Composite antibacterial peptide and application thereof in agricultural field

Abstract

The invention belongs to the field of agricultural biological agents, and particularly relates to a composite antibacterial peptide and application thereof in the agricultural field. The composite antibacterial peptide disclosed by the invention is prepared from the following components in parts by mass: 20 to 50 parts of antibacterial peptide, 300 to 400 parts of yeast peptide, 100 to 200 parts of amino acid, 15 to 25 parts of yeast oligosaccharide and 5 to 20 parts of nucleotide. Through the synergistic effect of the antibacterial peptide, the yeast peptide, the amino acid, the yeast oligosaccharide and the nucleotide, a multi-dimensional antibacterial system is constructed, the antibacterial peptide is modified through the Tween, and the antibacterial effect is improved. The raw materials used in the invention are safe and environment-friendly, the process stability is enhanced, and the production cost of the product can be reduced to a certain extent.

Description

Technical Field

[0001] The present invention belongs to the field of agricultural biological preparations, and in particular relates to a composite antimicrobial peptide and its application in the agricultural field. Background Art

[0002] The prevention and control of crop pathogens has always been a critical issue in agricultural production. While traditional chemical pesticides have achieved some success in disease control, they also pose a series of challenges, including environmental pollution, food safety risks, and the development of antibiotic resistance. In recent years, with the advancement of biotechnology, antimicrobial peptides, as natural bioactive substances, have gained increasing attention. Antimicrobial peptides offer advantages such as widespread availability, strong antimicrobial activity, and a low resistance to antibiotic resistance, making them an ideal green alternative to pesticides.

[0003] Antimicrobial peptides (AMPs) are natural immune molecules with advantages such as broad-spectrum antibacterial properties and low resistance to drug resistance. Patent CN110684092A discloses an antimicrobial peptide and its application. The invention uses the transcriptome data of leaves induced by powdery mildew fungus of wheat SN6306 combined with bioinformatics methods to obtain antimicrobial peptide genes. By transforming Escherichia coli to express the target protein, the obtained antimicrobial peptide can be used to prepare inhibitors against Fusarium graminearum and powdery mildew of wheat. It is a raw material for antifungal inhibitors with great potential utilization value. However, its stability is unclear and its antibacterial effect needs to be improved. Patent CN119039405A also discloses an antimicrobial peptide and its application. Through a large number of sequence analyses and tests, the invention discovered a short peptide with antagonistic fungal function in the Atp2β subunit protein of the extracellular protein of Rhodopseudomonas palustris. The prepared antimicrobial peptide can significantly inhibit the formation of appressorium of rice blast fungus and its pathogenicity on the host. While this short peptide has antibacterial properties against rice, its effects on other crops have not been reported. Furthermore, the lack of breakthroughs in large-scale production technology for natural antimicrobial peptides has hampered their commercial application.

[0004] Yeast extracts (such as yeast peptides and yeast oligosaccharides) are rich in amino acids, nucleotides and functional polysaccharides, and have both nutritional supplement and immune regulatory functions in agriculture. Studies have shown that β-glucan oligosaccharides can activate the plant immune system (such as inducing defense enzyme activity) and enhance crop disease resistance. However, the traditional yeast hydrolysis process has problems such as low yield of target oligosaccharides and insufficient β-glucan oligosaccharide content, and the yeast enzymatic hydrolysis technology needs to be optimized. In addition, in the existing technology, the combination of antimicrobial peptides and yeast extracts still has problems such as poor stability and insufficient synergistic effect.

[0005] Therefore, the development of an antimicrobial peptide that is safe, environmentally friendly, stable, and universal is of great significance to the development of agriculture. Summary of the Invention

[0006] The present invention discloses a composite antimicrobial peptide and its application. Through the synergistic effects of antimicrobial peptides, yeast peptides, amino acids, yeast oligosaccharides, and nucleotides, a multidimensional antimicrobial system is constructed. The antimicrobial peptide is modified with Tween to enhance its antimicrobial efficacy. Furthermore, the raw materials used in the present invention are safe and environmentally friendly, and the process stability is enhanced, which can reduce product production costs to a certain extent.

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

[0008] The first aspect of the present invention provides a composite antimicrobial peptide comprising the following components in parts by mass:

[0009] 20-50 parts of antimicrobial peptide, 300-400 parts of yeast peptide, 100-200 parts of amino acid, 15-25 parts of yeast oligosaccharide, and 5-20 parts of nucleotide.

[0010] In some embodiments, the preparation method of the antimicrobial peptide is:

[0011] The Tween aqueous solution is dropped into the buffer solution of the PR39 antimicrobial peptide, stirred at 20-30° C. and 150-250 rpm for 25-40 minutes, then ultrasonically treated at 15-25° C. for 5-10 minutes, and the obtained mixture is dried to obtain the antimicrobial peptide.

