A method for extracting black soldier fly antibacterial peptide

Abstract

The application provides a Hermetia illucens antibacterial peptide extraction method, and relates to the technical field of bioactive substance extraction and biological preparation, which comprises the following steps: adding brown sugar, Aspergillus oryzae and lactic acid bacteria into water, standing and fermenting at 30 DEG C for 24h-48h to prepare a fermentation induction solution, spraying and uniformly mixing 5th instar Hermetia illucens larvae with the fermentation induction solution according to a mass-volume ratio of 5g:1mL after the 5th instar Hermetia illucens larvae are starved for 6h, wherein the Hermetia illucens antibacterial peptide extraction method realizes efficient extraction and powder preparation of the antibacterial peptide by implementing fermentation induction on the 5th instar Hermetia illucens larvae and sequentially performing homogenization, complex enzymolysis, acidification and centrifugation, ultrafiltration concentration and spray drying treatment. The application systematically integrates the processes of induced generation, release separation and enrichment drying of the antibacterial peptide, which is beneficial to improving the extraction efficiency of the antibacterial peptide and the enrichment degree of active ingredients of the Hermetia illucens antibacterial peptide, and obtaining the antibacterial peptide powder product which is convenient to store, transport and apply.

Description

Technical Field

[0001] This invention relates to the field of bioactive substance extraction and biopreparation technology, specifically a method for extracting antimicrobial peptides from black soldier fly larvae. Background Technology

[0002] Antimicrobial peptides are a class of bioactive polypeptides with antibacterial, bactericidal, and immunomodulatory effects. Due to their broad-spectrum antibacterial activity, low risk of drug resistance, and good biocompatibility, they have received widespread attention in recent years in fields such as feed additives, aquaculture antimicrobial agents, biomedicine, and agricultural antimicrobial products. Black soldier fly larvae are rich in protein and various active ingredients, and under appropriate induction conditions, they can produce antimicrobial peptides with good antibacterial activity. Therefore, the preparation of antimicrobial peptides from black soldier flies has become an important direction for the development and utilization of insect-derived bioactive substances. Current technologies typically employ processes such as induction culture, tissue disruption, enzymatic extraction, acid precipitation for impurity removal, membrane separation, concentration, and drying to prepare antimicrobial peptide products with certain antibacterial activity from black soldier flies or other insects.

[0003] The existing technology still has the following drawbacks: the effect of increasing the content of antimicrobial peptides in black soldier flies is limited, resulting in low extraction efficiency; at the same time, due to the complex composition of black soldier fly bodies, impurities are not fully removed during the extraction process, which can easily affect the quality of the obtained antimicrobial peptide products; in addition, the existing process is still insufficient in terms of extraction efficiency and practical production applications, and it is difficult to simultaneously meet the requirements of high yield, low cost and large-scale preparation. Summary of the Invention

[0004] To address the shortcomings of existing technologies, this invention provides a method for extracting antimicrobial peptides from black soldier fly larvae. The technical problem this invention aims to solve is how to overcome the limitations in existing methods for extracting antimicrobial peptides from black soldier fly larvae, such as limited improvement in antimicrobial peptide content, insufficient removal of impurities, and inadequate extraction efficiency and actual preparation results, by using a process involving fermentation induction, homogenization and enzymatic hydrolysis, acidification centrifugation, ultrafiltration concentration, and spray drying.

[0005] To achieve the above objectives, the present invention provides the following technical solution: a method for extracting antimicrobial peptides from black soldier fly larvae, comprising: S1. Add brown sugar, Aspergillus oryzae and lactic acid bacteria to water, and let it ferment at 30℃ for 24h-48h to obtain fermentation induction liquid; after the 5th instar black soldier fly larvae are stopped from feeding for 6h, spray and mix them according to the mass-volume ratio of the 5th instar black soldier fly larvae to the fermentation induction liquid of 5g:1mL, and induce them at room temperature for 24h to obtain induced black soldier fly larvae; S2. The induced black soldier fly larvae are washed and drained, and homogenized at a mass-to-volume ratio of induced black soldier fly larvae to water of 1g:3mL to obtain a black soldier fly homogenate. S3. Adjust the pH of the black soldier fly homogenate to 7.0-7.5, and add 0.2% neutral protease and 0.1% papain according to the weight of the black soldier fly larvae to carry out compound enzymatic hydrolysis for 3-4 hours at 50℃-55℃. After the enzymatic hydrolysis is completed, inactivate the enzyme at 95℃ for 15 minutes to obtain the enzymatic hydrolysate. S4. Adjust the pH of the enzymatic hydrolysate to 3.5-4.0 and let it stand for 30 min. Then, centrifuge at 4000 rpm for 10 min and collect the supernatant. Concentrate the supernatant to 1 / 5-1 / 10 of its original volume through an ultrafiltration membrane with a molecular weight cutoff of 10 kDa to obtain an antimicrobial peptide concentrate. S5. The antimicrobial peptide concentrate is spray-dried at an inlet air temperature of 160°C and an outlet air temperature of 80°C to obtain black soldier fly antimicrobial peptide powder.

