Antimicrobial peptides and their preparation and application

Abstract

The present invention relates to an antimicrobial polypeptide and its preparation and application. The amino acid sequence of the antimicrobial polypeptide is shown in SEQ ID No.1. The preparation method of the antimicrobial polypeptide comprises the step of fermenting the shell of Procambarus clarkii with Candida tropicalis and Bacillus licheniformis. The antimicrobial peptide provided by the present invention is prepared by fermenting the shell of Procambarus clarkii with Candida tropicalis and Bacillus licheniformis, which can effectively inhibit Vibrio. It can be added as a feed additive to the basic feed for aquaculture, which can effectively reduce the harm caused by Vibrio to farmed animals. In addition, the inventors have found that the antimicrobial peptide of the present invention also has a significant inhibitory effect on other Gram-negative bacteria, such as Edwardsiella and Streptococcus agalactiae, and can also be used for the prevention and treatment of diseases caused by other Gram-negative bacteria.

Description

Technical Field

[0001] The invention relates to the technical field of aquaculture, and relates to an antimicrobial peptide and a preparation and application thereof. Background Art

[0002] Epidemic and explosive deaths of economic animals such as marine farmed fish, shellfish and crustaceans are mostly caused by Vibrio, including Vibrio parahaemolyticus, Vibrio splendidus, Vibrio anguillarum, Vibrio alginolyticus, Vibrio harveyi, Vibrio cholerae, Vibrio vulnificus, Aeromonas hydrophila, etc. Vibrio disease causes huge economic losses to the aquaculture industry, and the disease can also lead to the death of wild marine fish, shellfish and crustaceans, thus threatening marine natural biological resources.

[0003] At present, the use of antibiotics is still an important means to solve Vibrio disease in aquatic animals. It can have a good therapeutic effect in the short term and greatly reduce the economic losses of the aquaculture industry. However, due to the widespread and long-term use of antibiotics in disease prevention and control, the types and numbers of drug-resistant Vibrio continue to increase, posing a hidden danger to human food safety.

[0004] It is reported that although there are many types of antimicrobial peptides from bacteria, plants, insects, fish, mollusks, crustaceans, amphibians, and mammals, their antibacterial effects are not ideal. Therefore, how to provide an effective means for inhibiting Vibrio is a technical problem that needs to be solved urgently. Summary of the invention

[0005] Based on the above background technology, the main purpose of the present invention is to provide an antibacterial polypeptide, which can effectively inhibit Vibrio.

[0006] The purpose of the present invention can be achieved through the following technical solutions:

[0007] An antibacterial polypeptide, the amino acid sequence of the antibacterial polypeptide is shown as SEQ ID No.1.

[0008] The method for preparing the antibacterial polypeptide as described above comprises the step of fermenting the shell of Procambarus clarkii with Candida tropicalis and Bacillus licheniformis.

[0009] In one embodiment, the Candida tropicalis comprises Candida tropicalis ACCC20005.

[0010] In one embodiment, the Bacillus licheniformis comprises Bacillus licheniformis ACCC11091.

[0011] In one embodiment, the preparation method comprises: preparing a fermentation medium with Procambarus clarkii shells, and inoculating the tropical yeast and Bacillus licheniformis into the fermentation medium for fermentation.

[0012] In one embodiment, the step of preparing the fermentation medium comprises:

[0013] Obtaining the shell powder of Procambarus clarkii, mixing it with water, homogenizing it, and sterilizing it to prepare a shell homogenate solution;

[0014] mixing the chitosan homogenate and the culture matrix to prepare the fermentation medium;

[0015] The culture medium contains peptone, yeast extract, sodium chloride, glucose and water.

[0016] In one embodiment, the amount of the chitin powder corresponding to 1L of the chitin homogenate liquid is 80g to 120g.

[0017] In one embodiment, the homogenization conditions include: temperature of 75°C to 85°C, pressure of 20MPa to 30MPa, rotation speed of 2500rpm to 3500rpm, and duration of 5min to 15min.

[0018] In one embodiment, the amount of the chitin homogenate solution used per 1L of the culture matrix is ​​80mL to 120mL.

