Antibacterial peptide as well as extraction method and application thereof
Antimicrobial peptides were extracted from cotton bollworms using quick-freeze-drying and acid extraction techniques, solving the problem of high extraction costs for insect antimicrobial peptides and achieving low-cost industrial production and efficient extraction.
Patent Information
- Application Number
- CN202511129096.6
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- Filing Date
- 2025-08-13
- Publication Date
- 2025-11-28
AI Technical Summary
Existing technologies for extracting antimicrobial peptides from insects are costly and require sophisticated equipment, making industrial-scale production impossible. Furthermore, genetically modified technology faces safety concerns and has low public acceptance.
The cotton bollworm bodies were treated by quick-freezing and freeze-drying. Extraction was carried out using an acidic extractant and a hydrolytic enzyme inhibitor combined with ammonium sulfate, PEG400 and ethanol. Separation was performed by nanofiltration membrane, which simplifies the process steps and reduces equipment requirements and production costs.
It achieves efficient extraction of antimicrobial peptides, significantly reduces production costs, simplifies process steps, enables industrial production, significantly improves extraction efficiency, and solves the technical challenges of industrial production that have not been addressed in existing technologies.
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Abstract
Description
TECHNICAL FIELD
[0001] The present application relates to the field of biotechnology, in particular to an antibacterial peptide and its extraction method and application. BACKGROUND
[0002] The pathogen of Citrus Huanglongbing (HLB) is Candidatus Liberibacter, a gram-negative bacteria which is difficult to be cultured artificially. The transmission of HLB mainly includes grafting seedling transmission and citrus psyllid transmission. The symptoms of citrus infection are initially manifested as stunted plant, difficult to sprout, and leaf mottling and yellowing; at the mature stage, it is manifested as yellowing, green fruit skin (also known as "red-nosed fruit"), bitter taste of fruit pulp, and serious impact on commercial value. Usually, citrus is yellow and withered within a few years after the onset, resulting in absolute production and absolute loss.
[0003] Antibacterial peptides are widely present in the immune defense system of animals and plants, and are usually composed of 20 to 100 amino acids. When animals and plants are subjected to biological invasion or non-biological condition stimulation, antibacterial peptide substances can be rapidly produced to inhibit or kill the invading pathogenic organisms. The main mechanism of action of antibacterial peptides is through interaction with the cell membrane of pathogenic bacteria, including forming aggregates with phospholipid molecules on the cell membrane of pathogenic bacteria, penetrating the cell membrane, forming ring pores or ion leakage channels, affecting the permeability of the cell membrane of pathogenic bacteria, and achieving antibacterial or bactericidal effect. It is also found that antibacterial peptides can enter cells and interfere with cell metabolism by specific binding to intracellular targets, achieving the purpose of inhibiting and killing bacteria. Because of the special bactericidal mechanism of antibacterial peptides, it is considered that it is not easy to produce drug resistance.
[0004] Research reports that transgenic expression of antibacterial peptide D2A21 in citrus can make citrus resistant to canker disease. Overexpression of Thionins in citrus can improve the resistance of citrus to canker disease and Huanglongbing disease. Huang found that a heat-stable antibacterial peptide (SAMP) was expressed in citrus varieties resistant to Huanglongbing disease by comparing the gene expression differences between citrus varieties resistant to Huanglongbing disease and susceptible to Huanglongbing disease. The SAMP contains 69 amino acids, can quickly kill Liberibacter crescens, a strain of Liberibacter that can be cultured in vitro, and can inhibit the invasion of CLas. Injection of SAMP into the trunk of Huanglongbing disease trees found that SAMP not only effectively reduced the content of CLas in citrus trees and alleviated the symptoms of Huanglongbing disease, but also induced the immune system of citrus. SAMP is heat stable. Leaf spraying SAMP can be effectively absorbed by citrus leaves, conducted along the vascular bundle of citrus, and existed in the tree for at least a week. Supratim Basu et al. used a computer to construct a chimeric polypeptide library of antibacterial peptides from two different sources, then tested the antibacterial activity and toxicity of the polypeptides, and screened high-activity and non-toxic antibacterial peptide molecules. Then they tested the bactericidal ability of the antibacterial peptides on Huanglongbing disease bacteria on in vitro infected Huanglongbing disease leaves, and found that the antibacterial peptides not only completely killed the Huanglongbing disease bacteria in the leaves, but also improved the endogenous immunity of the leaf tissue. Cao Peng cloned and expressed the antibacterial peptides MaSAMP (Stable antimicrobial peptide), HBD-4 (Homo sapiens defensin beta4A), and CB (Cecropin B) genes; based on Agrobacterium rhizogenes-mediated hairy root induction transformation, the proliferation pattern of CLas in citrus roots at the early stage of infection and the optimal monitoring index were explored, and a rapid evaluation system for HLB resistance was established; further application of the established system to evaluate the HLB resistance of the above three antibacterial peptides showed that the CLas content in the three antibacterial peptide expression plants was significantly lower than that in the control plants, indicating that MaSAMP, HBD-4 and CB had a significant inhibitory effect on the growth and reproduction of CLas.
[0005] Although the prior art shows that antibacterial peptides can effectively prevent and control citrus Huanglongbing disease, these technologies all use transgenic technology to express antibacterial peptides and perform extraction and purification. Due to the small molecular weight of antibacterial peptides, they are easily hydrolyzed by hydrolases, and the yield is low and the cost is high by using transgenic technology. After bacterial expression, a large amount of waste liquid and solid is produced, and further purification is required after expression, which usually requires the use of gel chromatography, preparative liquid chromatography, Ni column adsorption and elution, enzyme digestion and other means. The technology used is complex, the equipment investment is large, and the materials used are expensive, further increasing the production cost of antibacterial peptides.
