Biological glue for sampling pesticide residues on surfaces of fruits and vegetables

Abstract

The invention provides biological glue for sampling pesticide residues on the surfaces of fruits and vegetables, the biological glue mainly comprises the following components: casein, a dispersant, a cross-linking agent, biological starch and a preservative, and the feeding mass ratio of the biological starch to the casein is 0.15-0.25. The biogel can effectively maintain the activity of cholinesterase used for rapid pesticide residue detection at present, and has good adhesion to pesticide residues on the surfaces of fruits and vegetables. Experimental results show that the biogel has higher detection sensitivity in actual pesticide residue rapid detection application than a standard detection method. The biogel disclosed by the invention is safe and non-toxic, and has a wide application prospect.

Description

Technical Field

[0001] The invention relates to a bio-glue, in particular to a bio-glue used for sampling pesticide residues on the surface of fruits and vegetables, belonging to the technical field of analysis and detection sample extraction. Background Art

[0002] Currently, the primary method for rapid detection of pesticide residues in fruits and vegetables in China utilizes the inhibitory effects of organophosphorus and carbamate pesticides on cholinesterase. This is primarily used for on-site food safety inspections at farmers' markets, where rapid cholinesterase test cards are used for colorimetric detection. Laboratories typically employ spectrophotometric colorimetric detection. In the simple practical application of the cholinesterase inhibition method for detecting pesticide residues on fruits and vegetables, sampling for pesticide residues on fruit and vegetable surfaces is typically performed by soaking the plants in water or aqueous solutions to avoid inactivation of cholinesterase by organic solvents. However, due to the poor water solubility of most pesticides, traditional sampling methods result in low extraction efficiency, significantly reducing detection sensitivity and easily leading to missed detections, resulting in food safety vulnerabilities. Therefore, a method for efficiently extracting pesticide residues from fruit and vegetable surfaces is urgently needed. The adhesive method is a non-destructive surface cleaning or sample extraction method, but there are currently no reports of using the adhesive method for extracting pesticide residues from fruit and vegetable surfaces.

[0003] Currently, common bioadhesives on the market are mostly used for traditional applications such as bonding and sealing (a biocollagen composition extracted from bullfrog and its preparation method, CN119424661A; a medical dressing containing collagen and its preparation method, CN119679996A; a bioadhesive and a bio-wood and grass health board using the bioadhesive, CN119639409A; a method for preparing bioadhesive water, CN119463805A; and bioadhesive and its preparation method and application, CN119564919A). However, little research has been conducted on their ability to protect enzyme activity. When traditional chemical adhesives come into contact with cholinesterase, they may inhibit enzyme activity due to interactions between their chemical components or affect the enzyme's catalytic efficiency due to physical structural limitations. Therefore, developing a bioadhesive that can both meet surface adhesion requirements and maintain cholinesterase activity holds significant promise for rapid detection of pesticide residues in fruits and vegetables. Summary of the Invention

[0004] The purpose of the present invention is to provide a bio-glue for sampling pesticide residues on the surface of fruits and vegetables. The bio-glue can not only provide effective adhesion and extraction of pesticide residues on the surface of fruits and vegetables, but also effectively protect the activity of cholinesterase, thereby improving the sensitivity and accuracy of subsequent detection.

[0005] The technical solutions of the present invention are as follows:

[0006] In a first aspect, the present invention provides a bio-glue for sampling pesticide residues on the surface of fruits and vegetables, wherein the bio-glue is mainly composed of the following components: casein, a dispersant, a cross-linking agent, biological starch and a preservative, wherein the mass ratio of biological starch to casein is 0.15-0.25.

[0007] Furthermore, the biological starch is corn starch.

[0008] Furthermore, the cross-linking agent is zinc oxide or zinc acetate or a combination thereof. More preferably, the cross-linking agent consists of zinc oxide and zinc acetate in a mass ratio of 1:1.

[0009] Furthermore, based on the total mass of biological starch and casein, the cross-linking agent accounts for 4%-6%.

