Alligator blood-derived antibacterial peptide, low-temperature enzymatic preparation method and application thereof

Abstract

The application belongs to the field of animal source antibacterial peptides, and discloses a crocodile blood source antibacterial peptide and a low-temperature enzymolysis preparation method and application thereof, the molecular weight of the crocodile blood source antibacterial peptide is 2.8+ / -0.2 kDa, and the isoelectric point is 8.8-9; the preparation method comprises the steps of raw material pretreatment, low-temperature homogenization, low-temperature enzymolysis, ultrafiltration purification, concentration, pre-freezing and freeze-drying; the process is mild, the activity retention rate is high, the obtained antibacterial peptide has strong inhibitory activity on staphylococcus aureus, escherichia coli and candida albicans, and can be used for preparing medicines, food preservatives and the like.

Description

Technical Field

[0001] This invention belongs to the field of animal-derived antimicrobial peptides, specifically relating to an antimicrobial peptide derived from crocodile blood, its low-temperature enzymatic hydrolysis preparation method, and its application. Background Technology

[0002] Crocodile-derived antimicrobial peptides refer to polypeptides with antimicrobial activity extracted from crocodile blood or other tissues or obtained through enzymatic hydrolysis. They are widely used in pharmaceuticals, food preservation, animal feed additives, and cosmetics. As an ancient species, crocodiles possess unique immune-active components in their blood. Common preparation methods include direct extraction of natural antimicrobial peptides or enzymatic hydrolysis of blood proteins followed by separation and purification to obtain active peptide products. These antimicrobial peptides typically exhibit broad-spectrum antimicrobial activity, inhibiting Gram-positive bacteria, Gram-negative bacteria, and some fungi, with a low risk of developing drug resistance.

[0003] The preparation of antimicrobial peptides often employs enzymatic hydrolysis of animal blood or protein raw materials. This method is low-cost and uses widely available raw materials, but it generally suffers from process limitations. Conventional high-temperature or crude enzymatic hydrolysis processes easily disrupt the spatial conformation of peptides, resulting in low retention of antimicrobial activity; the separation and purification process is complex and costly, making it difficult to scale up industrially; the resulting products have poor stability, are easily inactivated at room temperature, have poor solubility, and often carry the off-flavor of the raw materials, limiting practical applications.

[0004] Existing publicly available crocodile-derived antimicrobial peptides generally suffer from a wide molecular weight distribution and a narrow antimicrobial spectrum. Due to the non-specificity of protease hydrolysis, the resulting products are often complex mixtures of peptide segments of varying lengths, with molecular weights ranging from hundreds to tens of thousands of Daltons. This wide distribution leads to a low proportion of effective active ingredients and high purification costs. Furthermore, these antimicrobial peptides often exhibit strong activity only against certain Gram-positive or Gram-negative bacteria, showing limited antimicrobial efficacy against fungi or other drug-resistant bacteria, making them ineffective against mixed infections. Summary of the Invention

[0005] The purpose of this invention is to provide an antimicrobial peptide derived from crocodile blood and its low-temperature enzymatic hydrolysis preparation method. The antimicrobial peptide has a stable source, high antibacterial activity, small molecular weight, and good storage stability.

[0006] Another objective of this invention is to provide the application of crocodile blood-derived antimicrobial peptides in the preparation of antimicrobial drugs.

[0007] To achieve the above objectives, the technical solution adopted by the present invention is as follows:

[0008] This invention provides an antimicrobial peptide derived from crocodile blood. The amino acid sequence of the antimicrobial peptide is shown in SEQ ID NO:1, the molecular weight is 2.8±0.2kDa, and the isoelectric point is 8.8-9.0.

[0009] This invention provides a low-temperature enzymatic hydrolysis method for preparing the above-mentioned crocodile blood-derived antimicrobial peptides, the low-temperature enzymatic hydrolysis method comprising the following steps:

[0010] 1) Mix crocodile whole blood with sterile purified water at a mass ratio of 1:3-5, and perform shearing homogenization and homogenization to obtain crocodile blood homogenate;

[0011] 2) Adjust the pH of the crocodile blood homogenate to 5.5-8.0, add the complex protease for low-temperature enzymatic hydrolysis, and after the reaction is completed, heat up to inactivate the enzyme, cool and centrifuge to collect the supernatant to obtain the crude antimicrobial peptide extract.

