Pesticide residue degradation composite enzyme based on Delftia delfinii and preparation method and equipment of pesticide residue degradation composite enzyme

Abstract

The invention relates to the field of biodegradation, in particular to a pesticide residue degradation composite enzyme based on delftia and a preparation method and equipment thereof.The pesticide residue degradation composite enzyme comprises a smashing cylinder, the bottom of the smashing cylinder penetrates through and is fixedly connected with a feeding cylinder, the bottom of the feeding cylinder is connected with a screening assembly, and supporting blocks are fixedly connected to the four corners of the bottom of the smashing cylinder; a first motor is fixedly connected to the top of the smashing barrel, the output end of the first motor penetrates through the smashing barrel to be fixedly connected with a smashing rod, smashing blades are fixedly connected to the outer portion of the smashing rod, a constant-temperature shaking table is arranged on the right side of the smashing barrel, and a fermentation tank is arranged on the right side of the constant-temperature shaking table; the top of the fermentation tank is fixedly connected with a second motor. The degradation rates of the enzyme for organophosphorus pesticides such as phoxim, dichlorvos and dimethoate with the concentration of 200 mg / L within 48 hours reach 93.8%, 92.5% and 91.2% respectively and are far higher than those of mainstream biodegradable products (72%-80%) in the market, and the degradation period is only 5 days and is shortened by 83% compared with that of a traditional enzyme.

Description

Technical Field

[0001] This invention relates to the field of biodegradation technology, specifically to a pesticide residue degradation complex enzyme based on Delftobacterium, and its preparation method and equipment. Background Technology

[0002] With the development of intensive and large-scale agriculture, organophosphorus pesticides are widely used in the cultivation of fruits and vegetables such as citrus, broccoli, and tomatoes due to their high insecticidal efficiency and rapid action. However, organophosphorus pesticides are insoluble in water, difficult to degrade, and have strong penetrability. Long-term residues in fruits and vegetables not only lead to frequent food safety problems, but also accumulate through the food chain, damaging the human nervous system and inducing cancer, seriously threatening food safety and public health.

[0003] my country has strict standards for pesticide residue limits, stipulating that the detection limit for organophosphates in vegetables is 0.03 mg / kg, for stem and root vegetables it is 0.05 mg / kg, and for leeks it is specifically labeled as 0.1 mg / kg. However, existing pesticide residue degradation technologies are insufficient to meet practical needs, and there are three major pain points: Poor environmental performance: Chemical degradation agents contain strong oxidizing components. Long-term use leads to soil acidification, microbial community imbalance, secondary residues, and damage to the agricultural ecological environment. Low efficiency and long cycle: Conventional biodegradable products have a degradation rate of only 72%-80% for high concentrations (200mg / L) of organophosphorus compounds, and the fermentation cycle is as long as 10-30 days, which cannot meet the needs of rapid circulation of fruits and vegetables. High cost: The price of mainstream biodegradable agents on the market is as high as 37,000 yuan per ton, and the cost of pesticide residue treatment per mu for farmers exceeds 500 yuan, resulting in great economic pressure and making it difficult to promote on a large scale.

[0004] Microbial degradation has become a core direction for pesticide residue control due to its environmental friendliness and thoroughness. However, existing strains generally suffer from low degradation efficiency, weak environmental adaptability (such as narrow pH tolerance range and high temperature sensitivity), and dependence on high-cost synthetic culture media. Therefore, developing a high-efficiency, low-cost, environmentally adaptable, and waste-recycling-enabled pesticide residue degradation compound enzyme is of great practical significance for ensuring food safety and promoting green agriculture. Summary of the Invention

[0005] To address the shortcomings of existing technologies, this invention provides a pesticide residue degradation compound enzyme based on Delftobacterium, along with its preparation method and equipment, which solves the problems of poor environmental performance, low efficiency, high cost, and weak adaptability of existing pesticide residue degradation products.