[0012] In some embodiments, the Tween consists of Tween 20 and Tween 80 in a mass ratio of 1:(0.85-1.3).

[0013] The present application can solve the problem of insufficient permeability of PR39 antimicrobial peptide in plant cuticle by regulating the mass ratio of Tween 20 and Tween 80.

[0014] In some embodiments, the mass ratio of Tween to PR39 type antimicrobial peptide is (5-10):1.

[0015] Preferably, the PR39 antimicrobial peptide used contains 39 amino acid residues, 22 proline residues and 11 arginine residues.

[0016] In some embodiments, the β-glucan oligosaccharide in the yeast oligosaccharide is ≥ 60%.

[0017] In some embodiments, the yeast oligosaccharide is prepared by:

[0018] (1) Yeast autolysis: Use citric acid to adjust the pH of the yeast suspension to 4.5-5.5, then add oligoxylose, soybean lecithin hydrolysate and Aspergillus niger acid protease, and react at 35-45°C for 2-8 hours to obtain yeast autolyzed milk;

[0019] (2) Yeast enzymatic hydrolysis: first add mannanase and β-glucanase to the yeast autolyzed milk in step (1) for enzymatic hydrolysis, then add protease for enzymatic hydrolysis, adjust the system pH and enzymatic hydrolysis temperature during the enzymatic hydrolysis process, terminate the enzyme activity, and dry to obtain yeast oligosaccharides.

[0020] The present application adopts specific oligoxylose as raw material, which can protect β-bonds through hydrogen bonding in the enzymatic hydrolyzate.

[0021] In some embodiments, the amount of xylo-oligosaccharide used in step (1) is 1-3% of the dry weight of the yeast suspension; the amount of soy lecithin hydrolysate used is 0.5-1.5% of the volume of the yeast suspension.

[0022] In some embodiments, the Aspergillus niger acid protease is 0.1-0.3% of the dry weight of the yeast suspension.

[0023] In some embodiments, the dosage of the mannanase, β-glucanase, and protease is 0.5-1.5%, 0.5-2.0%, and 0.3-1.0% of the dry weight of the yeast autolyzed milk, respectively.

[0024] The second aspect of the present invention provides an application of the composite antimicrobial peptide described in the above scheme in the agricultural field, wherein the composite antimicrobial peptide is applied to the leaves and / or roots of crops; the dosage for the leaves is 30-50 mL / mu, and the dosage for the roots is 500-1000 mL / mu.

[0025] PR39 antimicrobial peptide, a cationic antimicrobial peptide, has significant inhibitory effects on Gram-negative bacteria and fungi, but its solubility and permeability in complex environments are insufficient, requiring modification to improve its performance. To overcome these problems, the present invention uses Tween 20 and Tween 80 to treat PR39 antimicrobial peptide. On the one hand, the amphiphilic structure of Tween can encapsulate the antimicrobial peptide molecules, reducing their intermolecular hydrophobic interactions, inhibiting aggregation and precipitation, and significantly improving its dispersibility in aqueous phases. On the other hand, the hydrophobic end of Tween can insert into the phospholipid bilayer of microbial cell membranes, altering membrane fluidity and permeability. This effect synergizes with the cationic properties of PR39 antimicrobial peptide: the antimicrobial peptide adsorbs to the negatively charged bacterial cell membrane through electrostatic interactions, while the Tween disrupts the membrane integrity, promoting the antimicrobial peptide's insertion into the membrane to form pores, accelerating the leakage of intracellular substances, and improving antimicrobial properties. In addition, the specific ratio of Tween 20 to Tween 80 (1:0.85-1.3) optimizes the hydrophilic-lipophilic balance value, which can not only ensure water solubility but also effectively penetrate into the plant cuticle, and more significantly improve the transmembrane efficiency and antibacterial properties of the antimicrobial peptide.

