Method for preparing theanine sustained-release microcapsule for improving the pesticide tolerance of pollinating insects, method for applying the same, and pollinating insect bait

Abstract

This invention belongs to the field of theanine sustained-release capsules and ecological agriculture application technology, specifically relating to a method for preparing, applying, and feeding theanine sustained-release microcapsules to enhance pesticide tolerance in pollinating insects. The method includes a pollinating insect protectant, the active ingredient of which is theanine; the pollinating insect protectant aqueous solution is slowly added dropwise to a sodium alginate aqueous solution under stirring to obtain a preliminary theanine mixture; the preliminary theanine mixture is added dropwise to a calcium chloride solution coagulation bath, where the droplets immediately cross-link upon contact with calcium ions to form calcium alginate gel microspheres; the calcium alginate gel microspheres are filtered out and gently rinsed with deionized water to remove excess calcium ions from the surface, obtaining theanine sustained-release microcapsules; this method addresses the problem of rapidly increasing the active ingredient of theanine in pollinating insects to achieve the required protective concentration in a short period before they face the threat of pesticide spraying.

Description

Technical Field

[0001] This invention belongs to the field of theanine sustained-release capsules and ecological agriculture application technology, specifically involving a method for preparing theanine sustained-release microcapsules, an application method, and a feed for pollinating insects to enhance their pesticide tolerance. Background Technology

[0002] Pollinating insects are a core component of the global agricultural ecosystem, with approximately 75% of crops relying on them for pollination, directly impacting food security and biodiversity. However, the widespread use of chemical pesticides in modern agricultural production, especially neonicotinoids and organophosphates, while effectively controlling pests and diseases, causes irreversible damage to the nervous system of pollinating insects. This leads to motor impairments, olfactory learning and memory decline, and abnormal social behavior in insects such as bees. Global pollinating insect populations are declining at a rate of 3%-5% annually, becoming a pressing global ecological crisis that urgently needs to be addressed.

[0003] In the existing technology, pesticide protection measures for pollinating insects mainly include: (1) limiting the time and dosage of pesticide application to reduce non-target exposure; (2) developing alternative products of low-toxicity pesticides; and (3) cultivating pesticide-resistant pollinating insect varieties. However, the above methods have obvious limitations: it is difficult to balance the needs of pest and disease control and crop yield when limiting pesticide application; the research and development cycle of low-toxicity pesticides is long and the cost is high; and the cultivation of pesticide-resistant varieties is prone to ecological risks.

[0004] Existing pollinator insect protection technologies mostly focus on passive protective measures such as pesticide application timing and physical isolation, lacking effective solutions for actively enhancing insect resistance. Some chemical antidotes pose a risk of secondary toxicity, while the protective effects of natural plant extracts in practical ecological agriculture applications are unstable, hindering the formation of a standardized application system. Therefore, developing a green, safe, efficient, and stable pollinator insect protection technology has become a key breakthrough in balancing agricultural production and ecological protection.

[0005] Studies have shown that theanine, an amino acid naturally found in tea, has the effects of regulating neurotransmitters, relieving oxidative stress, and enhancing the resistance of organisms. Chinese Patent 202410428757.4 discloses an application of theanine, in which theanine, as the only active ingredient, can promote normal bumblebee nest building and egg laying, improve flight ability, and enhance olfactory learning and memory. Experiments have shown that theanine can effectively prevent damage caused by pesticides. It was tested by directly feeding the insects with syrup containing theanine and sugar solution containing imidacloprid and theanine over a 20-day experimental period. However, its widespread application in ecological agriculture has obvious shortcomings: (1) Theanine is metabolized quickly in insects (half-life < 4 hours), requiring frequent feeding to maintain an effective concentration, resulting in high field operation costs and poor compliance; (2) Protection strategies mainly focus on remedial measures after pesticide exposure ("post-poisoning repair") or theanine mixed with pesticides, with limited protective effects; (3) The protection efficiency of using theanine sugar solution alone has a bottleneck and is difficult to meet the protection needs in complex field environments. Currently, there are no pesticide protection products for pollinating insects based on non-toxic neuroprotective mechanisms that can be widely used in ecological agriculture. There is an urgent need to develop a green, efficient, and scalable pollinating insect protection solution. Summary of the Invention

[0006] The technical problem to be solved by the present invention is to provide a method for preparing theanine slow-release microcapsules, an application method, and a feed for pollinating insects to enhance their pesticide tolerance, so as to solve the problem of rapidly increasing the active ingredient of theanine in pollinating insects in a short period of time to achieve the required protective concentration before they are about to face the threat of pesticide spraying.

