is of great significance for improving the health level of black sea bream farming, reducing antibiotic use, and promoting the green development of aquaculture. Summary of the Invention

[0007] The purpose of this invention is to provide a polypeptide with the effect of preventing and controlling bacterial diseases in black sea bream and its application, thereby making up for the shortcomings of the prior art.

[0008] The present invention first provides a polypeptide with antibacterial activity, which is prepared by cutting black sea bream tissue material into small pieces, adding extraction buffer to homogenize, and then purifying the homogenate by ultrafiltration fractionation, gel filtration chromatography and reversed-phase high-performance liquid chromatography.

[0009] Furthermore, the amino acid sequence of the polypeptide is LLGDFFRKSLKGLLKRF (SEQ ID NO: 1).

[0010] The present invention also provides an application of the aforementioned polypeptide in the reprocessing of antimicrobial products.

[0011] The antibacterial product is an antibacterial product against Vibrio alginolyticus, Vibrio harveyi, Vibrio vulnificus, and Aeromonas hydrophila.

[0012] The polypeptides provided by this invention can also be used to prepare feed additives.

[0013] In another aspect, the present invention provides a feed additive prepared by mixing the above-mentioned antimicrobial peptide with a feed-acceptable carrier;

[0014] The feed-grade acceptable carrier comprises microcrystalline cellulose, vitamin C, and sodium alginate.

[0015] In another aspect, the present invention provides a fish feed in which the above-mentioned polypeptide is added.

[0016] Furthermore, the fish feed mentioned is black sea bream feed.

[0017] The polypeptides provided by this invention have good antibacterial properties and exhibit excellent antibacterial effects against Vibrio alginolyticus, Vibrio harveyi, Vibrio vulnificus, and Aeromonas hydrophila, which are the main pathogenic bacteria of black sea bream. The feed additives prepared by this invention can be used to prevent and control bacterial diseases of black sea bream. When applied to black sea bream farming, they can significantly enhance the non-specific immunity of black sea bream, improve the survival rate after challenge, and have no negative impact on the normal growth of black sea bream, thus showing good economic and application prospects. Attached Figure Description

[0018] Figure 1 : Gel filtration chromatography results of peptides;

[0019] Figure 2 : Graph showing the purification results of the polypeptide by reversed-phase high-performance liquid chromatography;

[0020] Figure 3 : Immune index charts for each group of black sea bream;

[0021] Figure 4 : Graph showing mortality rates of black sea bream in each feeding group after infection and challenge;

[0022] Figure 5 : Growth performance indicators of black sea bream in different treatment groups. Detailed Implementation

[0023] The present invention will be further described in detail below with reference to specific embodiments.

[0024] Example 1: Extraction, separation, purification and structural identification of antimicrobial peptides

[0025] 1.1 Preparation of crude extract of tissue protein

[0026] Skin mucus, gill tissue, and intestinal tissue were collected from healthy black sea bream (Acanthopagrus schlegelii) at a marine aquaculture farm in Ningbo City, Zhejiang Province. Mucus was scraped from the sea bream's body surface using a sterile scraper, and three volumes of ice-cold extraction buffer (50 mM Tris-HCl, pH 7.4, containing 150 mM NaCl, 1 mM EDTA, and 1 mM PMSF) were added. The samples were homogenized in an ice bath (10,000 rpm, 1 min, 30 s intervals, repeated 3 times). 50 g of each tissue sample was weighed, chopped, and then added to five volumes of ice-cold extraction buffer. The samples were homogenized using a tissue homogenizer (IKA T25) in an ice bath (12,000 rpm, 2 min, 30 s intervals, repeated 3 times). Live samples were immediately frozen in liquid nitrogen after collection and stored at -80°C after being transported back to the laboratory for later use.

[0027] After extraction by stirring at 4℃ for 2 h, the homogenate was centrifuged at 12,000 ×g for 30 min at 4℃, and the supernatant was collected. The supernatant was filtered through a 0.22 μm filter membrane for sterilization to obtain the crude tissue protein extract. The protein concentration of the crude extract was determined using a BCA protein quantification kit. The protein concentration of the crude extract from animal tissue was approximately 8.2–11.5 mg / mL. All crude extracts were aliquoted and stored at -80℃ for later use.