[0026] In order to improve the purity of β-glucan oligosaccharides in yeast oligosaccharides and ensure the maximization of biological activity, the present invention first improves the autolysis efficiency of yeast through a synergistic wall-breaking mechanism: Aspergillus niger acid protease, oligoxylose and soybean lecithin hydrolysate are added during the autolysis process of step (1); Aspergillus niger acid protease has a high efficiency in decomposing structural glycoproteins cross-linked with mannan and β-glucan in the yeast cell wall, destroys the protein-polysaccharide network structure of the cell wall, and accelerates autolysis; phosphatidylcholine in the soybean lecithin hydrolysate dissolves cell membrane lipids; the two synergistically accelerate the release of cell contents; and oligoxylose promotes the water absorption and swelling of yeast cells by regulating the osmotic pressure of the system, reduces the mechanical strength of the cell wall, shortens the autolysis time, and saves time and cost. Then, in step (2), functional enzymes are added to the autolyzed milk for enzymatic hydrolysis: mannanase and β-glucanase are preferentially added to decompose the mannan and β-glucan in the outer layer of the yeast, and then protease is added to treat the protein to avoid the destruction of the β-bond during the enzymatic hydrolysis process; the added oligoxylose can also serve as an auxiliary agent, combining with the β-glucan through hydrogen bonding to form steric hindrance protection, reducing the breakage of the β-bond during the enzymatic hydrolysis process, and increasing the content of β-glucan oligosaccharides.

[0027] The mechanism of action of the antimicrobial peptides of the present invention is as follows: the surface of bacterial cell membranes generally exhibits a negative charge, and antimicrobial peptides carry a +2-+9 charge. Due to the adsorption effect of the charge, they bind to the cell membrane, and the hydrophobic end of the peptide inserts into the lipid bilayer, further inducing membrane lysis or increasing membrane permeability; the antimicrobial peptides bind to lipid II on the cell wall, inhibiting the transport of subunits on the cell wall and preventing cell wall synthesis; some antimicrobial peptides pass through the cell membrane and are absorbed by the bacteria, where they bind to nucleic acid sites in the bacteria and prevent DNA synthesis and protein transcription.

[0028] The antimicrobial peptides of the present invention have the following advantages: 1. They have a wide range of effects and are highly targeted at preventing and controlling a variety of pathogens, making them superior to traditional agents. 2. Compared with traditional agents that have mixing contraindications, antimicrobial peptides can be mixed with a variety of agricultural products and are flexible to use. 3. They are more stable than traditional agents that are greatly affected by the environment. 4. Due to their unique mechanism of action, antimicrobial peptides are less likely to cause microorganisms to develop drug resistance, and their long-lasting effect is stronger than traditional agents. 5. Antimicrobial peptides are not only antibacterial, but can also regulate crop growth, with comprehensive efficacy far exceeding that of traditional agents. 6. They are non-toxic and residue-free, safe for humans, the environment, and beneficial organisms, can ensure the quality of agricultural products, and do not have the disadvantages of traditional agents that are prone to hidden dangers due to improper use.

[0029] Compared with the prior art, the present invention has the following beneficial effects:

[0030] 1. This invention constructs a multidimensional antimicrobial system through the synergistic effects of antimicrobial peptides, yeast peptides, amino acids, yeast oligosaccharides, and nucleotides. The antimicrobial peptides are modified with Tween to enhance their antimicrobial efficacy. Furthermore, the raw materials used in this invention are safe and environmentally friendly, and the process stability is enhanced, which can reduce product production costs to a certain extent.

[0031] 2. The present invention optimizes the preparation process and parameters to prepare yeast oligosaccharides, which significantly improves the purity of β-glucan oligosaccharides in yeast oligosaccharides.

[0032] 3. The present invention modifies the antimicrobial peptide by adding Tween 20 and Tween 80 in a specific ratio, which can not only ensure its water solubility but also enable it to effectively penetrate into the plant cuticle, significantly improving the transmembrane efficiency and antibacterial activity of the antimicrobial peptide. BRIEF DESCRIPTION OF THE DRAWINGS

[0033] Figure 1 This is a diagram showing the leaf wetting effect of the composite antimicrobial peptide in Experiment 4 (the left side shows the composite antimicrobial peptide of Example 3, and the right side shows the imported product purchased);

[0034] Figure 2 This is a diagram showing the leaf penetration effect of the composite antimicrobial peptide in Experiment 5 (the left side shows the composite antimicrobial peptide of Example 3, and the right side shows the imported product purchased);

[0035] Figure 3 This is a diagram of the state after the composite antimicrobial peptide is mixed with other reagent solutions in Experiment 6;

[0036] Figure 4 、 5 This is a picture of wheat growth status after the first foliar spraying in Experiment 8;

[0037] Figure 6 、 7 This is a diagram of wheat growth status after the second root irrigation in Experiment 8;

[0038] Figure 8 This is a graph showing the overall growth of wheat after two applications in Experiment 8;

[0039] Figure 9 、 10 The data comparison chart and analysis bar chart of the average height and average growth height of wheat at different time points in Experiment 8;

[0040] Figure 11 This is a comparison of wheat growth in each treatment group in Experiment 8. DETAILED DESCRIPTION

[0041] Various exemplary embodiments of the present invention are now described in detail. This detailed description should not be considered as a limitation of the present invention, but should be understood as a more detailed description of certain aspects, characteristics and embodiments of the present invention. It should be understood that the terms described in the present invention are only for describing particular embodiments and are not intended to limit the present invention. In addition, for the numerical ranges in the present invention, it should be understood that each intermediate value between the upper and lower limits of the range is also specifically disclosed. Each smaller range between the intermediate value within any stated value or stated range and any other stated value or intermediate value within the range is also included in the present invention. The upper and lower limits of these smaller ranges may be independently included or excluded within the scope.