[0010] Furthermore, the dispersant is urea or trisodium phosphate or a combination thereof. More preferably, the dispersant consists of urea and trisodium phosphate in a mass ratio of 9:2.

[0011] Furthermore, based on the total mass of biological starch and casein, the dispersant accounts for 45%-48%.

[0012] Furthermore, the preservative is nisin.

[0013] Furthermore, based on the total mass of biological starch and casein, the preservative content is above 50 mg / kg, more preferably, the preservative content is 50-60 mg / kg.

[0014] Furthermore, the bio-glue also includes water, and the water content is 64-68% of the mass of the bio-glue.

[0015] In a second aspect, the present invention provides a method for preparing the bioglue according to the first aspect, comprising the following steps:

[0016] In a reaction vessel equipped with a stirrer, thermometer and reflux device, first add deionized water and dispersant and stir until completely dissolved;

[0017] Under slow stirring, add zinc oxide, biological starch and casein in sequence, raise the temperature to 80±2℃, and keep the temperature to react for more than 1.5 hours;

[0018] Add zinc acetate solution to the reaction system and continue the reaction at 80±2℃ for more than 2 hours;

[0019] Cool the reaction system to 50-60°C, add 1-5 drops of polyoxypropylene glycerol ether and stir for 10 minutes;

[0020] The pH value of the system is adjusted to 7-7.5 with sodium hydroxide solution, and then nisin is added and stirred evenly to obtain the bio-glue.

[0021] In a third aspect, the present invention provides a use of the bio-glue described in the first aspect for sampling pesticide residues on the surface of fruits and vegetables.

[0022] In a fourth aspect, the present invention provides a rapid pesticide residue detection card, which has both sampling and detection functions, including an immobilized enzyme part for sampling and a color developer part for detection, wherein the immobilized enzyme part is obtained by coating the biological glue described in the first aspect on a test strip.

[0023] Furthermore, the color developer portion is obtained by coating a chemical color developer solution on a test strip, and the chemical color developer is some common cholinesterase color developers in the art.

[0024] Furthermore, the fixed enzyme part and the color developer part are located on the left and right sides of the test card. After sticking the fixed enzyme part on the left to the surface of fruits and vegetables, it is combined with the color developer part on the right to observe whether the pesticide residues exceed the standard.

[0025] Furthermore, the fixed enzyme part is located below the color developer part. After the fixed enzyme part located below is adhered to the surface of fruits and vegetables, it is combined with the color developer part above to observe whether the pesticide residue exceeds the standard.

[0026] The experiment on the effect of the bio-glue on the activity of cholinesterase shows that the bio-glue does not affect the activity of the enzyme, and in practical applications has better detection sensitivity than the existing cholinesterase inhibition detection method.

[0027] By optimizing the formula and process, effective protection of cholinesterase activity is achieved. The bio-glue has stable performance, good water resistance, and good adhesion to a variety of pesticides. It is suitable for rapid detection of pesticide residues in fruits and vegetables using the cholinesterase method.

[0028] Compared with the prior art, the present invention has the following advantages:

[0029] 1) Cholinesterase activity retention rate ≥ 95% (stored at 25°C for 30 days);

[0030] 2) Adhesion force reaches 12.5N / cm 2 , supports non-destructive sampling of various types of pesticide residues on the surface of fruits and vegetables;

[0031] 3) The detection sensitivity of the pesticide residue rapid test card can be increased by more than 10 times compared with the standard immersion sampling. BRIEF DESCRIPTION OF THE DRAWINGS

[0032] Figure 1 This is the test result diagram of the first batch of samples in Application Example 2.

[0033] Figure 2 This is the detection effect diagram of the second batch of samples in Application Example 2.