[0006] However, the production cost of the antibacterial peptide is high, which will greatly limit the application of the antibacterial peptide. Moreover, the plant transgenic technology is involved, and special evaluation procedures are required due to the safety of the transgenic plant. In addition, the transgenic plant is not easily accepted by the public in practical use.
[0007] The cotton bollworm (Helicoverpa armigera) is an important agricultural pest. It contains various antibacterial peptides such as cecropin, defensin and lebocin. These antibacterial peptides have broad-spectrum antibacterial activity and can effectively inhibit the growth of various pathogenic microorganisms. Studies have shown that the antibacterial peptides in the cotton bollworm play an important role in the immune response and can enhance its resistance to pathogenic microorganisms. Therefore, the extraction of antibacterial peptides from the cotton bollworm and their application in the prevention and treatment of citrus Huanglong disease have good application prospects.
[0008] There are some research reports on the extraction of antibacterial peptides from insects. Sun Long used the larvae of Tenebrio molitor Linnaeus and Blaps rhynchoptera Fairmaire as research materials, and induced them to produce antibacterial peptides by needle puncture with a mixture of Staphylococcus aureus and Escherichia coli. Different solutions were used to extract the antibacterial peptides in the body, and after multiple gel chromatography, reverse phase chromatography and high performance liquid chromatography, small molecule peptides with obvious inhibition of gram-positive bacteria were successfully obtained from the crude extracts of Tenebrio molitor Linnaeus and Blaps rhynchoptera Fairmaire larvae. However, this technology uses liquid nitrogen to grind the insect bodies, and then uses extraction solution to extract, and uses gel chromatography column, exclusion chromatography column and reverse phase chromatography column and other chromatography column methods to purify the antibacterial peptides. This technology is only suitable for laboratory use and is not suitable for industrial production. Moreover, liquid nitrogen is expensive and dangerous, and cannot be used for large-scale grinding of insect bodies. Gel chromatography column, exclusion chromatography column and reverse phase chromatography column have limited sample loading capacity. In order to increase the amount of separation in industrial scale-up, high equipment investment, larger chromatography columns, more expensive fillers and larger eluent volumes are required. The cost of insect antibacterial peptides extracted by this method is too high, and there is no practical economic value.
[0009] Other prior art documents, including Liu Yuan induces the production of antibacterial peptides by infecting Musca domestica larvae and Tenebrio molitor Linnaeus larvae and pupae with pathogenic bacteria through body wall injury, and performing crude extraction at 24h and 48h respectively, using filter paper method to determine the antibacterial effect of antibacterial peptide crude extract on Actinoplanes uzbekistanensis, also using gel chromatography column, exclusion chromatography column to purify the extracted insect antibacterial peptide, which has been described above that it is not suitable for industrial production. In summary, the existing technology has the technical defects of high production cost, high equipment demand, complex process, and inability to realize economic industrial production expansion. SUMMARY
[0010] In view of the defects of the prior art, the first object of the present application is to provide an antibacterial peptide extraction method which is simple in steps, low in equipment demand, low in production cost and easy to realize industrial production.
[0011] The second object of the present application is to provide an antibacterial peptide which can be used as a basic raw material for preventing and treating citrus Huanglongbing disease, and mixed with other functional additives to be used in various forms for preventing and treating citrus Huanglongbing disease.
[0012] The third object of the present application is to provide an application of antibacterial peptide.
[0013] The first technical solution adopted by the present application is: an antibacterial peptide extraction method, comprising the following steps:
[0014] S1, freeze the live cotton bollworms quickly, and transfer them to a freeze-drying device for freeze-drying after the live cotton bollworms are frozen;
[0015] S2, crush the freeze-dried cotton bollworm bodies to obtain cotton bollworm body powder;
[0016] S3, use an extractant added with a hydrolytic enzyme inhibitor to extract the cotton bollworm body powder;
[0017] S4, filter the mixture, and after filtering, wash the filter cake with the extractant and collect the filtrate;
[0018] S5, weigh the collected filtrate, add ammonium sulfate, polyethylene glycol PEG400 and ethanol, and then stir;
[0019] S6, filter the mixture obtained in S5 to collect the filtrate, and remove the ammonium sulfate, PEG400 and ethanol from the filtrate through a nanofiltration membrane filtration device, and retain the antibacterial peptide in the filtrate;
[0020] S7, freeze-dry the filtrate to obtain powdered cotton bollworm antibacterial peptide.
[0021] Further, in the above S1, the quick freezing is performed in a refrigerator at-20℃ to-80℃; in S2, the cotton bollworm body powder has a particle size of less than 200 mesh; in S3, the extractant is an aqueous solution containing an acid, and the acid is one or a mixture of several of formic acid, citric acid, acetic acid, trifluoroacetic acid, phosphoric acid, oxalic acid, and sulfuric acid; the hydrolytic enzyme inhibitor is a mixture of benzylsulfonyl fluoride and phenylthiourea, wherein the concentration of benzylsulfonyl fluoride is 10 mg / L-100 mg / L, and the concentration of phenylthiourea is 10 mg / L-100 mg / L; and the mass ratio of the extractant added with the hydrolytic enzyme inhibitor to the cotton bollworm body powder is 1:1-10:1.
[0022] Further, in the above S1, the quick freezing is performed in a refrigerator at-40℃; in S3, the acid is an aqueous solution containing 0.1%wt of formic acid, 0.2%wt of citric acid, and 0.1%wt of oxalic acid; in the hydrolytic enzyme inhibitor, the concentration of benzylsulfonyl fluoride is 50 mg / L, and the concentration of phenylthiourea is 50 mg / L; the mass ratio of the extractant added with the hydrolytic enzyme inhibitor to the cotton bollworm body powder is 2.5:1; and the extraction is performed at a temperature of 0-4℃ and under stirring for 0.5 to 3 hours.