[0042] Unless otherwise indicated, all technical and scientific terms used herein have the same meanings as those generally understood by those skilled in the art. Although only preferred methods and materials are described herein, any methods and materials similar or equivalent to those described herein may be used in the practice or testing of the present invention. Various modifications and variations may be made to the specific embodiments of the present invention description without departing from the scope or spirit of the present invention, as will be apparent to those skilled in the art. Other embodiments obtained from the present invention description will be apparent to those skilled in the art. This application description and examples are exemplary only.

[0043] The oligoxylose used in the present invention is XOS-95P; the activity of the Aspergillus niger acid protease used is 50,000 U / mg; the activities of the mannanase, β-glucanase and protease (neutral) used are all 50,000 U / g; the activity of the phospholipase C used is 50,000 U / g; the nucleotides and yeast peptides used are all yeast nucleoside peptides-YNP produced by the company; and the amino acids used are amino acid polypeptide stock solutions produced by the company.

[0044] Preparation Example 1

[0045] The preparation method of yeast oligosaccharide is as follows:

[0046] (1) Yeast autolysis: Use acetic acid to adjust the pH of the yeast suspension (dilute the yeast paste with sterile water to a 10% yeast suspension) to 4.5, then add 1% xylo-oligosaccharide of the dry weight of the yeast suspension, 0.5% soybean lecithin hydrolyzate of the yeast suspension, and 0.1% Aspergillus niger acid protease of the dry weight of the yeast suspension, and react at 35°C and 150 rpm for 8 h to obtain yeast autolyzed milk;

[0047] (2) Yeast enzymatic hydrolysis: first add 0.5% of the dry weight of mannanase and 0.5% of β-glucanase to the yeast autolyzed milk of step (1), mix them evenly, adjust the pH of the system to 5.5, control the enzymatic hydrolysis temperature at 45°C, and react for 2 hours; then add 0.3% of the dry weight of protease, mix them evenly, adjust the pH of the system to 6.0 again, maintain the enzymatic hydrolysis temperature at 48°C, continue the reaction for 3 hours, raise the temperature to 90°C, maintain for 10 minutes to terminate the enzyme activity, concentrate under reduced pressure, and spray dry to obtain yeast oligosaccharides.

[0048] Preparation Example 2

[0049] The preparation method of yeast oligosaccharide is as follows:

[0050] (1) Yeast autolysis: The pH of the yeast suspension (the yeast paste was diluted with sterile water to a 15% yeast suspension) was adjusted to 5.5 with citric acid, and then 3% of the dry weight of the yeast suspension was added with xylo-oligosaccharide, 1.5% of the yeast suspension volume of soy lecithin hydrolysate, and 0.3% of the dry weight of the yeast suspension was added with Aspergillus niger acid protease. The mixture was reacted at 45°C and 100 rpm for 2 h to obtain yeast autolyzed milk.

[0051] (2) Yeast enzymatic hydrolysis: first add 1.5% and 2.0% of the dry weight of mannanase and β-glucanase to the yeast autolyzed milk of step (1), mix them evenly, adjust the pH of the system to 6.0, control the enzymatic hydrolysis temperature at 50°C, and react for 2 hours; then add 1.0% of the dry weight of protease, mix them evenly, adjust the pH of the system to 6.8 again, maintain the enzymatic hydrolysis temperature at 50°C, continue the reaction for 3 hours, heat to 90°C, maintain for 10 minutes to terminate the enzyme activity, concentrate under reduced pressure, and spray dry to obtain yeast oligosaccharides.

[0052] Preparation Example 3

[0053] The preparation method of yeast oligosaccharide is as follows:

[0054] (1) Yeast autolysis: The pH of the yeast suspension (the yeast paste was diluted with sterile water to a 12.5% ​​yeast suspension) was adjusted to 5.0 with citric acid, and then 2% of the dry weight of the yeast suspension was added with oligosaccharides, 1% of the yeast suspension volume of soy lecithin hydrolysate, and 0.2% of the dry weight of the yeast suspension was added with Aspergillus niger acid protease. The mixture was reacted at 40°C and 125 rpm for 5 h to obtain yeast autolyzed milk.