[0019] In one embodiment, each 1 L of the culture matrix contains 12 g to 18 g of the peptone, 4.5 g to 5.5 g of the yeast extract, 4.5 g to 5.5 g of the sodium chloride and 12 g to 18 g of the glucose.

[0020] In one embodiment, the shell powder is shell powder that has passed through a 250-350 mesh sieve.

[0021] In one embodiment, the fermentation conditions include: a temperature of 28°C to 32°C and a fermentation time of 40h to 50h.

[0022] In one embodiment, during the fermentation process, the inoculation concentration of the tropical Candida is (1-3)×10 10 cfu / mL, or / and the inoculation concentration of the Bacillus licheniformis is (2-4)×10 10 cfu / mL.

[0023] In one embodiment, the preparation method further comprises a step of purifying the fermentation product obtained by fermentation, and the purification step comprises centrifuging the fermentation product and ultrafiltering the obtained supernatant.

[0024] In one embodiment, the preparation method further comprises the step of drying the filtrate obtained by ultrafiltration, and the drying method is spray drying.

[0025] A feed comprising a feed additive and a basic feed, wherein the feed additive comprises the antimicrobial peptide as described above.

[0026] In one embodiment, each 1 kg of the feed contains 3 g to 8 g of the antimicrobial peptide.

[0027] Compared with the prior art, the present invention has the following beneficial effects:

[0028] The present invention provides an antimicrobial peptide, which is prepared by fermenting the carapace of Procambarus clarkii with Candida tropicalis and Bacillus licheniformis, and can effectively inhibit Vibrio. It is added to the basic feed as a feed additive for aquaculture, which can effectively reduce the harm caused by Vibrio to farmed animals. In addition, the inventors found that the antimicrobial peptide of the present invention also has a significant inhibitory effect on other Gram-negative bacteria (such as Edwardsiella and Streptococcus agalactiae), and can also be used for the prevention and treatment of diseases caused by other Gram-negative bacteria. The antimicrobial peptide provided by the present invention can increase the permeability of bacterial cell membranes, making it easier to enter the bacterial body, thereby acting on the target point inside the bacteria. BRIEF DESCRIPTION OF THE DRAWINGS

[0029] In order to more clearly illustrate the specific implementation methods of the present invention or the technical solutions in the prior art, the drawings required for use in the specific implementation methods or the description of the prior art will be briefly introduced below. Obviously, the drawings described below are some implementation methods of the present invention. For ordinary technicians in this field, other drawings can be obtained based on these drawings without paying creative work.

[0030] Figure 1 This is a picture of a breeding pond where the breeding effect of the antimicrobial peptide obtained in one embodiment of the present invention is verified;

[0031] Figure 2 This is an anatomical diagram of a cultured pond mullet using the antimicrobial peptide obtained in one embodiment of the present invention. DETAILED DESCRIPTION

[0032] For ease of understanding of the present invention, the present invention will be described in more detail below. However, it should be understood that the present invention can be implemented in many different forms and is not limited to the embodiments or examples described herein. On the contrary, the purpose of providing these embodiments or examples is to make the understanding of the disclosure of the present invention more thorough and comprehensive.

[0033] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as those generally understood by those skilled in the art of the present invention. The terms used herein in the specification of the present invention are only for the purpose of describing specific implementation or embodiment, and are not intended to limit the present invention. The optional scope of the term "and / or" used herein includes any one of two or more related listed items, and also includes any and all combinations of related listed items, and the combinations include any two related listed items, any more related listed items, or all related listed items.

[0034] In the present invention, “first aspect”, “second aspect”, “third aspect”, etc. are used for descriptive purposes only and cannot be understood as indicating or implying relative importance or quantity, nor can they be understood as implicitly indicating the importance or quantity of the indicated technical features.

[0035] In the present invention, the technical features described in an open manner include closed technical solutions composed of the listed features, and also include open technical solutions containing the listed features.

[0036] In the present invention, reference to a numerical range includes both endpoints of the numerical range unless otherwise specified.

[0037] The percentage content involved in the present invention, unless otherwise specified, refers to mass percentage for solid-liquid mixing and solid-solid mixing, and refers to volume percentage for liquid-liquid mixing.

