Zanthoxylum bungeanum leaf antibacterial peptide as well as preparation method and application thereof
By using alkali-soluble acid precipitation method and ultrasonic reaction, antibacterial peptides of simmer leaf with significant antibacterial activity were prepared, which solved the existing antibiotic resistance problem, achieved effective inhibition of Staphylococcus aureus and E. coli, and improved the cytocompatibility of silver-loaded antibacterial preparations.
Patent Information
- Application Number
- CN202510309452.6
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- Filing Date
- 2025-03-17
- Publication Date
- 2025-06-06
AI Technical Summary
Existing antibiotics face drug resistance problems and seek a new antibacterial substance to replace traditional antibiotics.
The crude protein of simmering leaves was extracted by alkali-soluble acid precipitation method, and the antibacterial peptide of simmering leaves was prepared by ultrasonic reaction and enzymatic decomposition. The method includes a multi-step extraction and processing process to obtain an antibacterial peptide of the sauerkum leaf with significant antibacterial activity.
The extracted antibacterial peptide of the pepper leaf has significant antibacterial activity, which can replace the healthy livestock and poultry aquaculture antibiotics and bulk fruits with bio-preservation and freshness. After the preparation of silver-loaded antibacterial preparations, the toxic effect of AgNO3 on cells was improved and cell compatibility was improved.
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Abstract
Description
Technical Field
[0001] The present invention belongs to the technical field of plant peptides and biological antibacterial materials, and more specifically, relates to an antibacterial peptide from Zanthoxylum bungeanum leaves and a preparation method and application thereof. Background Art
[0002] Antimicrobial peptides ( Antimicrobial Peptides , AMPs) are a class of natural defense molecules widely present in organisms. They have broad-spectrum antimicrobial activity and can inhibit or kill pathogens such as bacteria, fungi, viruses and parasites. Unlike traditional antibiotics, antimicrobial peptides mainly exert their effects by destroying the integrity of microbial cell membranes, interfering with intracellular metabolism or regulating host immune responses. Due to their unique mechanism of action, it is difficult for microorganisms to develop resistance to them, so they are considered to be a potential alternative solution to the problem of antibiotic resistance. Plants have formed a complex defense system during the long process of evolution, and antimicrobial peptides are an important component of it. Antimicrobial peptides have been isolated from a variety of plants, such as thionins, defensins and lipid transfer proteins (LTPs). For example, allicin in garlic and capsaicin in peppers both exhibit significant antimicrobial activity. These plant-derived antimicrobial peptides not only have inhibitory effects on pathogenic microorganisms, but also have multiple functions such as anti-inflammatory, antioxidant and wound healing. Zanthoxylum bungeanum ( Zanthoxylum spp .) is a traditional Chinese medicinal and edible plant, and its leaves, fruits and seeds are rich in bioactive ingredients. Studies have shown that Zanthoxylum bungeanum leaves are rich in terpenes, phenolic acid compounds and specific antimicrobial peptides. Zanthoxylum bungeanum leaf antimicrobial peptides not only have a significant inhibitory effect on common pathogens such as Staphylococcus aureus and Escherichia coli, but also show potential activity against drug-resistant strains. Its mechanism of action may be related to destroying bacterial cell membranes, inhibiting biofilm formation or interfering with DNA replication. In addition, the natural source and low cytotoxicity of Zanthoxylum bungeanum leaf antimicrobial peptides provide advantages for their application in food preservation and medicine. Summary of the invention
[0003] An object of the present invention is to solve at least the above problems and / or disadvantages and to provide at least the advantages which will be described hereinafter.
[0004] In order to achieve these purposes and other advantages according to the present invention, a method for preparing antimicrobial peptides from Zanthoxylum bungeanum leaves is provided, characterized in that it comprises the following steps: Step 1, extracting crude protein from Zanthoxylum bungeanum leaves by alkali dissolution and acid precipitation method, drying the crude protein from the Zanthoxylum bungeanum leaves to obtain crude protein freeze-dried powder; preparing a Zanthoxylum bungeanum leaf protein solution by using a phosphate buffer having a pH of 9.0; Step 2, placing the Zanthoxylum bungeanum leaf protein solution in a multi-purpose constant temperature ultrasonic reactor for ultrasonic reaction; Step 3: After the ultrasonic reaction is completed, add protease and mix well, adjust the pH, and place in a water bath to complete enzymatic hydrolysis; Step 5: After the enzymatic hydrolysis is completed, the enzyme is inactivated, the mixture is cooled to room temperature, centrifuged, and the supernatant is taken to obtain Zanthoxylum bungeanum leaf antimicrobial peptides.