[0055] (2) Yeast enzymatic hydrolysis: first add 1% of the dry weight of mannanase and 1% of β-glucanase to the yeast autolyzed milk of step (1), mix them evenly, adjust the pH of the system to 6, control the enzymatic hydrolysis temperature at 45°C, and react for 2 hours; then add 0.5% of the dry weight of protease, mix them evenly, adjust the pH of the system to 6.5 again, maintain the enzymatic hydrolysis temperature at 48°C, continue the reaction for 3.5 hours, raise the temperature to 90°C, maintain for 10 minutes to terminate the enzyme activity, concentrate under reduced pressure, and spray dry to obtain yeast oligosaccharides.

[0056] Preparation Example 4

[0057] The preparation method of yeast oligosaccharide is the same as that of Preparation Example 3, except that xylo-oligosaccharide is not added.

[0058] Preparation Example 5

[0059] The preparation method of yeast oligosaccharide is the same as that of Preparation Example 3, except that soybean lecithin hydrolysate is not added.

[0060] Preparation Example 6

[0061] The preparation method of yeast oligosaccharides is the same as that of Preparation Example 3, except for step (2): yeast enzymolysis: 1%, 1%, and 0.5% of the dry weight of mannanase, β-glucanase, and protease are added to the yeast autolyzed milk in step (1) at the same time, mixed evenly, the pH of the system is adjusted to 6, the enzymolysis temperature is controlled at 45°C, the reaction is carried out for 5.5 hours, the temperature is raised to 90°C, maintained for 10 minutes to terminate the enzyme activity, concentrated under reduced pressure, and spray-dried to obtain yeast oligosaccharides.

[0062] Preparation Example 7

[0063] The preparation method of yeast oligosaccharide is the same as that of Preparation Example 3, except that the Aspergillus niger acid protease used is 0.5% of the dry weight of the yeast suspension.

[0064] Preparation Example 8

[0065] The preparation method of yeast oligosaccharide is the same as that of Preparation Example 3, except that an equal amount of Aspergillus oryzae acid protease with the same activity is used instead of Aspergillus niger acid protease.

[0066] Preparation Example 9

[0067] The preparation method of antimicrobial peptide is as follows:

[0068] 100 mL of 5 wt% Tween aqueous solution was added dropwise to 100 mL of 10 mg / mL Tris-HCl buffer solution (pH = 7) of PR39 antimicrobial peptide, stirred at 20°C and 150 rpm for 40 min, then ultrasonically treated at 25°C for 5 min, and the resulting mixture was spray-dried to obtain the antimicrobial peptide.

[0069] The Tween used was composed of Tween 20 and Tween 80 with a mass ratio of 1:0.85.

[0070] Preparation Example 10

[0071] The preparation method of antimicrobial peptide is as follows:

[0072] 100 mL of a 10 wt% Tween aqueous solution was added dropwise to 100 mL of a 10 mg / mL Tris-HCl buffer solution (pH = 7) of a PR39 antimicrobial peptide, stirred at 30°C and 250 rpm for 25 min, then ultrasonically treated at 15°C for 10 min, and the resulting mixture was spray-dried to obtain the antimicrobial peptide.

[0073] The Tween used was composed of Tween 20 and Tween 80 with a mass ratio of 1:1.3.

[0074] Preparation Example 11

[0075] The preparation method of antimicrobial peptide is as follows:

[0076] 100 mL of 7.5 wt% Tween aqueous solution was added dropwise to 100 mL of 10 mg / mL Tris-HCl buffer solution (pH = 7) of PR39 antimicrobial peptide, stirred at 25°C and 200 rpm for 30 min, then ultrasonically treated at 20°C for 8 min, and the resulting mixture was spray-dried to obtain the antimicrobial peptide.

[0077] The Tween used was composed of Tween 20 and Tween 80 in a mass ratio of 1:1.

[0078] Preparation Example 12

[0079] The preparation method of the antimicrobial peptide is substantially the same as that of Preparation Example 11, except that an equal amount of Tween 20 is used instead of Tween 80.

[0080] Preparation Example 13

[0081] The preparation method of the antimicrobial peptide is substantially the same as that of Preparation Example 11, except that an equal amount of Tween 80 is used instead of Tween 20.

[0082] Preparation Example 14

[0083] The preparation method of the antimicrobial peptide is substantially the same as that of Preparation Example 11, except that the Tween used is composed of Tween 20 and Tween 80 at a mass ratio of 1:1.5.

[0084] Preparation Example 15

[0085] The preparation method of the antimicrobial peptide is substantially the same as that of Preparation Example 11, except that the concentration of the Tween aqueous solution used is 12 wt %.