[0023] Further, in the above S5, after the addition of ammonium sulfate, polyethylene glycol PEG400, and ethanol, the concentration of ammonium sulfate in the mixture is 1.0M-4.0M, the concentration of PEG400 is 0.5%-20%wt, and the concentration of ethanol is 1%wt to 20%wt; and the stirring is performed at 0-4℃ for 30 minutes.
[0024] Further, in the above S5, after the addition of ammonium sulfate, polyethylene glycol PEG400, and ethanol, the concentration of ammonium sulfate in the mixture is about 1M, the concentration of PEG400 is 2%wt, and the concentration of ethanol is 3%wt.
[0025] Further, in the above S4, the extractant is the same as that in S3 but does not contain the hydrolytic enzyme inhibitor; and in S6, the molecular weight cut-off of the nanofiltration membrane is 1000 Da.
[0026] The second technical solution adopted by the present application is to provide an antibacterial peptide extracted by the extraction method of the antibacterial peptide in the first technical solution.
[0027] The third technical solution adopted by the present application is the application of the antibacterial peptide as described in the second technical solution, which comprises a pharmaceutical composition for preventing and treating citrus Huanglongbing prepared based on the antibacterial peptide, which comprises the above antibacterial peptide provided by the present application and one or more of a plant nutrient fertilizer, a plant macroelement supplement, a plant microelement supplement, a chelating agent, a surfactant, a plant growth regulator, a preservative, and an antifreezing agent.
[0028] Furthermore, the plant nutrient fertilizer in the above-mentioned pharmaceutical composition includes one or more of urea, ammonium sulfate, potassium dihydrogen phosphate, dipotassium hydrogen phosphate, monoammonium phosphate, diammonium hydrogen phosphate, and ammonium sulfate, with a content of 0.1% wt-20% wt; the plant macronutrient supplement includes one or more of magnesium sulfate, magnesium chloride, calcium chloride, and calcium nitrate, with a content of 1% wt-20% wt; the plant micronutrient supplement includes one or more of zinc sulfate, ferrous sulfate, manganese sulfate, boric acid, and ammonium molybdate, with a content of 0.1% wt-5% wt; and the chelating agent... The product contains EDTA disodium salt and / or potassium citrate, at a content of 1% wt-5% wt; surfactants include sodium dodecylbenzenesulfonate and / or alkyl glycosides, at a content of 0.1% wt-5% wt; plant growth regulators include one or more of humic acid, sodium nitrate, naphthaleneacetic acid, indoleacetic acid, gibberellin, and brassinolide, at a content of 0.01% wt-20% wt; preservatives include sodium benzoate and / or potassium sorbate, at a content of 0.1% wt-4% wt; and antifreeze is glycerol, at a content of 1% wt-10% wt.
[0029] Furthermore, the drug composition for controlling citrus Huanglongbing is applied to citrus plants or root soil through foliar spraying, trunk injection, and root irrigation to control citrus Huanglongbing.
[0030] The beneficial effects of the above technical solution are as follows:
[0031] 1. The method for extracting antimicrobial peptides from cotton bollworms in this invention is simple, easy to operate and control, and does not require complex equipment. Compared with existing methods, it greatly shortens the extraction time and significantly reduces extraction time, labor and equipment costs. More importantly, it can achieve industrial-scale mass production for use in the research and development of citrus Huanglongbing control products that require low-cost supply.
[0032] 2. In this invention, the method for extracting antimicrobial peptides from cotton bollworms uses rapid freezing of the insect body followed by freeze-drying instead of the traditional tissue homogenization method. This overcomes the shortcomings of tissue homogenization, which involves a large amount of tissue fluid containing numerous active enzymes. These active hydrolytic enzymes hydrolyze the target antimicrobial peptides, resulting in low extraction yield and low antimicrobial activity of the extracted peptides. This invention employs rapid low-temperature cooling of the insect body followed by freeze-drying. This method effectively inhibits various hydrolytic enzymes within the insect body through low-temperature freeze-drying. During dehydration, enzyme activity is inhibited, effectively protecting the integrity of the antimicrobial peptides to be extracted and significantly improving the extraction efficiency.
[0033] 3. In the method for extracting antimicrobial peptides from cotton bollworms according to the present invention, the extraction liquid (extract) employs a mixed acid extraction technique. The extractant is an aqueous solution containing acid, preferably a mixed acid solution, specifically an aqueous solution containing 0.1% wt formic acid, 0.2% wt citric acid, and 0.1% wt oxalic acid. The purpose of oxalic acid is to remove calcium ions, forming calcium oxalate precipitate. Oxalic acid can also significantly inhibit the hydrolytic activity of some hydrolytic enzymes. Citric acid can neutralize and complex some metal ions such as calcium and magnesium ions, providing acidity and also inhibiting some hydrolytic enzymes. Formic acid is the main extractant, providing acidity. This extractant is a mixed solution. The principle behind the extraction liquid is that antimicrobial peptides are mostly polypeptides containing basic amino acids such as lysine and arginine. Antimicrobial peptides can dissolve in acidic solutions, while some other proteins are insoluble in acidic solutions. Simultaneously, a hydrolytic enzyme inhibitor is added to the extraction liquid. The effect of the hydrolytic enzyme inhibitor is to inhibit hydrolytic enzymes, protecting the extracted target antimicrobial peptides from hydrolysis. The extraction process is kept at around 0°C to minimize the activity of hydrolytic enzymes and reduce the hydrolysis of antimicrobial peptides by enzymes.