[0012] 3) The crude extract of antimicrobial peptides is subjected to ultrafiltration, the permeate is collected, and the permeate is concentrated under vacuum to obtain concentrated antimicrobial peptide solution;

[0013] 4) Add a freeze-drying protectant to the concentrated antimicrobial peptide solution and freeze-dry to obtain crocodile blood-derived antimicrobial peptides.

[0014] In step 1), the crocodile whole blood is derived from farmed adult Siamese crocodiles.

[0015] In step 1), the temperature of the shear homogenizer is 0-4℃, the processing time is 10-20 min, and the rotation speed is 8000-10000 r / min; the pressure of the homogenization process is 20-50 MPa, the temperature is 0-4℃, and the process is repeated 1-3 times.

[0016] In step 2), the enzymatic hydrolysis reaction is carried out at a temperature of 35-45°C for 12 hours. The complex protease is a mixture of neutral protease and papain, and the mass ratio of neutral protease to papain in the complex protease is 1:0.5-2.

[0017] In step 2), the temperature for inactivating the enzyme is 85-90℃, and the holding time is 10-15 min.

[0018] In step 3), the ultrafiltration pressure is 0.1-0.2 MPa; the vacuum concentration temperature is 30-50℃, and the vacuum degree is ≤0.15 MPa.

[0019] In step 4), the amount of the freeze-drying protectant added is 5%-10% of the volume of the concentrated antimicrobial peptide liquid, and the freeze-drying protectant is a mannitol ethanol solution with a mass fraction of 5%.

[0020] In step 4), the freeze drying includes a primary drying section and a secondary drying section. The primary drying section is characterized by a vacuum of 10-20 Pa, a temperature of -5-15℃, and a holding time of 12-24 h. The secondary drying section is characterized by a temperature of 15-30℃ and a holding time of 48 h.

[0021] The present invention also provides the application of the above-mentioned crocodile blood-derived antimicrobial peptides in the preparation of antimicrobial drugs, wherein the antimicrobial drugs are used to inhibit Staphylococcus aureus, Escherichia coli or Candida albicans.

[0022] Compared with the prior art, the present invention has the following advantages:

[0023] 1. To address the problem of low utilization rate of crocodile blood resources, this solution improves the release efficiency and recovery rate of functional peptides in blood by combining low-temperature enzymatic hydrolysis with ultrafiltration separation, thereby achieving high-value utilization of raw materials;

[0024] 2. To address the problem of antimicrobial peptide structure destruction and low activity retention caused by conventional high-temperature enzymatic hydrolysis, this method adopts low-temperature targeted enzymatic hydrolysis at 30-50℃ to reduce peptide spatial conformation destruction, thereby improving activity retention.

[0025] 3. To address the problem that existing processes are complex and difficult to scale up industrially, this solution adopts a composite protease system combined with ultrafiltration and freeze-drying processes to reduce complex chromatography steps, thereby simplifying the process and improving scalability; the preparation process only includes homogenization, enzymatic hydrolysis, ultrafiltration and freeze-drying, which has strong process continuity and is suitable for large-scale production;

[0026] 4. To address the issues of poor product stability and easy inactivation, this solution maintains the stability of the peptide structure by low-temperature concentration and the addition of mannitol as a freeze-drying protectant, thereby improving storage and usage stability.

[0027] 5. To address the issues of narrow antibacterial spectrum and unstable antibacterial effect, this method obtains small molecule antimicrobial peptides with good membrane action ability by controlling enzymatic hydrolysis conditions and molecular weight distribution. The obtained antimicrobial peptides show inhibitory effects on Staphylococcus aureus, Escherichia coli and Candida albicans, with inhibition zone diameter ≥15 mm. Attached Figure Description

[0028] Figure 1 This is a plate validation diagram showing the minimum inhibitory concentration (MIC) of crocodile blood-derived antimicrobial peptides against Staphylococcus aureus. Detailed Implementation

[0029] To make the objectives, technical solutions, and advantages of the embodiments of the present invention clearer, the technical solutions of the embodiments of the present invention will be clearly and completely described below in conjunction with the embodiments of the present invention. Obviously, the described embodiments are only some embodiments of the present invention, not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention.

[0030] Unless otherwise specified, all test materials and reagents used in the following examples are commercially available.

[0031] The amino acid sequence of the crocodile blood-derived antimicrobial peptide of this invention is as follows:

[0032] AAVCKVKKFVKKLKVGLKIIKGVLKII, as shown in SEQ ID NO:1.