[0086] Preparation Example 16

[0087] The preparation method of the soybean lecithin hydrolyzate used in Preparation Example 1-8 is as follows:

[0088] Lecithin was dispersed in Tris-HCl buffer (pH = 8.0) to prepare a 5 wt% suspension, to which phospholipase C (dosage: 200 U / g lecithin) was added. The mixture was stirred at 40°C for 4 h, the enzyme was inactivated in a boiling water bath for 10 min, and the suspension was cooled to room temperature to obtain soybean lecithin hydrolyzate.

[0089] Example 1

[0090] A composite antimicrobial peptide, comprising the following components in parts by mass:

[0091] 20 parts of antimicrobial peptides, 300 parts of yeast peptides, 100 parts of amino acids, 15 parts of yeast oligosaccharides, and 5 parts of nucleotides.

[0092] The yeast oligosaccharide used was obtained from Preparation Example 1; the antimicrobial peptide used was obtained from Preparation Example 9.

[0093] The above components are mixed in sequence to obtain a composite antimicrobial peptide.

[0094] Example 2

[0095] A composite antimicrobial peptide, comprising the following components in parts by mass:

[0096] 50 parts of antimicrobial peptides, 400 parts of yeast peptides, 200 parts of amino acids, 25 parts of yeast oligosaccharides, and 15 parts of nucleotides.

[0097] The yeast oligosaccharide used was obtained from Preparation Example 2; the antimicrobial peptide used was obtained from Preparation Example 10.

[0098] The above components are mixed in sequence to obtain a composite antimicrobial peptide.

[0099] Example 3

[0100] A composite antimicrobial peptide, comprising the following components in parts by mass:

[0101] 35 parts of antimicrobial peptides, 350 parts of yeast peptides, 150 parts of amino acids, 20 parts of yeast oligosaccharides, and 10 parts of nucleotides.

[0102] The yeast oligosaccharide used was obtained from Preparation Example 3; the antimicrobial peptide used was obtained from Preparation Example 11.

[0103] The above components are mixed in sequence to obtain a composite antimicrobial peptide.

[0104] Comparative Example 1

[0105] A composite antimicrobial peptide, the specific implementation manner is substantially the same as that of Example 3, except that yeast oligosaccharide is not added.

[0106] Performance Testing

[0107] Experiment 1: Test of yeast oligosaccharide content

[0108] The yeast oligosaccharide content obtained in Preparation Examples 1-8 was determined using high-performance liquid chromatography (HPLC). The specific testing method was referenced in Patent CN 115895901 A. The oligosaccharide content was analyzed using the 1.3.5 GPC method described in "Structural Characterization and Transdermal Transmission of β-Glucolosaccharides Prepared from Fermentation Broth of Engineered Bacteria Pichia Pastoris." The test data are shown in Table 1.

[0109] Table 1

[0110]

[0111] As shown in Table 1, the yeast oligosaccharides obtained in Preparation Examples 1-3 have the highest β-glucan content. Compared with Preparation Example 3, the β-glucan content in Preparation Examples 4-7 is reduced to varying degrees due to changes in preparation parameters. In Preparation Example 8, Aspergillus oryzae acid protease is used, and its promoting effect on the production of β-glucan in yeast oligosaccharides is weaker than that of Aspergillus niger acid protease.

[0112] Experiment 2: Testing the antimicrobial efficacy of antimicrobial peptides against crop pathogens

[0113] The specific test steps refer to patent CN 118994346 A. Each group was repeated three times and the average value was taken. The test data are shown in Table 2.

[0114] Table 2

[0115]

[0116] As shown in Table 2, the antimicrobial peptides obtained in Preparation Examples 9-11 exhibit excellent antimicrobial efficacy, with MIC values ​​against Pseudomonas aeruginosa, Fusarium wilt, and Rhizoctonia solani significantly lower than the MIC values ​​of the unmodified PR39 antimicrobial peptide against these three pathogens. Compared to Preparation Example 11, the Tween compositions used in Preparation Examples 12-14 were modified, resulting in a reduced modification effect on the PR39 antimicrobial peptides and, consequently, increased MIC values ​​for each antimicrobial peptide against these three pathogens.

[0117] Experiment 3: Stability test of antimicrobial peptides

[0118] The antimicrobial peptide from Preparation Example 11 was treated at different temperatures and pH levels for 30 minutes and then subjected to antimicrobial activity testing according to the procedures in Experiment 2. MIC values ​​were used to demonstrate the antimicrobial stability of the peptide. Three replicates were performed for each group, and the average value was calculated. Specific test data are shown in Table 3.