[0006] To achieve the above objectives, the present invention provides the following technical solution: a pesticide residue degradation compound enzyme based on Delftobacterium and its preparation method and equipment, comprising a crushing cylinder, a feeding cylinder connected through and fixedly connected to the bottom of the crushing cylinder, a screening component connected to the bottom of the feeding cylinder, support blocks fixedly connected to the four corners of the bottom of the crushing cylinder, and the bottom of the support blocks connected to the screening component, a first motor fixedly connected to the top of the crushing cylinder, a crushing rod fixedly connected to the output end of the first motor through the crushing cylinder, crushing blades fixedly connected to the outside of the crushing rod, a constant temperature shaking table arranged on the right side of the crushing cylinder, and a generator arranged on the right side of the constant temperature shaking table. The fermentation tank has a second motor fixedly connected to its top. A stirring rod is fixedly connected to the output end of the second motor, passing through the fermentation tank. Stirring blades are fixedly connected to the outer ring of the stirring rod. A weighing assembly is installed on the right side of the fermentation tank. An oxygen cylinder is located on the right side of the fermentation tank, with a gas pipe fixedly connected to its output end. A temperature sensor is fixedly connected to the inner wall of the fermentation tank. A heating wire is fixedly connected to the bottom of the fermentation tank. A dissolved oxygen electrode is fixedly connected to the inner wall of the fermentation tank. A controller is fixedly connected to the front end of the fermentation tank. A vacuum concentrator is located on the right side of the fermentation tank, and an encapsulation machine is located to the right of the vacuum concentrator.

[0007] Preferably, the screening assembly includes a screening box, the top of which is connected to and fixedly connected to the feed cylinder, support plates are fixedly connected to both sides inside the screening box, springs are fixedly connected to both sides of the top of the support plates, a mounting frame is fixedly connected to the top of the springs, a screen plate is fixedly connected inside the mounting frame, a vibration motor is fixedly connected to the bottom of the mounting frame, and a discharge port is opened on the left side of the screening box.

[0008] Preferably, the weighing component includes a mounting plate, which is fixedly connected to the outer wall of the fermenter. A weighing sensor is fixedly connected to the top of the mounting plate, and a weighing pan is fixedly connected to the top of the weighing sensor.

[0009] Preferably, the end of the trachea passes through and is fixedly connected to the fermenter, and an electromagnetic valve is installed on the outside of the trachea.

[0010] Preferably, the fermenter is fixedly connected to four corners at the bottom and to a feed pipe at the top.

[0011] Preferably, a collection box is provided on the left side of the screening box, and the collection box is located below the discharge port.

[0012] Preferably, a through groove is provided on the right side of the screening box, and a receiving box is slidably connected in the through groove.

[0013] Preferably, a pesticide residue degradation compound enzyme based on Dellfordia sp. comprises the following raw materials: 30%-40% Scutellaria baicalensis residue, 5%-8% hydrolyzed sucrose, 10%-15% Dellfordia sp. D39 inoculum, 0.5%-1% metal ion compound solution, and 36%-54.5% sterile water.

[0014] Preferably, the pesticide residue degradation complex enzyme based on Delftobacterium and its preparation method are characterized by the following steps: S1. Raw material pretreatment: The Scutellaria baicalensis residue was ultra-finely pulverized to 200 mesh at a low temperature of -5℃ to 0℃ (to increase the specific surface area and break the cell wall fiber structure). 0.5% of the raw material weight of the compound enzyme (cellulase: pectinase = 1:1) was added, and the mixture was enzymatically hydrolyzed in a constant temperature water bath at 45℃ for 5-6 hours to promote the dissolution of active ingredients and improve the subsequent microbial utilization efficiency.

[0015] S2, Seed culture: Delfordia D39 was inoculated into LB medium (10 g / L peptone, 5 g / L yeast extract, 10 g / L NaCl, pH 7.0) and incubated at 30-32°C on a shaker at 150 rpm for 48 hours, with hydrolyzed sucrose (5% concentration) added every 12 hours to ensure a viable count of 10-1. 8 S3, raw material pretreatment, with a concentration of CFU / mL or higher.

[0016] S3, Three-stage Precision Fermentation: Enzyme activity peak induction stage: The pretreated Scutellaria baicalensis residue and seed liquid were mixed at a weight ratio of 3:1, transferred to a fermenter, and the temperature was controlled at 35℃ and dissolved oxygen at 30% or higher. 200 mg / L phoxim was added as a substrate to induce the expression of degradation genes and cultured for 24 hours. Synergistic fermentation stage: Add metal ion complex solution to the fermenter, maintain temperature at 35℃ and dissolved oxygen at 25%-30%, and continue fermentation for 48 hours. During this period, sample and test enzyme activity every 24 hours to ensure that the activity of organophosphorus hydrolase is stable above 120U / mL. Post-processing stage: Low-temperature vacuum concentration at below 40℃ (concentrated to 1 / 3 of the original volume), adding Scutellaria baicalensis polysaccharide (concentration 2%) as wall material for microencapsulation, to produce liquid enzyme, extending the shelf life to more than 12 months.

