Method for extracting crucian scale antibacterial peptide through ultrasonic-assisted enzymolysis and process optimization method thereof

Abstract

The invention relates to a method for extracting crucian scale antibacterial peptide through ultrasonic-assisted enzymolysis and a process optimization method thereof. Compared with the prior art, the method has the advantages that the fish scales of the crucian are used as raw materials, ultrasonic-assisted protease is used for performing enzymolysis on the fish scales, the antibacterial diameter of escherichia coli and the short peptide recovery rate of enzymolysis are used as evaluation indexes, the optimal enzymolysis process is analyzed, the enzymolysis yield of the antibacterial peptide is increased, and the antibacterial peptide with relatively high antibacterial activity is obtained; and an effective theoretical basis is provided for effective utilization of freshwater fish leftovers and development of antibacterial peptides.

Description

Technical Field The invention relates to the field of antimicrobial peptide preparation, and in particular to a method for extracting antimicrobial peptides from crucian carp scales with ultrasound-assisted enzymatic hydrolysis and a process optimization method thereof. Background Art Antimicrobial peptides are low molecular weight natural polypeptides with strong immunomodulatory functions. They are distributed in aquatic products and other animals and plants, and have the characteristics of strong activity, wide range of functions, and broad-spectrum bactericidal. They are also called antibiotic peptides, which are low molecular weight peptides with a molecular weight of less than 5000 and an amino acid number of less than 100. Antimicrobial peptides are cationic particle bioactive peptides. Most organisms can use their own antimicrobial peptides to resist the first barrier of external invasion. According to Xu Yuchen [1] According to a study conducted by the University of California, Berkeley, antimicrobial peptides extracted from hybrid spotted sea bass have a significant inhibitory effect on Shewanella putrefaciens. [2] The antimicrobial peptides isolated from yellow croaker from distiller's grains showed good inhibitory effects on foodborne pathogenic bacteria Vibrio parahaemolyticus. These studies have shown that antimicrobial peptides can have a good inhibitory effect on microorganisms. my country is a major aquaculture country, and the processing of fish and aquatic products produces a large number of by-products. If these by-products are not properly disposed of, they will not only pollute the ecological environment, but also endanger human health, so they should be used to their full potential. Studies have found that fish waste is rich in protein and peptide substances, which have potential application prospects in the development of functional foods and drugs. [3] . Antimicrobial peptides in fish have certain medical value and can play a role in immune protection due to their low molecular weight, good heat resistance, broad antimicrobial ability, and low resistance to drug resistance. In recent years, more and more fish antimicrobial peptides have been extracted and studied. Enzymatic hydrolysis is a commonly used method for preparing protein hydrolysates, which can better decompose and obtain active antimicrobial peptides. However, there is also a problem of low enzymatic hydrolysis efficiency of antimicrobial peptides. This is because simple enzymatic hydrolysis has low reaction efficiency and low substrate conversion rate, resulting in low final product yield. References are as follows:

[0001] Xuelei Gai. Mining and activity research of antimicrobial peptides from Antarctic krill[D]. Qilu University of Technology, 2023.DOI:10.27278 / d.cnki.gsdqc.2023.000455.

[0002] Wang Wei. Research on the multifunctionalization and antibacterial properties of antimicrobial peptides[D]. Changchun University of Technology, 2023.DOI:10.27805 / d.cnki.gccgy.2023.000422.

