Tenebrio molitor antibacterial peptide as well as preparation method and application thereof

Abstract

The invention relates to the technical field of agricultural fertilizers. More specifically, the invention relates to an extraction method of tenebrio molitor antibacterial peptide and application of the tenebrio molitor antibacterial peptide in preparation of tea tree anthracnose resistant fertilizer. According to the preparation method, the colletotrichum camelliae liquid is taken as an induction factor, the ultrasonic induction technology is utilized to strengthen the absorption of tenebrio molitor to the induction factor, meanwhile, a supercritical / ethanol cosolvent extraction process and a bionic affinity chromatography purification process are combined, the efficient extraction of the antibacterial peptide is realized, and the obtained antibacterial peptide has a relatively good inhibition effect on the colletotrichum camelliae. A tea tree anthracnose resisting leaf fertilizer is further prepared through the tenebrio molitor antibacterial peptide, the leaf fertilizer has a good prevention and treatment effect on tea tree anthracnose caused by camellia colletotrichum spinosus, meanwhile, tea growth can be promoted, and the yield reduction caused by anthracnose and nutrition deficiency of tea tree crops can be effectively reduced.

Description

Technical Field

[0001] This invention relates to the field of agricultural fertilizer technology. More specifically, this invention relates to a method for extracting antimicrobial peptides from yellow mealworms and their application in the preparation of fertilizers resistant to anthracnose in tea trees. Background Technology

[0002] my country is the world's earliest country to discover and utilize tea trees. As an important economic crop, tea is widely cultivated in southern and southwestern my country. Tea anthracnose is caused by *Colletotrichum candida* (a fungus). Colletotrichum camelliae Tea anthracnose, triggered by high temperature and humidity, can cause leaf blight, branch necrosis, and even plant death, resulting in a 20%-50% reduction in tea yield and severely impacting tea quality and industry profitability. For a long time, chemically synthesized pesticides have been the primary means of controlling this disease. However, their overuse has led to increasing pathogen resistance, a heightened risk of pesticide residue exceeding standards, and continuous pollution of the tea garden's ecological environment, failing to meet the requirements of modern green and sustainable agricultural development. Developing safe, efficient, and environmentally friendly green control technologies for tea anthracnose has become an urgent need for the industry.

[0003] Antimicrobial peptides (AMPs), a class of naturally occurring immunomodulatory substances widely found in organisms, have shown great potential in the field of biological control of plant diseases due to their advantages such as broad-spectrum antibacterial activity (inhibiting bacteria, fungi, and viruses), low likelihood of inducing drug resistance in pathogens (multiple targets and unique mechanisms of action), and rapid action (destroying pathogen cell membranes within minutes). Studies have shown that antimicrobial peptides can bind to components such as phospholipids and chitin on pathogen cell membranes, forming transmembrane ion channels, leading to leakage of cell contents or collapse of membrane potential, ultimately achieving sterilization. Some antimicrobial peptides can also enter cells to inhibit key physiological processes such as nucleic acid replication and protein synthesis, enhancing their antibacterial effect. However, limited sources, low extraction efficiency, and poor stability restrict their application in field disease control.

[0004] Yellow mealworms, an important resource insect in my country, are rich in antimicrobial peptides, which are immunomodulatory substances. Induction by pathogens can significantly enhance the expression of specific antimicrobial peptides. They also offer advantages such as low breeding costs (feed mainly consists of wheat bran and corn), short production cycle (larval stage approximately 40 days), and no risk of disease outbreaks, making them ideal raw materials for large-scale antimicrobial peptide production. Current methods for preparing antimicrobial peptides from yellow mealworms primarily involve needle puncture to destroy host tissue, followed by pathogen infection to induce the production of antimicrobial peptides in the hemolymph. Extraction mainly employs enzymatic hydrolysis or chemical extraction. The needle puncture method is cumbersome, easily causing larval death, and the infiltration efficiency of the bacterial solution through the needle hole is low. Enzymatic hydrolysis or chemical extraction is costly, resulting in low production efficiency and high production costs for antimicrobial peptides, hindering their application and promotion in agricultural disease control. Summary of the Invention

[0005] To overcome the shortcomings of the prior art, this invention first provides a method for preparing antimicrobial peptides from mealworms. The second objective of this invention is to provide the application of the mealworm antimicrobial peptides obtained by the above extraction method in the preparation of fertilizers resistant to anthracnose in tea trees. This invention utilizes ultrasonic induction technology to enhance the absorption of inducing factors of *Colletotrichum camelliae* by mealworms. During the feeding process of mealworms, active tea extracts are added to enhance the immune response of mealworms to *Colletotrichum camelliae*, thereby increasing the yield of antimicrobial peptides. Simultaneously, supercritical fluid extraction is combined with... The ethanol co-solvent extraction process enables the efficient extraction of antimicrobial peptides. To address the limitations of single-function and insufficient synergistic effects of antimicrobial peptides, they are further formulated into foliar fertilizers, achieving synergistic effects of "nutrient supply and disease control," and providing an innovative solution for the green control of anthracnose in tea trees.