[0017] Working Principle: First, the Scutellaria baicalensis residue is fed into the grinding cylinder through the top feeding cylinder. Then, the grinding cylinder is placed inside a laboratory chiller (not shown in the diagram) to maintain a low-temperature environment and prevent the degradation of heat-sensitive active ingredients in the residue. The first motor at the top is started, driving the grinding rod and externally fixed grinding blades to rotate at high speed. The grinding blades, through shearing action, pulverize the Scutellaria baicalensis residue into 200-mesh ultrafine particles, simultaneously breaking down the cellulose skeleton of the plant cell walls, making it easier to release intracellular active substances. The pulverized residue particles enter the screening assembly below through the feeding cylinder 16 at the bottom of the grinding cylinder 11. The vibrating motor at the bottom of the mounting frame is started. The high-frequency vibration generated by the vibrating motor causes the screen plate to shake up and down. Residue particles that meet the 200-mesh requirement fall through the screen plate into the receiving box below. Coarse particles that do not meet the standard are discharged from the discharge port on the left side of the inclined screen plate to the collection box, and can be re-injected into the grinding cylinder for secondary pulverization, ensuring that all pre-treated residue particles meet the 200-mesh requirement. The 200-mesh sieving residue of Scutellaria baicalensis was transferred to an external constant temperature water bath (not shown in the figure). A compound enzyme (cellulase: pectinase = 1:1) was added at 0.5% of the raw material weight, and the mixture was enzymatically hydrolyzed in a 45℃ constant temperature water bath for 5-6 hours. During this process, cellulase hydrolyzes the cellulose chains in the cell walls of the residue, and pectinase decomposes the pectin binding substances between cells. Together, they break down the dense structure of the residue, increasing the release rate of active ingredients by more than 30%. Simultaneously, large polysaccharides are broken down into small oligosaccharides, providing an easily available carbon source for the subsequent metabolism of Delfordia D39. The LB medium (not shown in the figure) was prepared according to the formula (10g / L peptone, 5g / L yeast extract, 10g / L NaCl). After adjusting the pH to 7.0, Delfordia D39 was inoculated into the medium and dispensed into Erlenmeyer flasks (the flask openings were sealed with breathable membranes to ensure dissolved oxygen). The Erlenmeyer flasks were then placed in the shaking tray of a constant temperature shaker. Turn on the thermostatic shaker and set the temperature to 30℃-32℃ (the optimal growth temperature for *Delfordia* D39). The shaking rate should be 150 rpm. This 150 rpm shaking rate creates a continuous vortex motion in the culture medium within the Erlenmeyer flask. This ensures even distribution of nutrients (peptone, yeast extract) and prevents localized nutrient depletion. Furthermore, the agitation increases the surface area of ​​the medium in contact with air, raising the dissolved oxygen concentration (3-5 times higher than in static culture), thus meeting the aerobic metabolic needs of the strain. During cultivation, open the thermostatic shaker door every 12 hours and add 5% hydrolyzed sucrose (as a readily available carbon source) to the Erlenmeyer flask to prevent the strain from entering a death phase due to insufficient carbon source. After 48 hours of cultivation, the viable count of *Delfordia* D39 in the seed culture should stably reach 10⁻⁶. 8A concentration of CFU / mL or higher ensures sufficient high-activity microbial inoculum for subsequent fermentation. Pretreated Scutellaria baicalensis residue and seed culture are added to the fermenter at a weight ratio of 3:1 via the feed pipe at the top. The seed culture can be directly transferred from the constant-temperature shaker, while the Scutellaria baicalensis residue is added quantitatively through the receiving box. For precise control of the weight ratio, a weighing assembly on the right side of the fermenter can be used: the mounting plate is fixed to the outer wall of the fermenter, and the top weighing sensor works with the weighing pan. The Scutellaria baicalensis residue and seed culture are weighed separately on the weighing pan to ensure an accurate 3:1 weight ratio. The second motor is then started, driving the stirring rod and outer ring blades to rotate (initial stirring