Compound enzyme agent for removing pesticide residues on vegetables and fruits, and preparation method and application thereof

Abstract

This invention provides a compound enzyme agent for removing pesticide residues from fruits and vegetables, its preparation method, and its application. The preparation method includes the following steps: 1) adding an aqueous solution of calcium ions to a mixture comprising a compound enzyme and PBS buffer to obtain a raw material solution; allowing the raw material solution to stand at 25-40°C for 12-24 hours to obtain enzyme-inorganic hybrid nanoflower particles; 2) coating the enzyme-inorganic hybrid nanoflower particles with an alkaline buffer salt to obtain a compound enzyme agent; wherein the compound enzyme includes carboxylesterase, organophosphatase, protease, and / or amylase; and the pH of the PBS buffer is 6.5-7.8. The compound enzyme agent prepared by this method can remove a variety of common pesticides with a removal rate of not less than 89%, ensuring food safety.

Description

Technical Field

[0001] This invention relates to a compound enzyme agent, its preparation method and application, and more particularly to a compound enzyme agent for removing pesticide residues from fruits and vegetables, its preparation method and application. Background Technology

[0002] Pesticides are widely used agents for controlling biological hazards in agriculture and forestry, making a significant contribution to ensuring the quantity and quality of agricultural products such as fruits and vegetables. However, while bringing benefits, pesticide residues on the surface of fruits and vegetables have also caused serious food safety problems.

[0003] To address the problem of pesticide residues, detergents using enzymes to degrade pesticide residues on the surface of fruits and vegetables have appeared on the market. However, most of these enzymes can only remove certain single types of pesticides. With the continuous upgrading of pesticide products, existing enzymes are far from sufficient to remove multiple pesticide residues from the surface of fruits and vegetables. Furthermore, these enzymes are easily deactivated during use, failing to achieve true pesticide residue removal.

[0004] Therefore, how to comprehensively and effectively remove pesticide residues from the surface of fruits and vegetables has become a problem to be solved. Summary of the Invention

[0005] To address the aforementioned problems, this invention provides a method for preparing a composite enzyme agent for removing pesticide residues from fruits and vegetables. By immobilizing the composite enzyme to form enzyme-inorganic hybrid nanoflower particles, and then encapsulating the enzyme-inorganic hybrid nanoflower particles with an alkaline buffer salt, a comprehensive and efficient removal of pesticide residues from the surface of fruits and vegetables can be achieved, ensuring food safety.

[0006] The present invention also provides a compound enzyme agent, which is prepared by the above preparation method. This compound enzyme agent can be used to remove a variety of common pesticides, and the removal effect is significant.

[0007] The present invention further provides a compound enzyme detergent containing the above-mentioned compound enzyme, which can remove a variety of common pesticides more thoroughly.

[0008] The present invention also provides a method for removing pesticide residues from fruits and vegetables. When fruits and vegetables are treated with the above-mentioned compound enzyme agent or compound enzyme detergent, the pesticide removal rate on the surface of fruits and vegetables can reach more than 90%.

[0009] This invention provides a method for preparing a compound enzyme agent for removing pesticide residues from fruits and vegetables, comprising the following steps:

[0010] 1) Add an aqueous solution of calcium ions to a mixture including the complex enzyme and PBS buffer to obtain a raw material solution; let the raw material solution stand at 25-40℃ for 12-24h to obtain enzyme-inorganic hybrid nanoflower particles.

[0011] 2) The enzyme-inorganic hybrid nanoflower particles were coated with alkaline buffer salt to obtain a composite enzyme agent;

[0012] The complex enzyme includes carboxylesterase, organophosphatase, protease and / or amylase; the pH of the PBS buffer is 6.5-7.8.

[0013] According to one embodiment of the present invention, the carboxylesterase includes porcine liver esterase, and the enzyme activity of the porcine liver esterase is 5000-9000 U / g;

[0014] The activity of the organophosphatase is 30,000-40,000 U / g;

[0015] The protease includes at least one of papain and bromelain, wherein the enzyme activity of the papain is 8000-18000 U / g and the enzyme activity of the bromelain is 15000-28000 U / g.

[0016] The amylase includes glucoamylase, and the enzyme activity of the glucoamylase is 3000-9000 U / g.

[0017] According to one embodiment of the present invention, the mass ratio of the carboxylesterase, organophosphatase, papain, bromelain, and amylase is (0.5-1):(1-2):(0.5-2.5):(1.5-2.5):(1-5).

[0018] According to one embodiment of the present invention, the mass-to-volume ratio of the complex enzyme to the PBS buffer is (0.2-0.4) g: (0.9-1.1) L.

[0019] According to one embodiment of the present invention, the molar mass ratio of the calcium ions to the complex enzyme is (0.03-0.07) mol: (0.2-0.4) g.

[0020] According to one embodiment of the present invention, the mass ratio of the enzyme-inorganic hybrid nanoflower particles to the alkaline buffer salt is 0.3-1.8:2.

[0021] The present invention also provides a compound enzyme preparation, which is prepared by any of the above methods.

[0022] The present invention further provides a compound enzyme detergent, wherein the compound enzyme detergent comprises the above-mentioned compound enzyme agent.

[0023] According to one embodiment of the present invention, the compound enzyme detergent further includes water, and the compound enzyme agent is mixed with water at a mass ratio of 1:500-800.

[0024] The present invention also provides a method for removing pesticide residues from the surface of fruits and vegetables, wherein the fruits and vegetables are treated with the above-mentioned compound enzyme agent or compound enzyme detergent;

[0025] The pesticides include carbamates and pyrethroids.

[0026] In the preparation method provided by this invention, a complex enzyme is immobilized using calcium phosphate crystals formed by calcium ions and PBS buffer to prepare enzyme-inorganic hybrid nanoflower particles. These particles are then further coated with alkaline buffer salts to prepare a composite enzyme agent. This composite enzyme agent can remove a variety of common pesticides with a removal rate of not less than 89%, ensuring food safety. Detailed Implementation

[0027] To enable those skilled in the art to better understand the present invention, the present invention will be further described in detail below. The specific embodiments listed below are merely descriptions of the principles and features of the present invention, and the examples are only for explaining the present invention and are not intended to limit the scope of the present invention. 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.

[0028] The first aspect of this invention provides a method for preparing a compound enzyme agent for removing pesticide residues from fruits and vegetables, comprising the following steps:

[0029] 1) Add an aqueous solution of calcium ions to a mixture including the complex enzyme and PBS buffer to obtain a raw material solution; let the raw material solution stand at 25-40℃ for 12-24h to obtain enzyme-inorganic hybrid nanoflower particles.

[0030] 2) Enzyme-inorganic hybrid nanoflower particles were coated with alkaline buffer salts to obtain a composite enzyme agent;

[0031] The complex enzyme includes carboxylesterase, organophosphatase, protease and / or amylase; the pH of the PBS buffer is 6.5-7.8.