[0007] The technical solution of this invention to solve the above-mentioned technical problems is as follows: A method for preparing theanine sustained-release microcapsules to enhance pesticide tolerance in pollinating insects, comprising the following steps:

[0008] Step (1) Dissolve the pollinator insect protectant in deionized water and stir until completely dissolved to obtain an aqueous solution of the pollinator insect protectant. The active ingredient of the pollinator insect protectant includes theanine. Slowly add sodium alginate to deionized water and dissolve completely to obtain an aqueous solution of sodium alginate. Step (2) The pollinating insect protectant aqueous solution is slowly added dropwise to the sodium alginate aqueous solution while stirring to obtain theanine initial mixture; Step (3) The theanine initial mixture is added dropwise to the coagulation bath of calcium chloride solution. The droplets cross-link immediately after contacting calcium ions to form calcium alginate gel microspheres. Step (4) Filter out the calcium alginate gel microspheres and gently rinse with deionized water to remove excess calcium ions on the surface, thereby obtaining theanine sustained-release microcapsules.

[0009] The present invention provides a method for preparing theanine sustained-release microcapsules to enhance pesticide tolerance in pollinating insects, as described above. Further, it includes step (5) immersing the initial theanine sustained-release microcapsule sample in a chitosan acetate solution for electrostatic adsorption coating to form a polyelectrolyte composite membrane on the surface of the theanine sustained-release microcapsules. The microcapsules are then sieved, washed with water, and freeze-dried to obtain theanine sustained-release microcapsules coated with a chitosan layer. The present invention utilizes the electrostatic interaction between the amino groups of chitosan and the carboxyl groups of calcium alginate to form a polyelectrolyte composite membrane. The membrane formation principle is: calcium alginate (-COO-) + chitosan (-NH3+) → polyelectrolyte composite membrane.

[0010] The present invention provides a method for preparing theanine sustained-release microcapsules to enhance pesticide tolerance in pollinating insects. Further, the mass-volume concentration of the pollinating insect protectant aqueous solution is 5%-30%; the mass-volume concentration of the sodium alginate aqueous solution is 1%-3%; and the mass-volume concentration of the calcium chloride solution is 1%-2%.

[0011] The present invention discloses a method for preparing theanine sustained-release microcapsules to enhance pesticide tolerance in pollinating insects, as described above. Further, the degree of cross-linking of the sodium alginate is controlled by Ca... 2+ The concentration is 1-2% w / v, preferably Ca. 2+ The concentration is 1.5% w / v.

[0012] The present invention provides a method for preparing theanine sustained-release microcapsules to enhance pesticide tolerance in pollinating insects. Further, the chitosan acetate solution has a mass-volume concentration of 0.2-1% w / v, preferably 0.5% w / v; that is, 0.2-1g of chitosan is dissolved in 100mL of 1% glacial acetic acid solution, i.e., the concentration is 0.2-1% w / v; the chitosan acetate solution is stirred overnight, and the pH is adjusted to 5.5±0.1 with NaOH.

[0013] The present invention provides a method for preparing theanine sustained-release microcapsules to enhance pesticide tolerance in pollinating insects. Further, step (2) of slowly adding the capsules under stirring is described as adding the capsules slowly at a rate of 1-2 mL / min using an injection pump under gentle stirring conditions of 400-600 rpm.

[0014] The present invention provides a method for preparing theanine sustained-release microcapsules to enhance pesticide tolerance in pollinating insects. Further, in step (3), the coagulation bath is pre-cooled to 4°C; the addition of theanine initial mixture is done by adding the injection pump needle at a height of about 5-10 cm from the liquid surface, to ensure that the droplets form a spherical shape in the air before falling into the coagulation bath.

