Method for degrading pesticide residues in fruits and vegetables through cooperation of ultrasonic waves and natural enzymes

Abstract

The invention relates to the technical field of pesticide residue treatment, in particular to a method for degrading pesticide residues in fruits and vegetables through cooperation of ultrasonic waves and natural enzymes. The method for degrading the pesticide residues in the fruits and the vegetables through the cooperation of the ultrasonic waves and the natural enzymes comprises the step that in the presence of pulse ultrasonic waves, the natural oxidoreductase is used for treating the fruit and vegetable products, wherein the pulse ultrasonic waves and the natural oxidoreductase have a synergistic effect, so that the degradation of the pesticide residues is realized; the frequency of the pulse ultrasonic waves is 20-100 kHz, the power density is 0.1-1.0 W / mL, a pulse working mode is adopted, and the duty ratio is 20-80%. According to the method, the pulse ultrasonic waves with specific parameters are cooperated with the natural oxidoreductase, so that hydrophobic pesticides such as pyrethroids and organophosphorus which are generally residual and are difficult to remove in fruits and vegetables can be efficiently degraded within a short time.

Description

Technical Field

[0001] This invention relates to the field of pesticide residue treatment technology, specifically to a method for the degradation of pesticide residues in fruits and vegetables by ultrasound in conjunction with natural enzymes. Background Technology

[0002] Pesticide residues are one of the core issues affecting the food safety of fruits, vegetables, and their processed products. Currently, pyrethroids and organophosphates are widely used globally, and their residue detection rates are the highest. In addition, the residues of neonicotinoid insecticides and various fungicides are also receiving increasing attention. Many of these pesticides have high hydrophobicity and chemical stability, and conventional water washing has limited removal rates, making it difficult to meet increasingly stringent food safety standards.

[0003] Currently, the technologies for removing pesticide residues from fruits and vegetables are mainly divided into physical, chemical, and biological methods, but all have significant limitations: (1) Physical methods: such as ultrasonic treatment, the extreme conditions generated by its cavitation effect can destroy some pesticide molecules. However, the efficiency of using ultrasonic treatment alone to degrade stubborn organochlorine pesticides is low, and to achieve a high degradation rate, high energy input and long-term treatment are often required, which may lead to damage to fruit and vegetable tissues, loss of nutrients (such as heat-sensitive vitamin C), and excessive energy consumption. (2) Chemical methods: such as using oxidants such as ozone, hydrogen peroxide, or persulfate. Although these methods can improve degradation efficiency, there is a risk of introducing chemical residues, generating potentially toxic byproducts (such as bromate), and potentially changing the original flavor and color of food, which does not conform to the consumption trend of "clean label". (3) Biological methods: mainly using specific microorganisms or enzymes for degradation. Natural redox enzymes have the advantages of high catalytic specificity, mild reaction conditions, and environmental friendliness. However, in complex fruit and vegetable matrices, the use of enzymes alone has obvious bottlenecks: First, the mass transfer efficiency between strongly hydrophobic pesticides and enzymes in the aqueous phase is extremely low, making effective contact difficult; second, natural phenols, organic acids and other components present in fruit and vegetable juices may inhibit enzyme activity; third, the enzyme degradation kinetics of some structurally stable pesticides is slow and cannot meet the efficiency requirements of actual processing.

[0004] In view of this, the present invention is proposed. Summary of the Invention

[0005] The present invention aims to solve at least one of the above technical problems and provides a method for the degradation of pesticide residues in fruits and vegetables by ultrasound-assisted natural enzymes.

[0006] To achieve the above objectives, the technical solution adopted by the present invention is as follows: A method for degrading pesticide residues in fruits and vegetables using ultrasound in conjunction with natural enzymes includes: treating the fruits and vegetables with natural oxidoreductases in the presence of pulsed ultrasound; wherein the pulsed ultrasound and the natural oxidoreductases work synergistically to degrade the pesticide residues; the frequency of the pulsed ultrasound is 20-100 kHz, the power density is 0.1-1.0 W / mL, a pulse working mode is adopted, and the duty cycle is 20-80%.