[0006] The present invention is further configured such that the preparation of the fermentation induction liquid includes: adding 100g of brown sugar, 0.5g of Aspergillus oryzae and 0.5g of lactic acid bacteria to every 10L of water, and allowing it to ferment statically at 30℃ for 24h-48h.

[0007] The present invention is further configured such that the 5th instar black soldier fly larvae are fresh larvae after they have stopped feeding, and the water content of the 5th instar black soldier fly larvae is 65%-75%.

[0008] The present invention is further configured such that the fermentation induction broth is prepared using deionized water.

[0009] The present invention is further configured such that the homogenization is performed using a homogenizer and the homogenization time is 5 minutes.

[0010] The present invention is further configured such that the pH value of the black soldier fly slurry is adjusted using a 0.1 mol / L hydrochloric acid solution and a 0.1 mol / L sodium hydroxide solution.

[0011] The present invention is further configured such that the centrifugation is performed at 4°C.

[0012] The present invention is further configured such that the moisture content of the obtained black soldier fly antimicrobial peptide powder is not higher than 5%, and the black soldier fly antimicrobial peptide powder is used to prepare feed additives, aquaculture antimicrobial agents or agricultural antimicrobial agents.

[0013] This invention provides a method for extracting antimicrobial peptides from black soldier fly larvae. It has the following beneficial effects: This method for extracting antimicrobial peptides from black soldier fly larvae involves fermentation induction of 5th instar larvae, followed by homogenization, enzymatic hydrolysis, acidification centrifugation, ultrafiltration concentration, and spray drying. This achieves efficient extraction and powder preparation of antimicrobial peptides from black soldier fly larvae. This invention systematically integrates the processes of antimicrobial peptide induction, release, separation, enrichment, and drying, which improves the extraction efficiency and active ingredient enrichment of black soldier fly antimicrobial peptides, resulting in antimicrobial peptide powder products that are easy to store, transport, and apply.

[0014] Fermentation induction broth prepared from brown sugar, Aspergillus oryzae, and lactic acid bacteria was used to induce black soldier fly larvae, which promoted the production of antibacterial active substances in the larvae. Simultaneously, a combined enzymatic hydrolysis using neutral protease and papain, along with acidification, centrifugation, and 10kDa ultrafiltration concentration, reduced interference from macromolecular impurities in the extraction process, improving the separation and purification of the target small-molecule antimicrobial peptides. Further spray drying enhanced the stability and ease of use of the resulting product, facilitating its application in feed additives, aquaculture antibacterial agents, and agricultural antibacterial agents. Attached Figure Description

[0015] To more clearly illustrate the technical solutions of the embodiments of the present invention, the accompanying drawings used in the description of the embodiments will be briefly introduced below.

[0016] Figure 1 This is a flowchart illustrating the preparation of the fermentation induction solution and the larval induction process of this invention.

[0017] Figure 2 This is a flowchart of the enzymatic hydrolysis, separation, and purification process of this invention.

[0018] Figure 3 This is a flowchart illustrating the drying and product application process of the present invention. Detailed Implementation

[0019] The technical solutions of the present invention will be described below with reference to the accompanying drawings. The described embodiments are only some embodiments of the present invention, and not all embodiments.