[0028] 1.2 Enrichment of small molecule polypeptide components

[0029] The crude extract was fractionated using a tangential flow ultrafiltration system (Millipore, Pellicon 3), employing ultrafiltration membrane modules with molecular weight cutoffs of 10 kDa and 3 kDa to collect fractions with molecular weights between 3 and 10 kDa and <3 kDa. These two molecular weight ranges represent the most common distribution ranges for antimicrobial peptides. The collected fractions were lyophilized and concentrated, then reconstituted with sterile water to a protein concentration of 5 mg / mL to obtain enriched peptide fractions for subsequent separation.

[0030] 1.3 Isolation and purification of peptides

[0031] 1.3.1 Gel filtration chromatography

[0032] Take 200 mg each of the above-mentioned enriched peptide fractions (3~10 kDa and <3 kDa) lyophilized powder, dissolve them separately in 5 mL of 20 mM PBS, and load them onto a Sephadex G-25 gel filtration chromatography column (GE Healthcare, 2.6 cm × 80 cm) pre-equilibrated with the same buffer. Elute at a flow rate of 1.0 mL / min, monitoring with a UV detector at 280 nm and 214 nm wavelengths, collecting one tube every 3 mL. Collect the elution profile based on the 214 nm (peptide bond absorption peak) elution profile. Figure 1 Each elution peak component was freeze-dried and concentrated separately.

[0033] 1.3.2 Purification by reversed-phase high-performance liquid chromatography

[0034] Each component obtained by gel filtration chromatography was reconstituted with 0.1% TFA-water solution and then finely separated using reversed-phase high-performance liquid chromatography (RP-HPLC, Agilent 1260 Infinity II). The chromatographic conditions were as follows:

[0035] Chromatographic column: Zorbax SB-C18 column (4.6 mm × 250 mm, 5 μm, Agilent)

[0036] Mobile phase A: 0.1% trifluoroacetic acid (TFA) aqueous solution

[0037] Mobile phase B: 0.1% TFA-acetonitrile solution

[0038] Elution gradient: 0–5 min, 5% B; 5–60 min, 5%–60% B linear gradient; 60–65 min, 60%–95% B; 65–70 min, 95% B; 70–75 min, 95%–5% B

[0039] Flow rate: 1.0 mL / min

[0040] Detection wavelengths: 214 nm and 280 nm

[0041] Injection volume: 200 μL

[0042] Each elution peak was collected separately according to its peak number, and the solvent was removed by lyophilization to obtain a series of purified peptide samples. A total of four elution peaks were obtained from the black sea bream tissue. Figure 2 (These are numbered BdP-01 to BdP-04 respectively).

[0043] 1.4 Mass Sequencing and Peptide Library Construction

[0044] 1.4.1 Mass spectrometry analysis conditions

[0045] 0.5 mg of each of the purified peptide samples was dissolved in 0.5 mL of 0.1% formic acid-water solution. Molecular weight and amino acid sequence analysis were performed using liquid chromatography-tandem mass spectrometry (LC-MS / MS, Thermo Scientific Q Exactive HF-X). Chromatographic conditions: A C18 column (75 μm × 150 mm, 3 μm) was used; mobile phase A was 0.1% formic acid-water, and mobile phase B was 0.1% formic acid-acetonitrile, with gradient elution. Mass spectrometry was performed in positive ion mode, with a scan range of m / z 100–3000.

[0046] 1.4.2 Sequencing Results and Peptide Library

[0047] LC-MS / MS analysis, combined with a search of the UniProt database using the Mascot search engine, successfully identified the complete amino acid sequence of the peptide. The peptide sequence information is as follows: BdP-01: Gly-Leu-Leu-Lys-Leu-Leu-Lys-Lys-Phe-Leu-Lys; BdP-02: Phe-Lys-Leu-Leu-Leu-Lys-Lys-Leu-Phe-Lys-Ser-Leu-Leu-Lys; BdP-03: Leu-Leu-Gly-Asp-Phe-Phe-Arg-Lys-Ser-Leu-Lys-Gly-Leu-Leu-Leu-Lys-Arg-Phe; BdP-04: Ile-Lys-Leu-Leu-Lys-Lys-Leu-Phe-Lys-Lys-Leu.