[0034] Figure 3This is the detection effect diagram of the third batch of samples in Application Example 2. DETAILED DESCRIPTION

[0035] Example 1:

[0036] In a 250mL four-necked round-bottom flask equipped with a stirrer, thermometer, and reflux device, first add 100g of deionized water, 3.2g of trisodium phosphate, and 14.5g of urea and stir until completely dissolved. Then, under slow stirring, add 0.8g of zinc oxide, 4.8g of corn starch, and 32g of casein (the mass ratio of the materials is 0.15) in sequence, heat to (80±2)°C, and keep the reaction for 1.5 hours. Add the pre-dissolved zinc acetate solution (0.8g of zinc acetate + 15g of deionized water) to the reaction system, and continue to react at (80±2)°C for 2 hours. Cool the reaction system to 50°C-60°C, add a few drops of polyoxypropylene glycerol ether (defoaming agent), and stir for 10 minutes. Adjust the pH of the system to 7-7.5 with pre-dissolved sodium hydroxide solution (1.0g of sodium hydroxide + 5g of deionized water). Finally, add 2mg of nisin and stir evenly to obtain the No. 1 bio-glue.

[0037] Comparative Example 1:

[0038] The other steps were the same as in Example 1, except that the mass ratio of corn starch to casein was reduced to 0.14, resulting in Bio-Glue No. 2. Compared with Bio-Glue No. 1, Bio-Glue No. 2 had a lower starch content and higher cost. A more significant change was that the glue strands became longer, which easily formed spherical agglomerates during the adhesive removal process on fruit and vegetable surfaces, affecting the removal of pesticide residues. Furthermore, the water resistance of the glue film was reduced.

[0039] Comparative Example 2:

[0040] The other steps were the same as in Example 1, except that the mass ratio of corn starch to casein was increased to 0.26 to obtain No. 3 bio-glue. Compared with No. 1 bio-glue, No. 3 bio-glue had a significantly increased starch content and a reduced cost. However, as the casein content decreased too much, the effect of the glue on the removal of pesticide residues, especially organophosphorus pesticide residues, decreased.

[0041] Comparative Example 3:

[0042] The other steps were the same as in Example 1, except that the cross-linking agent mass ratio was reduced from 4.3% to 3.5%, yielding Bio-Glue No. 4. Compared to Bio-Glue No. 1, Bio-Glue No. 4 exhibited a lower degree of cross-linking, increased water solubility, decreased water resistance, prolonged drying time, and significantly decreased viscosity, resulting in insufficient adhesion to pesticide residues on fruit and vegetable surfaces.

[0043] Comparative Example 4:

[0044] The other steps were the same as in Example 1, except that the crosslinking agent mass ratio was increased from 4.3% to 6.5%, yielding Bio-glue No. 5. Compared to Bio-glue No. 1, Bio-glue No. 5 exhibited a higher degree of crosslinking, significantly increased water resistance, a shorter drying time, and a significant increase in glue viscosity due to the significantly increased crosslinking agent mass ratio. However, the glue's viscosity continued to increase significantly with prolonged storage time, and within the typical storage period (12 months), it formed a hard lump and could not be used normally.

[0045] Comparative Example 5:

[0046] The other steps were the same as in Example 1, except that the mass ratio of the dispersant was reduced from 48% to 44%, yielding Bio-glue No. 6. Compared to Bio-glue No. 1, Bio-glue No. 6, due to the reduced mass ratio of the dispersant, was insufficient to fully disrupt the hydrogen bonds within the casein molecules, fully exposing the reactive and polar groups. This hindered the subsequent cross-linking reaction, reduced the bonding strength, and affected the subsequent sampling and collection effect.

[0047] Comparative Example 6:

[0048] The other steps were the same as in Example 1, except that the dispersant feed ratio was increased from 48% to 49%, yielding Bio-glue No. 7. Compared to Bio-glue No. 1, Bio-glue No. 7 exhibited a significant increase in solution alkalinity due to the significantly increased dispersant feed ratio, requiring readjustment of the acidity in subsequent reactions. This resulted in a series of adverse effects, such as increased operating and feed costs and difficulty controlling the cross-linking reaction.

[0049] Comparative Example 7:

[0050] The other steps are the same as those in Example 1, except that the mass ratio of the preservative nisin is changed from 54 mg / kg to 40 mg / kg to obtain bio-glue No. 8. Compared with bio-glue No. 1, bio-glue No. 8 has a lower mass ratio of the preservative, which causes the product to become moldy during normal storage period and conditions, affecting product quality. Continuing to increase the amount of preservative to far more than 50 mg / kg does not significantly increase the storage period, but increases the cost significantly. Other types of preservatives, such as phenol or benzoic acid chemical preservatives, may cause adverse effects on cholinesterase activity and possible interference with detection in this product, so they are not considered and no relevant experimental verification is performed.