[0034] After extraction with the extractant, some basic proteins from the insect body, which are soluble in acidic solutions, are also extracted. These proteins mix with the antimicrobial peptides from the cotton bollworm, the target of this invention. Because the antimicrobial peptides have small molecular weights and high solubility in salt and PEG solutions, while proteins have low solubility in these solutions and tend to precipitate. Therefore, this invention ultimately uses a precipitant with approximately 1M ammonium sulfate, 2% wt PEG400, and 3% wt ethanol to precipitate the insect's basic proteins, while the antimicrobial peptides remain in solution. This invention has found that if the insect's basic proteins are not precipitated, these basic proteins will partially or significantly decompose the antimicrobial peptides during subsequent ultrafiltration, resulting in lower activity of the collected antimicrobial peptides. This may be because these insect basic proteins contain hydrolytic enzymes. These enzymes are inhibited in the extractant, but during ultrafiltration, the use of deionized water washes away the acid and inhibitors, allowing these hydrolytic enzymes to regain some activity and partially hydrolyze the antimicrobial peptides.
[0035] 4. The pharmaceutical composition for controlling citrus Huanglongbing based on cotton bollworm antimicrobial peptides of the present invention provides a source of plant element supplementation by adding other components. The purpose of adding the chelating agent is to improve the solubility of trace or medium elements, so that precipitation is less likely to occur; the purpose of adding the surfactant is to increase the ability of cotton bollworm antimicrobial peptides to penetrate leaves and improve the spreading properties of the drug; the role of the plant growth regulator is to promote plant growth; the role of the preservative is to inhibit the activity of microorganisms in the sample and improve the shelf life of the antimicrobial peptides. Detailed Implementation
[0036] To enable those skilled in the art to better understand the present invention, the present invention will be further described in detail below with reference to specific embodiments.
[0037] Example 1
[0038] A method for extracting antimicrobial peptides:
[0039] Live cotton bollworms were flash-frozen at -40°C for 10 hours. Once frozen solid, the bollworms were transferred to a freeze-drying apparatus. The insects were then freeze-dried. 1000g of the freeze-dried bollworms were weighed and pulverized into powder smaller than 200 mesh.
[0040] Prepare 2500 g (2500 ml) of an aqueous solution containing 0.1% wt formic acid, 0.2% wt citric acid, 0.1% wt oxalic acid, 50 mg / L benzyl sulfonyl fluoride, and 50 mg / L phenylthiourea. Add the lyophilized cotton bollworm powder as described above. Extract by stirring in an ice bath at 0°C for 1.5 hours.
[0041] The mixture was filtered through a 400-mesh sieve. After filtration, the filter cake was washed three times with 600 ml of an ice-water solution containing 0.1% wt formic acid, 0.2% wt citric acid, and 0.1% wt oxalic acid. The filter cake was gently agitated during washing. The filtrate was collected. After collection, the filter cake was washed with a small amount of tap water, dried, and prepared as an insect protein feed material. The filter cake consisted of bollworm tissue and insoluble protein.
[0042] The collected filtrate was weighed (3050 g). 403 g of ammonium sulfate, 61 g of polyethylene glycol (PEG400), and 92 g of ethanol were added, resulting in a final ammonium sulfate concentration of approximately 1 M, a PEG400 concentration of 2% wt, and an ethanol concentration of 3% wt. The mixture was stirred for 30 minutes in an ice bath at 0°C. A precipitate formed, consisting of bollworm contaminants present in the extract. The bollworm antimicrobial peptides, due to their positive charge and small molecular weight, remained soluble in the extract.
[0043] The filter cake was removed by vacuum filtration, and the cake contained impurities and proteins. The filtrate was collected. The filtrate was then passed through a nanofiltration membrane to remove ammonium sulfate, PEG400, and ethanol, retaining the antimicrobial peptides. The nanofiltration membrane had a molecular weight cutoff of 1000 Da. The filter cake was lightly washed with water, dried, and prepared as an insect protein feed ingredient.
[0044] The filtrate of desulfurized ammonium sulfate, PEG and ethanol was freeze-dried to obtain 89g of cotton bollworm antimicrobial peptide powder.
[0045] Comparative Example 1
[0046] A method for extracting antimicrobial peptides:
[0047] The method of directly grinding and homogenizing cotton bollworm bodies was adopted. Specifically, 2000g of cotton bollworm bodies were weighed and homogenized. Heat was generated during the homogenization process, and the temperature was controlled as much as possible between 0-4℃. The subsequent processing methods were the same as in Example 1.
[0048] Comparative Example 2
[0049] A method for extracting antimicrobial peptides:
[0050] The same method as in Example 1 was used, except that 0.2% wt citric acid, 0.1% wt oxalic acid, 50 mg / L benzyl sulfonyl fluoride, and 50 mg / L phenylthiourea were not added. Extraction was performed only with 0.4% wt formic acid aqueous solution. The pretreatment method and subsequent treatment method were the same.
[0051] Comparative Example 3
[0052] A method for extracting antimicrobial peptides:
[0053] The same method as in Example 1 was used, except that the precipitant with a final ammonium sulfate concentration of approximately 1M, a PEG400 concentration of 2%wt, and an ethanol concentration of 3%wt was not used to precipitate the insect basic protein.
[0054] The antimicrobial peptide powders obtained in each embodiment were weighed, and then a 500 mg / L antimicrobial peptide solution was prepared using deionized water. Using Bacillus subtilis as an indicator bacterium, the filter paper method was employed. 10 μL of the antimicrobial peptide solution was dropped onto a 9 mm diameter filter paper disc, and the mixture was incubated at 30°C for 24 h. The inhibition zone was then measured. The results are shown in Table 1. Here, the diameter of the inhibition zone includes the 9 mm diameter of the filter paper disc. If the diameter of the inhibition zone is 9 mm, there is no antimicrobial activity.