[0048] 1.5 Chemical Synthesis

[0049] The above-mentioned peptides were chemically synthesized using the solid-phase peptide synthesis method (Fmoc chemical method) (commissioned to Shanghai Jier Biochemical Co., Ltd.). After purification by RP-HPLC, the purity was ≥95%. The molecular weight was identified by mass spectrometry as consistent with that of the natural extract. The synthesized peptides were lyophilized and stored for later use.

[0050] Example 2: Screening of peptide antibacterial activity and determination of the optimal antibacterial peptide

[0051] 2.1 Experimental strains

[0052] The pathogens used in the experiment were Vibrio alginolyticus (ATCC 17749), Vibrio harveyi (ATCC 33868), Vibrio vulnificus (ATCC 27562), and Aeromonas hydrophila (ATCC 7966), all purchased from the American Type Culture Collection (ATCC). All strains were activated and cultured to the logarithmic growth phase using MH broth at 30°C and 180 r / min before use.

[0053] 2.2 Determination of Minimum Inhibitory Concentration of Peptide

[0054] The MIC values ​​of the above 10 polypeptides against four major pathogenic bacteria of black sea bream (Vibrio alginolyticus, Vibrio harveyi, Vibrio vulnificus, and Aeromonas hydrophila) were determined using the micro-broth dilution method.

[0055] (1) Add 100 μL of MH broth culture medium to each well of a 96-well sterile microplate.

[0056] (2) Prepare a stock solution of 512 μg / mL with sterile water for the polypeptide. Add 100 μL of the stock solution to the first well and perform serial dilution by 2 times so that the final concentrations of the polypeptide in each well are 256, 128, 64, 32, 16, 8, 4, 2, 1 and 0.5 μg / mL respectively.

[0057] (3) Adjust the bacterial solutions of each indicator bacteria to 1×10⁻⁶ with MH broth. 6 Add 100 μL of bacterial culture to each well at a concentration of CFU / mL to achieve a final bacterial concentration of 5 × 10⁻⁶ CFU / mL. 5 CFU / mL.

[0058] (4) Set up a positive control (containing 100 μL of bacterial culture + 100 μL of MH broth) and a negative control (containing 200 μL of MH broth). Set up 3 replicate wells for each concentration.

[0059] (5) Place the microplate in a 30°C constant temperature incubator for 24 h and use the lowest drug concentration at which no bacterial growth (no turbidity) is observed by the naked eye as the MIC value.

[0060] (6) The MIC values ​​of tetracycline hydrochloride against each indicator bacteria were determined simultaneously for comparison.

[0061] Table 1: Minimum inhibitory concentration (MIC) of peptides (unit: μg / mL)

[0062]

[0063] As shown in Table 1, the MIC determination results of the 10 candidate peptides indicate that BdP-03 has the lowest MIC value against the four pathogens and the strongest overall antibacterial activity, comparable to or better than the positive control drug tetracycline hydrochloride. Therefore, BdP-03 was identified as the candidate antimicrobial peptide with the strongest antibacterial properties and named BdAMP-1, with the following amino acid sequence: Leu-Leu-Gly-Asp-Phe-Phe-Arg-Lys-Ser-Leu-Lys-Gly-Leu-Leu-Lys-Arg-Phe (SEQ ID NO: 1).

[0064] 2.3 Hemolytic Activity Assay

[0065] To evaluate the safety of BdAMP-1 on black sea bream erythrocytes, hemolytic activity was determined.

[0066] (1) Preparation of erythrocytes from black sea bream: Anticoagulated blood was collected from the tail vein of healthy black sea bream (approximately 200 g in weight), washed three times with sterile PBS (pH 7.2), and centrifuged at 300 × g for 5 min each time. The supernatant was discarded. The erythrocytes were resuspended in PBS to prepare a 2% (v / v) erythrocyte suspension.