speed set to 200-250 r / min) to ensure uniform mixing of the materials. The controller sets the target temperature to 35℃. A temperature sensor monitors the tank temperature in real time. If the temperature drops below 35℃, the controller automatically activates the heating wire at the bottom of the tank until the temperature rises back to 35℃. Simultaneously, the oxygen cylinder is turned on, and sterile oxygen is introduced into the tank through a tubing. An external solenoid valve regulates the oxygen flow rate. A dissolved oxygen electrode monitors the dissolved oxygen level in the tank in real time, and the controller adjusts the solenoid valve opening based on the dissolved oxygen data to ensure the dissolved oxygen level remains stable above 30%. 200 mg / L of phoxim is added to the tank through the feed pipe. Phoxim acts as an inducer, binding to regulatory proteins in *Delfordia diffusa* D39, relieving inhibition of the organophosphorus hydrolase gene, activating gene transcription and translation, and promoting the strain to synthesize a large amount of organophosphorus hydrolase. Continuous culture for 24 hours completes the peak enzyme activity induction. A metal ion complex solution is added to the fermenter through the feed pipe. Metal ions act as coenzymes for organophosphorus hydrolases, increasing enzyme catalytic efficiency by 20%-25%. Simultaneously, the stirring rate was reduced to 150-200 r / min via the controller (to avoid excessive shear force damaging the strain), and the dissolved oxygen level was adjusted to 25%-30% (slightly lower than the induction phase to balance dissolved oxygen and substrate consumption), while the temperature was maintained at 35℃. During the cultivation process, organophosphorus hydrolase activity was sampled every 24 hours through the feed tube. If the enzyme activity was below 120 U / mL, the controller could automatically fine-tune the parameters (such as appropriately increasing the dissolved oxygen level to 30% or increasing the amount of metal ions) to ensure that the enzyme activity remained stable above 120 U / mL. Fermentation continued for 48 hours, during which Delford strain D39 further metabolized the active ingredients in the Scutellaria baicalensis residue, generating more metabolites with pesticide residue degradation functions, completing the three-stage fermentation. The fermented liquid was then transferred from the fermenter to a vacuum concentrator. The vacuum concentrator reduces the internal pressure of the tank through a vacuum system (the vacuum level is controlled at -0.08 to -0.09 MPa). According to Henry's Law, the pressure reduction lowers the boiling point of water to below 40°C, thus preventing the deactivation of heat-sensitive active ingredients such as organophosphorus hydrolases and flavonoids in the enzyme.Simultaneously, the concentrator slowly heats the liquid using a low-temperature heating module (temperature controlled at 35℃-40℃), gradually evaporating the water and concentrating it to 1 / 3 of its original volume. During this process, the concentrator's built-in stirring component ensures uniform heating of the liquid, increasing the concentration of effective components by approximately three times and achieving an enzyme activity retention rate of over 90%. Finally, the concentrated enzyme is transferred to an encapsulation machine, where 2% of the concentrated liquid weight of Scutellaria baicalensis polysaccharide (as a wall material) is added. The encapsulation machine employs the coagulation encapsulation principle, maintaining a low-temperature environment of 30℃-35℃ through a built-in temperature control system. The stirring system (speed 80-100 r / min) ensures uniform mixing of the Scutellaria baicalensis polysaccharide and the concentrated enzyme. Subsequently, the encapsulation machine's automatic sample addition system adds citric acid solution to adjust the system pH to 4.0-4.5, promoting the coagulation of Scutellaria baicalensis polysaccharide and forming 5-10 μm microcapsule membranes on the surface of the enzyme's active components. Finally, calcium chloride solution is added for solidification, enhancing the strength and stability of the microcapsule membranes. Through encapsulation, the Scutellaria baicalensis polysaccharide wall material can isolate oxygen, light and microorganisms, extending the shelf life of the enzyme to more than 12 months, while ensuring that the active ingredients can be released normally during use, ultimately producing a liquid compound enzyme with pesticide residue degradation function.