[0051] Application Example 1: Effect of Biogel on Cholinesterase Activity in Photometric Detection

[0052] Experimental purpose: To verify the effect of the bio-glue of the present invention on the activity of cholinesterase in the photometric detection method.

[0053] Experimental Materials:

[0054] The bioglue of the present invention (prepared according to Example 1).

[0055] Cholinesterase (AChE) solution.

[0056] Acetylthiocholine (ATCh) served as the substrate.

[0057] 5,5'-Dithiobisnitrobenzoic acid (DTNB) was used as a color developing agent.

[0058] Dichlorvos standard solution.

[0059] UV-visible spectrophotometer.

[0060] Experimental steps:

[0061] Enzyme activity assay:

[0062] Prepare the cholinesterase reaction system: add the cholinesterase solution, acetylthiocholine and 5,5'-dithiobisnitrobenzoic acid into the reaction system.

[0063] The absorbance change of the reaction system was measured at a wavelength of 412 nm, and the activity of cholinesterase was calculated.

[0064] Biogel treatment group:

[0065] The bioglue of the present invention is added into the reaction system to allow the bioglue to fully contact with the cholinesterase.

[0066] Using the same concentration of biogel solution as a reference, the absorbance change was measured under the same conditions and the cholinesterase activity was calculated.

[0067] Inhibitor control group:

[0068] A standard solution of dichlorvos was added to the reaction system to determine the inhibition of cholinesterase activity.

[0069] Bioglue + inhibitor group:

[0070] The bio-glue of the present invention and a dichlorvos standard solution were added to the reaction system simultaneously, and the activity inhibition of cholinesterase was measured.

[0071] Experimental results:

[0072] Table 1: Effect of adding biogel to the cholinesterase reaction system on enzyme activity

[0073] Standard reaction system set Join the biogel treatment group Absorbance (N=5) 0.919±0.02 0.920±0.03

[0074] Table 2: Effect of biogel on cholinesterase inhibition assay

[0075] Absorbance (N=5) Cholinesterase inhibition rate (N=5) Inhibitor control group 0.846±0.05 69.09±1.02％ Bioglue + inhibitor group 0.848±0.06 69.12±1.05％

[0076] Enzyme activity determination: As shown in Table 1, the cholinesterase activity of the biogel-treated group was comparable to that of the untreated group, indicating that biogel had no significant effect on cholinesterase activity.

[0077] Inhibitor control group: As shown in Table 2, the dichlorvos standard solution significantly inhibited the activity of cholinesterase and the absorbance decreased.

[0078] Bioglue + inhibitor group: As shown in Table 2, in the presence of bioglue, the degree of inhibition of cholinesterase was comparable to that of the inhibitor control group, indicating that bioglue did not affect the inhibitory effect of dichlorvos on cholinesterase.

[0079] Conclusion: The biogel of the present invention does not affect the activity of cholinesterase in the photometric detection method, nor does it affect the inhibitory effect of the inhibitor on the enzyme activity. It is suitable for detection methods related to the cholinesterase inhibition method.

[0080] Application Example 2: Effect of Bio-Glue on Cholinesterase Activity in the Application of Pesticide Residue Rapid Test Card

[0081] Experimental purpose: To verify the effect of the bio-glue of the present invention on cholinesterase activity in the detection of pesticide residue rapid test cards and the effect of the bio-glue on the sampling of pesticide residues on the surface of fruits and vegetables.

[0082] Experimental Materials:

[0083] The bioglue of the present invention (prepared according to Example 1).

[0084] Pesticide residue rapid test card (detection target is organophosphorus pesticides, such as dichlorvos).

[0085] Green vegetable samples.

[0086] Dichlorvos standard solutions (low and high concentrations).

[0087] Cleaning fluid, water.