[0067] (2) Hemolysis test: Take a 96-well U-shaped plate and add 100 μL of red blood cell suspension to each well. Add different concentrations of BdAMP-1 solution to make the final concentrations 2, 4, 8, 16, 32, 64, 128, 256, and 512 μg / mL, respectively, and make up the total volume of each well to 200 μL. Set up a positive control group and a negative control group. Set up 3 replicate wells for each group.

[0068] (3) Incubate the 96-well plate at 37°C for 1 h, then centrifuge at 4°C and 1,000 × g for 5 min. Carefully aspirate 150 μL of the supernatant into a new 96-well plate and measure the OD using a microplate reader. 570 value.

[0069] (4) Formula for calculating hemolysis rate: Hemolysis rate (%) = (OD of experimental group) 570 – Negative control group OD 570 ) / (Positive control group OD 570 – Negative control group OD 570 ) × 100%

[0070] Within a BdAMP-1 concentration range of 2–64 μg / mL, the hemolysis rate was less than 0.5%; when the concentration increased to 128 μg / mL, the hemolysis rate was 1.2%; and at the highest tested concentration of 512 μg / mL, the hemolysis rate was 3.8%. These results indicate that BdAMP-1 exhibits extremely low hemolytic activity (<0.5%) at its MIC concentration, demonstrating good biocompatibility against black sea bream erythrocytes, and a therapeutic index (HCI) of [missing value]. 10 A / MIC greater than 128 indicates a wider safety range.

[0071] 2.4 Stability Evaluation

[0072] To investigate the stability of BdAMP-1 in practical applications, the retention rate of its antibacterial activity under different conditions was measured.

[0073] (1) Thermal stability: BdAMP-1 solution (128 μg / mL) was placed in water baths at 40℃, 60℃, 80℃ and 100℃ for 30 min, respectively. After cooling, the diameter of the inhibition zone against Vibrio alginolyticus was measured. The activity retention rate was calculated with the untreated group as the control.

[0074] (2) pH stability: The pH of BdAMP-1 solution was adjusted to 3.0, 5.0, 7.0, 9.0 and 11.0 respectively with HCl or NaOH. After being placed at room temperature for 2 h, the pH was adjusted back to 7.0 and the antibacterial activity was measured.

[0075] (3) Protease tolerance: BdAMP-1 solution was mixed with trypsin, pepsin and proteinase K at a mass ratio of 10:1, incubated at 37℃ for 2 h, and the enzyme reaction was terminated by heating at 95℃ for 10 min. The antibacterial activity was then measured.

[0076] Thermal stability: After treatment at 40℃~80℃ for 30 min, the activity retention rate was >96%; after treatment at 100℃ for 30 min, the activity retention rate was 85.6%, indicating that BdAMP-1 has good thermal stability and can withstand the high temperature during feed pelleting.

[0077] pH stability: After treatment at pH 3.0 to 9.0 for 2 h, the activity retention rate was >92%; after treatment at pH 11.0 for 2 h, the activity retention rate was 75.3%, indicating that BdAMP-1 is stable under weakly acidic to weakly alkaline conditions and is suitable for functioning in the intestinal environment of fish.

[0078] Protease tolerance: After treatment with trypsin and pepsin, the activity retention rates were 72.5% and 78.3%, respectively; after treatment with proteinase K, the activity retention rate was only 25.6%. This indicates that BdAMP-1 has a certain degree of tolerance to digestive tract proteases, which is beneficial for maintaining its activity in the intestine and can be degraded in the environment, reducing the risk of residue.

[0079] Example 3: Preparation of feed additive containing antimicrobial peptide BdAMP-1 and evaluation of its feeding effect on black sea bream.