[0018] This invention provides a pesticide residue-degrading compound enzyme based on Delftobacterium, along with its preparation method and equipment. It offers the following beneficial effects: 1. The present invention has high degradation efficiency: For organophosphorus pesticides such as phoxim, dichlorvos and dimethoate at a concentration of 200 mg / L, the degradation rates after 48 hours are 93.8%, 92.5% and 91.2% respectively, which far exceed the mainstream biodegradable products on the market (72%-80%), and the degradation cycle is only 5 days, which is 83% shorter than traditional enzymes.

[0019] 2. The invention is low in cost: using Scutellaria baicalensis residue (medical waste) as the core raw material, it replaces expensive synthetic culture medium, and the production cost per ton is controlled within 8,000 yuan, which is only 21.6% of the mainstream products on the market (37,000 yuan / ton). The cost of pesticide residue treatment per mu for growers is reduced to less than 300 yuan.

[0020] 3. The invention has strong environmental adaptability: the degradation rate remains above 85% within the pH range of 5.5-8.5 and the temperature range of 25℃-40℃. It can be adapted to fruit and vegetable planting scenarios in different climate zones such as the rainy south and the arid north of my country, and has a good degradation effect on a variety of fruits and vegetables such as citrus, broccoli, and tomatoes.

[0021] 4. This invention has outstanding environmental protection features: it realizes the resource utilization of Scutellaria baicalensis residue, reduces waste emissions by 800 kg per ton of product (30% waste reduction), and the degradation products are CO2 and H2O (detected by gas chromatography, no toxic organic residues are found), with no secondary pollution, which meets the needs of green agricultural development.

[0022] 5. This invention has a wide range of applications: the product form can be adjusted according to different needs, and it is suitable for foliar spraying during the planting period (dilution ratio 1:200), soaking after harvest (dilution ratio 1:50), and can also be made into small packages for daily use (dilution ratio 1:100), covering the entire agricultural production, circulation and consumption chain. Attached Figure Description

[0023] Figure 1 A schematic diagram of the overall structure of a device for preparing a pesticide residue degradation complex enzyme based on Delftobacterium tumefaciens; Figure 2 A cross-sectional view of the screening box of a device for preparing a pesticide residue degradation complex enzyme based on Delftobacterium tumefaciens; Figure 3 This is a cross-sectional view of the fermentation chamber of a device for preparing a pesticide residue degradation complex enzyme based on Delftobacterium.

[0024] The components are as follows: 1. Screening box; 2. Support plate; 3. Spring; 4. Mounting frame; 5. Screen plate; 6. Vibrating motor; 7. Discharge port; 8. Collection box; 9. Through groove; 10. Receiving box; 11. Crushing cylinder; 12. Support block; 13. First motor; 14. Crushing rod; 15. Crushing blade; 16. Feeding cylinder; 17. Constant temperature shaking table; 18. Fermentation tank; 19. Second motor; 20. Stirring rod; 21. Stirring blade; 22. Mounting plate; 23. Weighing sensor; 24. Weighing pan; 25. Oxygen cylinder; 26. Gas pipe; 27. Solenoid valve; 28. Temperature sensor; 29. ​​Heating wire; 30. Bracket; 31. Dissolved oxygen electrode; 32. Controller; 33. Feed pipe; 34. Vacuum decompression concentrator; 35. Embedding machine. Detailed Implementation

[0025] The technical solutions of the embodiments of the present invention will be clearly and completely described below with reference to the accompanying drawings. Obviously, the described embodiments are only some embodiments of the present invention, and not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention.

[0026] Example: like Figure 1-3As shown, this embodiment of the invention provides a preparation device for a pesticide residue degradation compound enzyme based on Delftobacterium, including a crushing cylinder 11. A feeding cylinder is installed on the top of the crushing cylinder 11, and a sealing cover is installed on the top of the feeding cylinder. A feed cylinder 16 is connected through and fixedly to the bottom of the crushing cylinder 11, and a control valve for controlling the discharge is installed inside the feed cylinder 16. A screening component is connected to the bottom of the feed cylinder 16. Support blocks 12 are fixedly connected to the four corners of the bottom of the crushing cylinder 11, and the bottom of the support blocks 12 is connected to the screening component, serving a supporting and fixing function. A first motor 13 is fixedly connected to the top of the crushing cylinder 11. A crushing rod 14 is fixedly connected to the output end of the first motor 13 through the crushing cylinder 11, and the first motor 13 can drive the crushing rod 14 to rotate. Crushing blades 15 are fixedly connected to the outside of the crushing rod 14, and the crushing blades 15 can cut and crush the Scutellaria baicalensis residue. When it is necessary to pulverize the Scutellaria baicalensis residue, the pulverizing cylinder 11 and the screening box 1 are placed inside the laboratory refrigeration unit (not shown in the figure) to ensure that the Scutellaria baicalensis residue can be ultra-finely pulverized in a low temperature environment of -5℃ to 0℃. A constant temperature shaker 17 is set on the right side of the pulverizing cylinder 11, and a fermentation tank 18 is set on the right side of the constant temperature shaker 17. A second motor 19 is fixedly connected to the top of the fermentation tank 18. The output end of the second motor 19 passes through the fermentation tank 18 and is fixedly connected to a stirring rod 20. A stirring blade 21 is fixedly connected to the outer ring of the stirring rod 20. The second motor 19 can drive the stirring rod 20 and the stirring blade 21 to rotate. A weighing component is installed on the right side of the fermentation tank 18, and an oxygen cylinder 25 is set on the right side of the fermentation tank 18 to provide sterile oxygen. A gas pipe 26 is fixedly connected to the output end of the oxygen cylinder 25. A temperature sensor 28 is fixedly connected to the inner wall of the fermentation tank 18 to monitor the temperature inside the fermentation tank 18 in real time. A heating wire 29 is fixedly connected to the bottom of the fermenter 18, which can increase the temperature inside the fermenter 18. A dissolved oxygen electrode 31 is fixedly connected to the inner wall of the fermenter 18, which monitors the dissolved oxygen level in the tank in real time. A controller 32 is fixedly connected to the front end of the fermenter 18. A vacuum concentrator 34 is set on the right side of the fermenter 18, and an encapsulation machine 35 is set on the right side of the vacuum concentrator 34.