[0015] This invention discloses a method for preparing theanine sustained-release microcapsules to enhance pesticide tolerance in pollinating insects, as described above. Further, the pollinating insect protectant comprises 50-70% theanine, 10-30% rosmarinic acid, and 5-20% oregano oil by weight. This invention's combined protectant employs a multi-target synergistic endogenous insect protection system: theanine regulates neurotransmitters, rosmarinic acid modulates metabolic pathways, and oregano oil enhances pharmacokinetics. This system is designed based on precise regulation of insect physiological and biochemical pathways, aiming to overcome the bottleneck of limited protective efficiency due to the limited loading capacity of single components, and achieving "multi-dimensional, multi-level, and highly synergistic" protection for pollinating insects facing chemical pesticide stress.

[0016] Theanine's main target is the GABA receptor in the central nervous system, where it acts as a "sedative" and "antioxidant." By mimicking GABA, it pre-stabilizes the neuronal cell membrane potential, increases the nerve excitation threshold, and directly antagonizes the excessive nerve excitation caused by neonicotinoids and pyrethroid pesticides. At the same time, its strong free radical scavenging ability provides the first line of defense against pesticide-induced oxidative stress. Rosmarinic acid, as a metabolic detoxification regulator, primarily targets phase I metabolic enzymes such as cytochrome P450 enzymes (CYP450s), acting as a "metabolic switch." Many pesticides (such as organophosphates and some neonicotinoids) have low toxicity themselves, but the products of their activation by CYP450s in insects are the real sources of toxicity. Rosmarinic acid, by reversibly inhibiting the activity of key CYP450 subtypes, slows down the activation and metabolism of pesticides, reduces the instantaneous concentration of highly toxic intermediates, and buys valuable time for the body's detoxification system (such as the II-binding reaction), thus transforming "acute high-toxicity shock" into "chronic low-toxicity load." Oregano essential oil, as a bioavailability enhancer, primarily targets the intestinal epithelial cell membrane and intercellular tight junctions of insects, acting as a "Trojan horse" and "penetration promoter." Its main active components, carvacrol and thymol, are natural membrane-active substances. They can temporarily and reversibly alter the fluidity and permeability of the lipid bilayer of epithelial cell membranes and may slightly perturb intercellular junctions, thereby significantly increasing the efficiency of theanine and rosmarinic acid in penetrating the intestinal barrier and entering the hemolymph. This solves the key bottleneck of low bioavailability of the protectant itself, ensuring that sufficient active ingredients can exert their effects at the target site.

[0017] The three work synergistically to form a positive reinforcement loop: oregano essential oil enhances the intestinal absorption of theanine and rosmarinic acid → the theanine in the hemolymph reaches an effective concentration, stabilizing the nervous system and initiating antioxidant defense in advance → at the same time, sufficient rosmarinic acid inhibits pesticide activation metabolism and reduces the formation of toxic products → the reduction of toxic products, in turn, reduces the stress on the nervous and oxidative systems, making the protective effect of theanine more significant → the entire system achieves comprehensive interception of pesticide toxicity pathways at a lower individual dose, including "upstream inhibition" (metabolic inhibition), "midstream buffering" (neuroprotection), and "downstream clearance" (antioxidant).

[0018] Since oregano essential oil is the oil phase, and theanine and rosmarinic acid are water-soluble, an emulsion concentrate can be prepared. Process flow: a. Dissolve theanine and rosmarinic acid in an appropriate amount of deionized water, heating to 60℃ and stirring until completely dissolved, as the aqueous phase. b. Mix oregano essential oil with a safe, biodegradable emulsifier (such as polyoxyethylene castor oil derivative, 5% dosage), as the oil phase; c. Under high-speed shearing (8000 rpm), slowly add the oil phase to the aqueous phase, continuing shearing for 15 minutes to form a primary emulsion; d. Using a high-pressure homogenizer, the mixture can be circulated three times at 50 MPa pressure to obtain a stable nanoemulsion with a particle size less than 200 nm. The primary emulsion or the stable nanoemulsion can be slowly added dropwise to a sodium alginate aqueous solution under stirring to obtain a preliminary theanine mixture.

[0019] The present invention provides a method for preparing theanine sustained-release microcapsules to enhance pesticide tolerance in pollinating insects. Further, the pollinating insect protectant comprises the following raw materials by weight percentage: 65% theanine, 20% rosmarinic acid, and 15% oregano essential oil.