[0006] To achieve the above objectives, the technical solution adopted by the present invention is as follows: This invention provides a method for preparing antimicrobial peptides from yellow mealworms, comprising the following steps: S1. The *Colletotrichum camelliae* strain was cultured to obtain *Colletotrichum camelliae* cells, which were then diluted with 0.9% physiological saline to a cell concentration of [missing value]. Bacterial suspension; S2. Extract active substances from fresh tea leaves to obtain tea active extract; S3. Cultivate mealworm larvae and add 0.2-0.5% of tea active extract by weight to the feed. When the larvae reach a body length of 10-15 mm, induce the production of antimicrobial peptides by ultrasound. Place the treated larvae in a sterile environment and induce culture at 26-30℃ for 3 days. S4. Extract antimicrobial peptides from the yellow mealworms induced and cultured in S3, thus obtaining the yellow mealworm antimicrobial peptides.

[0007] Furthermore, in the method for preparing a mealworm antimicrobial peptide, S1 specifically includes: S1.1, prepare PDA medium, adjust its pH to 7.0±0.2, and sterilize at 121℃ for 20 min; S1.2, inoculate Guignardia c. sp. camelliae strain into PDA medium after sterilization treatment in S1.1, and cultivate at 25℃ in dark for 5-7d until the colony diameter reaches 3-3.5 cm; S1.3, transfer the mycelium in PDA medium to liquid PDB medium, and cultivate at 25℃ with a rotation speed of 150±5 rpm for 3d to obtain Guignardia c. sp. camelliae cell suspension; S1.4, centrifuge the Guignardia c. sp. camelliae cell suspension obtained in S1.3 at 4℃ and 1000×g for 5 min, collect the cell precipitate at the bottom, and wash with 0.1 mol / L PBS at 4℃; then pass the washed cell suspension through a sterile cell sieve, collect the filtrate, centrifuge at 4℃ and 1000×g for 3 min, and remove the supernatant to obtain the purified cell precipitate; S1.5, add sterile normal saline pre-cooled to 4℃ to the purified cell precipitate obtained in S1.4, and dilute to a cell concentration of 1×108 cells / mL; to obtain the cell suspension.

[0008] Further, in the preparation method of the yellow mealworm antibacterial peptide, S2 specifically comprises: S2.1, dry fresh tea leaves at 60℃±2℃ for 24h, crush to 60-80 mesh, and mix with 60% ethanol at a solid-liquid ratio of 1:30 (w / v), and then ultrasonically treat at 50±1℃ to obtain an extraction liquid, wherein 0.1% ascorbic acid by mass of the 60% ethanol is added; S2.2, evaporate and concentrate the extraction liquid obtained in S2.1 to 10% of the original volume, purify through a macroporous resin AB-8 column, then elute with 70% ethanol, and finally freeze-dry to obtain the tea leaf active extract.

[0009] Further, in the preparation method of the yellow mealworm antibacterial peptide, S3 specifically comprises: S3.1, feed yellow mealworm larvae at 26-30℃ and 70% humidity, and the feed used for feeding includes the following components in mass percentage: wheat bran 75-80%, corn flour 18-20%, yeast powder 5-8%, and tea leaf active extract 0.2-0.5%; S3.2, when the body length of the larvae reaches 10-15 mm, the yellow mealworms are starved for 12 h, and then placed in a closed container with humidity ≥ 95% and temperature 4℃, and a misting airflow containing the bacterial suspension obtained in S1.5 is introduced, and the yellow mealworms are treated for 3-5 min under the conditions of ultrasonic frequency 20-40 kHz, power density 1.0-1.5 W / cm², and duty cycle 50-70%, and then the yellow mealworms are transferred to a sterile feeding box and cultured for another 3 d under the conditions of S3.1.

[0010] Further, in the preparation method of the yellow mealworm antibacterial peptide, S4 specifically comprises: S4.1, stop feeding the yellow mealworms for 1 d, and then transfer the starved yellow mealworms to liquid nitrogen for quick freezing for 1 min, and then freeze them at -80℃ for 2 h, and then use a low-temperature ball mill to grind the yellow mealworms into fine powder under the condition of -20℃, and then add pre-cooled extraction buffer to the yellow mealworm powder at a volume-to-mass ratio of 100:1 (mL / g), and then perform low-temperature centrifugation, and then collect the supernatant and pass it through a 0.22 μm PVDF filter membrane to obtain a filtrate containing yellow mealworm hemolymph; S4.2, collect the filtrate obtained in S4.1, mix it with pre-cooled phosphate buffer with pH 7.4 and concentration 0.1 mol / L at a mass ratio of 1:3, and then stand it at 4℃ for 30 min, and then add polyethylene glycol 6000 with concentration 0.1% (w / v) in an amount equal to the volume of the filtrate, and then perform centrifugal treatment, and then collect the supernatant; S4.3, perform supercritical ethanol co-solvent extraction on the supernatant collected in S4.2 to obtain a crude extract; S4.4, purify the crude extract obtained in S4.3 by biomimetic affinity chromatography, and then freeze-dry it to obtain an antibacterial peptide powder, which is the yellow mealworm antibacterial peptide.

[0011] Further, in the preparation method of the yellow mealworm antibacterial peptide, S4.3 specifically comprises: add anhydrous ethanol in an amount of 5-10% of the volume of the supernatant obtained in S4.2 as an entraining agent, mix uniformly, and then extract under the conditions of pressure 25-35 MPa, temperature 40-45℃, flow rate 15-20 L / h for 90-120 min. The extraction phase enters separation kettle I, which is treated under the conditions of pressure 6-8 MPa and temperature 35℃, so as to precipitate fat-soluble impurities, and the remaining fluid enters separation kettle II, which is treated under the conditions of pressure 4-5 MPa and temperature 25℃, so as to collect an ethanol-water solution containing the antibacterial peptide, and then concentrate the separation liquid to 1 / 5 of the original volume by rotary evaporation at 40℃, and then freeze-dry it to obtain a crude extract.