[0032] In step 1), the complex enzyme includes carboxylesterase and organophosphatase, and further includes at least one of protease and amylase; PBS buffer refers to phosphate buffer, including Na2HPO4, KH2PO4, NaCl and KCl, specifically, the pH of the PBS buffer is 6.5-7.8.

[0033] In step 2), the alkaline buffer salt is a food-grade organic or inorganic salt, which is not further limited in this invention. Exemplarily, it includes carbonates, citrates, etc. Further, the alkaline buffer salt includes one or more of calcium, sodium, and potassium salts. For example, the alkaline buffer salt can be one or more of sodium citrate, potassium citrate, sodium bicarbonate, potassium chloride, magnesium carbonate, potassium carbonate, sodium chloride, and calcium bicarbonate.

[0034] This invention involves adding an aqueous solution of calcium ions to a mixture of a complex enzyme and a PBS buffer solution with a pH of 6.5-7.8, and allowing it to stand at 25-40°C for 12-24 hours. This process forms inorganic particles loaded with the complex enzyme. Initially, these inorganic particles appear as "petals." As the "petals" continue to form and accumulate anisotropically, they eventually become nanoflower particles, i.e., the aforementioned enzyme-inorganic hybrid nanoflower particles.

[0035] Subsequently, in step 2), the enzyme-inorganic hybrid nanoflower particles are coated with alkaline buffer salt to obtain the composite enzyme of the present invention.

[0036] When the composite enzyme prepared by the above method is used to treat fruits and vegetables with residues of different types of pesticides, the pesticide removal rate on the surface of the fruits and vegetables is no less than 89%, and the pesticide content in the washing solution is extremely low. Based on this phenomenon, the inventors analyzed the pesticide removal mechanism and believe that it may be due to the following: Firstly, the composite enzyme has a broad spectrum of degradation for most pesticides on fruits and vegetables (such as carbamates and pyrethroids), and can degrade multiple pesticides on the surface of fruits and vegetables, reducing pesticide residues on the surface of fruits and vegetables and in the washing solution; secondly, there is a hydrophobic-hydrophilic interaction between the composite enzyme and the pesticide, which can also promote the detachment of pesticides from the surface of fruits and vegetables, thus achieving the removal of pesticide residues on the surface of fruits and vegetables.

[0037] On the other hand, the process of forming enzyme-inorganic hybrid nanoflower particles described above is actually a process of efficiently immobilizing the complex enzyme and preventing its inactivation. Specifically, phosphate ions in the PBS buffer combine with calcium ions to generate Ca3(PO4)2 crystals for immobilizing the complex enzyme. The calcium ions in these crystals complex with the amide groups in the protein molecular backbone of the complex enzyme (the complexation product is the enzyme-inorganic hybrid nanoflower particle), thereby achieving the immobilization of the complex enzyme, reducing the number of easily inactivated free enzymes, and ensuring the high activity of the complex enzyme. At the same time, the enzyme-inorganic hybrid nanoflower particles have a larger specific surface area and lower mass transfer restriction. Compared to remaining on the surface of fruits and vegetables, pesticides tend to move towards the enzyme-inorganic hybrid nanoflower particles. Therefore, pesticides detach from the surface of fruits and vegetables and are adsorbed by the enzyme-inorganic hybrid nanoflower particles. The adsorbed pesticides can be degraded by the complex enzyme or coated in the enzyme-inorganic hybrid nanoflower particles, further reducing the pesticide content on the surface of fruits and vegetables and in the washing solution.

[0038] In addition, some pesticides are easily decomposed by alkali and cannot be detected. Therefore, the presence of the above-mentioned alkaline buffer salts can also promote the removal of pesticide residues on the surface of fruits and vegetables.

[0039] It is worth mentioning that the above preparation method also helps to solve environmental pollution problems. Specifically, based on the selection of the above-mentioned complex enzyme, it is not necessary to add surfactants to promote pesticide dissolution during the preparation of the complex enzyme agent. Therefore, the pollution problem caused by pesticide residues in the washing liquid after traditional surfactant washing is avoided. In the process of immobilizing the complex enzyme, the above-mentioned PBS buffer is selected to disperse the complex enzyme, avoiding the environmental pollution caused by the use of organic solvents in traditional methods. In addition, the enzyme-inorganic hybrid nanoflower particles are insoluble, and the phosphorus element from the PBS buffer is coated in the enzyme-inorganic hybrid nanoflower particles, avoiding water pollution caused by the use of soluble phosphates.

[0040] It should be noted that the pH of the PBS buffer, the temperature and time of the settling process in step 1), and the coating with alkaline buffer salt in step 2) are crucial to the pesticide removal efficiency of the composite enzyme. The pH of the PBS buffer provides a suitable pH environment for the composite enzyme, ensuring its high activity in combining with the Ca3(PO4)2 crystals. The temperature and time of the settling process ensure that the composite enzyme is highly active and uniformly dispersed on the surface of the Ca3(PO4)2 crystals and forms a complex with them, resulting in enzyme-inorganic hybrid nanoparticles with a uniform particle size of 100-450 nm. This avoids pesticide removal rates being affected by inactivation or uneven dispersion of the composite enzyme. The coating with alkaline buffer salt provides a suitable pH environment for pesticide removal and also dissolves the pesticide. The microbial indicators (total bacterial count and coliform count) in the composite enzyme prepared by the above method are lower than the content requirements in the national standard GB / T 24691-2009, ensuring food safety.

[0041] In the specific preparation process, this invention does not impose any further limitations on steps 1) and 2). For example, the various raw material enzymes in step 1) can be obtained directly from commercial sources, and any enzyme will not affect the quality of fruits and vegetables after treatment with pesticides; when preparing the mixture, it can be obtained by adding a compound enzyme to PBS buffer; this invention does not impose any special limitations on the specific implementation of the coating treatment in step 2). For example, after mixing an aqueous solution of alkaline buffer salt with enzyme-inorganic hybrid nanoflower particles, the mixture can be dried in a fluidized bed to achieve coating of the enzyme-inorganic hybrid nanoflower particles with alkaline buffer salt. Furthermore, when the pH of the aqueous solution of alkaline buffer salt is controlled at 9-10, the activity of the enzyme in the enzyme-inorganic hybrid nanoflower particles can be maximized during the coating treatment, thereby helping to further improve the removal effect of pesticides; for transportation, storage, or use requirements, in step 2), the process parameters of the coating treatment can be controlled to ensure that the water content of the compound enzyme is less than 10%. Furthermore, the granular compound enzyme can be prepared into tablets by means of compression or other methods.