[0038] The percentage concentrations involved in the present invention, unless otherwise specified, refer to the final concentration. The final concentration refers to the proportion of the added component in the system after the addition of the component.

[0039] The temperature parameters in the present invention, if not specifically limited, allow for both constant temperature treatment and treatment within a certain temperature range. The constant temperature treatment allows the temperature to fluctuate within the precision range controlled by the instrument.

[0040] The Procambarus clarkii in the present invention, commonly known as crayfish, can live in an environment with poor water quality and has a low prevalence of disease. Even if the disease occurs, the appearance of the crayfish is still intact. With the increase in market demand in recent years, the breeding volume of Procambarus clarkii (crayfish) has also increased year by year. The crayfish itself can tolerate rough feeding and rough breeding, which proves that the crayfish body itself, especially the body surface, has the ability to resist bacterial diseases. Therefore, the present invention extracts antibacterial substances from the surface of its carapace to kill Gram-negative bacteria including Vibrio parahaemolyticus, Edwardsiella, Aeromonas hydrophila, etc., to replace the use of antibiotics and ensure the food safety of aquatic animals.

[0041] The present invention uses the unusable shell of Procambarus clarkii as raw material, ensuring sufficient raw materials for preparing antimicrobial peptide products, preventing and treating diseases such as hepatopancreatic atrophy, enteritis and sudden death of shrimps and crabs caused by Vibrio, improving the immunity of shrimps and crabs, preventing and treating diseases such as enteritis, body rot, nodules and the like caused by Aeromonas hydrophila, Edwardsiella and the like, and improving the health of fish bodies.

[0042] In a first aspect, the present invention provides an antibacterial polypeptide, the amino acid sequence of the antibacterial polypeptide is shown as SEQ ID No.1.

[0043] The antimicrobial peptide provided by the present invention is prepared by fermenting the crayfish shell with tropical Candida and Bacillus licheniformis, which can effectively inhibit Vibrio. It is added to the basic feed as a feed additive for aquaculture, which can effectively reduce the harm caused by Vibrio to farmed animals. In addition, the inventors found that the antimicrobial peptide of the present invention also has a significant inhibitory effect on other Gram-negative bacteria (such as Edwardsiella and Streptococcus agalactiae), and can also be used for the prevention and treatment of diseases caused by other Gram-negative bacteria. The antimicrobial peptide provided by the present invention can increase the permeability of bacterial cell membranes, making it easier to enter the bacteria, thereby acting on the target points inside the bacteria.

[0044] In a second aspect, the present invention provides a method for preparing the antimicrobial polypeptide, comprising the step of fermenting the shell of Procambarus clarkii with Candida tropicalis and Bacillus licheniformis.

[0045] In one example, the tropical Candida includes tropical Candida ACCC20005, which can be purchased from Shanghai Yansheng Biochemical Reagent Co., Ltd.

[0046] In one example, the Bacillus licheniformis includes Bacillus licheniformis ACCC11091, which can be purchased from Shanghai Xuanya Biotechnology Co., Ltd.

[0047] In one example, the preparation method includes: preparing a fermentation medium using Procambarus clarkii shells, and inoculating the tropical yeast and Bacillus licheniformis into the fermentation medium for fermentation.

[0048] In one example, the steps of preparing the fermentation medium include:

[0049] Obtaining the shell powder of Procambarus clarkii, mixing it with water, homogenizing it, and sterilizing it to prepare a shell homogenate solution;

[0050] mixing the chitosan homogenate and the culture matrix to prepare the fermentation medium;

[0051] The culture medium contains peptone, yeast extract, sodium chloride, glucose and water.

[0052] In one example, the amount of the shell powder corresponding to 1L of the shell homogenate liquid is 80g to 120g, for example, the amount of the shell powder corresponding to 1L of the shell homogenate liquid is 80g, 85g, 90g, 95g, 100g, 105g, 110g, 115g, or 120g.

[0053] In the process of preparing the chitosan homogenate liquid, the present invention does not specifically limit the type of water used, for example, distilled water may be used.

[0054] In the process of preparing the chitosan homogenate liquid, the present invention does not particularly limit the sterilization method, for example, pasteurization can be used.