[0012] Further, in the preparation method of the yellow mealworm antibacterial peptide, S4.4 specifically comprises: S4.4.1, the purified bacteria body precipitate obtained in S1.4 is mixed with NaOH solution at a material to liquid ratio of 1:20 (w / v), and is oscillated in a water bath at 100°C for 1h, and the supernatant is removed by centrifugation, and the process is repeated multiple times until the mycelium is translucent; S4.4.2, the mycelium treated in S4.4.1 is mixed with 1 mol / L hydrochloric acid at a material to liquid ratio of 1:5 (w / v), and is stirred magnetically at room temperature for 2h, and is washed with water to neutral after centrifugation; S4.4.3, the mycelium treated in S4.4.2 is dehydrated with 50% ethanol, 70% ethanol and 95% ethanol for 1h respectively, and is ground through a 200 mesh sieve after vacuum drying, to obtain chitin powder of A. camphorata; S4.4.4, amino agarose microspheres with a particle size of 100-200μm are placed in 0.1 mol / L solution, and hydrobromic acid is added at a mass to volume ratio of 10:1 (mg / mL) for ice bath reaction for 15min, and then the reaction is terminated quickly with cold pure water, and the activated microspheres are obtained after drying at 60°C; S4.4.5, the chitin powder of A. camphorata obtained in S4.4.3 is dissolved in and , and the concentrations of and in the mixed solution are 0.1 mol / L and 0.2 mol / L respectively, and then the activated microspheres obtained in S4.4.4 are added, and the coupling is oscillated at 4°C for 12h, wherein the mass ratio of the chitin powder of A. camphorata and the activated microspheres is 5:1, to obtain a coupling body; S4.4.6, the residual active sites on the coupling body obtained in S4.4.5 are blocked by 1 mol / L ethanol for 2h, and then the coupling body is washed with PBS buffer and is placed in 20% ethanol and is stored at 4°C; S4.4.7, the coupling body treated in S4.4.6 is subjected to biomimetic affinity chromatography operation, and the chromatography liquid is freeze-dried to obtain an antibacterial peptide powder.

[0013] Further, in the preparation method of the antibacterial peptide of the yellow mealworm, S4.4.7 specifically comprises: S4.4.7.1, equilibrium stage: Tris-HCl+NaCl buffer solution is added to the coupling body treated in S4.4.6 at a flow rate of 1.0 mL / min, wherein the concentration of Tris-HCl is 20mmol / L, and the concentration of NaCl is 0.5mol / L; S4.4.7.2, sample loading stage: The supercritical / ethanol co-solvent extraction crude extract obtained in S4.3 is added at a flow rate of 0.5 mL / min; S4.4.7.3, washing miscellaneous stage: Add 0.1% Triton X-100 equilibration liquid at a flow rate of 1.5 mL / min; S4.4.7.4, elution stage: Add 0.1 mol / L Gly-HCl at a flow rate of 0.8 mL / min; S4.4.7.5, regeneration stage: Add urea+NaOH eluent to remove residual impurities at a flow rate of 1.0 mL / min, wherein the concentration of urea is 6 mol / L, and the concentration of NaOH is 0.5 mol / L; S4.4.7.6, after the eluent is completely added, immediately neutralize with 1 mol / L Tris-HCl, and after concentration by ultrafiltration, freeze-drying to obtain the antibacterial peptide powder.

[0014] The application also provides a Tenebrio molitor antibacterial peptide prepared by any of the above methods.

[0015] The application also provides an anti-tea tree anthracnose foliar fertilizer, comprising the following components in percentage by weight: Tenebrio molitor antibacterial peptide 5-8%, algalin 10-15%, trace elements 5-10%, potassium dihydrogen phosphate 30-40%, ammonium sulfate 35-45%; The sum of the mass percentages of the above components is 100%; wherein the trace elements include Zn, Fe, Mn, Cu, B and Mo.

[0016] Further, in the anti-tea tree anthracnose foliar fertilizer, the mass ratio of Zn, Fe, Mn, Cu, B and Mo in the trace elements is 1:2-3:1-2:0.2:0.5:0.1.

[0017] The application has the following beneficial effects: 1. The application breaks through the limitations of traditional needle puncture method by combining ultrasonic induction technology with tea active substances to induce immune response of Tenebrio molitor. The mechanical vibration of ultrasonic waves can cause micro-damage (similar to micropores of needle puncture) to the body wall (such as elytra and dorsal plate) or blood cell membrane of Tenebrio molitor, forming a temporary channel to promote the entry of antigens (such as mycelium fragments and cell wall components) in the Colletotrichum camelliae liquid into the blood cavity. The cavitation effect of ultrasonic waves can stimulate the activity of blood cells (such as plasma cells and granulocytes), enhancing their recognition and phagocytosis capacity of pathogens. Tea active ingredients synergistically activate key transcription factors of the immune pathway, and directionally express specific antibacterial peptides against Colletotrichum camelliae, with a yield of 0.35 mg / g of insect body (60% higher than needle puncture method), and an insect body survival rate of >95%, effectively improving the content of antibacterial peptides in the insect body.