[0005] Preferably, in the step 1, the specific method of extracting crude protein from Zanthoxylum bungeanum leaves by alkali dissolution and acid precipitation method comprises: S1. Rinse fresh Zanthoxylum bungeanum leaves with deionized water for several times to remove surface impurities, drain and dry in a 40-50°C oven to constant weight; crush the dried Zanthoxylum bungeanum leaves through a 60-mesh sieve, take Zanthoxylum bungeanum leaf powder and add n-hexane or petroleum ether, stir and defat for 2-4h, repeat several times, centrifuge at 4000rpm for 10 min to remove fat-soluble impurities, and air-dry to obtain defatted Zanthoxylum bungeanum leaf powder; the dosage ratio of Zanthoxylum bungeanum leaf powder to n-hexane or petroleum ether is 1-10mg:10mL; S2, adding defatted Zanthoxylum bungeanum leaf powder to 0.1-0.3 mol / L NaOH solution, adjusting the pH to 9.0-10.0, and obtaining a defatted Zanthoxylum bungeanum leaf powder solution; the amount ratio of defatted Zanthoxylum bungeanum leaf powder to NaOH solution is 1 mg: 10-20 mL; S3, placing the defatted Zanthoxylum bungeanum leaf powder solution in a 50-60°C water bath and stirring continuously for 1-2 hours, then centrifuging at 6000-9000 rpm for 20-40 minutes, and collecting the supernatant; S4. Place the supernatant in an ice bath, slowly add 1 mol / L HCl or citric acid solution, stirring while adding, and adjust the pH to 4.0-4.5; let stand at 4℃ for 1h, centrifuge at 6000-9000rpm for 15min, discard the supernatant, collect the precipitate, wash it several times with pre-cooled anhydrous ethanol, and obtain the crude protein precipitate of Zanthoxylum bungeanum leaf after centrifugation.
[0006] Preferably, in the step 1, the drying method for drying the crude protein from Zanthoxylum bungeanum leaf to obtain the crude protein freeze-dried powder is spray drying or vacuum freeze drying.
[0007] Preferably, in the step 2, the ultrasonic reaction parameters are: under the condition of medium temperature of 40-60°C, pulse ultrasound for 15s as one cycle, working for 10s and stopping for 5s; ultrasonic power of 150-800W, and ultrasonic treatment time of 10-50min.
[0008] Preferably, in step three, the protease is alkaline protease Alcalase, the pH adjustment range is 8-11, the amount of alkaline protease Alcalase is 100-1000 U / g, the enzymolysis temperature is 40-60° C., and the enzymolysis time is 50-80 min.
[0009] Preferably, in step 4, the enzyme inactivation temperature is 90-110° C. and the enzyme inactivation time is 5-20 min.
[0010] A Zanthoxylum bungeanum leaf antimicrobial peptide is prepared by the above-mentioned Zanthoxylum bungeanum leaf antimicrobial peptide preparation method.
[0011] An application of an antimicrobial peptide from Zanthoxylum bungeanum leaf, wherein the antimicrobial peptide from Zanthoxylum bungeanum leaf is applied to replace antibiotics for healthy breeding of livestock and poultry and for biological preservation and freshness preservation of bulk fruits.
[0012] An application of a Zanthoxylum bungeanum leaf antimicrobial peptide, wherein the Zanthoxylum bungeanum leaf antimicrobial peptide is used to prepare a silver-loaded antimicrobial preparation; wherein the specific method for preparing the silver-loaded antimicrobial preparation with the Zanthoxylum bungeanum leaf antimicrobial peptide comprises: S11, AgNO 3 The solution was added dropwise to the antimicrobial peptide from Zanthoxylum bungeanum leaves, and sodium alginate solution was added, and ultrasonic dispersion was performed for 20 to 60 minutes at an ultrasonic frequency of 60 to 80 kHz to obtain a mixed solution; S12, raising the temperature of the mixture to 90°C at a rate of 5°C / min, stirring in a 90°C water bath in the dark for 60 min, and then cooling to 25°C at a rate of 5°C / min; S13, adding chitosan solution to the cooled mixed solution and stirring thoroughly, then dropping calcium chloride solution, stirring and collecting gel beads, and freeze-drying to obtain a silver-loaded antibacterial preparation.