[0042] Furthermore, considering factors such as cost, when coating the aforementioned enzyme-inorganic hybrid nanoflower particles with alkaline buffer salts, one or more of the following can be added to assist in the granulation of the enzyme-inorganic hybrid nanoflower particles: binder, filler, granulation solvent, and stabilizer. The binder can be one or more of polyvinylpyrrolidone, polyvinyl alcohol, hydroxypropyl cellulose, methyl cellulose, and carboxymethyl cellulose; the filler can be carbonate; the granulation solvent can be water and / or propylene glycol; and the stabilizer can be propylene glycol and / or polyethylene glycol.

[0043] In one specific embodiment, the carboxylesterase includes porcine liver esterase, and the enzyme activity of porcine liver esterase is 5000-9000 U / g (the enzyme activity of porcine liver esterase is 5000-9000 U per gram).

[0044] The enzyme activity of organic phosphatase is 30,000-40,000 U / g (the enzyme activity of each gram of organic phosphatase is 30,000-40,000 U).

[0045] The protease includes at least one of papain and bromelain, with papain having an enzyme activity of 8000-18000 U / g (8000-18000 U per gram of papain) and bromelain having an enzyme activity of 15000-28000 U / g (15000-28000 U per gram of bromelain).

[0046] Amylase includes glucoamylase, and the enzyme activity of glucoamylase is 3000-9000 U / g (the enzyme activity of each gram of glucoamylase is 3000-9000 U).

[0047] For porcine liver esterase, 1 U is equal to the amount of enzyme required to catalyze the production of 1 μmol α-naphthyl ester per minute using 1.0 mmol / L 1-naphthyl acetate as a substrate; for organophosphatase, 1 U is equal to the amount of enzyme required to catalyze the production of 1 μg p-nitrophenol per minute using 10 μg / mL disodium p-nitrophenyl phosphate as a substrate; for protease, 1 U is equal to the amount of enzyme required to hydrolyze 10 g / L casein per minute to produce 1 μg tyrosine; for amylase, 1 U is equal to the amount of enzyme required to liquefy 1 mg soluble starch into dextrin per minute.

[0048] According to the inventors' research, when using a complex enzyme with the aforementioned activity units, the mass ratio of carboxylesterase, organophosphatase, papain, bromelain, and amylase is (0.5-1):(1-2):(0.5-2.5):(1.5-2.5):(1-5). When using enzymes with the aforementioned activity as raw materials for the complex enzyme, the pesticide removal rate on the surface of fruits and vegetables can be further improved by ensuring that the ratio between the various enzyme raw materials meets the above-mentioned limits.

[0049] It should be noted that the above-mentioned limitations of the present invention are only used to limit the proportion of each raw material enzyme, and are not used to limit the composition of the complex enzyme. For example, when the complex enzyme only includes carboxylesterase, organophosphatase and amylase, the mass ratio between the three is the same as the above-mentioned limitation, that is, the mass ratio of carboxylesterase, organophosphatase and amylase is (0.5-1):(1-2):(1-5).

[0050] It is understood that when the enzyme activity of the raw material enzyme differs from the aforementioned enzyme activity, those skilled in the art can further determine the mass ratio between the various raw material enzymes in the compound enzyme based on the specific enzyme activity and the type of pesticide to be removed.

[0051] Further research revealed that when using a complex enzyme with the above-mentioned activity units and enzyme ratio, the mass-to-volume ratio of the complex enzyme to the PBS buffer is (0.2–0.4) g : (0.9–1.1) L.

[0052] When the mass-to-volume ratio of the complex enzyme to PBS buffer is too high, the activity of the complex enzyme cannot be maintained. This is because if the PBS buffer is too low, it cannot provide a suitable pH environment for the complex enzyme, nor can it provide sufficient calcium phosphate crystals for its fixation, ultimately leading to enzyme inactivation and affecting pesticide removal efficiency. Conversely, if the mass-to-volume ratio of the complex enzyme to PBS buffer is too low, it results in waste of PBS buffer. Therefore, considering both pesticide removal efficiency and economic factors, the mass-to-volume ratio of the complex enzyme to PBS buffer is controlled at (0.2–0.4) g : (0.9–1.1) L.

[0053] Furthermore, the molar mass ratio of calcium ions to complex enzymes is (0.03-0.07) mol: (0.2-0.4) g.

[0054] In practice, insufficient calcium ions may result in a low content of calcium phosphate crystals, preventing the complex enzyme from being fully immobilized and hindering pesticide removal. Therefore, the aforementioned molar ratio of calcium ions to the complex enzyme is used to ensure an excess of calcium ions relative to the complex enzyme.

[0055] In this invention, the mass ratio of enzyme-inorganic hybrid nanoflower particles to alkaline buffer salt is 0.3-1.8:2.

[0056] At this mass ratio, the enzyme-inorganic hybrid nanoflower particles are fully coated with alkaline buffer salt, avoiding waste caused by excessive alkaline buffer salt. On the one hand, it can provide a protective layer for the enzyme-inorganic hybrid nanoflower particles, which is beneficial to maintaining the activity of the enzyme; on the other hand, the alkaline buffer salt can provide a suitable pH environment for pesticide removal for the compound enzyme.

[0057] A second aspect of this invention provides a compound enzyme agent prepared using the above-described method. The microbial content of this compound enzyme agent meets the microbial indicators specified in GB / T 24691-2009.

[0058] A third aspect of this invention provides a compound enzyme detergent, comprising the aforementioned compound enzyme agent. This compound enzyme detergent exhibits significant removal efficiency when used to remove pesticides.

[0059] Furthermore, the compound enzyme detergent of the present invention also includes water. The present invention does not limit the mass ratio of compound enzyme agent to water. Exemplarily, the compound enzyme agent and water can be mixed at a mass ratio of 1:500-800. At this mass ratio, the aqueous solution formed by the dissolution of alkaline buffer salt on the surface of the compound enzyme agent can provide a suitable pH environment for pesticide removal, which is beneficial to pesticide removal.

[0060] A fourth aspect of this invention provides a method for removing pesticide residues from the surface of fruits and vegetables. The method involves treating the fruits and vegetables with the aforementioned compound enzyme agent or compound enzyme detergent. The pesticides include carbamates and pyrethroids. This invention does not limit the specific method of treating the fruits and vegetables; for example, pesticide residues can be removed by soaking for 5-10 minutes, achieving a removal rate of over 90%.

[0061] The present invention will now be described in more detail through specific embodiments.

[0062] Example 1

[0063] The compound enzyme in this embodiment was prepared by the following method:

[0064] 1) Porcine liver esterase, organophosphatase, papain, and glucoamylase were mixed in a mass ratio of 1:1:1.5:1 to obtain a compound enzyme; wherein the enzyme activity of porcine liver esterase was 5000 U / g, the enzyme activity of organophosphatase was 35000 U / g, the enzyme activity of papain was 10000 U / g, and the enzyme activity of glucoamylase was 7000 U / g.