[0018] 2. The application adopts ultrasonic synergistic supercritical / ethanol cosolvent extraction combined with biomimetic affinity chromatography purification technology: ultrasonic crushing strengthens the release of cell contents, supercritical / ethanol cosolvent efficiently recovers active substances under mild conditions; further, a biomimetic chromatography medium is constructed using Camellia chitin as a natural ligand, and the specific recognition mechanism of pathogenic bacteria-antimicrobial peptides is used to accurately capture the target peptide segment.

[0019] 3. The application prepares a leaf fertilizer against tea anthracnose by the above-mentioned yellow mealworm antimicrobial peptide, which shows good prevention and control effect on tea anthracnose caused by Camellia chitin, can promote tea growth, can effectively reduce the yield reduction of tea crops caused by anthracnose and nutrient deficiency, can improve the agronomic characters and quality of tea leaves, and can improve the economic value.

[0020] Other advantages, objects and features of the present application will be apparent from the following description, and will be understood by those skilled in the art upon reading and understanding the specification. BRIEF DESCRIPTION OF DRAWINGS

[0021] Figure 1 Flowchart of the preparation method of the yellow mealworm antimicrobial peptide described in the application; Figure 2 Comparison chart of scanning electron microscopy of ultrasonic treatment and untreated yellow mealworm body wall in the embodiment of the application. DETAILED DESCRIPTION

[0022] The application will be further described in detail below with reference to the examples, so that those skilled in the art can implement it according to the specification.

[0023] It should be noted that the experimental methods described in the following embodiments are all conventional methods unless otherwise specified, and the reagents and materials can be obtained from commercial channels unless otherwise specified.

[0024] Example 1 A preparation method of a yellow mealworm antimicrobial peptide, comprising the following steps: S1, culture of Camellia chitin pathogenic bacteria: S1.1, prepare PDA culture medium (potato concentration is 200 g / L, glucose concentration is 20 g / L, and agar concentration is 15 g / L), adjust its pH to 7.0±0.2, and sterilize at 121℃ for 20 min; S1.2, inoculate the Gueldenstaedtia lanceolata Colletotrichum strain into the PDA medium after sterilization treatment in S1.1, and cultivate at 25°C in dark for 5-7 days until the colony diameter reaches 3-3.5 cm. The Gueldenstaedtia lanceolata Colletotrichum used is from China General Microbiological Culture Collection Center, and the preservation time is December 1, 2016, and the preservation number is CGMCC NO. 3.17884. The strain can cause Gueldenstaedtia lanceolata to produce anthracnose.

[0025] S1.3, take the mycelium block (5 mm in diameter) of the mycelium in the PDA medium with a sterile puncher, transfer it to liquid PDB medium (potato concentration is 200 g / L, glucose concentration is 20 g / L), and cultivate at 25°C with a rotation speed of 150±5 rpm for 3 days to obtain Gueldenstaedtia lanceolata mycelium suspension; S1.4, centrifuge the Gueldenstaedtia lanceolata mycelium suspension at 4°C and 1000×g for 5 min, collect the mycelium precipitate at the bottom, and add 0.1M PBS at 4°C for washing. The mycelium suspension after washing is passed through a 100 μm sterile cell screen, the filtrate is collected, centrifuged at 4°C and 1000×g for 3 min, and the supernatant is discarded to obtain the purified mycelium precipitate; S1.5, add sterile normal saline pre-cooled to 4°C to the purified mycelium precipitate, and dilute to a mycelium concentration of 1×108 CFU / mL to obtain the Gueldenstaedtia lanceolata mycelium suspension.

[0026] S2, extract active substances from fresh tea leaves to obtain tea leaf active extract, and the main active ingredient tea polyphenol content is ≥85%: S2.1, dry the fresh tea leaves at 60°C±2°C for 24 h, crush to 60-80 mesh, and mix with 60% ethanol at a solid-liquid ratio of 1:30 (w / v), and then ultrasonically treat at 50±1°C with a power of 400 W and a frequency of 40 kHz for three times, each for 20 min, and cool for 5 min, to obtain the extraction liquid, wherein 0.1% ascorbic acid is added to the 60% ethanol to prevent oxidation; S2.2, evaporate and concentrate the extraction liquid obtained in S2.1 to 10% of the original volume, purify it through a macroporous resin AB-8 column, the loading flow rate is 2BV / h, the elution volume is 3BV, then elute with 70% ethanol, and finally freeze-dry to obtain the tea leaf active extract.

[0027] ​S3. Cultivate mealworm larvae at 26-30℃ and 70% humidity. The feed contains the following components by weight percentage: wheat bran (passed through an 80-mesh sieve) 76%, corn flour 18%, yeast powder 5.7%, and tea active extract powder 0.3%. When the larvae reach a body length of 13±1mm, take 1kg of healthy larvae and place them in a sealed container with humidity ≥95% and temperature 4℃. Pass an atomized airflow containing the bacterial suspension described in S1.5 and treat for 4min at an ultrasonic frequency of 30kHz and a power density of 1.2W / cm². Then transfer the mealworms to a sterile rearing box and continue to cultivate for 3 days under the conditions of S3.1.