[0003] Yang Chenyuan, Yu Zichuan, Qin Di, et al. Research progress on structural analysis, antibacterial mechanism and modification and application of antimicrobial peptides[J]. Acta Microbiologica Sinica, 2024, 64(07): 2242-2259. DOI: 10.13343 / j.cnki.wsxb.20230812. Summary of the invention The purpose of the present invention is to provide a method for extracting antimicrobial peptides from crucian carp scales by ultrasonic-assisted enzymatic hydrolysis and a process optimization method thereof. The present invention prepares antimicrobial active peptides by utilizing freshwater fish waste, and the obtained crucian carp scale antimicrobial peptides can be applied to functional food and drug research and development. The purpose of the present invention can be achieved by the following technical solutions: A method for extracting antimicrobial peptides from crucian carp scales by ultrasound-assisted enzymatic hydrolysis, the specific steps of which are as follows: S1. Pretreatment of crucian carp scales; S2, mixing the pretreated crucian carp scales obtained in step S1 with a NaOH solution, an EDTA-2Na solution and a citric acid solution in sequence to obtain a mixture; S3, adding protease to the mixture obtained in step S2, and obtaining an enzymatic solution by ultrasonic-assisted enzymatic hydrolysis; S4, centrifuging the enzymatic hydrolyzate obtained in step S3, taking the supernatant after centrifugation and concentrating it by rotary evaporation to obtain a crude antimicrobial peptide enzyme solution; S5, ultrafiltrating the crude antimicrobial peptide enzyme solution obtained in step S4 to obtain a crucian carp scale antimicrobial peptide filtrate, desalting, freezing, and lyophilizing in sequence to obtain a crucian carp scale antimicrobial peptide. Further, in step S1, the specific steps of pre-treating crucian carp scales are as follows: Take fresh crucian carp scales, wash them until there is no dirt or mucus on the surface, dry them in a forced air drying oven, and refrigerate them for later use. As a preferred technical solution, the drying temperature is 30-50°C. Furthermore, in step S2, the pretreated crucian carp scales obtained in step S1 are chopped up and added with NaOH solution, magnetically stirred, washed and then added with EDTA-2Na solution, ultrasonicated, washed and then added with citric acid solution, and heated in a water bath to obtain a mixture. As a preferred technical solution, crucian carp scales: NaOH solution: EDTA-2Na solution: citric acid solution = 4-6 g: 40-60 mL: 70-80 mL: 65-75 mL. As a preferred technical solution, the concentration of the NaOH solution is 0.4-0.6 mol / L, the concentration of the EDTA-2Na solution is 0.1-0.3 mol / L, and the mass volume fraction of the citric acid solution is 5%-15%. As a preferred technical solution, the magnetic stirring time is 2 to 4 hours, the ultrasonic time is 2 to 3 hours, the water bath heating time is 10 to 14 hours, and the water bath heating temperature is 40 to 60°C. Furthermore, in step S3, protease is added to the mixture obtained in step S2, and ultrasound is used to assist enzymatic hydrolysis for a period of time, and then the enzyme is inactivated in a water bath to obtain an enzymatic solution. As a preferred technical solution, the enzyme inactivation temperature is 70-90°C, and the enzyme inactivation time is 8-12 minutes. Furthermore, in step S4, the centrifugal temperature is 3-5°C, the centrifugal speed is 3000-5000 r / min, and the centrifugal time is 10-30 min. The rotary evaporation concentration temperature is 30-50°C. Furthermore, in step S5, the molecular weight of the crucian carp scale antimicrobial peptide filtrate is 3000-5000Da. As a preferred technical solution, the freezing temperature is -60°C to -80°C, and the freezing time is 20 to 28 hours; The freeze-drying temperature is -40°C to -60°C, and the freeze-drying time is 2 days to 4 days. In addition, the present invention also provides a process optimization method for the method of extracting antimicrobial peptides from crucian carp scales by ultrasound-assisted enzymatic hydrolysis, and the specific steps are as follows: (1) According to the above-mentioned method for ultrasonic-assisted enzymatic hydrolysis to extract antimicrobial peptides from crucian carp scales, the diameter of the inhibition zone of Escherichia coli and the recovery rate of short peptides after enzymatic hydrolysis were used as evaluation indicators to determine the most suitable protease for enzymatic hydrolysis; (2) Based on the protease determined in step (1), four factors affecting the extraction effect of crucian carp scale antimicrobial peptides were screened out; (3) using the diameter of the inhibition zone of Escherichia coli and the recovery rate of the short peptides after enzymatic hydrolysis as evaluation indicators, a single factor experiment was performed based on the four factors screened out in step (2); (4) Based on the single-factor experimental results of step (3), three factors that have a significant impact on the diameter of the inhibition zone of Escherichia coli and the recovery rate of short peptides after enzymatic hydrolysis