[0033] Example 1

[0034] A method for preparing antimicrobial peptides from crocodile blood via low-temperature enzymatic hydrolysis, the method comprising the following steps:

[0035] 1) Mix crocodile whole blood with sterile purified water at a mass ratio of 1:3, and shear homogenize at 10000 rpm for 10 min at 4℃. Then, homogenize at 30 MPa and 4℃ for two cycles to obtain crocodile blood homogenate.

[0036] 2) Adjust the pH of the crocodile blood homogenate to 5.5, add 2% of the total mass of the crocodile blood homogenate with a complex protease, wherein the mass ratio of neutral protease to papain in the complex protease is 1:1, and carry out the enzymatic hydrolysis reaction at 35℃ for 12 hours. After the reaction is completed, raise the temperature to 85℃ to inactivate the enzyme, maintain for 10 minutes, cool, centrifuge and collect the supernatant to obtain the crude extract of antimicrobial peptides.

[0037] 3) The crude antimicrobial peptide extract was ultrafiltered using an ultrafiltration membrane with a molecular weight cutoff of 5 kDa. The ultrafiltration pressure was controlled at 0.15 MPa. The permeate was collected and concentrated under vacuum at 45°C and 0.08 MPa to obtain a highly active concentrated antimicrobial peptide solution.

[0038] 4) Add 5% (v / v) of 5 wt% mannitol ethanol solution as a freeze-drying protectant to the concentrated antimicrobial peptide solution. After thorough stirring and dissolution, place it in a freeze dryer and pre-freeze it to -45°C. Then perform a first drying at a vacuum of 10 Pa, a drying temperature of -5°C, and a drying time of 12 h. Perform a second drying at 15°C for 48 h to obtain crocodile blood-derived antimicrobial peptides. The molecular weight of the antimicrobial peptides is 2.8 kDa, and the isoelectric potential is 8.9.

[0039] Example 2

[0040] A method for preparing antimicrobial peptides from crocodile blood via low-temperature enzymatic hydrolysis, the method comprising the following steps:

[0041] 1) Mix crocodile whole blood with sterile purified water at a mass ratio of 1:4, and shear homogenize at 8000 rpm for 10 min at 4℃. Then, homogenize at 30 MPa and 4℃ for two cycles to obtain crocodile blood homogenate.

[0042] 2) Adjust the pH of the crocodile blood homogenate to 5.5, add 3% of the total mass of the crocodile blood homogenate with a complex protease, wherein the mass ratio of neutral protease to papain in the complex protease is 1:1, and carry out the enzymatic hydrolysis reaction at a temperature of 40℃ for 12 hours. After the reaction is completed, raise the temperature to 85℃ to inactivate the enzyme, maintain for 10 minutes, cool, centrifuge and collect the supernatant to obtain the crude extract of antimicrobial peptides.

[0043] 3) The crude antimicrobial peptide extract was ultrafiltered using an ultrafiltration membrane with a molecular weight cutoff of 5 kDa, and the ultrafiltration pressure was controlled at 0.15 MPa. The permeate was collected and concentrated under vacuum at 45°C and 0.15 MPa to obtain a highly active concentrated antimicrobial peptide solution.

[0044] 4) Add 7.5% (v / v) of 5wt% mannitol ethanol solution as a freeze-drying protectant to the concentrated antimicrobial peptide solution. After thorough stirring and dissolution, place it in a freeze dryer and pre-freeze it to -45℃. Then perform a first drying at a vacuum of 15Pa, a drying temperature of -10℃, and a drying time of 18h. Perform a second drying at 20℃ for 48h to obtain crocodile blood-derived antimicrobial peptides. The molecular weight of the antimicrobial peptides is 2.8kDa, and the isoelectric potential is 8.9.

[0045] Example 3

[0046] A method for preparing the antimicrobial peptide derived from crocodile blood at low temperature via enzymatic hydrolysis, the method comprising the following steps:

[0047] 1) Mix crocodile whole blood with sterile purified water at a mass ratio of 1:5, and shear homogenize at 9000 rpm for 10 min at 4℃. Then, homogenize at 30 MPa and 4℃ for two cycles to obtain crocodile blood homogenate.