[0065] Dissolve Na2HPO4, KH2PO4, NaCl and KCl in water to form a PBS buffer with a pH of 7.4;

[0066] Dissolve 0.05 mol of calcium chloride to form an aqueous solution of calcium chloride;

[0067] An aqueous solution of calcium chloride was added to a mixture of the complex enzyme and PBS buffer to obtain a raw material solution; wherein the mass-to-volume ratio of the complex enzyme to the PBS buffer was 0.3 g: 1 L.

[0068] The molar ratio of calcium chloride to the complex enzyme is 0.05 mol: 0.3 g;

[0069] The raw material solution was allowed to stand at 25℃ for 24 hours. The precipitate generated by the raw material solution was collected by centrifugation (12000r / min, 10min). The precipitate was then washed three times with deionized water to obtain enzyme-inorganic hybrid nanoflower particles.

[0070] The particle size of the enzyme-inorganic hybrid nanoflower particles was measured to be 100-300 nm using a Malvern laser particle size analyzer.

[0071] 2) Dissolve sodium bicarbonate and sodium chloride in water at a mass ratio of 30:45 to form an aqueous solution containing an alkaline buffer salt of sodium bicarbonate and sodium chloride.

[0072] Enzyme-inorganic hybrid nanoflower particles were mixed with an aqueous solution of alkaline buffer salt, polyvinylpyrrolidone, and porous calcium carbonate. The mass ratio of enzyme-inorganic hybrid nanoflower particles to alkaline buffer salt was 1:2; the mass ratio of enzyme-inorganic hybrid nanoflower particles to polyvinylpyrrolidone was 2:5; and the mass ratio of enzyme-inorganic hybrid nanoflower particles to porous calcium carbonate was 10:23.

[0073] The mixture was dried in a fluidized bed at 50°C to obtain a composite enzyme with a water content of less than 10%.

[0074] Example 2

[0075] The compound enzyme in this embodiment was prepared by the following method:

[0076] 1) Porcine liver esterase, organophosphatase, bromelain, and glucoamylase were mixed in a mass ratio of 1:1:1:1 to obtain a compound enzyme; wherein the enzyme activity of porcine liver esterase was 5000 U / g, the enzyme activity of organophosphatase was 35000 U / g, the enzyme activity of bromelain was 20000 U / g, and the enzyme activity of glucoamylase was 7000 U / g.

[0077] Dissolve Na2HPO4, KH2PO4, NaCl and KCl in water to form a PBS buffer with a pH of 7.4;

[0078] Dissolve 0.05 mol of calcium chloride to form an aqueous solution of calcium chloride;

[0079] An aqueous solution of calcium chloride was added to a mixture of the complex enzyme and PBS buffer to obtain a raw material solution; wherein the mass-to-volume ratio of the complex enzyme to the PBS buffer was 0.3 g: 1 L.

[0080] The molar ratio of calcium chloride to the complex enzyme is 0.05 mol: 0.3 g;

[0081] The raw material solution was allowed to stand at 25℃ for 24 hours. The precipitate generated by the raw material solution was collected by centrifugation (12000r / min, 10min). The precipitate was then washed three times with deionized water to obtain enzyme-inorganic hybrid nanoflower particles.

[0082] The particle size of the enzyme-inorganic hybrid nanoflower particles was measured to be 250-400 nm using a Malvern laser particle size analyzer (Mastersizer3000).

[0083] 2) Dissolve sodium bicarbonate and sodium chloride in water at a mass ratio of 30:45 to form an aqueous solution containing an alkaline buffer salt of sodium bicarbonate and sodium chloride.

[0084] Enzyme-inorganic hybrid nanoflower particles were mixed with an aqueous solution of alkaline buffer salt, polyvinylpyrrolidone, and porous calcium carbonate. The mass ratio of enzyme-inorganic hybrid nanoflower particles to alkaline buffer salt was 1:2; the mass ratio of enzyme-inorganic hybrid nanoflower particles to polyvinylpyrrolidone was 2:5; and the mass ratio of enzyme-inorganic hybrid nanoflower particles to porous calcium carbonate was 10:23.

[0085] The mixture was dried in a fluidized bed at 50°C to obtain a composite enzyme with a water content of less than 10%.

[0086] Example 3

[0087] The compound enzyme in this embodiment was prepared by the following method:

[0088] 1) Porcine liver esterase, organophosphatase, papain, and glucoamylase were mixed in a mass ratio of 1:1:1.5:1 to obtain a compound enzyme; wherein the enzyme activity of porcine liver esterase was 5000 U / g, the enzyme activity of organophosphatase was 35000 U / g, the enzyme activity of papain was 10000 U / g, and the enzyme activity of glucoamylase was 7000 U / g.

[0089] Dissolve Na2HPO4, KH2PO4, NaCl and KCl in water to form a PBS buffer with a pH of 7.4;

[0090] Dissolve 0.05 mol of calcium chloride to form an aqueous solution of calcium chloride;

[0091] An aqueous solution of calcium chloride was added to a mixture of the complex enzyme and PBS buffer to obtain a raw material solution; wherein the mass-to-volume ratio of the complex enzyme to the PBS buffer was 0.2 g: 0.9 L.

[0092] The molar ratio of calcium chloride to the complex enzyme is 0.033 mol: 0.2 g;

[0093] The raw material solution was allowed to stand at 25℃ for 24 hours. The precipitate generated by the raw material solution was collected by centrifugation (12000r / min, 10min). The precipitate was then washed three times with deionized water to obtain enzyme-inorganic hybrid nanoflower particles.

[0094] The particle size of the enzyme-inorganic hybrid nanoflower particles was measured to be 200-350 nm using a Malvern laser particle size analyzer (Mastersizer3000).

[0095] 2) Dissolve sodium bicarbonate and sodium chloride in water at a mass ratio of 30:45 to form an aqueous solution containing an alkaline buffer salt of sodium bicarbonate and sodium chloride.

[0096] Enzyme-inorganic hybrid nanoflower particles were mixed with an aqueous solution of alkaline buffer salt, polyvinylpyrrolidone, and porous calcium carbonate. The mass ratio of enzyme-inorganic hybrid nanoflower particles to alkaline buffer salt was 1:2; the mass ratio of enzyme-inorganic hybrid nanoflower particles to polyvinylpyrrolidone was 2:5; and the mass ratio of enzyme-inorganic hybrid nanoflower particles to porous calcium carbonate was 10:23.

[0097] The mixture was dried in a fluidized bed at 50°C to obtain a composite enzyme with a water content of less than 10%.