were screened out for orthogonal experimental design to optimize the enzymatic hydrolysis process of crucian carp scale antimicrobial peptides. Furthermore, in step (2), the most suitable protease is pepsin. As a preferred technical solution, the enzymatic hydrolysis temperature of the pepsin is 37° C., and the enzymatic hydrolysis pH of the pepsin is 2.0. Furthermore, in step (2), the four factors affecting the extraction effect of antimicrobial peptides are: enzyme activity, substrate concentration, enzymatic hydrolysis time and ultrasonic power. The enzyme activity was set to 3000, 4000, 5000, 6000 and 7000 U / g of protease activity, The substrate concentrations were set to 50, 100, 150, 200 and 250 mg / mL of the mixture, The enzymolysis time was set to 2, 3, 4, 5 and 6 h, respectively. The ultrasonic powers were set to 90, 105, 120, 135 and 150 W respectively. Furthermore, in step (3), the method for determining the diameter of the inhibition zone of Escherichia coli is as follows: Using Escherichia coli as a tool bacterium, absorb the Escherichia coli suspension into the agar medium solution to prepare an agar medium mixed with the test bacteria; put a sterile Oxford cup in the plate, and pour the agar medium mixed with the test bacteria; after solidification, take out the Oxford cup, absorb the crucian carp scale antimicrobial peptide sample and put it into the hole; after static cultivation, measure the diameter of the inhibition zone, and use physiological saline as a blank control. Furthermore, in step (3), the method for determining the recovery rate of the enzymatically hydrolyzed short peptide is as follows: The protein content of crucian carp scales and crucian carp scale antimicrobial peptides was determined by referring to GB / T 5009.5-2016 Determination of protein in food (method 1) Kjeldahl nitrogen method, and the short peptide recovery rate was calculated according to the formula: In the formula: C is the mass concentration of protein in the crucian carp scale antimicrobial peptide sample, mg / L; V is the volume of the crucian carp scale antimicrobial peptide sample, L; M is the mass of protein in the crucian carp scale, mg. Furthermore, in step (4), three factors having a significant influence are: enzyme activity, enzymatic hydrolysis time and ultrasonic power. Furthermore, in step (4), the optimal enzymatic hydrolysis process of crucian carp scale antimicrobial peptide is as follows: enzymatic hydrolysis time 4h, ultrasonic power 120W, and enzyme activity 5000U / g. Compared with the prior art, the beneficial effects of the present invention are as follows: 1. The present invention uses crucian carp scales as raw materials, uses ultrasound-assisted protease to enzymolyze the scales, uses the antibacterial diameter size of Escherichia coli and the recovery rate of short peptides from enzymolysis as evaluation indicators, analyzes the optimal enzymolysis process, improves the enzymolysis yield of antimicrobial peptides, obtains antimicrobial peptides with strong antimicrobial activity, and provides an effective theoretical basis for the effective utilization of freshwater fish waste and the development of antimicrobial peptides. 2. The present invention uses crucian carp scales as raw materials, adopts ultrasonic-assisted enzymatic method to obtain crucian carp scale antibacterial polypeptide crude enzyme solution, and through single factor experiment and orthogonal optimization experiment, takes the diameter of inhibition zone and short peptide recovery rate as test indexes, and finally obtains the optimal enzymatic hydrolysis process conditions as follows: substrate concentration of 200 mg / mL, enzymatic hydrolysis time of 4 h, ultrasonic power of 120 W, and enzyme activity of 5000 U / g. Studies have shown that under this process, the antibacterial activity of the antibacterial peptide crude enzyme solution obtained by enzymatic hydrolysis against Escherichia coli can reach an inhibition zone diameter of 2.66 cm and a short peptide recovery rate of 22.52%. BRIEF DESCRIPTION OF THE DRAWINGS Figure 1 Schematic diagram of the effects of four proteases on the extraction of fish scale antimicrobial peptides; Figure 2 Schematic diagram of the effect of substrate concentration on the extraction of fish scale antimicrobial peptides; Figure 3 Schematic diagram of the effect of enzyme activity on the extraction of fish scale antimicrobial peptides; Figure 4 Schematic diagram of the effect of enzymatic hydrolysis time on the extraction of fish scale antimicrobial peptides; Figure 5 Schematic diagram of the effect of ultrasonic power on the extraction of fish scale antimicrobial peptides. DETAILED DESCRIPTION The present invention is described in detail below in conjunction with the accompanying drawings and specific embodiments. This embodiment is implemented based on the technical solution of the present invention, and provides a detailed implementation method and specific operation process, but the protection scope of the present invention is not limited to the following embodiments. In conjunction with the accompanying drawings, some embodiments of the present invention are