[0028] S4. Extract antimicrobial peptides from mealworms induced and cultured in S3: S4.1 Stop feeding mealworms for 1 day, transfer the empty mealworms to liquid nitrogen for quick freezing for 1 minute and then freeze them at -80℃ for 2 hours. Then grind the mealworms into fine powder at -20℃ using a low-temperature ball mill. Add HEPES buffer pre-cooled to 4℃ at a volume-to-mass ratio of 100:1 (mL / g) and centrifuge at low temperature. Take the supernatant and filter it through a 0.22μm PVDF membrane to obtain the filtrate containing mealworm hemolymph. S4.2. Collect the filtrate obtained in S4.1 and mix it with phosphate buffer (pH 7.4, concentration 0.1 mol / L, pre-cooled to 4°C) at a mass ratio of 1:3. After standing at 4°C for 30 min, add polyethylene glycol 6000 with a concentration of 0.1% (w / v) equal to the volume of the filtrate. After centrifugation, collect the supernatant. S4.3. The supernatant collected in S4.2 is subjected to supercritical fluid extraction. / Ethanol co-solvent extraction. The extraction method is as follows: 8% by volume of anhydrous ethanol is added to the supernatant obtained in S4.2 as an entrainer, mixed thoroughly, and then extracted at a pressure of 30 MPa and a temperature of 42℃. Extraction was performed at a flow rate of 18 L / h for 100 min. The extract phase was introduced into separation vessel I (pressure 7 MPa, temperature 35℃), where lipid-soluble impurities precipitated. The remaining fluid was introduced into separation vessel II (pressure 4 MPa, temperature 25℃), where an ethanol-water solution containing antimicrobial peptides was collected. The separated liquid was concentrated to 1 / 5 of its original volume by rotary evaporation at 40℃ and then freeze-dried to obtain the crude extract.

[0029] S4.4 The crude extract obtained in S4.3 is purified by biomimetic affinity chromatography and then freeze-dried at -60℃ to obtain antimicrobial peptide powder, which is the yellow mealworm antimicrobial peptide.

[0030] Appendix Figure 2 The images show electron micrographs of the body walls of sonicated and untreated mealworms. The sonicated body walls show more reversible micropores, and the analysis of the electron micrographs indicates that the pore diameter is about 5-10 μm and the number of pores is about 6 times that of the untreated group. Example

[0031] The antimicrobial peptide of Tenebrio molitor was extracted according to the method of Example 1, and then was prepared into a foliar fertilizer against the anthracnose of tea tree, which comprises the following ingredients in percentage by weight: The antimicrobial peptide of Tenebrio molitor 5%, alginin 10%, trace elements 8%, potassium dihydrogen phosphate 35%, ammonium sulfate 42%.

[0032] The main components of the trace elements are Zn, Fe, Mn, Cu, B and Mo, and the mass ratio among them is Zn:Fe:Mn:Cu:B:Mo = 1.5:2:2:0.2:0.5:0.1. Example

[0033] The antimicrobial peptide of Tenebrio molitor was extracted according to the method of Example 1, and then was prepared into a foliar fertilizer against the anthracnose of tea tree, which comprises the following ingredients in percentage by weight: The antimicrobial peptide of Tenebrio molitor 8%, alginin 12%, trace elements 8%, potassium dihydrogen phosphate 32%, ammonium sulfate 40%.

[0034] The main components of the trace elements are Zn, Fe, Mn, Cu, B and Mo, and the mass ratio among them is Zn:Fe:Mn:Cu:B:Mo = 1.5:2:2:0.2:0.5:0.1.

[0035] Comparative Example 1 In order to verify the advantages of the preparation method of the antimicrobial peptide of Tenebrio molitor, Comparative Example 1 was set. The preparation method of the antimicrobial peptide of Tenebrio molitor in Experimental Group 1 of Comparative Example 1 was the same as that of Example 1, and the control group 1 used the traditional needle pricking method for Tenebrio molitor culture, and the variety and culture method of Tenebrio molitor used, the extraction and purification method of the antimicrobial peptide were the same as those of Example 1. The main difference between Experimental Group 1 and Experimental Group 1 is that the sterile needle pricking method is used to damage the body surface, and then the bacterial solution is sprayed. The specific operation method is as follows: in the clean bench, 27G micro syringe needle (the needle tip is ground to be round and blunt) is soaked with 75% ethanol for 5 min, and the needle is lightly pricked on the elytra or back of the larva (avoiding the ganglion), forming a small hole with a diameter of about 0.1-0.2 mm; the insect body is immediately transferred to a closed container with humidity ≥95% and temperature 4℃, and the bacterial solution is sprayed according to the concentration of 1×108cfu / mL, 1×107cfu / mL, 1×106cfu / mL and 1×105cfu / mL, respectively, and the spraying amount is 5-10 mL / 100 larvae, so that the bacterial solution slowly penetrates into the blood cavity through the needle hole. Each group is repeated 3 times, and each repetition is 100 larvae. The antimicrobial peptide of Tenebrio molitor prepared in Example 1 and Comparative Example 1 is detected, and the survival rate of the insect body, the yield of the antimicrobial peptide and the inhibition zone size of the antimicrobial peptide against Guignardia sp. in Example 1 are obtained. The experimental results are shown in Table 1: Table 1 Comparison of experimental results of Experimental Group 1 and Control Group 1 ​

[0036] From the data in Table 1, it can be seen that the survival rate of the insect body and the yield of the antibacterial peptide in the experimental group 1 are superior to those in the control group 1. Meanwhile, the antibacterial peptide obtained in the experimental group 1 maintains the biological activity equivalent to that of the antibacterial peptide prepared by the traditional method.