[0055] Table 1. Results of antimicrobial peptide extraction from cotton bollworm treated with different methods.
[0056] Specific examples Cotton bollworm antibacterial peptide powder mass (g) Inhibition zone diameter (mm) Example 1 89 32 Comparative Example 1 42 10 Comparative Example 2 75 16 Comparative Example 3 112 15
[0057] Table 1 shows that the method of the present invention significantly improved the activity and yield of the extracted cotton bollworm antimicrobial peptides in each key control step. Although Example 3 yielded slightly more antimicrobial peptides, it contained a large amount of basic protein, and its activity was far less than that of the antimicrobial peptides extracted by the method of the present invention.
[0058] As mentioned earlier, rapid freezing and freeze-drying of cotton bollworm bodies significantly improved the extraction efficiency of antimicrobial peptides, ensuring their activity. Using a mixed acid with a proteolytic enzyme inhibitor as the extraction solution significantly reduced the decomposition of antimicrobial peptides during extraction and also improved their antimicrobial activity. The method of precipitating basic proteins using ammonium sulfate, PEG400, and ethanol significantly improved the antimicrobial activity of the extracted peptides; the reduced yield was due to the removal of basic proteins. Since basic proteins are not the antimicrobial peptides desired in this invention, the antimicrobial peptides obtained in Comparative Example 3 were impure.
[0059] Example 2
[0060] Determination of the inhibitory activity of extracted antimicrobial peptides from cotton bollworm against different bacteria.
[0061] Since the pathogen causing Huanglongbing (citrus greening disease) cannot be cultured artificially to date, its closely related bacteria, *Heterobacter tumefaciens* and *Rhizobium alfalfa*, were used as test bacteria. *Xanthomonas citrinum*, the causal agent of citrus canker, and *Bacillus subtilis* were also used as test bacteria.
[0062] Citrus canker pathogen (Xanthomonas citrus), Bacillus subtilis and tumefaciens were inoculated and cultured on LB medium (LB medium formula is 10 g / L tryptone, 5 g / L yeast extract, 10 g / L sodium chloride, final pH 7) at 30℃ with shaking at 180 rpm until the OD600 value was about 1.0. The culture was then diluted 50 times with sterile water to obtain the diluted bacterial solution for use.
[0063] Alfalfa rhizobia were cultured on YEM medium (yeast mannitol broth medium, with the following formula: yeast extract 1.0 g / L, mannitol 10 g / L, sodium chloride 0.1 g / L, magnesium sulfate heptahydrate 0.2 g / L, potassium dihydrogen phosphate 0.5 g / L, calcium carbonate 1.0 g / L, final pH 7) at 30℃ with shaking at 180 rpm until the OD600 value reached approximately 1.0. The culture was then diluted 50 times with sterile water to obtain the diluted bacterial solution for later use.
[0064] Weigh out the cotton bollworm antimicrobial peptide powder obtained in Example 1, dissolve it in deionized water, and prepare a cotton bollworm antimicrobial peptide stock solution with a concentration of 10 g / L.
[0065] Determination of the MIC (minimum inhibitory concentration) of cotton bollworm antimicrobial peptide against citrus canker pathogen (Xanthomonas citrus): Take 50 μL of cotton bollworm antimicrobial peptide 10 g / L (10000 mg / L) solution, add 950 μL of LB liquid medium, and dilute to a cotton bollworm antimicrobial peptide solution of 500 mg / L for later use.
[0066] The MIC values of the antimicrobial peptide against bollworm were tested in a row of wells of a 96-well plate. For example, row A has 12 wells: A1, A2, A3, A4, A5, A6, A7, A8, A9, A10, A11, and A12. Except for A2 and A12, 200 μL of sterile LB broth was added to each well. 200 μL of a 500 mg / L antimicrobial peptide solution was added to A2. 200 μL of the same solution was added to A3. The solution in A3 was mixed, resulting in a volume of 400 μL. 200 μL of this solution was then added to A4 and mixed. 200 μL of this solution was then added to A5, and this process was repeated to achieve a two-fold serial dilution to A11. Finally, 200 μL of this solution was added to A12.
[0067] Well A1 serves as a negative control, without the addition of bollworm antimicrobial peptide solution. 50 μL of *Xanthomonas citrus canker* (citrus xanthomonas) bacterial suspension is added to wells A1 through A11. Well A12 contains no bacterial suspension to be used.
[0068] After incubating the 96-well plate at 28°C for 48 hours, the absorbance of each well was measured at 595 nm using a microplate reader. Wells with bacterial growth showed high absorbance values, while wells without growth showed very low or near-zero absorbance values. The minimum inhibitory concentration (MIC) of the cotton bollworm antimicrobial peptide was defined as the concentration at which the pathogen could be inhibited. At this concentration, the absorbance was very low or near-zero. At the next two-fold dilution of this concentration, the pathogen showed significant absorbance values.
[0069] The determination of the MICs of cotton bollworm antimicrobial peptides against alfalfa rhizobia, Bacillus subtilis, and tumefaciens was similar to that against citrus canker pathogens (Xanthomonas citrinum). MICs for Bacillus subtilis and tumefaciens were determined using LB medium. MICs for alfalfa rhizobia were determined using YEM liquid medium instead of LB liquid medium.
[0070] The measured MIC values are shown in Table 2. As can be seen from Table 2, the antimicrobial peptides of cotton bollworm have a strong antibacterial ability.
[0071] Table 2. MIC values of antimicrobial peptides against bacteria from cotton bollworm.