described in detail below. In the absence of conflict, the following embodiments and features in the embodiments may be combined with each other. Example 1 This embodiment provides a method for extracting antimicrobial peptides from crucian carp scales by ultrasound-assisted enzymatic hydrolysis, and the specific steps are as follows: S1. Pretreatment of crucian carp scales; Take fresh crucian carp scales, wash them until there is no dirt or mucus on the surface, dry them in a forced air drying oven at 40°C, and refrigerate them for later use. S2, rough extraction: Weigh 5g of the pretreated dried crucian carp scales obtained in step S1, chop them into pieces, add 50mL of 0.5mol / LNaOH solution, stir magnetically for 3h, change the solution every 1.5h, and wash with distilled water until neutral. Then add 75mL of 0.2mol / LEDTA-2Na solution, place in an ultrasonic machine, set the ultrasonic power to 120W, treat for 2h, use ultrasonic to assist decalcification, and wash with distilled water until neutral. Add 70mL of 10% citric acid solution, place in a 50℃ constant temperature water bath to extract for 12h to obtain a mixture. S3, Enzymatic hydrolysis: Pepsin was added to the mixture obtained in step S2, and the enzymatic hydrolysis temperature was set to 37°C, the enzymatic hydrolysis pH was set to 2.0, the enzymatic hydrolysis time was set to 4 h, and an ultrasonic power of 120 W was used for ultrasonic-assisted enzymatic hydrolysis. The sample was placed in an 80°C water bath to inactivate the enzyme for 10 min to obtain an enzymatic hydrolyzate; S4, concentrated freeze-dried: The enzymatic hydrolysate obtained in step S3 is subjected to low-temperature centrifugation at 4000 r / min at 4°C for 20 min, and the supernatant is collected; the supernatant is concentrated by rotary evaporation at 40°C to obtain a crude enzyme solution of crucian carp scale antimicrobial peptide, which is frozen at -80°C for later use. S5. Inject the crude enzyme solution of antimicrobial peptide obtained in step S4 into the storage tank of the ultrafiltration separation device, use ultrafiltration membranes with pore sizes of 5 kDa and 3 kDa respectively to obtain crucian carp scale antimicrobial peptide filtrate with a molecular weight of 3000-5000 Da, desalt and freeze in a -70 ° C refrigerator for 24 h, and freeze-dry in a -50 ° C freeze dryer under vacuum decompression for 3 days to obtain crucian carp scale antimicrobial peptide. Example 2 This embodiment provides a process optimization method for extracting antimicrobial peptides from crucian carp scales by ultrasound-assisted enzymatic hydrolysis, and the specific steps are as follows: (1) Determination of protease The scales were enzymatically hydrolyzed according to the experimental process for extracting antimicrobial peptides from crucian carp scales in Example 1. The substrate concentration was 150 mg / mL, the enzyme activity was 5000 U / g, the enzymatic hydrolysis time was 4 h, the enzymatic hydrolysis pH and the enzymatic hydrolysis temperature were as shown in Table 1. Pepsin, trypsin, papain and neutral protease were used to prepare the crucian carp scale crude enzyme solution, and the inhibition zone diameter and the short peptide recovery rate were used as indicators to analyze and determine the most suitable protease. The antibacterial performance determination method in this embodiment is as follows: Using Escherichia coli as the tool bacteria, the antibacterial properties of the crude enzyme solution of antimicrobial peptides were determined by the double-layer Oxford cup inhibition zone method. 1 mL of Escherichia coli suspension (1×10 6 CFU / mL) in the agar medium solution, shake well to make an agar medium mixed with the test bacteria, put a sterile Oxford cup in the plate, pour the agar medium and shake well, after solidification, take out the Oxford cup, and draw 100μL of fish scale antimicrobial peptide crude enzyme solution sample into the well. After culturing at 37℃ for 24h, measure the diameter of the inhibition zone, and use physiological saline as a blank control. The determination method of the short peptide recovery rate in this embodiment is as follows: The protein content of fish scales and fish scale antimicrobial peptide solution was determined by referring to GB / T 5009.5-2016 Determination of protein in food Method 1 Kjeldahl nitrogen determination method. The recovery rate of short peptides was calculated according to the formula: In the formula: C is the mass concentration of protein in the fish scale antimicrobial peptide solution, mg / L; V is the volume of the fish scale antimicrobial peptide solution, L; M is the mass of protein in the fish scale. Table 1 Protease enzymatic conditions Since the cleavage sites of each protease are different, the peptides obtained by hydrolysis are also different. The effects of the four proteases on the extraction of antimicrobial peptides from crucian carp scales are as follows: Figure 1 As shown in the figure, compared with the other three proteases, the short peptide recovery rate obtained by pepsin hydrolysis was the highest at 18.6%, and the crude enzyme solution had the best