[0013] Preferably, in the S11, AgNO 3 The concentration of the solution is 0.5~60mmol / L, the concentration of the sodium alginate solution is 1~5wt%; the concentration of AgNO 3 The volume ratio of solution, Zanthoxylum bungeanum leaf antimicrobial peptide, and sodium alginate solution is 1:5-15:2-8; In S13, the concentration of the chitosan solution is 2-5wt%, the concentration of the calcium chloride solution is 0.1-1 mol / L; the volume ratio of the sodium alginate solution, the chitosan solution and the calcium chloride solution is 2-8:1-2:0.01-1; In S13, the freeze-drying temperature is -80 to -25°C, the vacuum degree is less than 10 Pa, and the freeze-drying time is 24 to 48 hours.
[0014] The present invention has at least the following beneficial effects: the antimicrobial peptide extracted from Zanthoxylum bungeanum leaves has significant antibacterial activity, and can be used to replace antibiotics for healthy livestock and poultry breeding and for biological preservation of bulk fruits; at the same time, after being used to prepare silver-loaded antibacterial preparations, the antibacterial activity is ensured, and the AgNO 3 The toxic effect on cells makes AgNO 3 After compounding, it has higher cell compatibility and can be used as a cell antibacterial agent.
[0015] Other advantages, objectives and features of the present invention will be embodied in part through the following description, and in part will be understood by those skilled in the art through study and practice of the present invention. DETAILED DESCRIPTION
[0016] The present invention is described in further detail below so that those skilled in the art can implement it according to the description.
[0017] It should be understood that the terms such as “having”, “including” and “comprising” used herein do not exclude the existence or addition of one or more other elements or combinations thereof.
[0018] Embodiment 1:
[0019] This embodiment provides a method for preparing antimicrobial peptides from Zanthoxylum bungeanum leaves, comprising the following steps: Step 1: extracting crude protein from Zanthoxylum bungeanum leaves by alkali dissolution and acid precipitation method, the specific method includes: S1. Rinse fresh Zanthoxylum bungeanum leaves with deionized water for several times to remove surface impurities, drain and dry in a 40°C oven to constant weight; crush the dried Zanthoxylum bungeanum leaves through a 60-mesh sieve, take 50 mg of Zanthoxylum bungeanum leaf powder and add it to 100 mL of n-hexane, stir and defat for 4 h, repeat several times, centrifuge at 4000 rpm for 10 min to remove fat-soluble impurities, and air-dry to obtain defatted Zanthoxylum bungeanum leaf powder; S2, adding 25 mg of defatted Zanthoxylum bungeanum leaf powder into 250 mL of 0.2 mol / L NaOH solution, adjusting the pH to 10.0, to obtain a defatted Zanthoxylum bungeanum leaf powder solution; S3, placing the defatted Zanthoxylum bungeanum leaf powder solution in a 60°C water bath and stirring continuously for 2 h, then centrifuging at 8000 rpm for 40 min, and collecting the supernatant; S4. Place the supernatant in an ice bath, slowly drop 1 mol / L citric acid solution, stir while adding, and adjust the pH to 4.0; let stand at 4°C for 1 hour, centrifuge at 8000rpm for 15 minutes, discard the supernatant, collect the precipitate, wash it with precooled anhydrous ethanol 5 times, and obtain the crude protein precipitate of Zanthoxylum bungeanum after centrifugation. Spray dry the crude protein precipitate of Zanthoxylum bungeanum to obtain the crude protein freeze-dried powder; use 100mL of phosphate buffer with a pH of 9.0 to prepare the extracted 10mg crude protein freeze-dried powder into a Zanthoxylum bungeanum protein solution; Step 2: placing the Zanthoxylum bungeanum protein solution in a multi-purpose constant temperature ultrasonic reactor, under the condition of medium temperature of 60°C, pulse ultrasound for 15 seconds as one cycle, working for 10 seconds and stopping for 5 seconds; ultrasonic power of 200W, ultrasonic treatment time of 30 minutes; Step 3: After the ultrasonic reaction is completed, add 500U / g alkaline protease Alcalase and mix well, adjust the pH to 11, and place in a water bath at 60°C to complete enzymatic hydrolysis for 60 minutes; Step 5: After the enzymatic hydrolysis is completed, the enzyme is inactivated at 95°C for 15 minutes, cooled to room temperature, centrifuged, and the supernatant is taken to obtain Zanthoxylum bungeanum leaf antimicrobial peptides.