[0055] In one example, the homogenization conditions include: temperature of 75°C to 85°C, pressure of 20MPa to 30MPa, rotation speed of 2500rpm to 35000rpm, and duration of 5min to 15min. For example, homogenization at 75°C, 30MPa and 2500rpm for 15min, homogenization at 85°C, 20MPa and 3500rpm for 5min, and homogenization at 80°C, 25MPa and 3000rpm for 10min.

[0056] In one example, the amount of the chitosan homogenate solution per 1L of the culture matrix is ​​80mL to 120mL. For example, the amount of the chitosan homogenate solution per 1L of the culture matrix is ​​80mL, 85mL, 90mL, 95mL, 100mL, 105mL, 110mL, 115mL, or 120mL.

[0057] In one example, each 1L of the culture matrix contains 12g to 18g of the peptone, 4.5g to 5.5g of the yeast extract, 4.5g to 5.5g of the sodium chloride, and 12g to 18g of the glucose. For example: each 1L of the culture matrix contains 12g of the peptone, 5.5g of the yeast extract, 4.5g of the sodium chloride, and 18g of the glucose; each 1L of the culture matrix contains 18g of the peptone, 5.5g of the yeast extract, 4.5g of the sodium chloride, and 12g of the glucose; each 1L of the culture matrix contains 15g of the peptone, 5g of the yeast extract, 5g of the sodium chloride, and 15g of the glucose.

[0058] The present invention does not particularly limit the type of water in the culture matrix, for example, deionized water may be used.

[0059] In one example, the shell powder is shell powder that has passed through a 250-mesh sieve to a 350-mesh sieve. For example, shell powder that has passed through a 250-mesh sieve, shell powder that has passed through a 300-mesh sieve, and shell powder that has passed through a 350-mesh sieve. It is understood that the shell powder of the present invention can be purchased or prepared.

[0060] In one example, the fermentation conditions include: a temperature of 28°C to 32°C and a duration of 40h to 50h. For example: fermentation at 28°C for 50h, fermentation at 32°C for 40h, and fermentation at 30°C for 48h.

[0061] In one example, during the fermentation process, the inoculation concentration of the tropical yeast is (1-3)×10 10 cfu / mL, for example, the inoculation concentration is 1×10 10 cfu / mL, 2×10 10 cfu / mL, 3×10 10 cfu / mL.

[0062] In one example, during the fermentation process, the inoculation concentration of the Bacillus licheniformis was (2-4)×10 10 cfu / mL. For example, the inoculation concentration is 2×10 10 cfu / mL, 3×10 10 cfu / mL, 4×10 10 cfu / mL.

[0063] In one example, the preparation method further includes a step of purifying the fermentation product obtained by fermentation, and the purification step includes centrifuging the fermentation product and ultrafiltration (0.01 μm filter membrane pore size) of the obtained supernatant. Further, the centrifugal conditions can be controlled at 10000rpm to 30000rpm, 10min to 30min, for example: centrifugation at 20000rpm for 20min, at 10000rpm for 30min, and at 30000rpm for 25min.

[0064] In one example, the preparation method further includes the step of drying the filtrate obtained by ultrafiltration, and the drying method is spray drying.

[0065] In a third aspect, the present invention provides a feed, comprising a feed additive and a basic feed, wherein the feed additive comprises the antimicrobial peptide.

[0066] In one example, the feed contains 3 to 8 g of the antimicrobial peptide per kg of feed, for example, 3 g of the antimicrobial peptide per kg of feed, 5 g of the antimicrobial peptide per kg of feed, 7 g of the antimicrobial peptide per kg of feed, and 8 g of the antimicrobial peptide per kg of feed.

[0067] The feed of the present invention may be fish feed, for example, feed for mullet farming.

[0068] Example 1. Antimicrobial peptides and their preparation

[0069] This embodiment provides an antimicrobial peptide and a preparation method thereof.