[0119] Table 3

[0120]

[0121] As shown in Table 3, the antimicrobial peptide prepared in Preparation Example 11 has good stability in the range of pH 5-7 and temperature 30-70°C.

[0122] Test 4: Wettability test of composite antimicrobial peptides

[0123] Depend on Figure 1 It can be seen that the composite antimicrobial peptide of Example 3 of the present invention is more evenly distributed on the plant leaf surface and has better wettability than the comparative product.

[0124] Experiment 5: Permeability test of composite antimicrobial peptides

[0125] Depend on Figure 2 It can be seen that the composite antimicrobial peptide of Example 3 of the present invention has better permeability on crop leaves than the comparative product.

[0126] Experiment 6: Stability test of composite antimicrobial peptides

[0127] The composite antimicrobial peptide of Example 3 was mixed with a stock solution of pesticides such as aqueous emulsion, suspension, aqueous solution, emulsifiable concentrate, or a 30-fold solution, and the mixture was allowed to stand for 1 hour.

[0128] Depend on Figure 3 It can be seen that the composite antimicrobial peptide of Example 3 of the present invention has good compatibility with various stock solutions, no precipitation occurs after standing for 1 hour, and excellent stability.

[0129] Experiment 7: Testing of the inhibition of rice blast fungus by composite antimicrobial peptides

[0130] Healthy rice seedlings grown for 14 days (variety Co39, 10 rice leaves per group, three replicates per group, and the average value) were selected. Spores were collected from the spore-forming plates and added to a sterile aqueous solution containing 2‰ gelatin to a final concentration of 5.0×10 4 / mL spore suspension, and then use a throat sprayer to evenly spray the spore suspension onto the rice leaves. The treatment group sprayed only with the spore suspension was used as the control group. The test group was divided into 4 treatment groups. The composite antimicrobial peptides prepared in Examples 1-3 and Comparative Example 1 were diluted 500 times to obtain antimicrobial peptide liquids. The antimicrobial peptide liquid was sprayed 24 hours after spraying the spore suspension, and the spray amount was 150mL / m 2 The rice was then transferred to a container at 28°C, in darkness, and with high humidity (RH>95%) for 24 h. The culture was then continued in a container with alternating light and dark cycles (12 h each) and high humidity (RH>95%). The experiment was terminated after 7 days, and the antibacterial activity was analyzed by counting the number of lesions per unit area. The specific test results are shown in Table 4.

[0131] Table 4

[0132] serial number control group Example 1 Example 2 Example 3 Comparative Example 1 Number of lesions / slice 10 2 1 1 4

[0133] As shown in Table 1, the composite antimicrobial peptides prepared in Examples 1-3 have good antimicrobial effects. However, compared with Example 3, in Comparative Example 1, no yeast oligosaccharide was added, and the inhibitory effect on pathogenic bacteria was weakened.

[0134] Experiment 8: Wheat growth promotion experiment

[0135] 1. Experimental purpose: To explore the effect of antimicrobial peptide preparations on the growth of wheat stems and leaves

[0136] 2. Test date: April 7, 2025

[0137] 3. Experimental location: Leyishi Bioassay Laboratory

[0138] 4. Materials and Methods

[0139] Test crop: wheat

[0140] Treatment reagents: pure water, universal complete nutrient solution, composite antimicrobial peptide (defense peptide) of Example 3, imported product 1, imported product 2

[0141] 5. Experimental design and treatment

[0142] The first processing operation is shown in Table 5, and the processing time is April 8; the second processing operation is shown in Table 6, and the processing time is April 15.

[0143] Table 5

[0144]

[0145] Table 6

[0146]

[0147] 6. Wheat growth record

[0148] 6.1 Wheat growth status

[0149] The growth status of wheat during the experimental period is shown in Figure 4-Figure 8 It can be seen that the composite antimicrobial peptide (defense peptide) has a significant promoting effect on the growth of wheat, and the effect is significantly better than that of a certain imported product 1 and a certain imported product 2.

[0150] 6.2 Records of wheat indicators

[0151] The wheat growth indicators during the experiment were shown in Figures 9-11 , we can get the following results:

[0152] Wheat growth rate (cm / 14 days): Group 1 (blank): 1.31→1.17; Group 2 (complex antimicrobial peptide): 2.99→2.62; Group 3 (imported product 1): 1.53→2.33; Group 4 (imported product 2): 1.51→1.79.

[0153] The final growth height of wheat (April 22): Group 1 (blank): 8.03cm; Group 2 (complex antimicrobial peptide): 11.91cm (the highest); Group 3 (imported product 1): 10.28cm; Group 4 (imported product 2): 9.32cm.