[0020] Embodiment 2:
[0021] This embodiment provides a method for preparing antimicrobial peptides from Zanthoxylum bungeanum leaves, comprising the following steps: Step 1: extracting crude protein from Zanthoxylum bungeanum leaves by alkali dissolution and acid precipitation method, the specific method includes: S1. Rinse fresh Zanthoxylum bungeanum leaves with deionized water several times to remove surface impurities, drain and dry in a 50°C oven to constant weight; crush the dried Zanthoxylum bungeanum leaves through a 60-mesh sieve, take 50 mg of Zanthoxylum bungeanum leaf powder and add it to 100 mL of n-hexane, stir and defat for 4 h, repeat several times, centrifuge at 4000 rpm for 10 min to remove fat-soluble impurities, and air-dry to obtain defatted Zanthoxylum bungeanum leaf powder; S2, adding 25 mg of defatted Zanthoxylum bungeanum leaf powder into 250 mL of 0.2 mol / L NaOH solution, adjusting the pH to 10.0, to obtain a defatted Zanthoxylum bungeanum leaf powder solution; S3, placing the defatted Zanthoxylum bungeanum leaf powder solution in a 60°C water bath and stirring continuously for 2 h, then centrifuging at 8000 rpm for 40 min, and collecting the supernatant; S4. Place the supernatant in an ice bath, slowly drop 1 mol / L HCl solution, stir while adding, and adjust the pH to 4.0; let stand at 4°C for 1 hour, centrifuge at 8000rpm for 15 minutes, discard the supernatant, collect the precipitate, wash it with pre-cooled anhydrous ethanol 5 times, and obtain the crude protein precipitate of Zanthoxylum bungeanum after centrifugation. Spray dry the crude protein precipitate of Zanthoxylum bungeanum to obtain the crude protein freeze-dried powder; use 250mL phosphate buffer PBS with a pH of 9.0 to prepare the extracted 10mg crude protein freeze-dried powder into a Zanthoxylum bungeanum protein solution; Step 2: placing the Zanthoxylum bungeanum protein solution in a multi-purpose constant temperature ultrasonic reactor, under the condition of medium temperature of 60°C, pulse ultrasound for 15s as one cycle, working for 10s and stopping for 5s; ultrasonic power of 500W, ultrasonic treatment time of 30min; Step 3: After the ultrasonic reaction is completed, add 600 U / g alkaline protease Alcalase and mix well, adjust the pH to 11, and place in a water bath at 60°C to complete enzymatic hydrolysis for 60 minutes; Step 5: After the enzymatic hydrolysis is completed, the enzyme is inactivated at 95°C for 15 minutes, cooled to room temperature, centrifuged, and the supernatant is taken to obtain Zanthoxylum bungeanum leaf antimicrobial peptides.
[0022] Embodiment 3:
[0023] This embodiment provides a method for preparing antimicrobial peptides from Zanthoxylum bungeanum leaves, comprising the following steps: Step 1: extracting crude protein from Zanthoxylum bungeanum leaves by alkali dissolution and acid precipitation method, the specific method includes: S1. Rinse fresh Zanthoxylum bungeanum leaves with deionized water for several times to remove surface impurities, drain and dry in a 40°C oven to constant weight; crush the dried Zanthoxylum bungeanum leaves through a 60-mesh sieve, take 50 mg of Zanthoxylum bungeanum leaf powder and add it to 100 mL of n-hexane, stir and defat for 4 h, repeat several times, centrifuge at 4000 rpm for 10 min to remove fat-soluble impurities, and air-dry to obtain defatted Zanthoxylum bungeanum leaf powder; S2, adding 25 mg of defatted Zanthoxylum bungeanum leaf powder into 250 mL of 0.1 mol / L NaOH solution, adjusting the pH to 10.0, to obtain a defatted Zanthoxylum bungeanum leaf powder solution; S3, placing the defatted Zanthoxylum bungeanum leaf powder solution in a 60°C water bath and stirring continuously for 2 h, then centrifuging at 8000 rpm for 40 min, and collecting the supernatant; S4. Place the supernatant in an ice bath, slowly drop 1 mol / L citric acid solution, stir while adding, and adjust the pH to 4.0; let stand at 4°C for 1 hour, centrifuge at 8000rpm for 15 minutes, discard the supernatant, collect the precipitate, wash it with pre-cooled anhydrous ethanol 5 times, and obtain the crude protein precipitate of Zanthoxylum bungeanum after centrifugation. Spray dry the crude protein precipitate of Zanthoxylum bungeanum to obtain the crude protein freeze-dried powder; prepare 5 mg of the extracted crude protein freeze-dried powder into Zanthoxylum bungeanum protein solution with 250 mL of phosphate buffer with a pH of 9.0; Step 2: placing the Zanthoxylum bungeanum protein solution in a multi-purpose constant temperature ultrasonic reactor, under the condition of medium temperature of 60°C, pulse ultrasound for 15s as one cycle, working for 10s and stopping for 5s; ultrasonic power of 650W, ultrasonic treatment time of 30min; Step 3: After the ultrasonic reaction is completed, add 800 U / g alkaline protease Alcalase and mix well, adjust the pH to 11, and place in a water bath at 60°C to complete enzymatic hydrolysis for 60 minutes; Step 5: After the enzymatic hydrolysis is completed, the enzyme is inactivated at 95°C for 15 minutes, cooled to room temperature, centrifuged, and the supernatant is taken to obtain Zanthoxylum bungeanum leaf antimicrobial peptides.