[0072]
[0073] Example 3
[0074] A pharmaceutical composition for preventing and controlling citrus Huanglongbing (HLB) comprises the following components:
[0075] 10g of cotton bollworm antimicrobial peptide powder
[0076] 50g of glycerin
[0077] 20g of disodium EDTA
[0078] 60g of potassium dihydrogen phosphate
[0079] 100g of humic acid
[0080] 60g of urea
[0081] Add 1000ml of water and stir to dissolve evenly.
[0082] Example 4
[0083] A pharmaceutical composition for preventing and controlling citrus Huanglongbing (HLB) comprises the following components:
[0084] 10g of cotton bollworm antimicrobial peptide powder
[0085] 20g of glycerin
[0086] 20g of disodium EDTA
[0087] 60g of magnesium chloride
[0088] Calcium nitrate 100g
[0089] 30g of urea
[0090] Alkyl glycoside 20g
[0091] Gibberellin 1g
[0092] Add 1000ml of water and stir to dissolve evenly.
[0093] Example 5
[0094] A pharmaceutical composition for preventing and controlling citrus Huanglongbing (HLB) comprises the following components: 10g of cotton bollworm antimicrobial peptide powder.
[0095] 30g of glycerin
[0096] Alkyl glycoside 20g
[0097] 80g of disodium EDTA
[0098] 20g of zinc sulfate
[0099] 5g ferrous sulfate
[0100] 5g of manganese sulfate
[0101] 3g of boric acid
[0102] Add 1000ml of water and stir to dissolve evenly.
[0103] Example 6
[0104] A pharmaceutical composition for preventing and controlling citrus Huanglongbing (HLB) comprises the following components: 10g of cotton bollworm antimicrobial peptide powder.
[0105] 20g of glycerin
[0106] 60g of disodium EDTA
[0107] 80g of diammonium hydrogen phosphate
[0108] 10g sodium benzoate
[0109] 10g potassium sorbate
[0110] Sodium dodecyl sulfate 5g
[0111] Add 1000ml of water and stir to dissolve evenly.
[0112] Example 7
[0113] A pharmaceutical composition for preventing and controlling citrus Huanglongbing (HLB) comprises the following components: 10g of cotton bollworm antimicrobial peptide powder.
[0114] 20g of glycerin
[0115] 80g of urea
[0116] 10g sodium benzoate
[0117] 10g potassium sorbate
[0118] Sodium dodecyl sulfate 5g
[0119] 1g of naphthaleneacetic acid
[0120] 5g of indoleacetic acid
[0121] Brassinolide 0.2g
[0122] 50g of anhydrous ethanol
[0123] Add 1000ml of water and stir to dissolve evenly.
[0124] Example 8
[0125] A pharmaceutical composition for preventing and controlling citrus Huanglongbing (HLB) comprises the following components:
[0126] 10g of cotton bollworm antimicrobial peptide powder
[0127] 10g of disodium EDTA
[0128] 20g of diammonium hydrogen phosphate
[0129] 40g of potassium dihydrogen phosphate
[0130] 15g sodium dodecyl sulfate
[0131] Sodium nitrophenolate 5g
[0132] 1g of naphthaleneacetic acid
[0133] Add 900g of diatomaceous earth and mix well.
[0134] Example 9
[0135] In 2023, Newhall navel orange trees infected with Huanglongbing (HLB) were treated with antimicrobial peptides for cotton bollworm in an orchard in Dayu County, Ganzhou, Jiangxi Province. The trees were 3-4 years old.
[0136] Treatment 1 involved spraying the leaves with a 1000 mg / L aqueous solution of cotton bollworm antimicrobial peptide. (The aqueous solution of cotton bollworm antimicrobial peptide was obtained by diluting the drug composition for controlling citrus Huanglongbing of Example 5 of this invention with water by 10 times, and the cotton bollworm antimicrobial peptide powder therein was the product prepared in Example 1 of this invention.)
[0137] Treatment 2 involved spraying the leaves with a 1000 mg / L antimicrobial peptide aqueous solution (the antimicrobial peptide aqueous solution was obtained by diluting the drug composition for controlling citrus Huanglongbing of Example 5 of this invention with water by 10 times, and the antimicrobial peptide powder was the product prepared in Example 1 of this invention), combined with drenching the roots with a 500 mg / L antimicrobial peptide aqueous solution (the antimicrobial peptide aqueous solution was obtained by diluting the drug composition for controlling citrus Huanglongbing of Example 3 of this invention with water by 10 times, and the antimicrobial peptide powder was the product prepared in Example 1 of this invention).
[0138] The control group used the same ingredients as in Example 5 above, but without the cotton bollworm antimicrobial peptide (specific formula: 30g glycerol, 20g alkyl glycoside, 80g disodium EDTA, 20g zinc sulfate, 5g ferrous sulfate, 5g manganese sulfate, 3g boric acid, dissolved in 1000ml water). The solution was diluted 10 times and sprayed. The first application was on July 27, 2023. The second application was on August 27, 2023.
[0139] Leaves were sampled on July 11, 2023, before the first spray; on August 23, 2023, after the first spray; and on October 1, 2023, after the second spray. These samples were sent to the Navel Orange Testing Center of Gannan Normal University to extract CLas DNA. The Ct value was measured using quantitative real-time PCR (GB / T28062-2011). A lower Ct value indicates a higher CLas bacterial content.
[0140] Collect 4-5 mature leaves from each of the five cardinal directions (east, south, west, north, and center) of diseased trees, for a total of 20-25 leaves per tree. Avoid cross-contamination between samples during collection. Send the collected samples to the laboratory for processing or temporarily store them at 4℃. Supplement the experimental area with normal water and fertilizer management and pesticide management (primarily for controlling citrus psyllids).