[0070] The preparation method of the antimicrobial peptide comprises the following steps:

[0071] (1) ultrasonically cleaning, drying, and crushing (passing through a 300-mesh sieve) the carapace of a Procambarus clarkii to obtain carapace powder;

[0072] (2) 100 g of shell powder was mixed with 1 L of distilled water, and homogenized and emulsified at 80° C. and 25 MPa for 10 min at 3000 rpm. After pasteurization, a shell homogenate liquid was obtained;

[0073] (3) mixing the chitosan homogenate and the culture matrix to prepare a fermentation medium, inoculating Candida tropicalis ACCC20005 and Bacillus licheniformis ACCC11091, and fermenting at 30° C. for 48 h to obtain a fermentation broth;

[0074] The inoculation concentration of tropical Candida was 2×10 10 cfu / mL, the concentration of Bacillus licheniformis was 3×10 10 cfu / mL; formula of culture medium: peptone 15g, yeast extract 5g, NaCl 5g, glucose 15g, deionized water to 1L;

[0075] (4) The fermentation broth was centrifuged at 20,000 rpm for 20 min, the supernatant was separated by membrane ultrafiltration and concentrated, and then spray-dried at 120° C. to obtain the antimicrobial peptide raw powder.

[0076] The antibacterial peptide obtained in this example was determined by acid hydrolysis by HPLC to consist of 26 amino acids. The sequence order was determined based on the elution peaks as shown in SEQ ID No. 1: tyr gln gly gly val ser ile tyr val ser aspasn his val ser leu asp pro ser ser gln ser tyr ile gly gly (i.e., YQGGVSIYVSDNHVSLDPSSQSYIGG).

[0077] Example 2: Verification of the antibacterial effect of antimicrobial peptides

[0078] The antibacterial activity of the antimicrobial peptide (100 mg / ml) obtained in Example 1 against four strains (Aeromonas hydrophila, Edwardsiella, Streptococcus agalactiae, and Vibrio parahaemolyticus) was verified by agar diffusion method (inhibition zone method).

[0079] At the same time, a blank group (numbered "A" in Table 1) and an antibiotic group (numbered "B" in Table 1) were set up, wherein the blank group used distilled water; the antibiotic was florfenicol (the concentration of the antimicrobial peptide was 100 μg / mL).

[0080] The results are shown in Table 1:

[0081] Table 1. Diameter of inhibition zone (mm)

[0082] serial number sample Aeromonas hydrophila Edwardsiella Streptococcus agalactiae Vibrio parahaemolyticus A Blank group - - - - B Antimicrobial peptides 8.39 8.73 6.48 14.01 C antibiotic 14.12 - 12.09 22.50

[0083] The results in Table 1 show that the antimicrobial peptides obtained in the present invention have obvious antibacterial effects on the four strains.

[0084] Example 3: Verification of breeding effect

[0085] Due to the high density of breeding, it is easy for water pollution to cause bacterial infection during the breeding process. Figure 1 ) encountered death, and after autopsy and testing, it was found that the death was caused by infection caused by Aeromonas hydrophila ( Figure 2 ). Four ponds with more serious mortality were selected and divided into two groups:

[0086] The experimental group used the antimicrobial peptide of Example 1 mixed with feed, with a dosage of 5 g / kg feed (i.e., 5 g of antimicrobial peptide was mixed into 1 kg of feed), twice a day, for 6 consecutive days;

[0087] The control group used 10% enrofloxacin 2g / kg feed (i.e. 1kg feed mixed with 2g of 10w / w% enrofloxacin) twice a day for 6 consecutive days according to the farmers' habits;

[0088] The number of deaths was recorded from the first day of use, and the results are shown in Table 2:

[0089] Table 2. Statistics of daily mortality of mullets in each group

[0090] Group Day 1 Day 2 Day 3 Day 4 Day 5 Day 6 Experimental Group 1 128 135 102 76 55 28 Experimental Group 2 135 128 98 64 47 25 Control group 1 124 115 88 54 33 26 Control group 2 130 112 85 55 47 25 Blank control group 146 148 143 139 141 138

[0091] From the perspective of the number of deaths, both groups of products have good control over the number of deaths, indicating that the antimicrobial peptide product can be used to treat Aeromonas hydrophila in pond mullets, with an effect close to that of antibiotics, and can serve as a substitute.

[0092] Example 4. Antimicrobial peptides and their preparation

[0093] This embodiment provides an antimicrobial peptide and a preparation method thereof.