[0024] Application Example 1: For the Zanthoxylum bungeanum leaf antimicrobial peptide extracted in Example 1, this application example provides a method for preparing a silver-loaded antimicrobial preparation with the Zanthoxylum bungeanum leaf antimicrobial peptide, which specifically includes: S11, 10mL 0.5mmol / L AgNO 3 The solution was added dropwise to 50 mL of Zanthoxylum bungeanum leaf antimicrobial peptide, and 20 mL of 3 wt% sodium alginate solution was added, and ultrasonic dispersion was performed for 60 min at an ultrasonic frequency of 50 kHz to obtain a mixed solution; S12, raising the temperature of the mixture to 90°C at a rate of 5°C / min, stirring in a 90°C water bath in the dark for 60 min, and then cooling to 25°C at a rate of 5°C / min; S13. Add 10 mL of 1 wt% chitosan solution to the cooled mixed solution and stir thoroughly, then drop 5 mL of 0.5 mol / L calcium chloride solution, stir and collect the gel beads, freeze-dry at -55°C and 2.2 Pa vacuum for 24 h to obtain a silver-loaded antibacterial preparation.
[0025] Application Example 2: For the Zanthoxylum bungeanum leaf antimicrobial peptide extracted in Example 1, this application example provides a method for preparing a silver-loaded antimicrobial preparation with the Zanthoxylum bungeanum leaf antimicrobial peptide, which specifically includes: S11, 25mL 0.8mmol / L AgNO 3 The solution was added dropwise to 150 mL of Zanthoxylum bungeanum leaf antimicrobial peptide, and 50 mL of 3 wt% sodium alginate solution was added, and ultrasonic dispersion was performed for 60 min at an ultrasonic frequency of 80 kHz to obtain a mixed solution. S12, raising the temperature of the mixture to 90°C at a rate of 5°C / min, stirring in a 90°C water bath in the dark for 60 min, and then cooling to 25°C at a rate of 5°C / min; S13, add 25 mL of 2 wt% chitosan solution to the cooled mixed solution, stir thoroughly, then dropwise add 10 mL of 0.5 mol / L calcium chloride solution, stir, collect gel beads, and freeze-dry at -25°C and 5 Pa vacuum to obtain a silver-loaded antibacterial preparation.
[0026] Comparative application example 1: This comparative application example provides a method for preparing a silver-loaded antibacterial preparation, comprising the following steps: S11, 10mL 0.5mmol / L AgNO 3 The solution was added dropwise to 20 mL of 3 wt% sodium alginate solution, and ultrasonically dispersed for 60 min at an ultrasonic frequency of 50 kHz to obtain a mixed solution; S12, raising the temperature of the mixture to 90°C at a rate of 5°C / min, stirring in a 90°C water bath in the dark for 60 min, and then cooling to 25°C at a rate of 5°C / min; S13. Add 10 mL of 1 wt% chitosan solution to the cooled mixed solution and stir thoroughly, then drop 5 mL of 0.5 mol / L calcium chloride solution, stir and collect the gel beads, freeze-dry at -55°C and 2.2 Pa vacuum for 24 h to obtain a silver-loaded antibacterial preparation.