[0141] The national standard GB / T28062-2011, "Real-time Fluorescent PCR Detection Method for Citrus Huanglongbing," was adopted. Leaf veins were cut from leaf samples and ground into powder using liquid nitrogen. Approximately 200 mg of the leaf vein powder was used to extract leaf vein DNA from the leaf samples using the CTAB-Triton method. PCR detection was performed using SYBR Green I fluorescent dye.
[0142] The primer pair CQULA04F sequence is: 5'-TGGAGGTGTAAAAGTTGCCAAA-3'; the CQULA04R sequence is: 5'-CCAACGAAAAGATCAGATATTCCTCA-3'. The amplified fragment is a specific target sequence of the ribosomal protein gene rplJ / rplL of *Candidatus liberibacter asiaticus*, with a size of 87 bp, and is a single copy fragment.
[0143] A 25 μL SYBR Green I fluorescent dye PCR reaction system was prepared, containing a final concentration of 1×SYBR Green I quantitative IQSYBR Green Supermix (Bio-RadUSA), 0.75 mmol / L primer pair CQULA04F / CQULA04R, and 2 mL template. Quantitative PCR was performed on a real-time PCR instrument. The reaction program was: pre-amplification at 95 °C for 1 min; denaturation at 95 °C for 15 s; annealing at 59 °C for 15 s; extension at 72 °C for 20 s; for 41 cycles. Multiple simultaneous fluorescence acquisitions were performed during the extension phase of each cycle. To verify the specificity of the amplification, melting curve analysis was performed immediately after amplification. The reaction program was: 95 °C for 30 s; 55 °C for 30 s; increasing the temperature by 0.5 °C for 1 s starting from 55 °C, holding for 1 s, for a total of 80 increases (up to 95 °C). The measured Ct values were recorded.
[0144] Table 3 shows the changes in Ct values in navel orange leaves infected with Huanglongbing after treatment with cotton bollworm antimicrobial peptides. Compared to the control group, treatment with cotton bollworm antimicrobial peptides significantly reduced the CLa content in the leaves of infected navel orange trees, as evidenced by increased Ct values. In some trees, no Huanglongbing CLa bacteria were detected in the leaves after treatment. A higher Ct value indicates a lower CLa bacteria content.
[0145] The experiment showed that treatment groups 1 and 2, treated with the cotton bollworm antimicrobial peptide, produced fruit with normal color change. The sugar content of navel oranges in treatment groups 1, 2, and the control group was measured using a handheld saccharimeter, and the results are shown in Table 4. The navel oranges in treatment groups 1 and 2, treated with the cotton bollworm antimicrobial peptide, achieved a sugar content of 16°Brix, while the navel oranges in the control group only had a sugar content of approximately 14°Brix.
[0146] Table 3. Changes in Ct values of navel orange leaves infected with Huanglongbing after treatment with antimicrobial peptides from cotton bollworm.
[0147]
[0148] Table 4. Sugar content of navel oranges infected with Huanglongbing after treatment with antimicrobial peptides from cotton bollworm.
[0149]
[0150] Example 10
[0151] A pharmaceutical composition for preventing and controlling citrus Huanglongbing (HLB) comprises the following components:
[0152] 15g of antibacterial peptide powder for cotton bollworm
[0153] 10g of urea
[0154] 10g of potassium dihydrogen phosphate
[0155] Gibberellin 0.1g
[0156] Dissolve in deionized water to a volume of 1000 ml.
[0157] Example 11
[0158] In 2024, a small-scale experiment was conducted at Minglin Orchard in Shanglin County, Nanning City, Guangxi Province, to treat Wogan citrus trees infected with Huanglongbing (HLB) with an antimicrobial peptide for bollworm. The treatment group was sprayed once each in April, June, and September with a 1000 mg / L antimicrobial peptide aqueous solution (the antimicrobial peptide aqueous solution was obtained by diluting the drug composition for controlling HLB of citrus in Example 5 of this invention 10 times with water, wherein the antimicrobial peptide powder was the product prepared in Example 1 of this invention); and in April and September, the trunks of Wogan citrus trees were injected with a 1500 mg / L antimicrobial peptide aqueous solution (obtained by diluting the drug composition for controlling HLB of citrus in Example 10 of this invention 10 times with water, wherein the antimicrobial peptide powder was the product prepared in Example 1 of this invention), with 250 ml injected per Wogan citrus tree.
[0159] The control group consisted of Wogan mandarin orange leaves treated with water. Fruit was harvested in January 2025. Yield was calculated.
[0160] An experiment conducted in Nanning, Guangxi in 2024 showed that injecting 250 ml of a 1500 mg / L antimicrobial peptide solution into the trunk of Wogan mandarin orange trees had no adverse effects on the trees. The antimicrobial peptide can be used for trunk injection. At harvest, the number of fruits infected with Huanglongbing (HLB) and the number of healthy fruits were calculated, and the healthy fruit rate was ≥90%. The yields of Wogan mandarins in the treatment and control groups are shown in Table 5. The yield per tree in the treatment group was 78.84 catties, while the yield per tree in the control group was only 29.33 catties. The control group mainly exhibited significant fruit drop, and most of the fruit was infected with HLB.
[0161] Table 5. Yield of Wogan oranges infected with Huanglongbing treated with aqueous solution of cotton bollworm antimicrobial peptide.
[0162]
[0163] The various embodiments of the present invention have been described above. These descriptions are exemplary and not exhaustive, nor are they limited to the disclosed embodiments. Many modifications and variations will be apparent to those skilled in the art without departing from the scope and spirit of the described embodiments. Therefore, the scope of protection of the present invention should be determined by the scope of the claims.