[0098] Example 4

[0099] The compound enzyme in this embodiment was prepared by the following method:

[0100] 1) Porcine liver esterase, organophosphatase, papain, and glucoamylase were mixed in a mass ratio of 1:1:1.5:1 to obtain a compound enzyme; wherein the enzyme activity of porcine liver esterase was 5000 U / g, the enzyme activity of organophosphatase was 35000 U / g, the enzyme activity of papain was 10000 U / g, and the enzyme activity of glucoamylase was 7000 U / g.

[0101] Dissolve Na2HPO4, KH2PO4, NaCl and KCl in water to form a PBS buffer with a pH of 7.4;

[0102] Dissolve 0.05 mol of calcium chloride to form an aqueous solution of calcium chloride;

[0103] An aqueous solution of calcium chloride was added to a mixture of the complex enzyme and PBS buffer to obtain a raw material solution; wherein the mass-to-volume ratio of the complex enzyme to the PBS buffer was 0.3 g: 1 L.

[0104] The molar ratio of calcium chloride to the complex enzyme is 0.07 mol: 0.3 g;

[0105] The raw material solution was allowed to stand at 25℃ for 24 hours. The precipitate generated by the raw material solution was collected by centrifugation (12000r / min, 10min). The precipitate was then washed three times with deionized water to obtain enzyme-inorganic hybrid nanoflower particles.

[0106] The particle size of the enzyme-inorganic hybrid nanoflower particles was measured to be 350-450 nm using a Malvern laser particle size analyzer (Mastersizer3000).

[0107] 2) Dissolve sodium bicarbonate and sodium chloride in water at a mass ratio of 30:45 to form an aqueous solution containing an alkaline buffer salt of sodium bicarbonate and sodium chloride.

[0108] Enzyme-inorganic hybrid nanoflower particles were mixed with an aqueous solution of alkaline buffer salt, polyvinylpyrrolidone, and porous calcium carbonate. The mass ratio of enzyme-inorganic hybrid nanoflower particles to alkaline buffer salt was 1:2; the mass ratio of enzyme-inorganic hybrid nanoflower particles to polyvinylpyrrolidone was 2:5; and the mass ratio of enzyme-inorganic hybrid nanoflower particles to porous calcium carbonate was 10:23.

[0109] The mixture was dried in a fluidized bed at 50°C to allow the alkaline buffer salt to fully coat the surface of the enzyme-inorganic hybrid nanoflower particles until a composite enzyme with a water content of less than 10% was obtained.

[0110] Example 5

[0111] The compound enzyme in this embodiment was prepared by the following method:

[0112] 1) Porcine liver esterase, organophosphatase, papain, and glucoamylase were mixed in a mass ratio of 1:1:1.5:1 to obtain a compound enzyme; wherein the enzyme activity of porcine liver esterase was 5000 U / g, the enzyme activity of organophosphatase was 35000 U / g, the enzyme activity of papain was 10000 U / g, and the enzyme activity of glucoamylase was 7000 U / g.

[0113] Dissolve Na2HPO4, KH2PO4, NaCl and KCl in water to form a PBS buffer with a pH of 7.0;

[0114] Dissolve 0.05 mol of calcium chloride to form an aqueous solution of calcium chloride;

[0115] An aqueous solution of calcium chloride was added to a mixture of the complex enzyme and PBS buffer to obtain a raw material solution; wherein the mass-to-volume ratio of the complex enzyme to the PBS buffer was 0.3 g: 1 L.

[0116] The molar ratio of calcium chloride to the complex enzyme is 0.05 mol: 0.3 g;

[0117] The raw material solution was allowed to stand at 25℃ for 24 hours. The precipitate generated by the raw material solution was collected by centrifugation (12000r / min, 10min). The precipitate was then washed three times with deionized water to obtain enzyme-inorganic hybrid nanoflower particles.

[0118] The particle size of the enzyme-inorganic hybrid nanoflower particles was measured to be 200-450 nm using a Malvern laser particle size analyzer (Mastersizer3000).

[0119] 2) Dissolve sodium bicarbonate and sodium chloride in water at a mass ratio of 30:45 to form an aqueous solution containing an alkaline buffer salt of sodium bicarbonate and sodium chloride.

[0120] Enzyme-inorganic hybrid nanoflower particles were mixed with an aqueous solution of alkaline buffer salt, polyvinylpyrrolidone, and porous calcium carbonate. The mass ratio of enzyme-inorganic hybrid nanoflower particles to alkaline buffer salt was 1:2; the mass ratio of enzyme-inorganic hybrid nanoflower particles to polyvinylpyrrolidone was 2:5; and the mass ratio of enzyme-inorganic hybrid nanoflower particles to porous calcium carbonate was 10:23.

[0121] The mixture was dried in a fluidized bed at 50°C to obtain a composite enzyme with a water content of less than 10%.

[0122] Example 6

[0123] The compound enzyme in this embodiment was prepared by the following method:

[0124] 1) Porcine liver esterase, organophosphatase, papain, and glucoamylase were mixed in a mass ratio of 1:1:1.5:1 to obtain a compound enzyme; wherein the enzyme activity of porcine liver esterase was 5000 U / g, the enzyme activity of organophosphatase was 35000 U / g, the enzyme activity of papain was 10000 U / g, and the enzyme activity of glucoamylase was 7000 U / g.

[0125] Dissolve Na2HPO4, KH2PO4, NaCl and KCl in water to form a PBS buffer with a pH of 7.4;

[0126] Dissolve 0.05 mol of calcium chloride to form an aqueous solution of calcium chloride;

[0127] An aqueous solution of calcium chloride was added to a mixture of the complex enzyme and PBS buffer to obtain a raw material solution; wherein the mass-to-volume ratio of the complex enzyme to the PBS buffer was 0.3 g: 1 L.

[0128] The molar ratio of calcium chloride to the complex enzyme is 0.05 mol: 0.3 g;

[0129] The raw material solution was allowed to stand at 20℃ for 12 hours. The precipitate generated by the raw material solution was collected by centrifugation (12000r / min, 10min). The precipitate was then washed three times with deionized water to obtain enzyme-inorganic hybrid nanoflower particles.

[0130] The particle size of the enzyme-inorganic hybrid nanoflower particles was measured to be 150-300 nm using a Malvern laser particle size analyzer (Mastersizer3000).

[0131] 2) Dissolve sodium bicarbonate and sodium chloride in water at a mass ratio of 30:45 to form an aqueous solution containing an alkaline buffer salt of sodium bicarbonate and sodium chloride.

[0132] Enzyme-inorganic hybrid nanoflower particles were mixed with an aqueous solution of alkaline buffer salt, polyvinylpyrrolidone, and porous calcium carbonate, wherein the mass ratio of enzyme-inorganic hybrid nanoflower particles to alkaline buffer salt was 1:2; then polyvinylpyrrolidone was added as a binder and porous calcium carbonate as a filler for fluidized bed granulation, wherein the mass ratio of enzyme-inorganic hybrid nanoflower particles to polyvinylpyrrolidone was 2:5 and the mass ratio of enzyme-inorganic hybrid nanoflower particles to porous calcium carbonate was 10:23.