[0027] Comparative application example 2: This comparative application example provides a method for preparing a silver-loaded antibacterial preparation, comprising the following steps: S11, 25mL 0.8mmol / L AgNO 3 The solution was added dropwise to 50 mL of 3 wt% sodium alginate solution, and ultrasonically dispersed for 60 min at an ultrasonic frequency of 80 kHz to obtain a mixed solution; S12, raising the temperature of the mixture to 90°C at a rate of 5°C / min, stirring in a 90°C water bath in the dark for 60 min, and then cooling to 25°C at a rate of 5°C / min; S13, add 25 mL of 2 wt% chitosan solution to the cooled mixed solution, stir thoroughly, then dropwise add 10 mL of 0.5 mol / L calcium chloride solution, stir, collect gel beads, and freeze-dry at -25°C and 5 Pa vacuum to obtain a silver-loaded antibacterial preparation.
[0028] The antibacterial activity of the antimicrobial peptides extracted from Zanthoxylum bungeanum leaves in Examples 1 to 3 was determined by the Oxford cup method. The antimicrobial peptides from Zanthoxylum bungeanum leaves were prepared into a 1 mg / mL solution using sterile water to obtain Staphylococcus aureus ( S. aureus , ATCC6538) and Escherichia coli ( E. coli , ATCC 25922) and the diameter of the inhibition zone were obtained in Table 1: Table 1 Diameters of inhibition zones of each sample against Staphylococcus aureus and Escherichia coli
[0029] It can be seen from the above table that the Zanthoxylum bungeanum leaf antimicrobial peptides extracted in Examples 1 to 3 have excellent antibacterial activity against Staphylococcus aureus and Escherichia coli.
[0030] The minimum inhibitory concentration (MIC) of the silver-loaded antibacterial preparations prepared in Application Example 1-Application Example 2 and Comparative Application Example 1-Corresponding Example 2 was determined respectively. The specific method is as follows: Dissolve 25 g of MH broth in 1000 mL of deionized water and sterilize by high pressure steam at 121°C for 15 min to obtain MH liquid medium. Dissolve 25 g of MH broth and 15 g of agar powder in 1000 mL of deionized water and sterilize by high pressure steam at 121°C for 15 min to obtain MH solid medium.
[0031] Disperse the sterilized silver-loaded antibacterial preparations prepared in Application Example 1-Application Example 2 and Comparative Application Example 1-Comparative Application Example 2 in sterile water to form a 4 mg / mL sample solution. A series of sample solutions were obtained by performing a 2-fold gradient dilution in a 96-well culture plate. Add 100 μL of MH liquid culture medium to the wells of the 96-well culture plate, then add 100 μL of the 4 mg / mL sample solution, and mix by repeatedly blowing to form a 2 mg / L sample solution; pipette 100 μL of the 2 mg / mL sample solution into the next well (containing 100 μL of MH liquid culture medium), and mix by repeatedly blowing to form a 1 mg / mL sample solution; repeat this until the last concentration is 0.0156 mg / mL, and pipette 100 μL of the liquid in the last concentration well and discard; repeat each sample 3 times. Then, add 100 μL of the diluted bacterial solution (Staphylococcus aureus or Escherichia coli bacterial solution) (10 5 -10 6 CFU / mL), at which time the sample concentration in each well, that is, the final sample concentration, is 2000, 1000, 500, 250, 125, 62.5, 31.3, 15.6 and 7.8 μg / mL. In addition, a negative control (only 200 μL of blank MH liquid culture medium) and a positive control (100 μL of bacterial liquid and MH liquid culture medium each) were made on the same culture plate. After the 96-well culture plate was placed in a 37 ℃ constant temperature incubator for 24 hours, the color of the culture medium was observed, and the minimum sample concentration at which the color of the culture medium was consistent with that of the negative control group was obtained. Table 2 is obtained: Table 2 Minimum inhibitory concentration of each sample
[0032] It can be seen from the above table that the silver-loaded antibacterial preparations prepared in Application Example 1 and Application Example 2 have lower antibacterial concentrations and stronger antibacterial abilities against Staphylococcus aureus and Escherichia coli.
[0033] The compatibility of the silver-loaded antibacterial preparations prepared in Application Example 1-Application Example 2 and Comparative Application Example 1-Comparative Application Example 2 with cells was determined respectively, and the specific method was as follows: Mouse embryonic fibroblasts (NIH3T3) were used as test cells in a medium containing 10% fetal bovine serum, 2.0×10 5 The complete cell culture medium was DMEM containing 100 U / L penicillin and 150 mg / L streptomycin.