Claims
1. A method for extracting antimicrobial peptides, characterized in that, Includes the following steps: S1. Quick-freeze live cotton bollworms. Once the live cotton bollworms are frozen, freeze-dry them to obtain freeze-dried cotton bollworm bodies. S2. The freeze-dried cotton bollworm body is crushed to obtain cotton bollworm body powder; S3. Extract the cotton bollworm body powder using an extractant containing a hydrolytic enzyme inhibitor to obtain a first mixture; S4. Filter the first mixture by suction filtration. After filtration, wash the filter cake with an extractant and collect the filtrate. S5. Weigh the collected filtrate, add ammonium sulfate, polyethylene glycol PEG400 and ethanol, and then stir to obtain a second mixture; S6. After filtering the second mixture, collect the filtrate, and remove ammonium sulfate, PEG400 and ethanol from the collected filtrate by passing it through a nanofiltration membrane filter, while retaining the antimicrobial peptides in the filtrate. S7. Freeze-dry the filtrate to obtain antimicrobial peptides.
2. The method for extracting antimicrobial peptides as described in claim 1, characterized in that, In S1, the quick-freezing is carried out in a refrigerator at -20°C to -80°C; In S2, the particle size of the bollworm body powder is less than 200 mesh; In S3, the extractant is an aqueous solution containing an acid, which is one or a mixture of several of formic acid, citric acid, acetic acid, trifluoroacetic acid, phosphoric acid, oxalic acid, and sulfuric acid; the hydrolytic enzyme inhibitor is a mixture of benzyl sulfonyl fluoride and phenylthiourea, wherein the concentration of benzyl sulfonyl fluoride is 10 mg / L-100 mg / L and the concentration of phenylthiourea is 10 mg / L-100 mg / L; the mass ratio of the extractant containing the hydrolytic enzyme inhibitor to the cotton bollworm body powder is 1:1-10:
1.
3. The method for extracting antimicrobial peptides as described in claim 2, characterized in that, In S1, quick-freezing is carried out in a -40°C freezer; In S3, the acid is an aqueous solution containing 0.1% wt formic acid, 0.2% wt citric acid, and 0.1% wt oxalic acid; the concentration of phenylmethylsulfonyl fluoride and phenylthiourea in the hydrolase inhibitor is 50 mg / L; the mass ratio of the extractant containing the hydrolase inhibitor to the cotton bollworm body powder is 2.5:1; the extraction is carried out at 0-4℃ with stirring for 0.5 to 3 hours.
4. The method for extracting antimicrobial peptides as described in claim 3, characterized in that, In step S5, after adding ammonium sulfate, polyethylene glycol PEG400 and ethanol, the concentration of ammonium sulfate in the mixture is 1.0M-4.0M, the concentration of PEG400 is 0.5%-20%wt, and the concentration of ethanol is 1%wt to 20wt%. The stirring is carried out at 0-4℃ for 30 minutes.
5. The method for extracting antimicrobial peptides as described in claim 4, characterized in that, In S5, after adding ammonium sulfate, polyethylene glycol PEG400 and ethanol, the concentration of ammonium sulfate in the mixture is approximately 1M, the concentration of PEG400 is 2wt%, and the concentration of ethanol is 3wt%.
6. The method for extracting antimicrobial peptides as described in claim 5, characterized in that, In S4, the extractant is the same as the extractant in S3 but does not contain hydrolytic enzyme inhibitors; In S6: The molecular weight cutoff of the nanofiltration membrane is 1000 Da.
7. An antimicrobial peptide extracted using the extraction method according to any one of claims 1-6, characterized in that, The antimicrobial peptide is a cotton bollworm antimicrobial peptide.
8. An application of the antimicrobial peptide as described in claim 7, characterized in that, A pharmaceutical composition for the prevention and control of citrus Huanglongbing is prepared based on the antimicrobial peptide. The pharmaceutical composition includes the antimicrobial peptide and also includes one or more of the following: plant nutrient fertilizer, plant medium element supplement, plant micro element supplement, chelating agent, surfactant, plant growth regulator, preservative and antifreeze agent.
9. The application of the antimicrobial peptide as described in claim 8, characterized in that, The plant nutrient fertilizer includes one or more of urea, ammonium sulfate, potassium dihydrogen phosphate, dipotassium hydrogen phosphate, monoammonium phosphate, diammonium hydrogen phosphate, and ammonium sulfate, and the content of the plant nutrient fertilizer in the pharmaceutical composition is 0.1%wt-20%wt. The plant micronutrient supplement includes one or more of magnesium sulfate, magnesium chloride, calcium chloride, and calcium nitrate, and the content of the plant micronutrient supplement in the pharmaceutical composition is 1%wt-20%wt. The plant trace element supplement includes one or more of zinc sulfate, ferrous sulfate, manganese sulfate, boric acid, and ammonium molybdate, and the content of the plant trace element supplement in the pharmaceutical composition is 0.1%wt-5%wt. The chelating agent includes disodium EDTA and / or potassium citrate, and the content of the chelating agent in the pharmaceutical composition is 1%wt-5%wt. The surfactants include sodium dodecylbenzenesulfonate and / or alkyl glycosides, and the surfactant content in the pharmaceutical composition is 0.1% wt to 5% wt. The plant growth regulator includes one or more of humic acid, sodium nitrate, naphthaleneacetic acid, indoleacetic acid, gibberellin, and brassinolide, and the content of the plant growth regulator in the pharmaceutical composition is 0.01%wt-20%wt. The preservatives include sodium benzoate and / or potassium sorbate, and the content of the preservatives in the pharmaceutical composition is 0.1%wt-4%wt; The antifreeze is glycerin, and the content of the antifreeze in the pharmaceutical composition is 1%wt-10%wt.
10. The application of the antimicrobial peptide as described in claim 8, characterized in that, The drug composition for controlling citrus Huanglongbing is applied to citrus plants or root soil by foliar spraying, trunk injection, and root irrigation to control citrus Huanglongbing.