[0007] Preferably, the natural redox enzyme is selected from at least one of laccase, peroxidase, carboxylesterase, and organophosphorus hydrolase.

[0008] Preferably, the peroxidase is horseradish peroxidase or soybean peroxidase.

[0009] Preferably, the natural redox enzyme is in the form of an immobilized enzyme, loaded onto magnetic nanoparticles, calcium alginate microspheres, or a porous ceramic carrier.

[0010] Preferably, the pesticide residue includes at least one of pyrethroids, organophosphates, neonicotinoids, and fungicides.

[0011] Preferably, the pesticide residue is a pyrethroid.

[0012] Preferably, the pulsed ultrasound treatment time is 5-60 min, the treatment temperature is 10°-40°C, and the pH value of the treatment environment is 3.5-7.0.

[0013] Preferably, the processing environment is a closed system, filled with inert gas to maintain a low-oxygen environment.

[0014] Preferably, the fruit and vegetable products are whole fruits, slices, fruit pulp, or pure fruit juice.

[0015] Preferably, after the synergistic treatment with pulsed ultrasound and natural redox enzymes, a static curing step is also included, with a curing time of 5-30 minutes.

[0016] Compared with the prior art, the present invention has the following beneficial effects: This invention achieves a synergistic effect beyond simple addition by combining pulsed ultrasound with natural redox enzymes using specific parameters. This results in the efficient degradation of hydrophobic pesticides such as pyrethroids and organophosphates, which are commonly found and difficult to remove from fruits and vegetables, within a short timeframe. The low-frequency pulsed ultrasound, through its mechanical stress, may induce reversible conformational flexibility in enzyme proteins. The localized extreme conditions generated by ultrasonic cavitation and free radicals can pre-activate or partially break stable bonds (such as ester bonds) in pesticide molecules. This precise bidirectional regulation of the enzyme and substrate forms the scientific basis for the efficient degradation of various complex pesticide structures using this method.

[0017] The entire process of this invention is carried out at room temperature or low temperature and near-neutral pH, falling under the category of non-thermal processing. This improves the retention rate of vitamin C in fruits and vegetables, reduces total phenol loss and color changes, and achieves no significant deterioration in sensory quality. Furthermore, the entire process does not add any exogenous chemical oxidants, relying solely on water and natural enzymes, eliminating the risk of secondary chemical contamination and meeting green processing and clean labeling requirements.

[0018] The pulsed ultrasonic equipment used in this invention is a mature technology with controllable modification costs, and it is easy to integrate with existing fruit and vegetable washing or processing production lines. The enzyme preparation can be immobilized for easy recovery and reuse, helping to control operating costs. This method provides fruit and vegetable processing enterprises with a new, efficient, safe, and environmentally friendly approach to pesticide residue control. Detailed Implementation

[0019] The technical solutions of the embodiments of the present invention will be clearly and completely described below with reference to the examples. 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 of ordinary skill in the art without creative effort are within the scope of protection of the present invention.

[0020] This invention provides a method for the degradation of pesticide residues in fruits and vegetables by ultrasound-assisted natural enzymes, comprising: treating the fruit and vegetable products with natural oxidoreductases in the presence of pulsed ultrasound; wherein the pulsed ultrasound and the natural oxidoreductases work synergistically to degrade the pesticide residues; the frequency of the pulsed ultrasound is 20-100 kHz, the power density is 0.1-1.0 W / mL, a pulse working mode is adopted, and the duty cycle is 20-80%.

[0021] The embodiments of the present invention creatively couple a pulsed ultrasonic field with specific parameters with a specific type of natural redox enzyme in the same treatment system to produce a synergistic degradation effect.