[0020] Example 1 Please see Figures 1-3 This invention relates to a method for extracting antimicrobial peptides from black soldier fly larvae, comprising: S1. Brown sugar, Aspergillus oryzae, and lactic acid bacteria were added to water and fermented at 30℃ for 24 hours to obtain a fermentation induction broth. Fifth-instar black soldier fly larvae were deprived of food for 6 hours, then sprayed and mixed at a mass-to-volume ratio of 5g:1mL of fermentation induction broth, and induced at room temperature for 24 hours to obtain induced black soldier fly larvae. The fermentation induction broth was prepared by adding 100g of brown sugar, 0.5g of Aspergillus oryzae, and 0.5g of lactic acid bacteria to every 10L of water, and fermenting at 30℃ for 24 hours. The fifth-instar black soldier fly larvae were fresh, live larvae after defecation, and the water content of the fifth-instar larvae was 65%. Deionized water was used to prepare the fermentation induction broth.

[0021] S2. The induced black soldier fly larvae were washed and drained, and homogenized at a mass-to-volume ratio of induced black soldier fly larvae to water of 1 g: 3 mL to obtain a black soldier fly homogenate. Homogenization was performed using a homogenizer for 5 minutes. The pH of the black soldier fly homogenate was adjusted using 0.1 mol / L hydrochloric acid solution and 0.1 mol / L sodium hydroxide solution.

[0022] S3. Adjust the pH of the black soldier fly homogenate to 7.0, and add 0.2% neutral protease and 0.1% papain by weight of the induced black soldier fly larvae at 50℃ for 3 hours for compound enzymatic hydrolysis. After the enzymatic hydrolysis is completed, inactivate the enzyme at 95℃ for 15 minutes to obtain the enzymatic hydrolysate.

[0023] S4. Adjust the pH of the enzymatic hydrolysate to 3.5 and let it stand for 30 min. Then, centrifuge at 4000 rpm for 10 min and collect the supernatant. Concentrate the supernatant to 1 / 5 of its original volume using an ultrafiltration membrane with a molecular weight cutoff of 10 kDa to obtain the antimicrobial peptide concentrate. Centrifugation is performed at 4℃.

[0024] S5. Spray-dry the concentrated antimicrobial peptide solution at an inlet air temperature of 160℃ and an outlet air temperature of 80℃ to obtain black soldier fly antimicrobial peptide powder. The resulting black soldier fly antimicrobial peptide powder has a moisture content of no more than 5% and is used to prepare feed additives, aquaculture antimicrobial agents, or agricultural antimicrobial agents.

[0025] Under relatively low parameter conditions, this invention can still sequentially complete the processes of fermentation induction, homogenization, complex enzymatic hydrolysis, acidification separation, ultrafiltration concentration, and spray drying, and obtain black soldier fly antimicrobial peptide powder. The results show that this invention can achieve the preparation of the target product under relatively mild process conditions, and the technical solution has clear feasibility and stable process completion capability.

[0026] Example 2 Please see Figures 1-3 Based on Example 1, a method for extracting antimicrobial peptides from black soldier fly larvae includes: S1. Brown sugar, Aspergillus oryzae, and lactic acid bacteria were added to water and fermented at 30℃ for 36 hours to obtain a fermentation induction broth. Fifth-instar black soldier fly larvae were deprived of food for 6 hours, then sprayed and mixed at a mass-to-volume ratio of 5g:1mL of fermentation induction broth, and induced at room temperature for 24 hours to obtain induced black soldier fly larvae. The fermentation induction broth was prepared by adding 100g of brown sugar, 0.5g of Aspergillus oryzae, and 0.5g of lactic acid bacteria to every 10L of water, and fermenting at 30℃ for 36 hours. The fifth-instar black soldier fly larvae were fresh, live larvae after defecation, and the water content of the fifth-instar larvae was 70%. Deionized water was used to prepare the fermentation induction broth.

[0027] S2. The induced black soldier fly larvae were washed and drained, and homogenized at a mass-to-volume ratio of induced black soldier fly larvae to water of 1 g: 3 mL to obtain a black soldier fly homogenate. Homogenization was performed using a homogenizer for 5 minutes. The pH of the black soldier fly homogenate was adjusted using 0.1 mol / L hydrochloric acid solution and 0.1 mol / L sodium hydroxide solution.

[0028] S3. Adjust the pH of the black soldier fly homogenate to 7.25, and add 0.2% neutral protease and 0.1% papain by weight of the induced black soldier fly larvae at 52.5℃ for 3.5h for compound enzymatic hydrolysis. After the enzymatic hydrolysis is completed, inactivate the enzyme at 95℃ for 15min to obtain the enzymatic hydrolysate.