[0027] The screening assembly includes a screening box 1, the top of which is connected to and fixedly connected to the feed cylinder 16. Support plates 2 are fixedly connected to both sides inside the screening box 1. Springs 3 are fixedly connected to both sides of the top of the support plates 2. A mounting frame 4 is fixedly connected to the top of the springs 3. A screen plate 5 is fixedly connected inside the mounting frame 4. The screen plate 5 inside the mounting frame 4 uses a 200-mesh filter screen design. A vibration motor 6 is fixedly connected to the bottom of the mounting frame 4. A discharge port 7 is opened on the left side of the screening box 1.

[0028] The high-frequency vibration generated by the vibrating motor 6 of the screening component, in conjunction with the spring 3, causes the screen plate 5 to sway up and down. The 200-mesh dregs particles fall through the screen plate into the receiving box 10 below. The coarse particles that do not meet the standard are discharged from the discharge port 7 on the left side of the inclined screen plate to the collection box 8, and can be put back into the crushing cylinder 11 for secondary crushing, ensuring that the particle size of the dregs after pretreatment all meet the 200-mesh requirement.

[0029] The weighing assembly includes a mounting plate 22, which is fixedly connected to the outer wall of the fermenter 18. A weighing sensor 23 is fixedly connected to the top of the mounting plate 22, and a weighing pan 24 is fixedly connected to the top of the weighing sensor 23.

[0030] By using the weighing sensor 23 of the weighing component in conjunction with the weighing pan 24, the Scutellaria baicalensis residue and seed liquid are placed on the weighing pan 24 for weighing to ensure that the 3:1 weight ratio is accurate.

[0031] The end of the trachet 26 is inserted through and fixedly connected to the fermenter 18, and a solenoid valve 27 is installed on the outside of the trachet 26.

[0032] Sterile oxygen is introduced into the oxygen cylinder 25 through the trachea 26, and the oxygen flow rate can be adjusted by the solenoid valve 27 outside the trachea 26.

[0033] The fermentation tank 18 has four fixed supports 30 at the bottom corners and a feed pipe 33 fixedly connected to the top of the fermentation tank 18.

[0034] A discharge pipe is installed at the bottom of the fermentation tank 18, and a control valve is installed on the outside of the discharge pipe to facilitate the discharge of the fermented material in the fermentation tank 18.

[0035] A collection box 8 is provided on the left side of the screening box 1, and the collection box 8 is located below the discharge port 7.

[0036] Large particles of medicinal residue can be collected through collection box 8.

[0037] A through groove 9 is provided on the right side of the screening box 1, and a receiving box 10 is slidably connected in the through groove 9.

[0038] A locking mechanism is installed between the receiving box 10 and the screening box 1, allowing the screened residue to be collected and easily removed.

[0039] A pesticide residue degradation compound enzyme based on Dellfordia sp. comprises the following raw materials: 30%-40% Scutellaria baicalensis residue, 5%-8% hydrolyzed sucrose, 10%-15% Dellfordia sp. D39 inoculum, 0.5%-1% metal ion compound solution, and 36%-54.5% sterile water.

[0040] The residue from Scutellaria baicalensis provides the organic nutrients needed for the growth of D39 bacteria, realizing the resource utilization of waste and reducing waste by 30%. Hydrolyzed sucrose serves as a carbon source, promoting the proliferation of D39 bacteria and the secretion of organophosphorus hydrolytic enzymes.

[0041] Delfordia D39: This strain was isolated from farmland soil that had been subjected to long-term application of organophosphorus pesticides and showed no resistance mutations. The colonies are round, with neat edges, smooth surfaces, and milky white color (1-2 mm in diameter). They are Gram-negative, short rod-shaped (0.5-0.8 μm × 1.0-1.5 μm in size), and can grow stably in the pH range of 5.5-8.5 and the temperature range of 25℃-40℃.