[0022] This invention is applicable to the degradation of pesticides with benzene rings, ester bonds, phospholipid bonds, and other groups in their molecular structure, such as currently widely used pyrethroids and organophosphate pesticides. It also shows good degradation potential for neonicotinoids and some fungicides. Its synergistic mechanism is mainly reflected in the following three interrelated levels: At the molecular level: reversible conformational regulation of enzyme proteins by ultrasound. The periodic mechanical stress (shear force, microjets) generated by specific low-frequency pulsed ultrasound in this invention can act on natural redox enzyme molecules in solution, inducing reversible and beneficial conformational dynamics. This adjustment may moderately loosen the higher-order structure of the enzyme protein, increasing its conformational flexibility and making previously buried active sites more easily exposed; simultaneously, it may fine-tune the orientation of key amino acid residues in the active site, optimizing its stereocompatibility with the aromatic ring structure in the target pesticide substrate molecule (such as the phenoxybenzyl group of pyrethroids and the pyridine ring of chlorpyrifos); for the hydrolysis of ester / phospholipid bonds, pre-activation mainly relies on the local extreme conditions (high temperature and high pressure, ·OH radicals) generated by ultrasonic cavitation, while the enzyme catalyzes subsequent oxidative ring-opening reactions for complete mineralization. Crucially, due to the use of a pulsed mode, the intervals between ultrasound actions provide a recovery period for the enzyme molecules, ensuring the reversibility of conformational changes and avoiding thermal denaturation and irreversible inactivation that could be caused by continuous ultrasound.

[0023] Interface and mass transfer level: Ultrasonic cavitation effect enriches and pre-activates substrates, while maximizing mass transfer. The transient microbubbles generated by ultrasonic cavitation and the local extreme high temperature and high pressure microenvironment formed during their collapse have the following effects: (1) The hydrophobic interface of the microbubbles can specifically enrich the highly hydrophobic target pesticides (such as organochlorine pesticides) in the fruit and vegetable system; (2) The local extreme conditions and the generated active free radicals (such as ·OH) can pre-activate the enriched pesticide molecules and weaken their stable chemical bonds (such as the ester bond of pyrethroids, the PO or P=S bond of organophosphorus pesticides); (3) The strong microjets and turbulence accompanying cavitation collapse can maximize the mass transfer and collision frequency between enzyme molecules and pesticide substrates in the entire reaction system, effectively solving the core bottleneck of low contact efficiency between hydrophobic pollutants and biocatalysts in the aqueous phase.

[0024] At the system level: an optimized reaction microenvironment is constructed. By controlling overall processing conditions (such as temperature and pH) and providing a suitable working environment for the enzyme, while utilizing the energy input of ultrasound to overcome some matrix inhibition effects, the efficient and stable synergistic reaction is ensured.

[0025] To achieve the aforementioned synergistic effect, the parameters of the pulsed ultrasound are set as follows: frequency of 20-100 kHz, power density of 0.1-1.0 W / mL, pulse working mode, and duty cycle of 20-80%. The power density is calculated based on the total volume of the treatment system; for example, a 500 mL treatment system corresponds to an ultrasound power of 50-500 W.

[0026] The preferred frequency range is low, between 20 and 60 kHz. Low-frequency ultrasound has a lower cavitation threshold, making it easier to generate a large number of cavitation bubbles and a violent collapse effect. This is beneficial for generating strong shear forces (for enzyme conformation regulation) and microjets (for enhanced mass transfer), while also generating a moderate amount of free radicals for substrate preactivation. High-frequency ultrasound (>200 kHz), on the other hand, tends to generate chemical free radicals, which may increase the risk of physical damage to enzymes, and the equipment cost is also higher.

[0027] The pulse mode and duty cycle (i.e., the proportion of ultrasonic emission time to one pulse cycle) also directly affect the processing effect. The duty cycle should be controlled between 20% and 80% (preferably 30% to 60%), such as working for 10 seconds and then pausing for 10 seconds. The core purpose is to provide sufficient energy during the ultrasonic working period to trigger conformational adjustment and cavitation effects; and to allow heat dissipation and partial relaxation recovery of the enzyme molecular structure during the pause period. This achieves synergistic effects while maximizing the protection of enzyme activity and stability, avoiding the cumulative thermal effects and excessive mechanical disturbances caused by continuous ultrasound.

[0028] A suitable power density ensures that a sufficiently strong cavitation effect is generated to achieve enrichment, preactivation, and enhanced mass transfer, while avoiding excessive power that could cause the solution temperature to rise too quickly (potentially leading to thermal inactivation of the enzyme) or generate excessive non-selective free radicals that attack the enzyme protein itself.