[0133] The above mixture was dried at 50°C using a fluidized bed (Glatt W500) to obtain a composite enzyme with a water content of less than 10%.

[0134] Comparative Example 1

[0135] The enzyme preparations in this comparative example were prepared using the following method:

[0136] 1) Porcine liver esterase was immobilized; the enzyme activity of porcine liver esterase was 5000 U / g.

[0137] 2) Dissolve Na2HPO4, KH2PO4, NaCl and KCl in water to form a PBS buffer with a pH of 7.4;

[0138] Dissolve 0.05 mol of calcium chloride to form an aqueous solution of calcium chloride;

[0139] An aqueous solution of calcium chloride was added to a mixture of porcine liver esterase and PBS buffer to obtain a raw material solution; wherein the mass-to-volume ratio of porcine liver esterase to PBS buffer was 0.3 g: 1 L.

[0140] The molar ratio of calcium chloride to porcine liver esterase was 0.05 mol: 0.3 g;

[0141] The raw material solution was allowed to stand at 20-30℃ for 12-24 hours. The precipitate generated by the raw material solution was collected by centrifugation (12000r / min, 10min). The precipitate was then washed three times with deionized water to obtain enzyme-inorganic hybrid nanoflower particles.

[0142] The particle size of the enzyme-inorganic hybrid nanoflower particles was measured to be 350-450 nm using a Malvern laser particle size analyzer (Mastersizer3000).

[0143] 2) Dissolve sodium bicarbonate and sodium chloride in water at a mass ratio of 30:45 to form an aqueous solution containing an alkaline buffer salt of sodium bicarbonate and sodium chloride.

[0144] Enzyme-inorganic hybrid nanoflower particles were mixed with an aqueous solution of alkaline buffer salt, polyvinylpyrrolidone, and porous calcium carbonate. The mass ratio of enzyme-inorganic hybrid nanoflower particles to alkaline buffer salt was 1:2; the mass ratio of enzyme-inorganic hybrid nanoflower particles to polyvinylpyrrolidone was 2:5; and the mass ratio of enzyme-inorganic hybrid nanoflower particles to porous calcium carbonate was 10:23.

[0145] The mixture was dried in a fluidized bed at 50°C to obtain an enzyme preparation with a water content of less than 10%.

[0146] Comparative Example 2

[0147] The enzyme preparations in this comparative example were prepared using the following method:

[0148] 1) Immobilize the organophosphatase; the enzyme activity of the organophosphatase is 35000 U / g;

[0149] 2) Dissolve Na2HPO4, KH2PO4, NaCl and KCl in water to form a PBS buffer with a pH of 7.4;

[0150] Dissolve 0.05 mol of calcium chloride to form an aqueous solution of calcium chloride;

[0151] An aqueous solution of calcium chloride was added to a mixture of organophosphatase and PBS buffer to obtain a raw material solution; wherein the mass-to-volume ratio of organophosphatase to PBS buffer was 0.3 g: 1 L.

[0152] The molar ratio of calcium chloride to organophosphatase was 0.05 mol: 0.3 g;

[0153] The raw material solution was allowed to stand at 20-30℃ for 12-24 hours. The precipitate generated by the raw material solution was collected by centrifugation (12000r / min, 10min). The precipitate was then washed three times with deionized water to obtain enzyme-inorganic hybrid nanoflower particles.

[0154] The particle size of the enzyme-inorganic hybrid nanoflower particles was measured to be 300-400 nm using a Malvern laser particle size analyzer (Mastersizer3000).

[0155] 2) Dissolve sodium bicarbonate and sodium chloride in water at a mass ratio of 30:45 to form an aqueous solution containing an alkaline buffer salt of sodium bicarbonate and sodium chloride.

[0156] Enzyme-inorganic hybrid nanoflower particles were mixed with an aqueous solution of alkaline buffer salt, polyvinylpyrrolidone, and porous calcium carbonate. The mass ratio of enzyme-inorganic hybrid nanoflower particles to alkaline buffer salt was 1:2; the mass ratio of enzyme-inorganic hybrid nanoflower particles to polyvinylpyrrolidone was 2:5; and the mass ratio of enzyme-inorganic hybrid nanoflower particles to porous calcium carbonate was 10:23.

[0157] The mixture was dried in a fluidized bed at 50°C to obtain an enzyme preparation with a water content of less than 10%.

[0158] Comparative Example 3

[0159] The enzyme preparations in this comparative example were prepared using the following method:

[0160] 1) Papain was immobilized; the enzyme activity of papain was 10000 U / g.

[0161] 2) Dissolve Na2HPO4, KH2PO4, NaCl and KCl in water to form a PBS buffer with a pH of 7.4;

[0162] Dissolve 0.05 mol of calcium chloride to form an aqueous solution of calcium chloride;

[0163] An aqueous solution of calcium chloride was added to a mixture of papain and PBS buffer to obtain a raw material solution; wherein the mass-to-volume ratio of papain to PBS buffer was 0.3 g: 1 L.

[0164] The molar ratio of calcium chloride to papain was 0.05 mol: 0.3 g;

[0165] The raw material solution was allowed to stand at 20-30℃ for 12-24 hours. The precipitate generated by the raw material solution was collected by centrifugation (12000r / min, 10min). The precipitate was then washed three times with deionized water to obtain enzyme-inorganic hybrid nanoflower particles.

[0166] The particle size of the enzyme-inorganic hybrid nanoflower particles was measured to be 200-300 nm using a Malvern laser particle size analyzer (Mastersizer3000).

[0167] 2) Dissolve sodium bicarbonate and sodium chloride in water at a mass ratio of 30:45 to form an aqueous solution containing an alkaline buffer salt of sodium bicarbonate and sodium chloride.

[0168] Enzyme-inorganic hybrid nanoflower particles were mixed with an aqueous solution of alkaline buffer salt, polyvinylpyrrolidone, and porous calcium carbonate. The mass ratio of enzyme-inorganic hybrid nanoflower particles to alkaline buffer salt was 1:2; the mass ratio of enzyme-inorganic hybrid nanoflower particles to polyvinylpyrrolidone was 2:5; and the mass ratio of enzyme-inorganic hybrid nanoflower particles to porous calcium carbonate was 10:23.

[0169] The mixture was dried in a fluidized bed at 50°C to obtain an enzyme preparation with a water content of less than 10%.