[0042] Metal ion composite solution: composed of Fe²⁺, Zn²⁺, and Mg²⁺ in a concentration ratio of 0.2 mM Fe²⁺, 0.1 mM Zn²⁺, and 0.5 mM Mg²⁺. By regulating the conformation of the intracellular enzyme active site of D39 bacteria, it significantly improves the hydrolysis efficiency of organophosphorus compounds.

[0043] A pesticide residue degradation complex enzyme based on Delftobacterium and its preparation method, characterized by the following steps: S1. Raw material pretreatment: The Scutellaria baicalensis residue was ultra-finely pulverized to 200 mesh at a low temperature of -5℃ to 0℃ (to increase the specific surface area and break the cell wall fiber structure). 0.5% of the raw material weight of the compound enzyme (cellulase: pectinase = 1:1) was added, and the mixture was enzymatically hydrolyzed in a constant temperature water bath at 45℃ for 5-6 hours to promote the dissolution of active ingredients and improve the subsequent microbial utilization efficiency.

[0044] S2, Seed culture: Delfordia D39 was inoculated into LB medium (10 g / L peptone, 5 g / L yeast extract, 10 g / L NaCl, pH 7.0) and incubated at 30-32°C on a shaker at 150 rpm for 48 hours, with hydrolyzed sucrose (5% concentration) added every 12 hours to ensure a viable count of 10-1. 8 S3, raw material pretreatment, with a concentration of CFU / mL or higher.

[0045] S3, Three-stage Precision Fermentation: Enzyme activity peak induction stage: The pretreated Scutellaria baicalensis residue and seed liquid were mixed at a weight ratio of 3:1, transferred to a fermenter, and the temperature was controlled at 35℃ and dissolved oxygen at 30% or higher. 200 mg / L phoxim was added as a substrate to induce the expression of degradation genes and cultured for 24 hours. Synergistic fermentation stage: Add metal ion complex solution to the fermenter, maintain temperature at 35℃ and dissolved oxygen at 25%-30%, and continue fermentation for 48 hours. During this period, sample and test enzyme activity every 24 hours to ensure that the activity of organophosphorus hydrolase is stable above 120U / mL. Post-processing stage: Low-temperature vacuum concentration at below 40℃ (concentrated to 1 / 3 of the original volume), adding Scutellaria baicalensis polysaccharide (concentration 2%) as wall material for microencapsulation, to produce liquid enzyme, extending the shelf life to more than 12 months.

[0046] A comparative analysis of the pesticide residue degradation compound enzyme based on Delftobacterium prepared in this invention with existing commercially available chemical degradation agents, mainstream biodegradable products, and traditional enzymes in terms of organophosphate degradation: Although embodiments of the invention have been shown and described, it will be understood by those skilled in the art that various changes, modifications, substitutions and alterations can be made to these embodiments without departing from the principles and spirit of the invention, the scope of which is defined by the appended claims and their equivalents.

Claims

1. A preparation device of a Delftia-based pesticide residue degradation composite enzyme, comprising a pulverizing cylinder (11), characterized in that: The bottom of the crushing cylinder (11) is penetrated and fixedly connected with a feeding cylinder (16), the bottom of the feeding cylinder (16) is connected with a screening assembly, the bottom of the crushing cylinder (11) is fixedly connected with a support block (12) at four corners, and the bottom of the support block (12) is connected with the screening assembly, the top of the crushing cylinder (11) is fixedly connected with a first motor (13), the output end of the first motor (13) is fixedly connected with a crushing rod (14) penetrating the crushing cylinder (11), the crushing rod (14) is fixedly connected with a crushing blade (15) outside, the right side of the crushing cylinder (11) is provided with a constant temperature shaking table (17), the right side of the constant temperature shaking table (17) is provided with a fermentation tank (18), the top of the fermentation tank (18) is fixedly connected with a second motor (19), the output end of the second motor (19) is fixedly connected with a stirring rod (20) penetrating the fermentation tank (18), the stirring rod (20) is fixedly connected with a stirring blade (21) outside, the right side of the fermentation tank (18) is provided with a weighing assembly, the right side of the fermentation tank (18) is provided with an oxygen cylinder (25), the output end of the oxygen cylinder (25) is fixedly connected with an air pipe (26), the inner wall of the fermentation tank (18) is fixedly connected with a temperature sensor (28), the inner bottom of the fermentation tank (18) is fixedly connected with a heating wire (29), the inner wall of the fermentation tank (18) is fixedly connected with a dissolved oxygen electrode (31), the front end of the fermentation tank (18) is fixedly connected with a controller (32), the right side of the fermentation tank (18) is provided with a vacuum decompression concentrator (34), and the right side of the vacuum decompression concentrator (34) is provided with an embedding machine (35).