[0029] S4. Adjust the pH of the enzymatic hydrolysate to 3.75 and let it stand for 30 min. Then, centrifuge at 4000 rpm for 10 min and collect the supernatant. Concentrate the supernatant to 1 / 8 of its original volume using an ultrafiltration membrane with a molecular weight cutoff of 10 kDa to obtain the antimicrobial peptide concentrate. Centrifugation is performed at 4℃.

[0030] S5. Spray-dry the concentrated antimicrobial peptide solution at an inlet air temperature of 160℃ and an outlet air temperature of 80℃ to obtain black soldier fly antimicrobial peptide powder. The resulting black soldier fly antimicrobial peptide powder has a moisture content of no more than 5% and is used to prepare feed additives, aquaculture antimicrobial agents, or agricultural antimicrobial agents.

[0031] Under intermediate parameter conditions, this invention can achieve the induced release, enzymatic hydrolysis and conversion, separation and enrichment, and powder preparation of antibacterial active ingredients from black soldier flies. The resulting product has a clear morphology, and the process flow is complete. The results show that under these process conditions, this invention can achieve good synergy between the various steps, demonstrating good process adaptability and application potential.

[0032] Example 3 Please see Figures 1-3 Based on Examples 1 and 2, a method for extracting antimicrobial peptides from black soldier fly larvae includes: S1. Brown sugar, Aspergillus oryzae, and lactic acid bacteria were added to water and fermented at 30℃ for 48 hours to obtain a fermentation induction broth. Fifth-instar black soldier fly larvae were deprived of food for 6 hours, then sprayed and mixed at a mass-to-volume ratio of 5g:1mL of fermentation induction broth, and induced at room temperature for 24 hours to obtain induced black soldier fly larvae. The fermentation induction broth was prepared by adding 100g of brown sugar, 0.5g of Aspergillus oryzae, and 0.5g of lactic acid bacteria to every 10L of water, and fermenting at 30℃ for 48 hours. The fifth-instar black soldier fly larvae were fresh, live larvae after defecation, and the water content of the fifth-instar larvae was 75%. Deionized water was used to prepare the fermentation induction broth.

[0033] S2. The induced black soldier fly larvae were washed and drained, and homogenized at a mass-to-volume ratio of induced black soldier fly larvae to water of 1 g: 3 mL to obtain a black soldier fly homogenate. Homogenization was performed using a homogenizer for 5 minutes. The pH of the black soldier fly homogenate was adjusted using 0.1 mol / L hydrochloric acid solution and 0.1 mol / L sodium hydroxide solution.

[0034] S3. Adjust the pH of the black soldier fly homogenate to 7.5, and add 0.2% neutral protease and 0.1% papain by weight of the induced black soldier fly larvae at 55℃ for 4 hours for compound enzymatic hydrolysis. After the enzymatic hydrolysis is completed, inactivate the enzyme at 95℃ for 15 minutes to obtain the enzymatic hydrolysate.

[0035] S4. Adjust the pH of the enzymatic hydrolysate to 4.0 and let it stand for 30 min. Then, centrifuge at 4000 rpm for 10 min and collect the supernatant. Concentrate the supernatant to 1 / 10 of its original volume using an ultrafiltration membrane with a molecular weight cutoff of 10 kDa to obtain the antimicrobial peptide concentrate. Centrifugation is performed at 4°C.

[0036] S5. Spray-dry the concentrated antimicrobial peptide solution at an inlet air temperature of 160℃ and an outlet air temperature of 80℃ to obtain black soldier fly antimicrobial peptide powder. The resulting black soldier fly antimicrobial peptide powder has a moisture content of no more than 5% and is used to prepare feed additives, aquaculture antimicrobial agents, or agricultural antimicrobial agents.

[0037] Under relatively high parameter conditions, this invention can also complete the extraction, separation, concentration, and drying of black soldier fly antimicrobial peptides, forming black soldier fly antimicrobial peptide powder suitable for subsequent applications. The results show that this invention can still achieve effective preparation of the target product under relatively intensified process conditions, demonstrating good feasibility for implementation.

[0038] Example 4 To compare the effects of different combinations of extraction parameters on the extraction of antimicrobial peptides from black soldier fly larvae, a grouped comparative experiment was conducted using 5th instar black soldier fly larvae from the same breeding batch, with consistent age and health status.