[0020] This invention also provides a method for preparing theanine slow-release microcapsules to enhance pesticide tolerance in pollinating insects, and a method for applying theanine slow-release microcapsules. The theanine slow-release microcapsule aqueous solution is prepared 24 hours before the planned pesticide spraying. This solution can be applied using an agricultural drone, spraying evenly at low altitude above crops or in areas where pollinating insects are active; or a wetting device containing theanine slow-release microcapsule aqueous solution can be placed near nectar-producing plants, allowing insects to naturally ingest the solution while collecting nectar. The concentration of theanine slow-release microcapsule aqueous solution is controlled at 0.5-2.0 mg / L.

[0021] This invention also provides a pollinating insect bait, including bee-type bait, bumblebee-type bait, and / or hoverfly-type bait. The bee-type bait comprises the theanine sustained-release microcapsules prepared by the method for improving pesticide tolerance of pollinating insects as described in claims 1 to 7, the theanine sustained-release microcapsules, sucrose, and pollen extract, which are mixed in proportion and dissolved in a sucrose aqueous solution with a concentration of 0.5%-1.5% to prepare a paste or liquid. The bumblebee-type bait comprises the theanine slow-release microcapsules prepared by the method described in 1 to 7 for improving the pesticide tolerance of pollinating insects, honey, and plant protein powder, mixed in proportion, dissolved in a sucrose aqueous solution with a concentration of 0.5%-1.5%, and placed in bumblebee hives or special feeding points in facility agriculture. The hoverfly bait includes the theanine slow-release microcapsules prepared by the method described in 1 to 7 for improving the pesticide tolerance of pollinating insects, the theanine slow-release microcapsules, yeast extract, and fructose, which are mixed in proportion and dissolved in a sucrose aqueous solution with a concentration of 0.5%-1.5%. The solution is then sprayed on the underside of plant leaves where adult hoverflies tend to gather or placed in a feeding tray.

[0022] In the pollinating insect bait described above, the mass ratio of theanine slow-release microcapsules to pollen extract is 6:4; the mass ratio of theanine slow-release microcapsules, honey, and plant protein powder is 5:3:2; or the mass ratio of theanine slow-release microcapsules, yeast extract, and fructose is 4:4:2.

[0023] The present invention has the following beneficial effects: This invention involves mixing sodium alginate solution with an aqueous solution of a pollinating insect protectant, then adding the mixture to a coagulation bath of calcium chloride solution to form calcium alginate gel microspheres coated with a high concentration of theanine. The surface of the calcium alginate gel microspheres is then coated with a chitosan layer to form chitosan-sodium alginate bilayer theanine sustained-release microcapsules.

[0024] The theanine sustained-release microcapsules of this invention consist of an inner sodium alginate sustained-release layer with release rate controlled by ionic cross-linking, and an outer pH-responsive chitosan layer that dissolves rapidly in a weakly alkaline environment (pH≈8). By precisely controlling the degree of sodium alginate cross-linking and the concentration of the chitosan acetate solution, under simulated midgut alkaline conditions, the cumulative release of theanine from these microcapsules can reach 60% in 12 hours and 90% in 24 hours, achieving continuous and stable release of theanine from pollinating insects for 12-24 hours. HPLC analysis shows that the microcapsule encapsulation efficiency is consistently ≥85%, with a drug loading as high as 29.8%.

[0025] The pollinating insect protectant of this invention comprises 50-70% theanine, 10-30% rosmarinic acid, and 5-20% oregano oil by weight. Rosmarinic acid (derived from plants such as rosemary) effectively inhibits the activity of key pesticide-metabolizing enzymes (such as cytochrome P450 enzymes) in pollinating insects, slowing down the activation and metabolism of pesticides and reducing the accumulation of toxic metabolites. Oregano oil (mainly containing carvacrol and thymol) has a strong permeation-enhancing effect, significantly improving the efficiency of theanine and rosmarinic acid in penetrating the insect intestinal epithelial cell barrier and increasing bioavailability. This invention is the first to propose and verify a three-in-one synergistic theoretical model of insect chemical protection: "neuroprotection-metabolic inhibition-permeation enhancement."

[0026] This invention forms a "neuroprotective barrier" to block the neurotoxicity of pesticides by administering theanine slow-release microcapsules 24 hours before planned pesticide spraying. Detailed Implementation

[0027] The principles and features of the present invention are described in conjunction with the following embodiments. The examples are only used to explain the present invention and are not intended to limit the scope of the present invention.