[0029] In some preferred embodiments, the natural oxidoreductases in this invention are selected from laccase, peroxidase, carboxylesterase, organophosphorus hydrolase, etc. These oxidoreductases can catalyze the oxidation reactions of various aromatic and halogenated aromatic compounds. Pyrethroids, organophosphorus compounds, and other common pesticide molecules widely contain aromatic structures such as benzene rings. Therefore, they have a high structural matching with the catalytic substrate of the enzyme, which constitutes the molecular basis for its efficient degradation.

[0030] Enzymes can be added in their conventional free form. To further improve the economy and practicality of the technology, this invention specifically covers the preparation of enzymes in the form of immobilized enzymes, such as those loaded onto magnetic nanoparticles, calcium alginate microspheres, or porous ceramics. Immobilized enzymes not only improve enzyme stability, but more importantly, they facilitate recovery and reuse after processing through methods such as magnetic separation or filtration. This provides a feasible solution for industrial continuous or batch processing and effectively controls costs.

[0031] In some preferred embodiments, the treatment temperature is 10-40°C and the pH is 3.5-7.0 to match the optimal activity range of natural oxidoreductases, while maintaining non-thermal or mild processing conditions that are friendly to the quality of fruits and vegetables. The treatment time can be flexibly adjusted according to the initial pesticide residue concentration and treatment intensity, for example, it can be 5-60 min.

[0032] The present invention employs a preferred auxiliary method: filling a closed system with an inert gas (such as nitrogen) to maintain a low-oxygen environment. Its main purpose is to reduce dissolved oxygen, thereby potentially decreasing the non-targeted oxidative loss of nutrients (such as vitamin C) by reactive oxygen species generated by ultrasonic cavitation, allowing energy to be more concentrated on the degradation of the target pesticide.

[0033] In some preferred embodiments, a static ripening step (5-30 min) may be introduced after active sonication-enzyme co-treatment. The purpose of this step is to allow the active intermediates formed in the intense sonic field to continue to undergo subsequent enzymatic or chemical transformation under relatively mild and static conditions, which may help improve the thoroughness and stability of the final degradation.

[0034] The method of this invention is applicable to various physical forms of fruit and vegetable products, including whole fruit, slices, pulp, or pure juice. For whole fruit and slices, the treatment is usually carried out in an aqueous medium; for pulp and juice, they can be directly used as the reaction system for treatment.

[0035] After co-treatment in an aqueous medium, the enzymes are typically washed away with water. When applied to fruit pulp and juice, the co-treatment step further includes enzyme inactivation and / or enzyme removal. Enzyme inactivation can be achieved through gentle heating (e.g., maintaining at 60-80°C for 1-5 minutes), adjusting the pH to the enzyme inactivation range, or pasteurization. Enzyme removal can be achieved through membrane filtration (e.g., ultrafiltration, nanofiltration), centrifugation (for immobilized enzymes or enzyme aggregates), or adsorption.

[0036] The following detailed description of the method and effect of ultrasound-assisted natural enzyme degradation of pesticide residues in fruits and vegetables is provided through several specific examples.

[0037] Example 1 Removal of cyhalothrin from strawberries This embodiment simulates the washing process of whole berries. Strawberries, with their complex skins and high pesticide residue levels, are ideal for this demonstration.

[0038] Fresh strawberries were purchased from the market and manually and evenly sprayed with a standard solution of cyhalothrin at an initial concentration of 1.5 mg / kg. The strawberries were then allowed to dry at room temperature to adhere. After drying, the following different treatments were performed.

[0039] Experimental group 1: In a buffer containing 5 U / mL laccase, pulsed sonication at 40 kHz with a duty cycle of 50% (5 s on, 5 s off) was applied simultaneously for 15 min.

[0040] Control group 1: Soaked in deionized water and manually stirred for 15 minutes.

[0041] Control group 2 (ultrasound alone): treated with pH 5.5 buffer for 15 min in a 40 kHz continuous ultrasonic cleaning tank.

[0042] Control group 3 (enzyme alone): Soaked in buffer containing laccase (5 U / mL) and gently stirred for 15 min.

[0043] Example 2: Removal of Chlorpyrifos from Tomatoes This embodiment verifies the effectiveness of the present invention in removing pesticides from the surface and possibly permeated epidermis of solanaceous vegetables.