[0048] 2) Adjust the pH of the crocodile blood homogenate to 5.5, add 4% of the total mass of the crocodile blood homogenate with a complex protease, wherein the mass ratio of neutral protease to papain in the complex protease is 1:1, and carry out the enzymatic hydrolysis reaction at a temperature of 45℃ for 12 hours. After the reaction is completed, raise the temperature to 85℃ to inactivate the enzyme, maintain for 10 minutes, cool, centrifuge and collect the supernatant to obtain the crude extract of antimicrobial peptides.

[0049] 3) The crude extract of antimicrobial peptides was ultrafiltered using an ultrafiltration membrane with a molecular weight cutoff of 5 kDa. The ultrafiltration pressure was controlled at 0.2 MPa. The permeate was collected and concentrated under vacuum at 45°C and 0.08 MPa to obtain a highly active concentrated antimicrobial peptide solution.

[0050] 4) Add 10% (v / v) of 5wt% mannitol ethanol solution as a freeze-drying protectant to the concentrated antimicrobial peptide solution. After thorough stirring and dissolution, place it in a freeze dryer and pre-freeze it to -45℃. Then perform a first drying at a vacuum of 20Pa, a drying temperature of -15℃, and a drying time of 24h. Perform a second drying at 30℃ for 48h to obtain crocodile blood-derived antimicrobial peptides. The molecular weight of the antimicrobial peptides is 2.8kDa, and the isoelectric potential is 8.9.

[0051] Comparative Example 1

[0052] A method for preparing an antimicrobial peptide derived from crocodile blood, specifically including the following steps:

[0053] The difference between this comparative example and Example 2 is that the temperature of the enzymatic hydrolysis reaction in step 2) is changed from 45°C to 55°C. The rest is the same as in Example 2. The molecular weight of the antimicrobial peptide is 2.97 kDa and the isoelectric potential is 8.8.

[0054] Comparative Example 2

[0055] A method for preparing an antimicrobial peptide derived from crocodile blood, specifically including the following steps:

[0056] The difference between this comparative example and Example 2 is that no freeze-drying protectant was added in step 4). The rest is the same as Example 2. The molecular weight of the antimicrobial peptide is 2.8 kDa and the isoelectric potential is 8.9.

[0057] Comparative Example 3

[0058] A method for preparing an antimicrobial peptide derived from crocodile blood, specifically including the following steps:

[0059] The difference between this comparative example and Example 2 is that step 3) is replaced by Sephadex G-25 gel chromatography with a column volume of 500 mL and a flow rate of 2 mL / min. The chromatographic solution is collected and concentrated under vacuum at 45°C and 0.15 MPa to obtain a highly active concentrated antimicrobial peptide solution. The rest is the same as in Example 2. The molecular weight of the antimicrobial peptide is 2.8 kDa and the isoelectric potential is 8.9.

[0060] Comparative Example 4

[0061] The comparative example is Alligatorin 6 from the Yangtze alligator, which is a single basic cationic antimicrobial peptide with a molecular weight of 3213.9 Da, published in Chinese Patent Publication No. 201410314114.3. It has inhibitory effects on Gram-positive and Gram-negative bacteria.

[0062] Comparative Example 5

[0063] This comparative example is the Siamese crocosin from the paper "Isolation and characterisation of crocosin, an antibacterial compound from crocodile (Crocodylus siamensis) plasma". It has a molecular weight >10 kDa and has inhibitory effects on Gram-positive and Gram-negative bacteria.

[0064] Test Example 1

[0065] Antibacterial activity assay

[0066] The antibacterial activity of the samples against Staphylococcus aureus, Escherichia coli and Candida albicans was determined by the Oxford cup agar diffusion method. The experimental method was performed in accordance with GB / T 39101-2020 "Determination of Antimicrobial Activity of Polypeptides - Inhibition Zone Method".

[0067] Minimum inhibitory concentration (MIC) determination

[0068] The minimum inhibitory concentrations of the samples against Staphylococcus aureus, Escherichia coli, and Candida albicans were determined using the micro-broth dilution method.

[0069] The minimum inhibitory concentration was determined by the microbroth dilution method, and the experimental method was performed in accordance with NY / T 4142-2022 "Technical Specification for Testing Antimicrobial Susceptibility of Animal-Derived Bacteria - Microbroth Dilution Method".

[0070] Table 1 shows the inhibition zone and minimum inhibitory concentration data for each embodiment and comparative example:

[0071] Table 1

[0072]

[0073] As can be seen from the data in Table 1, the crocodile blood-derived antimicrobial peptides prepared in each embodiment showed inhibitory effects on Staphylococcus aureus, Escherichia coli, and Candida albicans Gram-positive bacteria, Gram-negative bacteria, and fungi, with inhibition zone diameters ≥15 mm.