[0034] The silver-loaded antibacterial preparations prepared in Application Example 1-Application Example 2 and Comparative Application Example 1-Comparative Application Example 2 were dispersed in DMEM culture medium containing 10% fetal bovine serum, and after gradient dilution, sample solutions with silver concentrations of 12, 10, 8, 6, 4, 2, 1 and 0.5 μg / mL were obtained. NIH3T3 cells were cultured at 1.2×10 5 The cells were seeded into 96-well cell culture plates at a density of 1.54 × 10 cells / well and incubated at 37 °C with 5% CO 2 After culturing in a humidified incubator for 24 h, the culture medium was removed and 100 μL of sample solution was added for continued incubation. The control group was incubated with 100 μL of culture medium. After incubation for 24 h, the cell viability was tested by the MTT method. After the time was reached, 10 μL of thiazolyl blue (concentration of 5 mg / mL) reagent was added to each well and incubated for another 4 h. Then the culture medium in the well was carefully removed and 150 μL of dimethyl sulfoxide was added and incubated for another 15 min. After the incubation, the optical density value was read at 570 nm using a microplate reader. With silver concentration as the horizontal axis and cell viability as the vertical axis, the half-inhibitory concentration (IC50) of each silver-loaded antibacterial preparation on NIH3T3 cells was calculated by curve fitting. 50 ), we get Table 3: Table 3 The half inhibitory concentration of each sample on NIH3T3 cells
[0035] It can be seen from the above table that the silver-loaded antibacterial preparations prepared in Application Examples 1 and 2 have higher half inhibition concentrations and exhibit higher cell compatibility.
[0036] The number of devices and processing scales described here are used to simplify the description of the present invention. Applications, modifications and variations of the present invention will be obvious to those skilled in the art.
[0037] Although the embodiments of the present invention have been disclosed as above, they are not limited to the applications listed in the specification and the implementation modes, and they can be fully applied to various fields suitable for the present invention. For those familiar with the art, additional modifications can be easily implemented. Therefore, without departing from the general concept defined by the claims and the scope of equivalents, the present invention is not limited to the specific details and the examples shown and described herein.
Claims
1. A method for preparing an antimicrobial peptide from Zanthoxylum bungeanum leaves, characterized in that: The following steps are involved: Step 1, extracting crude protein from Zanthoxylum bungeanum leaves by alkali dissolution and acid precipitation method, drying the crude protein from the Zanthoxylum bungeanum leaves to obtain crude protein freeze-dried powder; preparing a Zanthoxylum bungeanum leaf protein solution by using a phosphate buffer having a pH of 9.0; Step 2, placing the Zanthoxylum bungeanum leaf protein solution in a multi-purpose constant temperature ultrasonic reactor for ultrasonic reaction; Step 3: After the ultrasonic reaction is completed, add protease and mix well, adjust the pH, and place in a water bath to complete enzymatic hydrolysis; Step 5: After the enzymatic hydrolysis is completed, the enzyme is inactivated, the mixture is cooled to room temperature, centrifuged, and the supernatant is taken to obtain Zanthoxylum bungeanum leaf antimicrobial peptides.
2. The method for preparing the antimicrobial peptide from Zanthoxylum bungeanum leaves as claimed in claim 1, wherein: In the step 1, the specific method of extracting crude protein from Zanthoxylum bungeanum leaves by alkali dissolution and acid precipitation method comprises: S1. Rinse fresh Zanthoxylum bungeanum leaves with deionized water for several times to remove surface impurities, drain and dry in a 40-50°C oven to constant weight; crush the dried Zanthoxylum bungeanum leaves through a 60-mesh sieve, take Zanthoxylum bungeanum leaf powder and add n-hexane or petroleum ether, stir and defat for 2-4h, repeat several times, centrifuge at 4000rpm for 10 min to remove fat-soluble impurities, and air-dry to obtain defatted Zanthoxylum bungeanum leaf powder; the dosage ratio of Zanthoxylum bungeanum leaf powder to n-hexane or petroleum ether is 1-10mg:10mL; S2, adding defatted Zanthoxylum bungeanum leaf powder to 0.1-0.3 mol / L NaOH solution, adjusting the pH to 9.0-10.0, and obtaining a defatted Zanthoxylum bungeanum leaf powder solution; the amount ratio of defatted Zanthoxylum bungeanum leaf powder to NaOH solution is 1 mg: 10-20 mL; S3, placing the defatted Zanthoxylum bungeanum leaf powder solution in a 50-60°C water bath and stirring continuously for 1-2 hours, then centrifuging at 6000-9000 rpm for 20-40 minutes, and collecting the supernatant; S4. Place the supernatant in an ice bath, slowly add 1 mol / L HCl or citric acid solution, stirring while adding, and adjust the pH to 4.0-4.5; let stand at 4℃ for 1h, centrifuge at 6000-9000rpm for 15min, discard the supernatant, collect the precipitate, wash it several times with pre-cooled anhydrous ethanol, and obtain the crude protein precipitate of Zanthoxylum bungeanum leaf after centrifugation.