[0037] Comparative Example 2 To verify the promoting effect of the feed containing the active extract of tea leaves on the production and antibacterial activity of the Tenebrio molitor antibacterial peptide, Comparative Example 2 is set. The preparation method of the Tenebrio molitor antibacterial peptide in the experimental group 2 of Comparative Example 2 is the same as that in Example 1, the variety and culture method of the Tenebrio molitor used in the control group 2 are the same as those in Example 1, and the antibacterial peptide extraction and purification method is the same as that in Example 1. The main difference between the experimental group 2 and the control group 2 is that the experimental group 2 is fed with ordinary feed (formula: wheat bran 76%, corn flour 18%, yeast powder 6%) without the addition of the active extract of tea leaves. The Tenebrio molitor antibacterial peptides obtained from the experimental group 2 and the control group 2 are detected to obtain the yield of the antibacterial peptide and the antibacterial circle size against Colletotrichum camelliae in Example 1, and the experimental results are shown in Table 2. Table 2 Influence of the active extract of tea leaves on the yield of the Tenebrio molitor antibacterial peptide

[0038] From the data in Table 2, it can be seen that the yield of the antibacterial peptide in the experimental group 2 is superior to that in the control group 2, which indicates that the addition of the active extract of tea leaves in the feed helps to improve the Tenebrio molitor. The active substances such as tea polyphenols and catechins in the active extract of tea leaves have an immunomodulatory effect, which can enhance the activity of Tenebrio molitor blood cells, promote the activation of key transcription factors in the immune pathway, and amplify the immune response signal to the pathogenic bacteria.

[0039] Comparative Example 3 To verify the actual prevention and treatment effect of the Tenebrio molitor antibacterial peptide foliar fertilizer provided by the application on the tea tree anthracnose and the growth promoting effect, a field comparison test is set. The conventional foliar fertilizer without antibacterial peptide is used as a control to evaluate the disease reduction effect and the promotion effect on the growth of tea leaves.

[0040] The tested tea tree is Xinyang No. 10 tea tree, which is planted in a tea garden in Xinyang City, Henan Province (north latitude 32°13', east longitude 114°05'), with a tree age of 5 years, a cultivation mode of double-row strip planting (row spacing 1.5 m, plant spacing 0.33 m), and no spraying of any fungicides before the test. The tea tree anthracnose (the pathogenic bacteria are Colletotrichum camelliae), and the incidence rate of the tea garden before the test is about 15% (disease leaf rate).

[0041] A randomized block design is adopted, with 50 tea trees in each group (3 times of repetition, a total of 150 tea trees). The foliar fertilizer is diluted by 1000 times, and the spraying amount of each group is 50 L.

[0042] Among them, the experimental group 3 sprays the yellow mealworm antibacterial peptide foliar fertilizer prepared in Example 2, the control group 3 sprays the foliar fertilizer containing 0.1% mass concentration of prochloraz (alginin 15%, trace elements 8%, potassium dihydrogen phosphate 35%, ammonium sulfate 42%); the control group 4 sprays the conventional foliar fertilizer not containing the yellow mealworm antibacterial peptide (alginin 15%, trace elements 8%, potassium dihydrogen phosphate 35%, ammonium sulfate 42%), and the blank group only sprays the same amount of water. On the 15th day after spraying the foliar fertilizer, the simulated rainfall of 5mm / h is sprayed to wash the drug.

[0043] On the 3rd day, the 7th day, the 14th day, the 21st day and the 28th day after spraying, the tea tree disease leaf rate (DR) and the leaf antibacterial peptide residual amount are respectively sampled and counted; on the 28th day after spraying, the 0-20cm surface soil of the tea garden (5 sample points in each group) is collected, and the number of bacteria, fungi and actinomycetes is determined by the dilution plate method; on the 28th day after spraying, 30 new shoots are randomly selected in each group, and the new shoot length (the length from the bud head to the top end), the single bud fresh weight (the fresh weight of 1 bud at the top end of each new shoot) and the leaf number are measured.

[0044] The antibacterial peptide residual amount is detected by high performance liquid chromatography, and the chromatographic conditions are as follows: C18 chromatographic column, mobile phase is acetonitrile:0.1% trifluoroacetic acid aqueous solution (30:70, v / v), flow rate is 1.0mL / min, detection wavelength is 214nm (antibacterial peptide characteristic absorption peak), and column temperature is 30℃.

[0045] Experimental results: The tea tree disease leaf rate at each time point after spraying and the tea leaf growth index on the 28th day after spraying are respectively shown in Tables 3 and 4: Table 3 Tea tree disease leaf rate (%) at each time point after spraying

[0046] Table 4 Tea leaf growth index on the 28th day after spraying

[0047] After spraying, the DR of the tea tree in the control group 4 and the blank group continues to rise over time (natural development of anthracnose), the DR in the control group 3 gradually decreases in the first 14 days, but rises after the 21st day, indicating that the prevention and treatment effect decreases after rainwater washing. In the experimental group 3, the DR begins to decrease significantly on the 3rd day, decreases to 8.23% on the 21st day (decreases by 47.3% compared with before spraying), and still maintains a low level of 7.69% on the 28th day, and the prevention and treatment effect on the tea tree anthracnose is better than that of the control groups 3, 4 and the blank group. On the 28th day after spraying, the tea leaf growth index in the experimental group is significantly better than that in the control group, the new shoot length in the experimental group increases by 19.4% compared with the control group, the single bud fresh weight increases by 18.2%, and the leaf number increases by 26.2%, indicating that the foliar fertilizer has a significant growth-promoting effect.