[0074] To visually verify the antibacterial effect of the antimicrobial peptides of this invention, the minimum inhibitory concentration of Staphylococcus aureus was visualized using the sample prepared in Example 2, employing the plate coating method. The results are as follows: Figure 1 As shown.

[0075] Depend on Figure 1It was observed that the negative control group plates were covered with Staphylococcus aureus colonies, while the gentamicin positive control group showed no visible colonies. When the concentration of the antimicrobial peptide of this invention was ≥1.2 mg / mL, the growth of Staphylococcus aureus was completely inhibited, and no visible colonies were observed on the plates. When the concentration was below 1.2 mg / mL, colonies began to appear on the plates, and the number of colonies gradually increased as the concentration of the antimicrobial peptide decreased. This result is consistent with the minimum inhibitory concentration determined by the microbroth dilution method in Table 1, further confirming the significant inhibitory activity of the antimicrobial peptide of this invention against Staphylococcus aureus.

[0076] Antimicrobial peptide yield and activity retention:

[0077] The yields and activity retention rates of each embodiment and comparative example were calculated using the following formula:

[0078] Yield = Mass of crocodile blood-derived antimicrobial peptides / Dry weight of crocodile whole blood × 100%;

[0079] Activity retention rate = Antimicrobial activity of crocodile blood-derived antimicrobial peptides against Staphylococcus aureus / Antimicrobial activity of crude antimicrobial peptide extract against Staphylococcus aureus × 100%;

[0080] Wherein, antibacterial activity = sample volume × (1 / MIC value)

[0081] The calculation results are shown in Table 2 below:

[0082] Table 2. Data on the yield and activity retention of antimicrobial peptides

[0083]

[0084] As can be seen from the data in Table 2, the antimicrobial peptides prepared by the method described in the examples all have an activity retention rate of over 90%.

[0085] Long-term storage stability:

[0086] The blood-derived antimicrobial peptides prepared in Example 2 and the comparative example were sealed in aluminum foil bags and stored at 25±2°C and 60%±5% relative humidity in the dark for 12 months. The MIC value against Staphylococcus aureus was measured monthly, and the activity loss rate was calculated. The activity loss rate data are shown in Table 3 below:

[0087] Table 3 Activity Loss Rate Data Table

[0088]

[0089] In Example 2 of this invention, the activity loss after 12 months of storage at room temperature was only 7.0%, which was significantly lower than that of Comparative Example 1 and Comparative Example 2, indicating that the synergistic effect of low temperature process and freeze-drying protectant significantly improved product stability.

[0090] As can be seen from Tables 1-3, in Comparative Example 1, the high temperature severely disrupted the spatial conformation of the antimicrobial peptide, resulting in a significant loss of its antimicrobial activity. The antimicrobial effect against Staphylococcus aureus, Escherichia coli, and Candida albicans was significantly weakened, and the minimum inhibitory concentration was much higher than in the examples.

[0091] Comparative Example 2, lacking a freeze-drying protectant, experienced partial destruction of the peptide structure during freeze-drying, resulting in a slight decrease in immediate activity and a significant deterioration in long-term stability. Although the immediate antibacterial activity was similar to that of Example 2, the activity loss was significantly greater after 12 months of storage.

[0092] Comparative Example 3 showed a significant loss of active peptides due to non-specific adsorption during gel chromatography, resulting in decreased antibacterial activity and a substantial increase in production costs. The antibacterial activity against all three strains was lower than in Examples 1-3, with a significantly increased minimum inhibitory concentration.

[0093] Hemolytic test:

[0094] Take 2% rabbit erythrocyte suspension, add 0.1, 0.5 and 1.0 mg / mL of the antimicrobial peptides described in Comparative Example 1 and Example 2 respectively, incubate at 37°C for 1 h, measure the amount of hemoglobin released, and calculate the hemolysis rate.

[0095] Hemolysis rate (%) = (OD sample − OD negative) / (OD positive − OD negative) × 100%;

[0096] Using 0.1% Triton X-100 as a positive control for 100% hemolysis and PBS as a negative control for 0% hemolysis, OD sample was the absorbance value of the test sample group; OD negative was the absorbance value of the negative control; OD positive was the absorbance value of the positive control. The calculation results are shown in Table 4.