antibacterial effect on Escherichia coli, with an inhibition zone diameter of 2.09±0.04cm. Although the short peptide recovery rate after trypsin hydrolysis reached 15.72%, the inhibition zone diameter of the hydrolyzate was only 0.26±0.05cm, indicating that trypsin could not effectively separate the antimicrobial peptides from crucian carp scales. Considering the short peptide recovery rate and antibacterial properties, pepsin can be selected as the enzyme for extracting antimicrobial peptides from fish scales. (2) Single factor experiment of enzymatic hydrolysis process The basic enzymatic hydrolysis process was 150 mg / mL substrate concentration, 5000 U / g enzyme activity, 120 W ultrasonic power, 4 h enzymatic hydrolysis time, 37 °C enzymatic hydrolysis temperature, and pH 2.0. Under the premise that other factors remained unchanged, the inhibition zone diameter and short peptide recovery rate were used as evaluation indicators to analyze the effects of enzyme activity (3000, 4000, 5000, 6000 and 7000 U / g), substrate concentration (50, 100, 150, 200 and 250 mg / mL), enzymatic hydrolysis time (2, 3, 4, 5 and 6 h), and ultrasonic power (90, 105, 120, 135 and 150 W) on the extraction of antimicrobial peptides from crucian carp scales. 1) Effect of substrate concentration on the extraction of fish scale antimicrobial peptides Depend on Figure 2 It can be seen that under the condition of fixed other enzymatic hydrolysis conditions, with the increase of substrate concentration, the recovery rate of short peptides showed an upward trend, and the diameter of the inhibition zone increased synchronously. The substrate concentration directly affects the contact area between the enzyme and the substrate. Therefore, the increase in substrate content makes the short peptides obtained by enzymatic hydrolysis, especially the antibacterial peptide fragments, increase accordingly, and the release rate of antimicrobial peptides is accelerated. When the substrate concentration reaches 200 mg / mL, the recovery rate of short peptides reaches a maximum of 17.05% ± 0.23, and the maximum diameter of the inhibition zone is 2.59 ± 0.04 cm, indicating that the reaction between the enzyme and the substrate reaches equilibrium, fully combines and acts, and the enzymatic hydrolysis reaction is more thorough. However, when the substrate concentration continues to increase, the recovery rate of short peptides decreases, and the diameter of the inhibition zone does not change much, which shows that the continued increase in substrate concentration does not promote the extraction of antimicrobial peptides. Therefore, the antimicrobial peptides obtained by enzymatic hydrolysis have the best antibacterial properties when the substrate concentration is 200 mg / mL. 2) Effect of enzyme activity on the extraction of fish scale antimicrobial peptides Depend on Figure 3 It can be seen that under the condition of fixed other enzymatic hydrolysis conditions, with the increase of enzyme activity, the recovery rate of short peptides and the diameter of the inhibition zone also gradually increased. When the enzyme activity was 5000U / g, the recovery rate of short peptides was 18.10%±0.17, and the maximum diameter of the inhibition zone was 2.18±0.13cm. The antibacterial effect of the antimicrobial peptides obtained by enzymatic hydrolysis reached the highest. However, as the enzyme activity continued to increase, the recovery rate of short peptides changed relatively slowly and did not continue to grow, while the diameter of the inhibition zone did not increase but decreased, indicating that the active sites of the enzyme and the substrate have reached saturation, and the isolated antimicrobial peptides will not continue to increase with the increase of the concentration of the added enzyme. The extra enzyme may further decompose the antimicrobial peptides into smaller molecular peptides or free amino acids, reducing the content of antimicrobial peptides with antibacterial properties, resulting in a downward trend in the diameter of the inhibition zone. 3) Effect of enzymatic hydrolysis time on the extraction of fish scale antimicrobial peptides Depend on Figure 4 It can be seen that under the condition of fixed other enzymatic hydrolysis conditions, the enzymatic hydrolysis time is the time for the enzyme to act on the substrate. Properly increasing the enzymatic hydrolysis time is conducive to the decomposition of the substrate by the enzyme and the release of more antimicrobial peptides. Therefore, in the enzymatic hydrolysis reaction of 2-4h, the recovery rate of short peptides and the diameter of the inhibition zone show an upward trend. When the enzymatic hydrolysis time is 4h, the recovery rate of short peptides is 18.11%±0.23, and the diameter of the inhibition zone is 2.09±0.13cm, both reaching the maximum value. However, as the enzymatic hydrolysis time continues to increase, the recovery rate of short peptides tends to be flat, and the diameter of the inhibition zone begins to decrease, indicating that the enzymatic hydrolysis reaction has reached a state of equilibrium at 4h, and the antimicrobial peptides in the substrate are completely decomposed. 