[0048] The HPLC detection result shows that the residual amount of the antibacterial peptide in the leaf is 12.3±1.1 mg / kg on the 3rd day after spraying, decreases to 8.7±0.9 mg / kg (degradation rate 29.3%) on the 7th day, 2.2±0.4 mg / kg on the 14th day, 1.5±0.2 mg / kg on the 21st day, and decreases to 0.8 mg / kg on the 28th day, indicating that the antibacterial peptide has good stability on the leaf.

[0049] On the 28th day after spraying, the number of soil bacteria , fungi , and actinomycetes of the experimental group has no significant difference (P>0.05) with that of the blank group (bacteria , fungi , and actinomycetes ), and the soil micro-ecological balance is not destroyed.

[0050] Although the embodiments of the present application have been disclosed as above, it is not limited to the application listed in the specification and the embodiments, and can be fully applied to various fields suitable for the present application, and other modifications can be easily realized by those skilled in the art, and therefore the present application is not limited to specific details and the embodiments shown and described herein, without departing from the general concept defined by the claims and the equivalent scope.

Claims

1. A method for preparing an antibacterial peptide of Tenebrio molitor, characterized by, The method comprises the following steps: S1, culturing Camellia thesaponin strain to obtain Camellia thesaponin bacteria, and diluting it with 0.9% physiological saline to a bacteria concentration of 1.0×108 / mL bacteria suspension; S2, extracting active substances from fresh tea leaves to obtain tea leaf active extract; S3, culturing Tenebrio molitor larvae and adding 0.2-0.5% of the tea leaf active extract in the mass of the feed for feeding, when the body length of the larvae reaches 10-15 mm, the larvae are treated by ultrasonic induction to produce antibacterial peptides, and the treated larvae are placed in a sterile environment and induced to culture at 26-30°C for 3 days; S4, extracting antibacterial peptides from the Tenebrio molitor induced and cultured in S3, to obtain the Tenebrio molitor antibacterial peptides.

2. The method for preparing a mealworm antimicrobial peptide as described in claim 1, characterized in that, S1 specifically comprises: S1.1, preparing PDA culture medium, adjusting the pH to 7.0±0.2, and sterilizing at 121°C for 20 min; S1.2, inoculating the Guignardia camelliae strain into the PDA culture medium after sterilization in S1.1, and culturing at 25°C in the dark for 5-7 days until the colony diameter reaches 3-3.5 cm; S1.3, transferring the mycelium in the PDA culture medium to a liquid PDB culture medium, and culturing at 25°C with a rotation speed of 150±5 rpm for 3 days to obtain a Guignardia camelliae cell suspension; S1.4, centrifuging the Guignardia camelliae cell suspension obtained in S1.3 at 4°C and 1000×g for 5 min, collecting the mycelium precipitate at the bottom, and washing with 0.1 mol / L PBS at 4°C; then, the washed mycelium suspension is passed through a sterile cell sieve, the filtrate is collected, centrifuged at 4°C and 1000×g for 3 min, and the supernatant is removed to obtain the purified mycelium precipitate; S1.5, sterile physiological saline solution pre-cooled to 4°C was added to the pellet of purified bacteria obtained in S1.4, and diluted to a bacterial concentration of 1 x 108CFU / ml and a bacterial suspension was obtained.

3. The method for preparing a mealworm antimicrobial peptide as described in claim 1, characterized in that, S2 specifically comprises: S2.1, drying fresh tea leaves at 60°C±2°C for 24 h, crushing to 60-80 mesh, mixing with 60% ethanol at a solid-liquid ratio of 1:30 (w / v), and ultrasonic treating at 50±1°C to obtain an extract, wherein 0.1% of ascorbic acid is added to the 60% ethanol; S2.2, evaporating and concentrating the extract obtained in S2.1 to 10% of the original volume, purifying through a macroporous resin AB-8 column, then eluting with 70% ethanol, and finally freeze-drying to obtain the tea leaf active extract.

4. The method for preparing a mealworm antimicrobial peptide as described in claim 1, characterized in that, S3 specifically comprises: S3.1, feeding Tenebrio molitor larvae at 26-30°C and 70% humidity, and the feed for feeding comprises the following components in mass percentage: wheat bran 75-80%, corn flour 18-20%, yeast powder 5-8%, and tea leaf active extract 0.2-0.5%; S3.2, when the body length of the larvae reaches 10-15 mm, starving for 12 h, placing the Tenebrio molitor in a closed container with humidity ≥95% and temperature 4°C, and introducing atomized gas flow containing the bacterial suspension obtained in S1.5, and treating under the conditions of ultrasonic frequency 20-40 kHz, power density 1.0-1.5 W / cm², and duty cycle 50-70% for 3-5 min, and then transferring the Tenebrio molitor to a sterile feeding box for further culturing for 3 days under the conditions of S3.

1.