[0170] Comparative Example 4

[0171] The enzyme preparations in this comparative example were prepared using the following method:

[0172] 1) Immobilize glucoamylase; wherein the enzyme activity of glucoamylase is 7000 U / g;

[0173] Dissolve Na2HPO4, KH2PO4, NaCl and KCl in water to form a PBS buffer with a pH of 7.4;

[0174] Dissolve 0.05 mol of calcium chloride to form an aqueous solution of calcium chloride;

[0175] An aqueous solution of calcium chloride was added to a mixture of glucoamylase and PBS buffer to obtain a raw material solution; wherein the mass-to-volume ratio of glucoamylase to PBS buffer was 0.3 g: 1 L.

[0176] The molar ratio of calcium chloride to glucoamylase was 0.05 mol: 0.3 g;

[0177] The raw material solution was allowed to stand at 20-30℃ for 12-24 hours. The precipitate generated by the raw material solution was collected by centrifugation (12000r / min, 10min). The precipitate was then washed three times with deionized water to obtain enzyme-inorganic hybrid nanoflower particles.

[0178] The particle size of the enzyme-inorganic hybrid nanoflower particles was measured to be 250-400 nm using a Malvern laser particle size analyzer (Mastersizer3000).

[0179] 2) Dissolve sodium bicarbonate and sodium chloride in water at a mass ratio of 30:45 to form an aqueous solution containing an alkaline buffer salt of sodium bicarbonate and sodium chloride.

[0180] Enzyme-inorganic hybrid nanoflower particles were mixed with an aqueous solution of alkaline buffer salt, polyvinylpyrrolidone, and porous calcium carbonate. The mass ratio of enzyme-inorganic hybrid nanoflower particles to alkaline buffer salt was 1:2; the mass ratio of enzyme-inorganic hybrid nanoflower particles to polyvinylpyrrolidone was 2:5; and the mass ratio of enzyme-inorganic hybrid nanoflower particles to porous calcium carbonate was 10:23.

[0181] The mixture was dried in a fluidized bed at 50°C to obtain an enzyme preparation with a water content of less than 10%.

[0182] Comparative Example 5

[0183] The enzyme preparations in this comparative example were prepared using the following method:

[0184] 1) Porcine liver esterase, organophosphatase, papain, and glucoamylase were mixed in a mass ratio of 1:1:1.5:1 to obtain a compound enzyme; wherein the enzyme activity of porcine liver esterase was 5000 U / g, the enzyme activity of organophosphatase was 35000 U / g, the enzyme activity of papain was 10000 U / g, and the enzyme activity of glucoamylase was 7000 U / g.

[0185] 2) Dissolve sodium bicarbonate and sodium chloride in water at a mass ratio of 30:45 to form an aqueous solution containing an alkaline buffer salt of sodium bicarbonate and sodium chloride.

[0186] The complex enzyme was mixed with an aqueous solution of alkaline buffer salt, polyvinylpyrrolidone, and porous calcium carbonate. The mass ratio of the complex enzyme to the alkaline buffer salt was 1:2; the mass ratio of the complex enzyme to polyvinylpyrrolidone was 2:5; and the mass ratio of the complex enzyme to porous calcium carbonate was 10:23.

[0187] The mixture was dried in a fluidized bed at 50°C to obtain an enzyme preparation with a water content of less than 10%.

[0188] Experimental Example 1

[0189] Microbial detection: The microbial content of the compound enzymes prepared in the examples and comparative examples was detected by the method of GB / T 24691-2009, and the results are shown in Table 1.

[0190] Table 1

[0191]

[0192] The reference value for coliform bacteria is ≤3, and the reference value for bacterial count is ≤1000.

[0193] As shown in Table 1, the most probable number of coliform bacteria and the number of bacterial groups in the compound enzyme prepared in the embodiments of the present invention both meet the standard of GB / T 24691-2009.

[0194] Experimental Example 2

[0195] The compound enzyme agent of the present invention and the enzyme preparation of the comparative example are used as detergents to remove pesticide residues on the surface of snow peas.

[0196] 1. Preparation of pesticide-coated snow peas

[0197] 1) Prepare snow peas of similar size, without obvious breaks, blemishes, insect holes, openings at the edges, or damage, and without pesticide dipping treatment.

[0198] 2) Prepare pesticide emulsions according to the method in Appendix A of GB / T24691-2022 and apply them to snow peas. The prepared pesticide emulsions include 50 wt.% cypermethrin and 50 wt.% propoxur.

[0199] 2. How to wash snow peas

[0200] Two sets of experiments were conducted in parallel.

[0201] Group 1: Includes four samples of snow peas, 80g each. One sample was soaked for 0 minutes (no treatment), the second sample was soaked in water for 5 minutes, the third sample was soaked in water for 10 minutes, and the fourth sample was soaked in water for 15 minutes. All washed snow peas and washing solutions were retained.

[0202] The second group consists of four samples of snow peas, each weighing 80g. One sample was soaked for 0 minutes (without any treatment), the second sample was soaked for 5 minutes using the compound enzyme agent of Example 1 of this invention, the third sample was soaked for 10 minutes using the compound enzyme agent of Example 1 of this invention, and the fourth sample was soaked for 15 minutes using the compound enzyme agent of Example 1 of this invention.

[0203] The detergents used in Examples 2-6 and Comparative Examples 1-5 were used to soak and wash the snow peas according to the method in Example 1. All washed snow peas and the washing solution were retained.

[0204] The specific washing methods described above shall be performed in accordance with Appendix A of GB / T24691—2022.

[0205] 3. Evaluation of pesticide residue removal effect on snow pea surface

[0206] Refer to Appendix A of GB / T24691—2022 for the evaluation method of pesticide removal effect of vegetable and fruit detergents. Take snow peas (including all samples before and after soaking) from the above two groups, and use homogenization, extraction and concentration to obtain the test solution for determining the pesticide residue on the surface of snow peas.

[0207] Pesticide residues in the test solution and washing solution were determined by high performance liquid chromatography (HPLC). The content of cypermethrin was calculated using the standard curve method in GB / 5009.110-2003, and the content of propoxur was calculated using the standard curve method in GB / T20769-2008. The results are shown in Tables 3 and 4. The pesticide removal rate on the surface of the snow peas was calculated based on the pesticide residue levels. The pesticide removal effect was evaluated by the ratio of the pesticide removal rate by the detergent to the pesticide removal rate by water. The pesticide removal rate on the surface of the snow peas, the pesticide residue rate in the washing solution, and the pesticide removal effect are shown in Table 5.

[0208] The reference conditions for high performance liquid chromatography are as follows:

[0209] Mobile phase A: Methanol: Water: Glacial acetic acid = 80:20:0.1;

[0210] Mobile phase B: Water;

[0211] The mobile phase gradient table is shown in Table 2;

[0212] Chromatographic column: C18 column, 250mm × 4.6mm (inner diameter), particle size 10μm, column temperature 30℃;

[0213] Injection volume: 20 μL;

[0214] Detector: Ultraviolet detector, wavelength 276nm;

[0215] Table 2 Mobile Phase Gradient Table

[0216] 0 60 40 0.7 8 100 0 1.0 25 60 40 0.7

[0217] Table 3. Residual amounts of cypermethrin on the surface of snow peas and in the washing solution.