[0154] Fresh and dry weight of wheat (g): Group 1 (blank): 2.93→0.5; Group 2 (complex antimicrobial peptide): 3.83→0.58 (heaviest); Group 3 (imported product 1): 3.49→0.54; Group 4 (imported product 2): 3.06→0.54.

[0155] From this, we can see that the first group (blank): plant height growth was small, stems were thin and sparse, leaves were light yellow, some leaves were withered and yellow, and the growth trend was the worst; the second group (complex antimicrobial peptide): plant height growth and biomass accumulation far exceeded those of the other groups, stems were thick, leaves were broad and numerous, dark green and shiny, and growth was lush and upright, with significant advantages; the third group (a certain imported product 1): plant height and biomass accumulation were inferior to the second group, the stems and leaves were in average condition, and the growth performance was better than the first and fourth groups; the fourth group (a certain imported product 2): plant height and biomass accumulation were limited, the plant appearance was similar to the third group, but the stems and leaves were slightly less lush, better than the first group.

[0156] In summary, the composite antimicrobial peptide treatment group of the present invention performed best in terms of plant height growth, fresh weight, and dry weight accumulation. This suggests that the composite antimicrobial peptide preparation significantly promotes wheat stem and leaf growth and has great potential for application in agricultural production growth promotion.

[0157] The above is only a preferred embodiment of the present invention and does not limit the present application in any form. Although the present application is disclosed as above with a preferred embodiment, it is not intended to limit the present application. Any technician familiar with this profession, without departing from the scope of the technical solution of the present application, makes some changes or modifications using the technical content disclosed above, which are equivalent to equivalent implementation cases. However, any simple modifications, equivalent changes and modifications made to the above embodiments based on the technical essence of the present invention without departing from the content of the technical solution of the present invention are still within the scope of the technical solution.

Claims

1. A composite antimicrobial peptide, characterized in that: Calculated by mass, it includes the following components: 20-50 parts of antimicrobial peptide, 300-400 parts of yeast peptide, 100-200 parts of amino acid, 15-25 parts of yeast oligosaccharide, and 5-20 parts of nucleotide.

2. The composite antimicrobial peptide according to claim 1, characterized in that The preparation method of the antimicrobial peptide is as follows: The Tween aqueous solution is dropped into the buffer solution of the PR39 antimicrobial peptide, stirred at 20-30° C. and 150-250 rpm for 25-40 minutes, then ultrasonically treated at 15-25° C. for 5-10 minutes, and the obtained mixture is dried to obtain the antimicrobial peptide.

3. The composite antimicrobial peptide according to claim 2, characterized in that: The Tween consists of Tween 20 and Tween 80 in a mass ratio of 1: (0.85-1.3).

4. The composite antimicrobial peptide according to claim 2, characterized in that The mass ratio of the Tween to the PR39 antimicrobial peptide is (5-10):

1.

5. The composite antimicrobial peptide according to claim 1, characterized in that The preparation method of the yeast oligosaccharide is as follows: (1) Yeast autolysis: Use citric acid to adjust the pH of the yeast suspension to 4.5-5.5, then add oligoxylose, soybean lecithin hydrolysate and Aspergillus niger acid protease, and react at 35-45°C for 2-8 hours to obtain yeast autolyzed milk; (2) Yeast enzymatic hydrolysis: first add mannanase and β-glucanase to the yeast autolyzed milk in step (1) for enzymatic hydrolysis, then add protease for enzymatic hydrolysis, adjust the system pH and enzymatic hydrolysis temperature during the enzymatic hydrolysis process, terminate the enzyme activity, and dry to obtain yeast oligosaccharides.

6. The composite antimicrobial peptide according to claim 5, characterized in that In the step (1), the amount of xylooligosaccharide used is 1-3% of the dry weight of the yeast suspension; the amount of soybean lecithin hydrolyzate used is 0.5-1.5% of the volume of the yeast suspension.

7. The composite antimicrobial peptide according to claim 5, characterized in that: The Aspergillus niger acid protease is 0.1-0.3% of the dry weight of the yeast suspension.

8. The composite antimicrobial peptide according to claim 5, characterized in that: The dosage of the mannanase, beta-glucanase and protease is 0.5-1.5%, 0.5-2.0% and 0.3-1.0% of the dry weight of the yeast autolyzed milk respectively.

9. The composite antimicrobial peptide according to claim 5, characterized in that: The β-glucan oligosaccharide in the yeast oligosaccharide is ≥60%.

10. Use of the composite antimicrobial peptide according to any one of claims 1 to 9 in the agricultural field, characterized in that: The composite antimicrobial peptide is applied to the leaves and / or roots of crops; the dosage for the leaves is 30-50 mL / mu, and the dosage for the roots is 500-1000 mL / mu.

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