[0094] The preparation method of the antimicrobial peptide comprises the following steps:

[0095] (1) ultrasonically cleaning, drying, and crushing (passing through a 350-mesh sieve) the carapace of a Procambarus clarkii to obtain carapace powder;

[0096] (2) 80 g of shell powder was mixed with 1 L of distilled water, and homogenized and emulsified at 2500 rpm for 15 min at a temperature of 75° C. and a pressure of 30 MPa, and then pasteurized to obtain a shell homogenate liquid;

[0097] (3) mixing the chitosan homogenate and the culture matrix to prepare a fermentation medium, inoculating Candida tropicalis ACCC20005 and Bacillus licheniformis ACCC11091, and fermenting at 28°C for 50 h to obtain a fermentation broth;

[0098] The inoculation concentration of tropical Candida was 1×10 10 cfu / mL, the concentration of Bacillus licheniformis was 2×10 10 cfu / mL; culture matrix formula: 18g peptone, 5.5g yeast extract, 4.5g sodium chloride, 12g glucose, dilute to 1L with deionized water;

[0099] (4) The fermentation broth was centrifuged at 10,000 rpm for 30 min, the supernatant was separated by membrane ultrafiltration and concentrated, and then spray-dried at 120° C. to obtain the antimicrobial peptide raw powder.

[0100] In this example, an antimicrobial peptide with an amino acid sequence as shown in SEQ ID No. 1 was obtained.

[0101] Example 5. Antimicrobial peptides and their preparation

[0102] This embodiment provides an antimicrobial peptide and a preparation method thereof.

[0103] The preparation method of the antimicrobial peptide comprises the following steps:

[0104] (1) ultrasonically cleaning, drying, and crushing (passing through a 250-mesh sieve) the carapace of a Procambarus clarkii to obtain carapace powder;

[0105] (2) 100 g of shell powder was mixed with 1 L of distilled water, and homogenized and emulsified at 85° C. and 20 MPa for 5 min at 3500 rpm. After pasteurization, a shell homogenate liquid was obtained;

[0106] (3) mixing the chitosan homogenate and the culture matrix to prepare a fermentation medium, inoculating Candida tropicalis ACCC20005 and Bacillus licheniformis ACCC11091, and fermenting at 32° C. for 40 h to obtain a fermentation broth;

[0107] The inoculation concentration of tropical Candida was 3×10 10 cfu / mL, the concentration of Bacillus licheniformis was 4×10 10 cfu / mL; culture matrix formula: 12g peptone, 5.5g yeast extract, 4.5g sodium chloride, 18g glucose, dilute to 1L with deionized water;

[0108] (4) The fermentation broth was centrifuged at 30,000 rpm for 25 min, the supernatant was separated by membrane ultrafiltration and concentrated, and then spray-dried at 120° C. to obtain the antimicrobial peptide raw powder.

[0109] In this example, an antimicrobial peptide with an amino acid sequence as shown in SEQ ID No. 1 was obtained.

[0110] In summary, the antimicrobial peptide provided by the present invention is prepared by fermenting the crayfish shell with tropical Candida and Bacillus licheniformis, which can effectively inhibit Vibrio, and can be added to the basic feed as a feed additive for aquaculture, which can effectively reduce the harm caused by Vibrio to farmed animals. In addition, the inventors found that the antimicrobial peptide of the present invention also has a significant inhibitory effect on other Gram-negative bacteria (such as Edwardsiella and Streptococcus agalactiae), and can also be used for the prevention and treatment of diseases caused by other Gram-negative bacteria.

[0111] The technical features of the above-described embodiments may be arbitrarily combined. To make the description concise, not all possible combinations of the technical features in the above-described embodiments are described. However, as long as there is no contradiction in the combination of these technical features, they should be considered to be within the scope of this specification.