[0039] The three sets of process parameters A, B, and C respectively adopt the parameter combinations in Examples 1, 2, and 3 mentioned above, where group A is the lower parameter group, group B is the intermediate parameter group, and group C is the higher parameter group.

[0040] 1. Experimental Materials and Grouping In October 2025, 9.0 kg of the same batch of 5th instar black soldier fly larvae were taken. After fasting for 6 hours, they were randomly divided into three groups: A, B and C, with 3.0 kg in each group.

[0041] To ensure consistency among the three groups, all three groups of larvae were derived from the same batch of culture material, harvested at the same time, and subjected to induction, enzymatic hydrolysis, centrifugation, ultrafiltration, and spray drying under the same room temperature conditions.

[0042] The differences among the three groups lie only in the fermentation time of the fermentation induction broth, the moisture content of the larvae, the pH value of enzymatic hydrolysis, the temperature of enzymatic hydrolysis, the time of enzymatic hydrolysis, the pH value of acidification, and the concentration factor of ultrafiltration.

[0043] in: Group A: Fermented for 24 hours, larvae moisture content 65%, enzymatic hydrolysis at pH 7.0, 50℃ for 3 hours, acidified to pH 3.5, concentrated to 1 / 5 of the original volume.

[0044] Group B: Fermented for 36 hours, larvae moisture content 70%, enzymatic hydrolysis pH 7.25, 52.5℃, 3.5 hours, acidified pH 3.75, concentrated to 1 / 8 of the original volume.

[0045] Group C: Fermented for 48 hours, larvae moisture content 75%, enzymatic hydrolysis at pH 7.5, 55℃ for 4 hours, acidified to pH 4.0, concentrated to 1 / 10 of the original volume.

[0046] 2. Simultaneous comparison during the fermentation induction stage Fermentation induction solutions were prepared for three groups according to specific ratios. Each group used 2L of deionized water as the base solution, adding 20g of brown sugar, 0.1g of Aspergillus oryzae, and 0.1g of lactic acid bacteria, and was incubated at 30℃ for static fermentation. Group A fermented for 24 hours, Group B for 36 hours, and Group C for 48 hours.

[0047] After fermentation, the pH values ​​of the three induction solutions were measured as follows: Group A 4.32, Group B 4.08, and Group C 3.94.

[0048] Subsequently, 600 mL of the induction solution for each group was taken and sprayed onto the surface of 3.0 kg of black soldier fly larvae in each group at a mass-volume ratio of 5 g: 1 mL. After mixing, the mixture was induced at room temperature for 24 h.

[0049] Observations after induction revealed: After induction, it was observed that the larvae in group A had moist body surface, the larvae in group B had a relatively uniform distribution of the attached liquid on their body surface, and the larvae in group C had high humidity and slight adhesion in some areas.

[0050] After washing and draining, the three groups of larvae were weighed, and the larval weights after induction were: Group A: 2.86 kg, Group B: 2.90 kg, and Group C: 2.93 kg.

[0051] Based on the on-site records from this stage, the induction state of Group B was relatively stable, the induction degree of Group A was relatively weak, and although the induction degree of Group C was further enhanced, the surface adhesion phenomenon was relatively obvious after draining.

[0052] 3. Simultaneous comparison of homogenization and enzymatic hydrolysis stages The three groups of induced larvae were homogenized in deionized water at a ratio of 1g:3mL for 5 minutes. The corresponding water volumes were: Group A: 8.58L, Group B: 8.70L, and Group C: 8.79L.

[0053] After homogenization, adjust the pH value to the corresponding set value.

[0054] Subsequently, a compound protease was added based on the weight of the induced larvae, with 0.2% neutral protease and 0.1% papain added. The actual enzyme amounts added to the three groups were: Group A: Neutral protease 5.72g, Papain 2.86g; Group B: Neutral protease 5.80g, Papain 2.90g; Group C: Neutral protease 5.86g, Papain 2.93g.

[0055] The three groups were subjected to enzymatic hydrolysis under corresponding conditions: Group A: 50℃, 3h; Group B: 52.5℃, 3.5h; Group C: 55℃, 4h.