4) Effect of ultrasonic power on the extraction of fish scale antimicrobial peptides Depend on Figure 5 It can be seen that the short peptide recovery rate and antibacterial activity of fish scale antimicrobial peptides show a trend of first increasing and then decreasing with the increase of ultrasonic power. In the ultrasonic range of 90W-120W, ultrasonic waves can significantly enhance the vibration power of water molecules, destroy the protein cells of fish scales, strengthen the reaction between proteases and substrates, improve the effect of enzymatic hydrolysis, and make the antimicrobial peptides in fish scales be quickly decomposed. Therefore, the short peptide recovery rate and the diameter of the inhibition zone are both increasing. When the ultrasonic power reaches 120W, the antibacterial activity of antimicrobial peptides and the short peptide recovery rate are maximized. However, when the ultrasonic power is too large, an acoustic wave barrier will be generated, hindering the transmission of sound waves, thereby destroying the structure of proteins in fish scales, and ultimately reducing the extraction of antimicrobial peptides. Therefore, the diameter of the inhibition zone and the recovery rate of short peptides show a downward trend. (3) Orthogonal optimization experiment of ultrasound-assisted enzymatic hydrolysis process Based on the results of single-factor experiments, the effects of various factors on the extraction of antimicrobial peptides were comprehensively considered. To obtain the best ultrasonic-assisted enzymatic hydrolysis process, enzymatic hydrolysis time, enzyme activity, and ultrasonic power were selected as experimental factors. Orthogonal L9(3 3 ) Orthogonal experimental design was used to optimize the enzymatic hydrolysis process of crucian carp scale antimicrobial peptides with inhibition zone diameter and short peptide recovery rate as indicators. Table 2 Orthogonal experimental design According to the results of the single factor experiment, compared with the other three factors, the effect of substrate concentration on the enzymatic hydrolysis effect is not very obvious. Therefore, the substrate concentration is fixed, and the three factors of enzymatic hydrolysis time, ultrasonic power and enzyme activity value are selected for orthogonal optimization test. The results are shown in Table 2. The results of the range analysis show that the effect of each factor on the diameter of the inhibition zone is enzyme activity > enzymatic hydrolysis time > ultrasonic power, and the effect on the recovery rate of short peptides is enzyme activity > enzymatic hydrolysis time > ultrasonic power. SPSS22.0 software was used for variance analysis. From the variance analysis results Table 3 and Table 4, it can be seen that the three factors have no significant differences in the diameter of the inhibition zone and the recovery rate of short peptides. Therefore, based on the results of the range analysis, the optimal process of ultrasonic-assisted enzymatic hydrolysis is determined to be enzymatic hydrolysis time 4h, ultrasonic power 120W, and enzyme activity 5000U / g. Table 3 Orthogonal test results Table 4 Orthogonal experiment variance results (inhibition zone diameter) Table 5 Orthogonal experiment variance results (short peptide recovery rate) (4) Verification experiment Under the optimal enzymatic hydrolysis process, a verification experiment was carried out in three parallel experiments. The experimental results are shown in Table 5. The average diameter of the inhibition zone was 2.66 cm, the calculated RSD value was 0.66%, the average short peptide recovery rate was 22.52%, and the RSD value was 1.64%. The experimental accuracy was high. Table 6 Verification experiment The invention uses crucian carp scales as raw materials, adopts ultrasonic-assisted enzymatic method to obtain crucian carp scale antibacterial polypeptide crude enzyme solution, and through single factor experiment and orthogonal optimization experiment, takes the diameter of inhibition zone and the recovery rate of short peptide as test indexes, and finally obtains the optimal enzymatic hydrolysis process conditions as follows: substrate concentration of 200 mg / mL, enzymatic hydrolysis time of 4 hours, ultrasonic power of 120 W, and enzyme activity of 5000 U / g. Studies have shown that under this process, the antibacterial activity of the antibacterial peptide crude enzyme solution obtained by enzymatic hydrolysis against Escherichia coli can reach an inhibition zone diameter of 2.66 cm and a short peptide recovery rate of 22.52%. The above description of the embodiments is to facilitate the understanding and use of the invention by those skilled in the art. It is obvious that those skilled in the art can easily make various modifications to these embodiments and apply the general principles described herein to other embodiments without creative work. Therefore, the present invention is not limited to the above embodiments, and 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 present invention should be within the scope of protection of the present invention.