[0028] Example 1 Weigh 10.0g of pollinating insect protectant (theanine), dissolve it in 80mL of 60℃ deionized water and stir magnetically (500rpm, 20min) until completely dissolved; weigh 2.0g of sodium alginate, slowly add it to 100mL of 50℃ deionized water and disperse it at 800rpm for 10min, let it stand to remove bubbles, and cool to room temperature; While gently stirring at 400-600 rpm, the theanine core material solution was slowly added dropwise to the sodium alginate solution using a syringe pump at a rate of 1-2 mL / min. After the addition was complete, stirring was continued for 30 minutes to ensure that the active ingredients were evenly dispersed in the sodium alginate solution to form a homogeneous initial mixture. Prepare a 1.5% (w / v) calcium chloride solution as a coagulation bath and pre-cool it to 4°C. Using a syringe or micro-injection pump, add the initial mixture to the coagulation bath at a constant drip rate (e.g., 20-40 drops / minute).

[0029] The needle is positioned approximately 5-10 cm above the liquid surface to ensure that the droplets form spherical shapes in the air before falling into the coagulation bath. Upon contact with calcium ions, the droplets immediately undergo cross-linking to form calcium alginate gel microspheres. The calcium alginate microspheres are then filtered out and gently rinsed twice with deionized water to remove excess calcium ions from the surface, yielding theanine sustained-release microcapsules.

[0030] Example 2 Dissolve 0.5g of chitosan in 100mL of 1% glacial acetic acid solution and stir overnight. Adjust the pH to 5.5±0.1 with NaOH to obtain a chitosan acetic acid solution.

[0031] The theanine sustained-release microcapsules prepared in Example 1 were immersed in a chitosan acetate solution and electrostatically coated by slow shaking at 50 rpm at room temperature for 20 minutes. After coating, the microcapsules were collected with a sieve and rinsed with plenty of deionized water until the rinsing solution was neutral. The wet microcapsules were placed in a freeze-drying tray and pre-frozen in an ultra-low temperature freezer at -80°C for at least 4 hours. The pre-frozen samples were transferred to a freeze dryer and dried for 24-48 hours under conditions of condenser temperature below -50°C and vacuum degree <10 Pa until completely dried. The microcapsules were collected through a 100-mesh sieve to obtain free-flowing white to off-white powdery double-layer sustained-release microcapsules, namely the theanine sustained-release microcapsules coated with a chitosan layer. The outer layer of the theanine sustained-release microcapsules coated with chitosan is pH-responsive chitosan (which dissolves rapidly in the weakly alkaline environment of the bee midgut at pH≈8), and the inner layer is a sodium alginate sustained-release layer. The release rate is controlled by ionic cross-linking.

[0032] Example 3 The theanine, rosmarinic acid, and oregano essential oil were mixed in a mass ratio of 60% to 15% to prepare a pollinating insect protectant. The theanine sustained-release microcapsules coated with chitosan were obtained according to Example 2.

[0033] Theanine sustained-release microcapsules coated with chitosan layers trigger the dissolution of the outer layer in the alkaline environment of the insect midgut, and the inner sodium alginate layer achieves gradient release of theanine through dissolution and diffusion (60% release in 12 hours and 90% release in 24 hours).

[0034] This invention mainly studies: 1. the encapsulation efficiency, drug loading, and microcapsule particle size of theanine sustained-release microcapsules; 2. the sustained-release characteristics under weakly alkaline conditions; and 3. the metabolic kinetics in bee colonies.

[0035] (I) Test results of theanine sustained-release microcapsules in Examples 1 to 3 1. Performance test results of theanine sustained-release microcapsules in Example 1: Encapsulation efficiency 86.5±1.5%; drug loading 20.2±0.3%; microcapsule particle size 85±15μm (≤100μm).

[0036] 2. Performance test results of theanine sustained-release microcapsules in Example 2: Encapsulation efficiency 87.3±1.5%; drug loading 19.2±0.3%; microcapsule particle size 80±15μm (≤100μm), suitable for insect ingestion, ensuring efficient delivery.

[0037] 3. Performance test results of theanine sustained-release microcapsules in Example 3: Encapsulation efficiency 86.5±1.5%, drug loading 28.7±1.2%, microcapsule particle size 45±5μm.