[0218]

[0219]

[0220] Table 4. Residues of carbendazim on the surface of snow peas and in the washing solution.

[0221]

[0222]

[0223] Table 5. Pesticide removal effect after 10 minutes of washing.

[0224]

[0225] As shown in Tables 3-5, when using the compound enzyme agent of the present invention to soak snow peas containing cypermethrin and propoxur, the pesticide removal rate on the surface of snow peas is significantly improved compared with soaking using water and a comparative detergent. This indicates that the compound enzyme agent prepared by immobilizing a compound enzyme has a better pesticide removal effect than enzyme preparations prepared by immobilizing a single enzyme or enzyme preparations prepared by not immobilizing a compound enzyme. Specifically, after soaking with the compound enzyme agent of the present invention, the pesticide removal rate on the surface of fruits and vegetables reaches over 96%, and the pesticide removal effect meets the requirements of national standard 24691-2022, P≥4 (P=removal rate of residual pesticide by fruit and vegetable cleaning agent sample solution / removal rate of residual pesticide by water). Furthermore, the residual amount of propoxur on the surface of snow peas after washing complies with national standard NY660-2003, and the residual amount of cypermethrin complies with national standard GB2763-2021.

[0226] Experimental Example 3

[0227] The composite enzymes used in the embodiments and comparative examples of this invention were used to wash celery to remove pesticide residues from the surface.

[0228] 1. Preparation of celery with pesticide-coated surface

[0229] 1) Prepare celery that is similar in size, without obvious breaks, blemishes, insect holes, openings at the edges, or damage, and has not been treated with pesticides.

[0230] 2) The pesticide emulsion was prepared and impregnated according to the method of Experimental Example 2. The pesticide emulsion consisted of 50 wt.% cypermethrin and 50 wt.% propoxur.

[0231] 2. Methods for washing celery

[0232] The washing method in this experiment differs from that in Experiment 2 in that the first group used an alkaline saline solution for soaking. The alkaline saline solution was prepared according to the usage requirements of a commercially available alkaline salt-based fruit and vegetable detergent.

[0233] 3. Evaluation of the effect of pesticide residue removal on celery surface

[0234] The pesticide residue levels on the celery surface and in the washing solution, the pesticide removal rate on the celery surface, and the pesticide residue rate in the washing solution in this experiment were evaluated using the same methods as in Experiment 2. The results are shown in Tables 6-8.

[0235] Table 6. Residual amounts of cypermethrin on the surface of celery and in the washing solution.

[0236]

[0237]

[0238] Table 7. Residues of carbendazim on the surface of celery and in washing solution.

[0239]

[0240]

[0241] Table 8. Pesticide removal rate after 10 minutes of washing.

[0242]

[0243]

[0244] The results above show that when celery containing cypermethrin and propoxur is soaked in the compound enzyme agent of the present invention, the pesticide removal rate on the surface of the celery is not less than 88%, which is significantly better than soaking in commercially available products and comparative detergents. Moreover, there are almost no pesticide residues in the washing solution after soaking. Furthermore, the residual amount of propoxur on the surface of the celery after washing meets the national standard NY660-2003, and the residual amount of cypermethrin meets the national standard GB2763-2021.

[0245] Finally, it should be noted that the technical solution of the present invention has been clearly and completely described above in conjunction with specific embodiments. Obviously, the described embodiments are only some embodiments of the present invention, 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.

Claims

1. A method for preparing a complex enzyme agent for removing pesticide residues on vegetables and fruits, characterized by, Includes the following steps: 1) Add an aqueous solution of calcium ions to a mixture including the complex enzyme and PBS buffer to obtain a raw material solution; let the raw material solution stand at 25-40℃ for 12-24h to obtain enzyme-inorganic hybrid nanoflower particles with a particle size of 100-450 nm; the phosphate group in the PBS buffer combines with calcium ions to generate Ca3(PO4)2 crystals for immobilizing the complex enzyme, and the calcium ions in the crystals complex with the amide groups in the protein molecular backbone of the complex enzyme to generate enzyme-inorganic hybrid nanoflower particles; 2) The enzyme-inorganic hybrid nanoflower particles are coated with an alkaline buffer salt to obtain a composite enzyme agent; wherein the pH of the aqueous solution of the alkaline buffer salt is 9-10. The complex enzyme is a carboxylesterase, an organophosphatase, a papain, a bromelain, and an amylase; the pH of the PBS buffer is 6.5-7.

8. The carboxylesterase includes porcine liver esterase, and the enzyme activity of the porcine liver esterase is 5000-9000 U / g; The activity of the organophosphatase is 30,000-40,000 U / g; The papain has an enzyme activity of 8000-18000 U / g, and the bromelain has an enzyme activity of 15000-28000 U / g. The amylase includes glucoamylase, and the enzyme activity of the glucoamylase is 3000-9000 U / g; The mass ratio of carboxylesterase, organophosphatase, papain, bromelain, and amylase is (0.5-1):(1-2):(0.5-2.5):(1.5-2.5):(1-5). The mass-to-volume ratio of the complex enzyme to the PBS buffer is (0.2~0.4) g : (0.9-1.1) L; The ratio of calcium ions to the complex enzyme is (0.03-0.07) mol : (0.2-0.4) g; When coating the enzyme-inorganic hybrid nanoflower particles with an alkaline buffer salt, one or more of the following are added to assist in the granulation of the enzyme-inorganic hybrid nanoflower particles: binder, filler, granulation solvent, and stabilizer; wherein the binder is one or more of polyvinylpyrrolidone, polyvinyl alcohol, hydroxypropyl cellulose, methyl cellulose, and carboxymethyl cellulose; the filler is a carbonate; the granulation solvent is water and / or propylene glycol; and the stabilizer is propylene glycol and / or polyethylene glycol. The mass ratio of the enzyme-inorganic hybrid nanoflower particles to the alkaline buffer salt is 0.3-1.8:

2.

2. A compound enzyme agent for removing pesticide residues from fruits and vegetables, characterized in that, It is prepared by the method described in claim 1.

3. A compound enzyme detergent for removing pesticide residues from fruits and vegetables, characterized in that, The compound enzyme detergent includes the compound enzyme agent as described in claim 2.

4. The complex enzyme detergent according to claim 3, characterized by, The compound enzyme detergent also includes water, and the compound enzyme agent is mixed with water at a mass ratio of 1:500-800.

5. A method for removing pesticide residues on the surface of vegetables and fruits, characterized by, Vegetables and fruits are treated with the compound enzyme agent as described in claim 2 or the compound enzyme detergent as described in claim 3 or 4; The pesticides include at least one of carbamates and pyrethroids.

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