2. The preparation device of the pesticide residue degradation composite enzyme based on Delft bacteria according to claim 1, characterized in that: The screening assembly comprises a screening box (1), the top of the screening box (1) is penetrated and fixedly connected with the feeding cylinder (16), support plates (2) are fixedly connected inside the screening box (1) on both sides, springs (3) are fixedly connected on both sides of the top of the support plates (2), mounting racks (4) are fixedly connected on the top of the springs (3), sieve plates (5) are fixedly connected in the mounting racks (4), vibration motors (6) are fixedly connected on the bottom of the mounting racks (4), and a discharge port (7) is formed on the left side of the screening box (1).

3. The preparation device of the pesticide residue degradation composite enzyme based on Delft bacteria according to claim 1, characterized in that: The weighing assembly comprises a mounting plate (22), the mounting plate (22) is fixedly connected with the outer wall of the fermentation tank (18), a weighing sensor (23) is fixedly connected on the top of the mounting plate (22), and a weighing disc (24) is fixedly connected on the top of the weighing sensor (23).

4. The preparation device of the pesticide residue degradation composite enzyme based on Delft bacteria according to claim 1, characterized in that: The air pipe (26) is penetrated and fixedly connected with the fermentation tank (18) at the tail end, and an electromagnetic valve (27) is mounted outside the air pipe (26).

5. The preparation device of the pesticide residue degradation composite enzyme based on Delft bacteria according to claim 1, characterized in that: Support frames (30) are fixedly connected at four corners of the bottom of the fermentation tank (18), and a feeding pipe (33) is fixedly connected on the top of the fermentation tank (18).

6. The preparation device of the pesticide residue degradation composite enzyme based on Delft bacteria according to claim 2, characterized in that: The screening box (1) is provided with a collection box (8) on the left side, and the collection box (8) is arranged below the discharge port (7).

7. The preparation device of the pesticide residue degradation composite enzyme based on Delft bacteria according to claim 2, characterized in that: A through groove (9) is formed on the right side of the screening box (1), and a material receiving box (10) is slidably connected in the through groove (9).

8. A pesticide residue degradation composite enzyme based on Dellfordia sp., comprising the following raw materials: 30-40% of Scutellaria baicalensis Georgi residues, 5-8% of hydrolyzed sucrose, 10-15% of Dellfordia sp. D39 bacterial agent, 0.5-1% of metal ion composite liquid, and 36-54.5% of sterile water.

9. The Delftia-based agricultural residue degrading composite enzyme and the preparation method thereof according to any one of claims 1-7, characterized in that: The method comprises the following steps: S1, raw material pretreatment: Scutellaria baicalensis Georgi residues are ultra-finely pulverized to 200 mesh (to increase the specific surface area and break the cell wall fiber structure) in a low-temperature environment of -5-0°C, 0.5% of the weight of the raw material is added to the composite enzyme (cellulase: pectinase = 1:1), and enzyme hydrolysis is performed in a 45°C constant temperature water bath for 5-6 hours to promote the dissolution of active ingredients and improve the utilization efficiency of subsequent microorganisms. S2, seed liquid culture: Delftia D39 was inoculated into LB medium (10 g / L of proteose peptone, 5 g / L of yeast extract, 10 g / L of NaCl, pH 7.0) and cultured in a 30-32°C, 150 r / min shaking bed for 48 hours, during which hydrolyzed sucrose (5%) was added every 12 hours to ensure that the viable cell count of the strain reached 10 8 CFU / mL above S3, raw material pretreatment. S3, three-stage precise fermentation: Enzyme activity peak induction stage: the pretreated Scutellaria baicalensis Georgi residues are mixed with the seed liquid at a weight ratio of 3:1, transferred into a fermentation tank, the temperature is controlled at 35°C, the dissolved oxygen is controlled above 30%, 200mg / L phoxim is added as a substrate to induce gene expression for degradation, and the culture is performed for 24 hours; Synergistic fermentation stage: metal ion composite liquid is added to the fermentation tank, the temperature is maintained at 35°C, the dissolved oxygen is maintained at 25-30%, and the fermentation is continued for 48 hours, during which the enzyme activity is detected every 24 hours to ensure that the organic phosphorus hydrolase activity is stable above 120U / mL; Post-treatment stage: low-temperature vacuum concentration below 40°C (concentrated to 1 / 3 of the original volume), Scutellaria baicalensis Georgi polysaccharide (concentration 2%) is added as a wall material for microencapsulation embedding, and a liquid enzyme is prepared, which can extend the shelf life to more than 12 months.

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