Claims

1. A method for extracting antimicrobial peptides from crucian carp scales by ultrasound-assisted enzymatic hydrolysis, characterized in that: The specific steps are as follows: S1. Pretreatment of crucian carp scales; S2, mixing the pretreated crucian carp scales obtained in step S1 with a NaOH solution, an EDTA-2Na solution and a citric acid solution in sequence to obtain a mixture; S3, adding protease to the mixture obtained in step S2, and obtaining an enzymatic solution by ultrasonic-assisted enzymatic hydrolysis; S4, centrifuging the enzymatic hydrolyzate obtained in step S3, taking the supernatant after centrifugation and concentrating it by rotary evaporation to obtain a crude antimicrobial peptide enzyme solution; S5, ultrafiltrating the crude antimicrobial peptide enzyme solution obtained in step S4 to obtain a crucian carp scale antimicrobial peptide filtrate, desalting, freezing, and freeze-drying in sequence to obtain a crucian carp scale antimicrobial peptide.

2. The method for extracting antimicrobial peptides from crucian carp scales by ultrasound-assisted enzymatic hydrolysis according to claim 1, characterized in that: In step S1, the specific steps of pre-treating crucian carp scales are as follows: Take fresh crucian carp scales, wash them until there is no dirt or mucus on the surface, dry them in a forced air drying oven, and refrigerate them for later use.

3. The method for extracting antimicrobial peptides from crucian carp scales by ultrasound-assisted enzymatic hydrolysis according to claim 1, characterized in that: In step S2, the pretreated crucian carp scales obtained in step S1 are chopped up, added with NaOH solution, magnetically stirred, washed, added with EDTA-2Na solution, ultrasonicated, washed, added with citric acid solution, and heated in a water bath to obtain a mixture.

4. The method for extracting antimicrobial peptides from crucian carp scales by ultrasound-assisted enzymatic hydrolysis according to claim 1, characterized in that: In step S3, protease is added to the mixture obtained in step S2, and ultrasonic-assisted enzymatic hydrolysis is performed for a period of time, and then the enzyme is inactivated by water bath to obtain an enzymatic solution.