[0112] The above-described embodiments only express several implementation methods of the present invention, which are convenient for understanding the technical solutions of the present invention in detail, but they cannot be understood as limiting the scope of protection of the invention patent. It should be pointed out that, for ordinary technicians in this field, several variations and improvements can be made without departing from the concept of the present invention, which all belong to the protection scope of the present invention. It should be understood that the technical solutions obtained by those skilled in the art through logical analysis, reasoning or limited experiments on the basis of the technical solutions provided by the present invention are all within the protection scope of the attached claims described in the present invention. Therefore, the protection scope of the patent of the present invention shall be based on the contents of the attached claims, and the description and drawings can be used to interpret the contents of the claims. Sequence Listing <110> Zhuhai Yulaiyuwang Biotechnology Co., Ltd. Guangdong Nutriera Group Co., Ltd. <120> Antimicrobial peptides and their preparation and application <160> 1 <170> SIPO Sequence Listing 1.0 <210> 1 <211> 26 <212> PRT <213> Procambarus clarkii <400> 1 Tyr Gln Gly Gly Val Ser Ile Tyr Val Ser Asp Asn His Val Ser Leu 1 5 10 15 Asp Pro Ser Ser Gln Ser Tyr Ile Gly Gly 20 25

Claims

1. An antimicrobial polypeptide, characterized in that: The amino acid sequence of the antibacterial polypeptide is shown in SEQ ID No.

1.

2. The method for preparing the antimicrobial polypeptide according to claim 1, characterized in that: The preparation method comprises the steps of fermenting the carapace of Procambarus clarkii with Candida tropicalis and Bacillus licheniformis; The tropical Candida is tropical Candida ACCC20005, and the Bacillus licheniformis is Bacillus licheniformis ACCC11091; The preparation method comprises: preparing a fermentation medium with the shell of Procambarus clarkii, inoculating the tropical Candida and Bacillus licheniformis into the fermentation medium for fermentation; The steps of preparing the fermentation medium include: Obtaining the shell powder of Procambarus clarkii, mixing it with water, homogenizing it, and sterilizing it to prepare a shell homogenate solution; mixing the chitosan homogenate and the culture matrix to prepare the fermentation medium; The culture medium comprises peptone, yeast extract, sodium chloride, glucose and water; The homogenization conditions include: temperature of 75°C to 85°C, pressure of 20MPa to 30MPa, rotation speed of 2500rpm to 3500rpm, and duration of 5min to 15min.

3. The method for preparing the antimicrobial polypeptide according to claim 2, characterized in that: The amount of the shell powder corresponding to each 1L of the shell homogenized liquid is 80g to 120g.

4. The method for preparing the antimicrobial polypeptide according to claim 2, characterized in that: The amount of the chitin homogenate solution used per 1L of the culture matrix is ​​80mL to 120mL.

5. The method for preparing the antimicrobial polypeptide according to claim 2, characterized in that: Each 1L of the culture matrix contains 12g to 18g of the peptone, 4.5g to 5.5g of the yeast extract, 4.5g to 5.5g of the sodium chloride and 12g to 18g of the glucose.

6. The method for preparing the antimicrobial polypeptide according to claim 2, characterized in that: The shell powder is shell powder that has passed through a 250-mesh sieve to a 350-mesh sieve.

7. The method for preparing an antimicrobial polypeptide according to any one of claims 2 to 6, characterized in that: The fermentation conditions include: temperature of 28°C to 32°C and duration of 40h to 50h.

8. The method for preparing an antimicrobial polypeptide according to any one of claims 2 to 6, characterized in that: During the fermentation process, the inoculation concentration of tropical Candida is (1-3)×10 10 cfu / mL.

9. The method for preparing an antimicrobial polypeptide according to any one of claims 2 to 6, characterized in that: The inoculation concentration of the Bacillus licheniformis is (2-4)×10 10 cfu / mL.

10. The method for preparing the antimicrobial polypeptide according to any one of claims 2 to 6, characterized in that: The preparation method further comprises a step of purifying the fermentation product obtained by fermentation, wherein the purification step comprises centrifuging the fermentation product and ultrafiltering the obtained supernatant.

11. The method for preparing the antimicrobial polypeptide according to claim 10, characterized in that: The preparation method further comprises the step of drying the filtrate obtained by ultrafiltration, and the drying method is spray drying.

12. A feed, characterized in that: The feed comprises a feed additive and a basal feed, and the feed additive comprises the antimicrobial peptide according to claim 1.

13. The feed according to claim 12, characterized in that Each 1kg of the feed contains 3g to 8g of the antimicrobial peptide.

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