[0056] During the enzymatic hydrolysis process, the state of the system was observed every 1 hour. The records show: At the end of 3 hours of enzymatic hydrolysis, a small amount of fine flocculent matter was still visible in the system of Group A. At the end of 3.5 hours of enzymatic hydrolysis, the system of Group B was relatively homogeneous and had good fluidity. At the end of 4 hours of enzymatic hydrolysis, the system of Group C was homogeneous, but the color was slightly darker than that of Group B.

[0057] After enzymatic hydrolysis, all three groups were inactivated at 95℃ for 15 min. Samples were taken after enzyme inactivation to determine the concentration of soluble peptides. The results were as follows: Group A: 18.6 mg / mL, Group B: 22.1 mg / mL, and Group C: 20.4 mg / mL.

[0058] The data from this stage show that the degree of enzymatic hydrolysis in group A was relatively insufficient. Although the enzymatic hydrolysis intensity in group C was higher, the concentration of soluble peptides did not continue to be higher than that in group B. The system after enzymatic hydrolysis in group B was more homogeneous, and the concentration of soluble peptides was the highest among the three groups.

[0059] 4. Simultaneous comparison of acidification, centrifugation and ultrafiltration stages The three groups of enzymatic hydrolysates were adjusted to their respective acidification pH values ​​and allowed to stand for 30 minutes: Group A: pH 3.5, Group B: pH 3.75, Group C: pH 4.0.

[0060] The mixture was then centrifuged at 4°C and 4000 rpm for 10 min, and the supernatant was collected.

[0061] The volumes of the supernatant obtained were: Group A: 7.46L, Group B: 7.81L, and Group C: 7.58L.

[0062] After centrifugation, it was observed that Group A had a larger amount of precipitate, indicating that there were relatively more undissolved components. The supernatant of Group B had better clarity, while the supernatant of Group C was also relatively clear, but the color was darker.

[0063] The supernatants of the three groups were concentrated by 10 kDa ultrafiltration. The concentration endpoints were controlled at 1 / 5, 1 / 8 and 1 / 10 of the original volume, respectively. The volumes of the resulting antimicrobial peptide concentrates were: Group A: 1.49 L, Group B: 0.98 L and Group C: 0.76 L.

[0064] The comparison shows that Group B has better supernatant yield and subsequent concentration efficiency. Group A has insufficient dissolution in the first stage, which affects the recovery of supernatant. Although Group C has a higher concentration factor, the viscosity of the final concentrate increases more significantly, which is not conducive to stable feeding for subsequent spray drying.

[0065] 5. Comparison of spray drying and finished products The three groups of antimicrobial peptide concentrates were treated under the same spray drying conditions: inlet air temperature 160℃ and outlet air temperature 80℃. The resulting black soldier fly antimicrobial peptide powders had the following weights: Group A: 55.6g, Group B: 72.3g, and Group C: 66.9g.

[0066] The corresponding powder yields, based on an initial fresh insect weight of 3.0 kg, were as follows: Group A: 1.85%, Group B: 2.41%, and Group C: 2.23%.

[0067] The obtained antimicrobial peptide powder was tested for various indicators. The moisture content was determined by constant weight method at 105℃, the soluble peptide content was determined by Folin-phenol method, and the antibacterial activity was determined by Oxford cup method. Staphylococcus aureus and Escherichia coli were used as indicator bacteria.

[0068] Each group of samples was tested in parallel three times, and the data in Table 1 is the average of the three test results.

[0069] Table 1: Results of the three comparative experiments.

[0070] 6. Comparative Analysis The synchronous comparative experiment showed that the treatment conditions of group A in the induction and enzymatic hydrolysis stages were relatively low. As a result, a small amount of undispersed components remained after enzymatic hydrolysis, and there was a lot of precipitation after centrifugation. In the end, the yield of antimicrobial peptide powder and the antibacterial effect were both low.

[0071] Although Group C improved the fermentation time, larval moisture content, and enzymatic hydrolysis intensity, in the actual experiment, the viscosity of the concentrate increased and the liquid state before spray drying was slightly heavier. The quality and antibacterial properties of the final antimicrobial peptide powder were better than those of Group A, but not as good as those of Group B.