[0038] Theanine sustained-release microcapsules are all spherical with a porous surface structure and a pore size of 0.5-2μm, which is conducive to sustained release. The encapsulation efficiency reflects the efficiency of the microcapsules in encapsulating theanine, the drug loading reflects the content of effective active ingredients, and the particle size affects the insect's feeding adaptation. Example 3 showed a significant increase in drug loading, which was attributed to the combined use of rosmarinic acid and oregano essential oil promoting multiphase synergistic precipitation and core densification. 1. Oregano essential oil and rosmarinic acid can form hydrogen bonds and hydrophobic interactions with theanine, inducing the three to co-precipitate in the early stage of emulsification, forming a high-density, multiphase composite core structure, which significantly increases the drug loading compared to Example 2.

[0039] 2. Adjusting the interfacial tension between oil and water to stabilize the droplet morphology: Oregano essential oil in the ternary components has natural surface activity, and rosmarinic acid also has a certain polarity. The two work together to reduce the interfacial tension of the system, making the initial droplets smaller and more evenly distributed, and finally forming microcapsules with a particle size of only 45±5μm, which is much smaller than 80±15μm in Example 2.

[0040] 3. Enhances continuous phase viscosity and inhibits microcapsule aggregation: The rigid molecular structure of rosmarinic acid can effectively increase the viscosity of the aqueous phase, slow down the collision and fusion rate of emulsion droplets, maintain the independence of microcapsules during the curing process, and ensure particle size uniformity.

[0041] 4. Optimize wall material deposition kinetics and coating density: The ternary system may locally regulate the pH or charge distribution of the microenvironment, promoting the more uniform deposition of positively charged chitosan on the surface of sodium alginate microspheres, forming a denser double-layer coating structure. Although the encapsulation efficiency decreased slightly (87.3% → 86.5%), the structural stability was not affected.

[0042] 5. Improve drug distribution and encapsulation pathways: The fat-soluble oregano essential oil promotes the enrichment of theanine at the oil-water interface, making it easier for the emerging sodium alginate gel network to capture, reducing drug loss and improving encapsulation efficiency.

[0043] (II) In vitro release kinetics tests in Examples 1 to 3 (simulating the midgut environment of bees) Objective: To verify the sustained-release properties of microcapsules under weakly alkaline conditions.

[0044] Materials and Methods: The microcapsules of Examples 1 to 3 were placed in a pH 8.0 phosphate buffer (simulating the midgut of a bee).

[0045] The sample was oscillated at a constant temperature of 37℃ and samples were taken at 1h, 2h, 4h, 6h, 8h, 12h, and 24h.

[0046] The concentration of theanine in the released solution was determined by HPLC, and the cumulative release rate was calculated. The results are shown in Table 1.

[0047] Table 1. Cumulative release curves for Examples 1 to 3

[0048] The fitted equation, the Higuchi model (R²=0.992), confirms the diffusion-controlled release mechanism.

[0049] The experimental design and operation specifications for bee colonies in this invention are as follows: sealed and clean beehive --> bee colony preparation --> experimental grouping --> exposure treatment --> behavioral observation --> data collection --> statistical analysis.

[0050] 1. Bee colony preparation: Bee source: Italian worker bees, 10-15 days old (main foraging bees) Standardized growth conditions for bees are shown in Table 2.

[0051] Table 2. Standardized growth conditions for bees

[0052] Selection and elimination criteria: body length <11mm or >13mm (developmental abnormality); wing edge defects (impaired flight ability); no response to stimuli (touch the antennae lightly with tweezers).

[0053] Pretreatment: Isolation rearing: Adapted to the environment in a dark box (25±1℃, 60%RH) for 24 hours; Selection criteria: Body length 12±1mm, intact wings, and no parasites (microscopic examination).

[0054] 2. Experimental Group Design The parameter settings for the experimental design group are shown in Table 3.

[0055] Table 3. Parameters of the Experimental Design Group

[0056] Exposure system setup: Customized feeding tower with 24-hole feeding unit --> metering pump --> UV protection shield Pesticide stock solution --> sucrose solution dilution --> thiamethoxam final concentration 0.1 mg / L --> exposure device Key operating parameters for exposure are shown in Table 4; thiamethoxam was administered via microcapsules of Examples 1-3 24 hours prior to exposure.