[0080] 3.1 Preparation of feed additives

[0081] BdAMP-1 lyophilized powder was thoroughly mixed with microcrystalline cellulose (MCC, Shanghai Changwei Pharmaceutical Excipients Technology Co., Ltd.) at a mass ratio of 1:100, and then 0.1% vitamin C and 1% sodium alginate were added by mass. The mixture was placed in a fluidized bed granulator (GEA, model MP-1), with the inlet air temperature set to 50℃, material temperature to 35℃, and atomization pressure to 1.2 bar. A suitable amount of deionized water was sprayed in as a wetting agent to obtain microcapsule particles with a particle size of 0.5~1.0 mm. HPLC analysis showed that the actual content of BdAMP-1 in this feed additive was 0.95% (w / w). The additive was sealed in packaging and stored at room temperature, protected from light, for later use.

[0082] 3.2 Experimental Design and Feeding Management

[0083] The black sea bream used in the experiment were purchased from an aquaculture farm in Ningbo City, Zhejiang Province. Their average weight was 20±2 g, and they were temporarily housed in a recirculating aquaculture system for 7 days to acclimatize. During this period, they were fed a basic commercial feed (crude protein ≥42%, crude fat ≥8%, Ningbo Tianbang Co., Ltd.) twice daily at a rate of 3% of their total body weight.

[0084] After the temporary holding period, 300 healthy and vigorous black sea bream were selected and randomly divided into 3 treatment groups, with 3 replicates in each group and 30 sea bream in each replicate. All groups were housed in identical fiberglass tanks (500 L capacity). The treatments for each group were as follows:

[0085] Control group: fed basal feed throughout the entire process;

[0086] Low-dose group: 0.2% (w / w) of the above feed additives were added to the basal diet;

[0087] High-dose group: 0.4% (w / w) of the above feed additives were added to the basal diet.

[0088] After thoroughly mixing the calculated feed additive powder with the base feed pellets, spray in a small amount of fish oil to increase adhesion, and let it air dry naturally before use. Prepare each group of feed weekly and store at 4℃. Feed continuously for 21 days, maintaining a water temperature of 24±2℃, dissolved oxygen >6.0 mg / L, pH 7.8~8.2, ammonia nitrogen <0.05 mg / L, and changing 30% of the total water volume daily.

[0089] 3.3 Immunological marker determination

[0090] After 21 days of feeding, nine fish were randomly selected from each group. After anesthetizing with MS-222 (100 mg / L), blood was collected from the tail vein. The blood samples were allowed to stand at 4°C for 2 h, then centrifuged at 3,000 ×g for 10 min to separate the serum, which was then stored at -80°C for later use.

[0091] The activities of lysozyme, superoxide dismutase and alkaline phosphatase were determined using a kit.

[0092] Table 2: Immune indicators of black sea bream in each group

[0093] control group 42.6 ± 5.2ᵃ 86.3 ± 7.4ᵃ 12.4 ± 1.8ᵃ low-dose group 68.5 ± 6.1ᵇ 112.5 ± 8.9ᵇ 18.7 ± 2.1ᵇ High-dose group 85.3 ± 7.3ᶜ 138.2 ± 10.3ᶜ 23.5 ± 2.5ᶜ

[0094] Table 2 and Figure 3 The results showed that, compared with the control group, the feed supplemented with BdAMP-1 significantly increased the activities of serum lysozyme, superoxide dismutase and alkaline phosphatase in black sea bream (P<0.05) in a dose-dependent manner, indicating that BdAMP-1 can effectively enhance the non-specific immune function of black sea bream.

[0095] 3.4 Immersion Challenge Experiment

[0096] After 21 days of feeding, the remaining black sea bream from each group underwent artificial infection experiments. The natural infection route was simulated using an immersion method: Vibrio alginolyticus (ATCC 17749) was inoculated into MH broth, incubated at 30°C for 18 h, washed with sterile seawater and resuspended, and the bacterial concentration was adjusted to 1×10⁻⁶. 8 CFU / mL. Each group of black sea bream was placed in a culture tank containing the bacterial solution for 60 minutes, with continuous oxygenation and a water temperature maintained at 24±1℃ during the immersion period. After immersion, the fish were transferred to a clean culture system and fed the corresponding experimental diet.