3. The method for preparing the antimicrobial peptide from Zanthoxylum bungeanum leaves as claimed in claim 1, wherein: In the step 1, the method for drying the crude protein from Zanthoxylum bungeanum leaves to obtain the crude protein freeze-dried powder is spray drying or vacuum freeze drying.
4. The method for preparing the antimicrobial peptide from Zanthoxylum bungeanum leaves as claimed in claim 1, wherein: In the step 2, the ultrasonic reaction parameters are: under the condition of medium temperature of 40-60°C, pulse ultrasound for 15s as one cycle, working for 10s and stopping for 5s; ultrasonic power of 150-800W, and ultrasonic treatment time of 10-50min.
5. The method for preparing the antimicrobial peptide from Zanthoxylum bungeanum leaves as claimed in claim 1, wherein: In the step three, the protease is alkaline protease Alcalase, the pH adjustment range is 8-11, the amount of alkaline protease Alcalase is 100-1000 U / g, the enzymolysis temperature is 40-60° C., and the enzymolysis time is 50-80 min.
6. The method for preparing the antimicrobial peptide from Zanthoxylum bungeanum leaves as claimed in claim 1, wherein: In the step 4, the enzyme inactivation temperature is 90-110° C., and the enzyme inactivation time is 5-20 min.
7. An antimicrobial peptide from Zanthoxylum bungeanum leaves, characterized in that The Zanthoxylum bungeanum leaf antimicrobial peptide is prepared by the method for preparing the Zanthoxylum bungeanum leaf antimicrobial peptide according to any one of claims 1 to 6.
8. A use of the Zanthoxylum bungeanum leaf antimicrobial peptide as claimed in claim 7, characterized in that: The Zanthoxylum bungeanum leaf antimicrobial peptide is used to replace antibiotics for healthy livestock and poultry breeding and for biological preservation of bulk fruits.
9. An application of the antimicrobial peptide from Zanthoxylum bungeanum leaves as claimed in claim 7, characterized in that: The Zanthoxylum bungeanum leaf antimicrobial peptide is used to prepare a silver-loaded antimicrobial preparation; wherein the specific method for preparing the silver-loaded antimicrobial preparation with the Zanthoxylum bungeanum leaf antimicrobial peptide comprises: S11, dropping the AgNO3 solution into the Zanthoxylum bungeanum leaf antimicrobial peptide, adding the sodium alginate solution, and ultrasonically dispersing for 20 to 60 min at an ultrasonic frequency of 60 to 80 kHz to obtain a mixed solution; S12, raising the temperature of the mixture to 90°C at a rate of 5°C / min, stirring in a 90°C water bath in the dark for 60 min, and then cooling to 25°C at a rate of 5°C / min; S13, adding chitosan solution to the cooled mixed solution and stirring thoroughly, then dropping calcium chloride solution, stirring and collecting gel beads, and freeze-drying to obtain a silver-loaded antibacterial preparation.
10. The use of the Zanthoxylum bungeanum leaf antimicrobial peptide according to claim 9, characterized in that: In the S11, the concentration of the AgNO3 solution is 0.5-60 mmol / L, and the concentration of the sodium alginate solution is 1-5 wt%; the volume ratio of the AgNO3 solution, the Zanthoxylum bungeanum leaf antimicrobial peptide, and the sodium alginate solution is 1:5-15:2-8; In S13, the concentration of the chitosan solution is 2-5wt%, the concentration of the calcium chloride solution is 0.1-1 mol / L; the volume ratio of the sodium alginate solution, the chitosan solution and the calcium chloride solution is 2-8:1-2:0.01-1; In S13, the freeze-drying temperature is -80 to -25°C, the vacuum degree is less than 10 Pa, and the freeze-drying time is 24 to 48 hours.