[0057] Table 4. Exposed Key Operating Parameters

[0058] Note: Each group is set up with 3 replicates (i.e., 300 bees per group, divided into 3 cages × 100 bees / cage) to avoid cross-contamination between cages.

[0059] 3. Core Indicator Collection Process 1) Survival rate monitoring Day 0 → Initialization: Mark the bee body (acrylic paint number); Daily 8:00 → Death Count: Remove dead bees and record their numbers; Daily at 17:00 → Replenish feed; Continue → Data recording: until day 7.

[0060] Key procedures: Near death: lying on its side, foot twitching, no response to light touch with forceps; Determination of dead bees: no respiratory movements (tracheal constriction observed under stereoscopic observation, trachea observation > 1 min under stereoscopic observation), no response to light touch.

[0061] 2) Flower Visit Rate Observation Flower preparation --> Alfalfa --> Soaking in 10% nectar extract; Test area layout --> 1m diameter circular area --> 20 simulated flowers evenly distributed; Observation method --> Release a single cage of bees --> Video analysis for 10 minutes.

[0062] 3) Data Collection Flower visiting definition: A bee lands on a flower and extends its proboscis for ≥3 seconds; Calculation formula: Flower visitation rate = Number of successful flower visits / Total number of released bees × 100%; Data collection and quality control: Daily operating procedures are shown in Table 5; survival rate of each group is dynamically recorded.

[0063] Table 5. Daily Operating Procedures (SOPs)

[0064] (III) Metabolomics experiments in Examples 4 to 6 1. Following the bee colony experimental design and operation specifications of this invention, the active ingredient of theanine was 1.5 mg / L, and pharmacokinetic and physiological damage indicators were detected.

[0065] 2. Detection Indicators and Methods 2.1 Pharmacokinetic parameters: HPLC determination of the active ingredient concentration of hemolymph theanine sustained-release microcapsules (time points: 0h, 4h, 8h, 12h, 24h) 2.2 Physiological damage: Acetylcholinesterase (AChE) activity assay (Ellman method) Liquid nitrogen quick-freezing of bees --> brain tissue homogenization --> AChE activity detection --> nAChR expression level.

[0066] 3. The test results are shown in Table 6.

[0067] Table 6. Physiological effects of Examples 4 to 6 on bees

[0068] As shown in Table 6, the experimental data revealed that administering theanine slow-release microcapsules (Examples 1 to 3 of this invention) 24 hours before planned pesticide spraying significantly improved the survival rate of bees 48 hours after pesticide exposure, significantly reduced the inhibition rate of acetylcholinesterase, and significantly improved bee pollination efficiency (p < 0.001). This indicates that administering theanine slow-release microcapsules 24 hours before planned pesticide spraying can effectively form a "neuroprotective barrier" to block pesticide neurotoxicity. The use of this invention before pesticide spraying effectively improves pesticide tolerance in pollinating insects.

Claims

1. A method for preparing theanine sustained-release microcapsules to enhance pesticide tolerance in pollinating insects, characterized in that, Includes the following steps: Step (1) Dissolve the pollinator insect protectant in deionized water and stir until completely dissolved to obtain an aqueous solution of the pollinator insect protectant. The active ingredient of the pollinator insect protectant includes theanine. Slowly add sodium alginate to deionized water and dissolve completely to obtain an aqueous solution of sodium alginate. Step (2) The pollinating insect protectant aqueous solution is slowly added dropwise to the sodium alginate aqueous solution while stirring to obtain theanine initial mixture; Step (3) The theanine initial mixture is added dropwise to the coagulation bath of calcium chloride solution. The droplets cross-link immediately after contacting calcium ions to form calcium alginate gel microspheres. Step (4) Filter out the calcium alginate gel microspheres and gently rinse with deionized water to remove excess calcium ions on the surface, thereby obtaining theanine sustained-release microcapsules.

2. The method for preparing theanine sustained-release microcapsules to enhance pesticide tolerance in pollinating insects according to claim 1, characterized in that, The method also includes step (5) immersing the theanine sustained-release microcapsules in a chitosan acetate solution for electrostatic adsorption coating, so as to form a polyelectrolyte composite film on the surface of the theanine sustained-release microcapsules, and then sieving, washing and freeze-drying to obtain theanine sustained-release microcapsules coated with chitosan.