[0097] The fish were observed for 14 consecutive days, with daily records of morbidity and mortality. Dead fish were dissected, and bacteria were isolated from the liver and kidneys for PCR identification, confirming the cause of death as Vibrio alginolyticus infection. Symptoms were also recorded, and the cumulative morbidity rate was calculated using the following formula:

[0098] Cumulative morbidity rate (%) = (Cumulative number of diseased fish / Total number of experimental fish) × 100%

[0099] Table 3

[0100]

[0101] From Table 3 and Figure 4 The results showed that on day 3 after infection with the virus, diseased individuals began to appear in the control group, with a sharp increase in incidence on day 5, reaching a cumulative incidence of 81.5% by day 14. In contrast, the incidence rates in the low-dose and high-dose groups were significantly lower, with cumulative incidence rates of 44.4% and 29.6% respectively on day 14. The results indicated that the incidence rates in both the low-dose and high-dose groups were significantly lower than those in the control group (P<0.01), and the incidence rate in the high-dose group was significantly lower than that in the low-dose group (P<0.05). This demonstrates that adding BdAMP-1 to the feed can significantly reduce the incidence rate after infection with *Vibrio alginolyticus* in black sea bream, effectively preventing bacterial diseases in black sea bream, and exhibiting a dose-response relationship within a certain range.

[0102] 3.5 Growth performance indicators

[0103] At the beginning and end of the feeding period, each group of black sea bream was weighed on an empty stomach to calculate the weight gain rate (WGR) and specific growth rate (SGR). The growth performance indicators for each group are shown in Table 4.

[0104] Table 4: Growth performance indicators of black sea bream in different treatment groups

[0105] control group 20.1 ± 1.8 35.8 ± 2.5 78.3 ± 6.2 2.78 ± 0.21 low-dose group 20.0 ± 1.9 36.4 ± 2.3 82.1 ± 5.7 2.89 ± 0.19 High-dose group 20.2 ± 1.7 37.3 ± 2.6 84.5 ± 6.1 2.95 ± 0.20

[0106] As shown in Tables 4 and 5, the weight gain rates of the low-dose group and the high-dose group were 82.1% and 84.5%, respectively, and the specific growth rates were 2.89% / d and 2.95% / d, respectively, which were slightly higher than those of the control group (78.3% and 2.78% / d). However, statistical analysis showed no significant difference in weight gain rate and specific growth rate between the experimental groups and the control group (P>0.05), indicating that the addition of BdAMP-1 to the feed had no adverse effect on the normal growth of black sea bream and had good application safety.

[0107] This invention obtains an endogenous antimicrobial peptide, BdAMP-1, from the tissues of black sea bream. This antimicrobial peptide, BdAMP-1, has strong inhibitory and killing activity against the main pathogenic bacteria of black sea bream. When fed as a feed additive, it can significantly enhance the non-specific immunity of black sea bream, improve the survival rate of black sea bream, and has no negative impact on the growth of the fish. It is a safe, efficient, and residue-free antibiotic alternative.

Claims

1. A polypeptide having antibacterial activity, characterized in that, The polypeptide is prepared by cutting black sea bream tissue material into small pieces, adding extraction buffer to homogenize, and then purifying the homogenate by ultrafiltration fractionation, gel filtration chromatography and reversed-phase high-performance liquid chromatography.

2. The polypeptide according to claim 1, characterized in that, The amino acid sequence of the polypeptide is SEQ ID NO:

1.

3. The application of the polypeptide reprocessing method according to claim 1 in the preparation of antibacterial products.

4. The application as described in claim 3, characterized in that, The antibacterial product is an antibacterial product against Vibrio alginolyticus, Vibrio harveyi, Vibrio vulnificus, and Aeromonas hydrophila.

5. The application of the polypeptide reprocessing feed additive according to claim 1.

6. A feed additive, characterized in that, The feed additive contains the polypeptide of claim 1 and a feed-acceptable carrier.

7. The feed additive as described in claim 6, characterized in that, The carrier comprises microcrystalline cellulose, vitamin C, and sodium alginate.

8. A fish feed, characterized in that, The fish feed contains the feed additive described in claim 6.

9. The fish feed as described in claim 8, characterized in that, The fish feed mentioned is black sea bream feed.

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