[0097] Table 4

[0098]

[0099] At all concentrations, the hemolysis rate of the antimicrobial peptides of this invention was less than 5%, which was much lower than that of the positive control and significantly lower than that of control sample 1, with p < 0.05, indicating that the antimicrobial peptides prepared by the low-temperature process have better safety.

[0100] In summary, compared with comparative examples 1-3, the technical solution of the present invention has significant advantages in terms of activity retention rate, long-term stability, production cost and safety, which fully demonstrates the effectiveness and practicality of the technical solution of the present invention.

[0101] It should be noted that the above embodiments are merely some preferred embodiments of the present invention, and not all embodiments. Obviously, based on the above embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort should fall within the scope of protection of the present invention.

[0102] The above description of the embodiments is intended to enable those skilled in the art to understand and use the invention. It will be apparent to those skilled in the art that various modifications can be made to these embodiments, and the general principles described herein can be applied to other embodiments without inventive effort. Therefore, the present invention is not limited to the above embodiments, and any improvements and modifications made by those skilled in the art based on the disclosure of the present invention without departing from the scope of the invention should be within the protection scope of the present invention.

Claims

1. A crocodile blood-derived antimicrobial peptide, characterized in that, The amino acid sequence of the antimicrobial peptide is shown in SEQ ID NO:1, the molecular weight is 2.8±0.2kDa, and the isoelectric point is 8.8-9.

0.

2. A method for preparing crocodile blood-derived antimicrobial peptides by low-temperature enzymatic hydrolysis as described in claim 1, characterized in that, The low-temperature enzymatic hydrolysis preparation method includes the following steps: 1) Mix crocodile whole blood with sterile purified water at a mass ratio of 1:3-5, and perform shearing homogenization and homogenization to obtain crocodile blood homogenate; 2) Adjust the pH of the crocodile blood homogenate to 5.5-8.0, add the complex protease to carry out a low-temperature enzymatic hydrolysis reaction. The reaction temperature of the low-temperature enzymatic hydrolysis is 35-45℃. After the reaction is completed, the enzyme is inactivated by heating, cooled and centrifuged to collect the supernatant to obtain the crude antimicrobial peptide extract. 3) The crude extract of antimicrobial peptides is subjected to ultrafiltration, the permeate is collected, and the permeate is concentrated under vacuum to obtain concentrated antimicrobial peptide solution; 4) Add a freeze-drying protectant to the concentrated antimicrobial peptide solution and freeze-dry to obtain crocodile blood-derived antimicrobial peptides.

3. The preparation method according to claim 2, characterized in that, In step 1), the crocodile whole blood is derived from farmed adult Siamese crocodiles.

4. The preparation method according to claim 2, characterized in that, In step 1), the temperature of the shear homogenizer is 0-4℃, the processing time is 10-20 min, and the rotation speed is 8000-10000 r / min; the pressure of the homogenization process is 20-50 MPa, the temperature is 0-4℃, and the process is repeated 1-3 times.

5. The preparation method according to claim 2, characterized in that, In step 2), the low-temperature enzymatic hydrolysis reaction takes 12 hours, the complex protease is a mixture of neutral protease and papain, and the mass ratio of neutral protease to papain in the complex protease is 1:0.5-2.

6. The preparation method according to claim 2, characterized in that, In step 2), the temperature for inactivating the enzyme is 85-90℃, and the holding time is 10-15 min.

7. The preparation method according to claim 2, characterized in that, In step 3), the ultrafiltration pressure is 0.1-0.2 MPa; the vacuum concentration temperature is 30-50℃, and the vacuum degree is ≤0.15 MPa.

8. The preparation method according to claim 2, characterized in that, In step 4), the amount of the freeze-drying protectant added is 5%-10% of the volume of the concentrated antimicrobial peptide liquid, and the freeze-drying protectant is a mannitol ethanol solution with a mass fraction of 5%.

9. The preparation method according to claim 2, characterized in that, In step 4), the freeze drying includes a primary drying section and a secondary drying section. The primary drying section is characterized by a vacuum of 10-20 Pa, a temperature of -5-15℃, and a holding time of 12-24 h. The secondary drying section is characterized by a temperature of 15-30℃ and a holding time of 48 h.

10. The application of the crocodile blood-derived antimicrobial peptide as described in claim 1 in the preparation of antimicrobial drugs, characterized in that, The antibacterial drug is used to inhibit Staphylococcus aureus, Escherichia coli, or Candida albicans.

Images