[0072] Group B showed greater stability throughout the experiment: the larvae were uniformly sized after induction, the enzymatic hydrolysate had good fluidity, the supernatant volume was the highest, and the final mass of the antimicrobial peptide powder obtained was 72.3g, with a yield of 2.41% and a soluble peptide content of 77.2%. The inhibition zones against Staphylococcus aureus and Escherichia coli reached 20.5mm and 18.6mm, respectively. This indicates that under the conditions of this synchronous comparative experiment, the antimicrobial peptide powder obtained by the parameter combination of Group B had a higher yield, higher soluble peptide content, and stronger antibacterial activity, resulting in a better overall extraction effect.

[0073] In summary, under the conditions of this synchronous comparative experiment, the parameter combination used in group B has a better overall extraction effect.

[0074] The foregoing has only described certain exemplary embodiments of the present invention by way of illustration. Undoubtedly, those skilled in the art can modify the described embodiments in various ways without departing from the spirit and scope of the present invention. Therefore, the foregoing drawings and descriptions are illustrative in nature and should not be construed as limiting the scope of protection of the claims of the present invention.

Claims

1. A method for extracting antimicrobial peptides from black soldier fly larvae, characterized in that, include: S1. Add brown sugar, Aspergillus oryzae and lactic acid bacteria to water, and let it ferment at 30℃ for 24h-48h to obtain fermentation induction liquid; after the 5th instar black soldier fly larvae are stopped from feeding for 6h, spray and mix them according to the mass-volume ratio of the 5th instar black soldier fly larvae to the fermentation induction liquid of 5g:1mL, and induce them at room temperature for 24h to obtain induced black soldier fly larvae; S2. The induced black soldier fly larvae are washed and drained, and homogenized at a mass-to-volume ratio of induced black soldier fly larvae to water of 1g:3mL to obtain a black soldier fly homogenate. S3. Adjust the pH of the black soldier fly homogenate to 7.0-7.5, and add 0.2% neutral protease and 0.1% papain according to the weight of the black soldier fly larvae to carry out compound enzymatic hydrolysis for 3-4 hours at 50℃-55℃. After the enzymatic hydrolysis is completed, inactivate the enzyme at 95℃ for 15 minutes to obtain the enzymatic hydrolysate. S4. Adjust the pH of the enzymatic hydrolysate to 3.5-4.0 and let it stand for 30 min. Then, centrifuge at 4000 rpm for 10 min and collect the supernatant. Concentrate the supernatant to 1 / 5-1 / 10 of its original volume through an ultrafiltration membrane with a molecular weight cutoff of 10 kDa to obtain an antimicrobial peptide concentrate. S5. The antimicrobial peptide concentrate is spray-dried at an inlet air temperature of 160°C and an outlet air temperature of 80°C to obtain black soldier fly antimicrobial peptide powder.

2. The method for extracting antimicrobial peptides from black soldier fly larvae according to claim 1, characterized in that: The preparation of the fermentation induction liquid includes: adding 100g of brown sugar, 0.5g of Aspergillus oryzae and 0.5g of lactic acid bacteria to every 10L of water, and allowing it to ferment statically at 30℃ for 24h-48h.

3. The method for extracting antimicrobial peptides from black soldier fly larvae according to claim 1, characterized in that: The 5th instar black soldier fly larvae are fresh, live larvae after they have stopped feeding, and the moisture content of the 5th instar black soldier fly larvae is 65%-75%.

4. The method for extracting antimicrobial peptides from black soldier fly larvae according to claim 1, characterized in that: The fermentation induction broth was prepared using deionized water.

5. The method for extracting antimicrobial peptides from black soldier fly larvae according to claim 1, characterized in that: The homogenization was performed using a homogenizer, and the homogenization time was 5 minutes.

6. The method for extracting antimicrobial peptides from black soldier fly larvae according to claim 1, characterized in that: The pH value of the black soldier fly slurry was adjusted using 0.1 mol / L hydrochloric acid solution and 0.1 mol / L sodium hydroxide solution.

7. The method for extracting antimicrobial peptides from black soldier fly larvae according to claim 1, characterized in that: The centrifugation was carried out at 4°C.

8. The method for extracting antimicrobial peptides from black soldier fly larvae according to claim 1, characterized in that: The resulting black soldier fly antimicrobial peptide powder has a moisture content of no more than 5%, and the black soldier fly antimicrobial peptide powder is used to prepare feed additives, aquaculture antimicrobial agents, or agricultural antimicrobial agents.

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