5. The method for preparing a mealworm antimicrobial peptide as described in claim 1, characterized in that, S4 specifically comprises: S4.1, stop feeding Tenebrio molitor for 1 day, transfer the empty stomach Tenebrio molitor to liquid nitrogen for 1 minute, then freeze at -80℃ for 2 hours, then grind the Tenebrio molitor into fine powder at -20℃ using a low-temperature ball mill, add pre-cooled extraction buffer to 4℃ at a volume-mass ratio of 100:1 (mL / g), and then centrifuge at low temperature to obtain the supernatant, and then pass the supernatant through a 0.22 μm PVDF filter membrane to obtain a filtrate containing Tenebrio molitor hemolymph; S4.2, collect the filtrate obtained in S4.1, mix with a pre-cooled phosphate buffer with a pH of 7.4 and a concentration of 0.1 mol / L at 4℃ at a mass ratio of 1:3, stand for 30 minutes at 4℃, then add polyethylene glycol 6000 with a concentration of 0.1% (w / v) in an amount equal to the volume of the filtrate, and then collect the supernatant after centrifugal treatment; S4.

3. Subjecting the supernatant collected in S4.2 to supercritical / ethanol cosolvent extraction to obtain a crude extract; S4.4, freeze-dry the crude extract obtained in S4.3 after biomimetic affinity chromatography purification to obtain an antibacterial peptide powder, which is the Tenebrio molitor antibacterial peptide.

6. The method for preparing a mealworm antimicrobial peptide as described in claim 5, characterized in that, S4.4 specifically comprises: S4.4.1, mix the purified bacteria body precipitate obtained in S1.4 with NaOH solution at a material-liquid ratio of 1:20 (w / v), and then oscillate in a water bath at 100℃ for 1 hour, and then remove the supernatant by centrifugation, and then repeat the operation for multiple times until the mycelium becomes translucent; S4.4.2, mix the mycelium treated in S4.4.1 with 1 mol / L hydrochloric acid at a material-liquid ratio of 1:5 (w / v), and then magnetically stir at room temperature for 2 hours, and then wash with water until neutral after centrifugal treatment; S4.4.3, sequentially dehydrate the mycelium treated in S4.4.2 using 50% ethanol, 70% ethanol and 95% ethanol for 1 hour each time, and then grind through a 200-mesh sieve after vacuum drying to obtain a chitin powder of A. camphorata; S4.4.4, the amino agarose microspheres with particle size of 100-200 μm were placed in 0.1 mol / L of solution, and then 15 min of ice bath reaction was carried out after adding hydrobromic acid at a mass-volume ratio of 10:1 (mg / mL), and the reaction was quickly terminated with cold pure water. After drying at 60°C, the activated microspheres were obtained. S4.4.5, dissolving the chitin powder of S4.4.3 in a mixed solution of and , wherein the concentrations of and in the mixed solution are 0.1 mol / L and 0.2 mol / L respectively, and then adding the activated microspheres of S4.4.4, oscillating and coupling at 4°C for 12 hours, wherein the mass ratio of the chitin powder of S4.4.3 and the activated microspheres is 5:1, to obtain the coupling product; S4.4.6, block the residual active sites of the conjugate obtained in S4.4.5 by 1 mol / L ethanol for 2 hours, then rinse with PBS buffer, and then store in 20% ethanol at 4℃; S4.4.7, perform biomimetic affinity chromatography on the conjugate treated in S4.4.6, and then freeze-dry the chromatography liquid to obtain an antibacterial peptide powder.

7. The method for preparing a mealworm antimicrobial peptide as described in claim 6, characterized in that, S4.4.7 specifically comprises: S4.4.7.1, equilibrium stage: Add Tris-HCl+NaCl buffer solution to the conjugate treated in S4.4.6 at a flow rate of 1.0 mL / min, wherein the concentration of Tris-HCl is 20 mmol / L, and the concentration of NaCl is 0.5 mol / L; S4.4.7.2, sample stage: The supercritical CO2 / ethanol co-solvent extract of the crude extract obtained in S4.3 was added at a flow rate of 0.5 mL / min / ethanol co-solvent extract of the crude extract obtained in S4.3 was added at a flow rate of 0.5 mL / min S4.4.7.3, washing stage: Add 0.1% Triton X-100 balanced liquid at a flow rate of 1.5 mL / min; S4.4.7.4, elution stage: Add 0.1 mol / L Gly-HCl at a flow rate of 0.8 mL / min; S4.4.7.5, regeneration stage: Add urea+NaOH eluent to remove residual impurities at a flow rate of 1.0 mL / min, wherein the concentration of urea is 6 mol / L, and the concentration of NaOH is 0.5 mol / L; S4.4.7.6, after the eluent is completely added, immediately neutralized with 1 mol / L Tris-HCl, concentrated by ultrafiltration, and freeze-dried to obtain the antibacterial peptide powder.

8. A Tenebrio molitor antimicrobial peptide, characterized in that, Prepared by the method of any one of claims 1-7.

9. A foliar fertilizer for tea anthracnose resistance, characterized by, Composed of the following ingredients by weight percentage: The composition is composed of the following ingredients by weight percentage: The Tenebrio antibacterial peptide 5-8%, alginin 10-15%, trace elements 5-10%, potassium dihydrogen phosphate 30-40%, ammonium sulfate 35-45% according to claim 8; The sum of the mass percentages of the above components is 100%. The trace elements include Zn, Fe, Mn, Cu, B and Mo.

10. The anti-Camellia leaf spot foliar fertilizer according to claim 9, wherein the tea plant is Camellia sinensis. The mass ratio of Zn, Fe, Mn, Cu, B and Mo in the trace elements is 1:2-3:1-2:0.2:0.5:0.

1. ​

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