3. The method for preparing theanine sustained-release microcapsules to enhance pesticide tolerance in pollinating insects according to claim 1, characterized in that, The pollinating insect protectant aqueous solution has a mass-volume concentration of 5%-30%; the sodium alginate aqueous solution has a mass-volume concentration of 1%-3%; the calcium chloride solution has a mass-volume concentration of 1%-2%; and the chitosan acetate solution is a chitosan acetate solution with a mass-volume concentration of 0.5g chitosan dissolved in 100mL of 1% glacial acetic acid solution. The chitosan acetate solution was stirred overnight, and the pH was adjusted to 5.5 ± 0.1 with NaOH.

4. The method for preparing theanine sustained-release microcapsules to enhance pesticide tolerance in pollinating insects according to claim 1, characterized in that, Step (2) refers to the slow addition under stirring conditions, which means adding slowly at a rate of 1-2 mL / min using a syringe pump under gentle stirring conditions of 400-600 rpm.

5. The method for preparing theanine sustained-release microcapsules to enhance pesticide tolerance in pollinating insects according to claim 4, characterized in that, In step (3), the coagulation bath is pre-cooled to 4°C; the addition of the theanine initial mixture is done by adding the solution drop by drop with the needle of the injection pump about 5-10 cm above the liquid surface, so as to ensure that the droplets form a spherical shape in the air before falling into the coagulation bath.

6. The method for preparing theanine sustained-release microcapsules for enhancing pesticide tolerance in pollinating insects according to any one of claims 1 to 5, characterized in that, The pollinating insect protectant comprises 50-70% theanine, 10-30% rosmarinic acid, and 5-20% oregano essential oil by weight.

7. The method for preparing theanine sustained-release microcapsules to enhance pesticide tolerance in pollinating insects according to claim 6, characterized in that, The pollinating insect protectant comprises the following ingredients by weight percentage: 65% theanine, 20% rosmarinic acid, and 15% oregano essential oil.

8. A method for preparing theanine sustained-release microcapsules to enhance pesticide tolerance in pollinating insects as described in claims 1 to 7, and a method for applying theanine sustained-release microcapsules, characterized in that, 24 hours before the planned pesticide spraying, prepare an aqueous solution of theanine slow-release microcapsules and spray it evenly at low altitude above the crops or in the area where pollinating insects are active; or set up a wetting device containing theanine slow-release microcapsule aqueous solution near nectar-producing plants so that insects can naturally ingest it when collecting nectar; the concentration of theanine slow-release microcapsule aqueous solution should be controlled at 0.5-2.0 mg / L.

9. A pollinating insect bait, characterized in that, This includes bee-type bait, bumblebee-type bait, and / or hoverfly-type bait. The bee-type bait comprises the theanine sustained-release microcapsules prepared by the method for improving pesticide tolerance of pollinating insects as described in claims 1 to 7, the theanine sustained-release microcapsules, sucrose, and pollen extract, which are mixed in proportion and dissolved in a sucrose aqueous solution with a concentration of 0.5%-1.5% to prepare a paste or liquid. The bumblebee-type bait comprises the theanine slow-release microcapsules prepared by the method described in 1 to 7 for improving the pesticide tolerance of pollinating insects, honey, and plant protein powder, mixed in proportion, dissolved in a sucrose aqueous solution with a concentration of 0.5%-1.5%, and placed in bumblebee hives or special feeding points in facility agriculture. The hoverfly bait includes the theanine slow-release microcapsules prepared by the method described in 1 to 7 for improving the pesticide tolerance of pollinating insects, the theanine slow-release microcapsules, yeast extract, and fructose, which are mixed in proportion and dissolved in a sucrose aqueous solution with a concentration of 0.5%-1.5%. The solution is then sprayed on the underside of plant leaves where adult hoverflies tend to gather or placed in a feeding tray.

10. The pollinating insect bait according to claim 1, characterized in that, The mass ratio of the theanine sustained-release microcapsules to pollen extract is 6:4; the mass ratio of theanine sustained-release microcapsules to honey and plant protein powder is 5:3:2; or the mass ratio of theanine sustained-release microcapsules to yeast extract and fructose is 4:4:2.

Images