5. The method for extracting antimicrobial peptides from crucian carp scales by ultrasound-assisted enzymatic hydrolysis according to claim 1, characterized in that: In step S5, the molecular weight of the crucian carp scale antimicrobial peptide filtrate is 3000-5000Da.

6. A process optimization method for extracting antimicrobial peptides from crucian carp scales by ultrasonic-assisted enzymatic hydrolysis, characterized in that: The specific steps are as follows: (1) According to the method for extracting antimicrobial peptides from crucian carp scales by ultrasound-assisted enzymatic hydrolysis as described in any one of claims 1 to 5, the diameter of the inhibition zone of Escherichia coli and the recovery rate of short peptides by enzymatic hydrolysis are used as evaluation indicators to determine the most suitable protease for enzymatic hydrolysis; (2) Based on the protease determined in step (1), four factors affecting the extraction effect of crucian carp scale antimicrobial peptides were screened out; (3) using the diameter of the inhibition zone of Escherichia coli and the recovery rate of the short peptides after enzymatic hydrolysis as evaluation indicators, a single factor experiment was performed based on the four factors screened out in step (2); (4) Based on the single-factor experimental results of step (3), three factors that have a significant impact on the diameter of the inhibition zone of Escherichia coli and the recovery rate of short peptides after enzymatic hydrolysis were screened out for orthogonal experimental design to optimize the enzymatic hydrolysis process of crucian carp scale antimicrobial peptides.

7. The process optimization method for extracting antimicrobial peptides from crucian carp scales by ultrasonic-assisted enzymatic hydrolysis according to claim 6, characterized in that: In step (2), the most suitable protease is pepsin.

8. The process optimization method for extracting antimicrobial peptides from crucian carp scales by ultrasonic-assisted enzymatic hydrolysis according to claim 6, characterized in that: In step (2), the four factors that affect the extraction of antimicrobial peptides are: enzyme activity, substrate concentration, enzymatic hydrolysis time and ultrasonic power. The enzyme activity was set to 3000, 4000, 5000, 6000 and 7000 U / g of protease activity, The substrate concentrations were set to 50, 100, 150, 200 and 250 mg / mL of the mixture, The enzymolysis time was set to 2, 3, 4, 5 and 6 h, respectively. The ultrasonic powers were set to 90, 105, 120, 135 and 150 W respectively.

9. The process optimization method for extracting antimicrobial peptides from crucian carp scales by ultrasonic-assisted enzymatic hydrolysis according to claim 8, characterized in that: In step (3), the method for determining the diameter of the inhibition zone of Escherichia coli is as follows: Using Escherichia coli as a tool bacterium, a suspension of Escherichia coli was sucked into an agar medium solution to prepare an agar medium mixed with the test bacteria; a sterile Oxford cup was placed in a plate, and the agar medium mixed with the test bacteria was poured; after solidification, the Oxford cup was taken out, and the crucian carp scale antimicrobial peptide sample was sucked into the hole; after static culture, the diameter of the inhibition zone was measured, and physiological saline was used as a blank control; In step (3), the method for determining the recovery rate of the enzymatically hydrolyzed short peptide is as follows: The protein content of crucian carp scales and crucian carp scale antimicrobial peptides was determined by referring to GB / T 5009.5-2016 Determination of protein in food (method 1) Kjeldahl nitrogen method, and the short peptide recovery rate was calculated according to the formula: In the formula: C is the mass concentration of protein in the crucian carp scale antimicrobial peptide sample, mg / L; V is the volume of the crucian carp scale antimicrobial peptide sample, L; M is the mass of protein in the crucian carp scale, mg.

10. The process optimization method for extracting antimicrobial peptides from crucian carp scales by ultrasonic-assisted enzymatic hydrolysis according to claim 8, characterized in that: In step (4), the three factors with significant influence are: enzyme activity, enzymatic hydrolysis time and ultrasonic power; In step (4), the optimal enzymatic hydrolysis process of crucian carp scale antimicrobial peptide is as follows: enzymatic hydrolysis time 4h, ultrasonic power 120W, and enzyme activity 5000U / g.

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