[0044] Ripe tomatoes were purchased from the market and manually and evenly sprayed with a standard solution of chlorpyrifos at an initial concentration of 2.0 mg / kg, resulting in surface residue. Several different treatments were then administered. After treatment, the tomato skin was rinsed and wiped with ultrapure water, and then a 1 mm thick layer of the epidermis was peeled off for pesticide extraction and testing to assess the removal capacity for epidermal penetration residues.

[0045] Experimental Group 2: The washing solution containing 10 U / mL horseradish peroxidase was subjected to pulsed ultrasound at 28 kHz with a duty cycle of 60% (6 s on, 4 s off) for 1 min. Then it was allowed to stand for 10 min to mature.

[0046] Control group 4: Rinse with running water and gently brush the surface with a soft brush for 1 minute.

[0047] Control group 5 (enzyme wash alone): Soaked and brushed with a washing solution containing peroxidase (10 U / mL) for 1 min.

[0048] Example 3 Synergistic removal of imidacloprid and pyraclostrobin from cabbage leaves This embodiment simulates the batch processing of leafy vegetables, while verifying the broad-spectrum removal capability of the present invention for mixed residues of different types of pesticides, and demonstrating the industrial application potential of immobilized enzymes.

[0049] Intact outer leaves of cabbage were purchased from the market and manually sprayed with a mixed standard solution of imidacloprid (a neonicotinoid) and pyraclostrobin (a methoxyacrylate fungicide), with an initial concentration of 1.0 mg / kg for both. Several different treatments were then administered. After each treatment, immobilized enzyme microspheres (which can be recycled) were separated using a filter, and the cabbage leaves were removed, drained, and sampled for testing of the residues of the two pesticides.

[0050] The laccase used below was immobilized on calcium alginate microspheres.

[0051] Experimental group 3: In a buffer containing 0.25 g immobilized laccase microspheres (enzyme activity loading of approximately 200 U / g), pulsed sonication at 35 kHz with a duty cycle of 40% (4 s on, 6 s off) was applied simultaneously for 10 min.

[0052] Control group 6 (traditional water washing): Rinse with clean water in a stirring tank for 10 minutes.

[0053] Control group 7 (sonication alone): In buffer solution, continuous sonication at 35 kHz was applied for 10 min.

[0054] Control group 8 (immobilized enzyme wash): Washed in buffer containing 0.25 g immobilized laccase microspheres (enzyme activity loading of approximately 200 U / g) with stirring for 10 min.

[0055] Experimental Example The samples treated in the above embodiments were subjected to pesticide degradation rate testing, vitamin C testing, color testing, and sensory evaluation. The specific methods are as follows: 1. Sample pretreatment Take the edible parts of the processed fruits and vegetables, rinse them quickly with deionized water, and blot dry with filter paper. Use a food-grade homogenizer to homogenize them thoroughly and set aside.

[0056] 2. Pesticide residue detection Extraction and purification: Accurately weigh 10.00 g of homogenized sample and process it using the modified QuEChERS method. Add 10 mL of acetonitrile, vortex for 2 min, then add an extraction salt packet containing 4 g anhydrous MgSO4, 1 g NaCl, 1 g sodium citrate, and 0.5 g disodium hydrogen citrate. After vigorous shaking, centrifuge at 4000 r / min for 5 min. Transfer 6 mL of the supernatant to a purification tube containing 150 mg anhydrous MgSO4 and 50 mg PSA, vortex, centrifuge, and filter the supernatant through a 0.22 μm filter membrane for analysis.

[0057] Pyrethroids, organophosphates, etc.: Analyzed using gas chromatography-tandem mass spectrometry (GC-MS / MS). The chromatographic column was HP-5MS UI (30 m × 0.25 mm × 0.25 μm); the temperature program was: initial temperature 60°C, gradually increased to 300°C; the ion source was an EI source; and the detection mode was multiple reaction monitoring (MRM).

[0058] Neonicotinoids, etc.: analyzed using liquid chromatography-tandem mass spectrometry (LC-MS / MS). The chromatographic column was a C18 column (100 mm × 2.1 mm, 1.8 μm); the mobile phase was 0.1% formic acid aqueous solution and methanol, with gradient elution; the ion source was an electrospray ionization (ESI) source; and the detection mode was MRM.

[0059] The external standard method was used for quantification. The formula for calculating the pesticide degradation rate is: Degradation rate (%) = [(C0 - C...] t [) / C0]×100%; where C0 is the average detection concentration (mg / kg) of pesticide in the blank control group sample, representing the initial pollution level; C t The average detection concentration (mg / kg) of pesticides in the samples of each treatment group.

[0060] 3. Vitamin C retention rate test High-performance liquid chromatography (HPLC) was used. 5.00 g of homogenized sample was weighed, extracted with 2% metaphosphoric acid solution, diluted to volume, filtered, and then injected for analysis. Chromatographic conditions: C18 column; mobile phase: 0.1% oxalic acid aqueous solution; detection wavelength: 254 nm.

[0061] The formula for calculating the vitamin C retention rate is: Retention rate (%) = (Ct / Cs) × 100%; where Cs is the average vitamin C content (mg / 100g) in the blank control group sample, and Ct is the average vitamin C content in each treatment group sample.

[0062] 4. Color (ΔE) detection The L (lightness), a (red-green value), and b* (yellow-blue value) of the processed vegetable samples were directly measured using a colorimeter, and the total color difference was calculated. At least 5 different points were measured for each sample, and the average value was taken.

[0063] The detection results of Examples 1-3 are shown in Tables 1-3.

[0064] Table 1 Detection results of Example 1 Table 2 Detection Results of Example 2 Table 3 Detection results of Example 3 .

[0065] The above results show that the pulsed ultrasound-assisted natural enzyme treatment method of the present invention has a significant degradation effect on various common pesticides, and can effectively maintain the vitamin C content in fruits and vegetables, and only causes slight color changes that are difficult to detect with the naked eye, thus achieving a balance between efficient residue removal and good quality preservation.

[0066] The above description is merely a preferred embodiment of the present invention and is not intended to limit the invention. Various modifications and variations can be made to the present invention by those skilled in the art. Any modifications, equivalent substitutions, improvements, etc., made within the spirit and principles of the present invention should be included within the scope of protection of the present invention.

Claims

1. A method for the synergistic degradation of pesticide residues in fruits and vegetables by ultrasound and natural enzymes, characterized in that, include: The fruit and vegetable products are treated with natural oxidoreductase in the presence of pulsed ultrasound; wherein the pulsed ultrasound and the natural oxidoreductase work synergistically to degrade the pesticide residues; the frequency of the pulsed ultrasound is 20-100 kHz, the power density is 0.1-1.0 W / mL, and a pulse working mode is adopted with a duty cycle of 20-80%.

2. The method as described in claim 1, characterized in that, The natural redox enzyme is selected from at least one of laccase, peroxidase, carboxylesterase, and organophosphorus hydrolase.

3. The method as described in claim 2, characterized in that, The peroxidase is horseradish peroxidase or soybean peroxidase.

4. The method as described in claim 1, characterized in that, The natural redox enzyme is in the form of an immobilized enzyme, loaded onto magnetic nanoparticles, calcium alginate microspheres, or porous ceramic carriers.

5. The method as described in claim 1, characterized in that, The pesticide residues include at least one of pyrethroids, organophosphates, neonicotinoids, and fungicides.

6. The method as described in claim 5, characterized in that, The pesticide residues mentioned are pyrethroids.

7. The method as described in claim 1, characterized in that, The pulsed ultrasound treatment time is 5-60 min, the treatment temperature is 10°-40°C, and the pH value of the treatment environment is 3.5-7.

0.

8. The method as described in claim 1, characterized in that, The processing environment is a closed system, filled with inert gas to maintain a low-oxygen environment.

9. The method as described in claim 1, characterized in that, The fruit and vegetable products mentioned are whole fruits, slices, fruit pulp, or pure fruit juice.

10. The method as described in claim 1, characterized in that, After the synergistic treatment with pulsed ultrasound and natural redox enzymes, a static curing step is also included, with a curing time of 5-30 minutes.

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