[0088] Experimental steps:

[0089] Sample preparation: Select fresh green vegetable samples and spray water, low-concentration dichlorvos solution (10 times lower than the detection limit of conventional rapid test cards) and high-concentration dichlorvos solution (10 times higher than the detection limit of conventional rapid test cards) on the first, second and third batches of samples respectively.

[0090] Standard test method:

[0091] Select a group of leaves from the same part of each batch of green vegetable samples, apply cleaning solution or clean water on the surface of the green vegetables and soak for 5 minutes, or rub the leaves against each other several times, then drop the soaking liquid onto the rapid test card detection piece and observe the color development.

[0092] Biogel detection method:

[0093] Another group of leaves were selected from the same part of each batch of green vegetable samples, and the bioglue of the present invention was used to stick to the leaf surface several times. Then, clear water was dripped onto the film, and then the water was transferred to the rapid test card to observe the color development.

[0094] Result judgment: The pesticide residue test result is judged according to the color of the rapid test card. Blue is negative, light blue is weak positive, and white is positive.

[0095] Experimental results:

[0096] The first batch of samples (spray water): the standard test method and the bio-glue test method both showed blue, indicating a negative result, such as Figure 1 As shown, Figure 1 From left to right in the middle are the results of the standard test method and the results of the bio-glue sampling test.

[0097] The second batch of samples (low concentration of dichlorvos): the standard detection method showed blue (no pesticide residues were detected), and the bio-glue detection method showed light blue (weak positive), indicating that the bio-glue detection method has higher sensitivity, such as Figure 2 As shown, Figure 2 From left to right in the middle are the results of the standard test method and the results of the bio-glue sampling test.

[0098] The third batch of samples (high concentration of dichlorvos): both the standard test method and the bio-gel test method showed white (positive), such as Figure 3 As shown, Figure 3 From left to right in the middle are the results of the standard test method and the results of the bio-glue sampling test.

[0099] Conclusion: The bioglue of the present invention does not affect the activity of cholinesterase in the detection of pesticide residue rapid test cards and has higher detection sensitivity than the standard detection method.

[0100] The above embodiments are preferred implementation modes of the present invention, but the implementation modes of the present invention are not limited to the above embodiments. Any other changes, modifications, substitutions, combinations, and simplifications that do not deviate from the spirit and principles of the present invention should be considered as equivalent replacement methods and are included in the scope of protection of the present invention.

Claims

1. A bio-glue for sampling pesticide residues on the surface of fruits and vegetables, characterized in that: The bio-glue mainly consists of the following components: casein, a dispersant, a cross-linking agent, biological starch and a preservative, wherein the mass ratio of the biological starch to the casein is 0.15-0.

25.

2. The bioglue according to claim 1, wherein The cross-linking agent is zinc oxide or zinc acetate or a combination thereof. More preferably, the cross-linking agent is composed of zinc oxide and zinc acetate in a mass ratio of 1:

1.

3. The bioglue according to claim 1, wherein Based on the total mass of biological starch and casein, the cross-linking agent accounts for 4%-6%.

4. The bioglue according to claim 1, wherein The dispersant is urea or trisodium phosphate or a combination thereof. More preferably, the dispersant is composed of urea and trisodium phosphate in a mass ratio of 9:

2.

5. The bioglue according to claim 1, wherein Based on the total mass of biological starch and casein, the dispersant accounts for 45%-48%.

6. The bioglue according to claim 1, wherein Based on the total mass of biological starch and casein, the preservative content is above 50 mg / kg, more preferably, the preservative content is 50-60 mg / kg.

7. A method for preparing the bioglue according to any one of claims 1 to 6, characterized in that: The steps include: In a reaction vessel equipped with a stirrer, thermometer and reflux device, first add deionized water and dispersant and stir until completely dissolved; Under slow stirring, add zinc oxide, biological starch and casein in sequence, raise the temperature to 80±2℃, and keep the temperature to react for more than 1.5 hours; Add zinc acetate solution to the reaction system and continue the reaction at 80±2℃ for more than 2 hours; Cool the reaction system to 50-60°C, add 1-5 drops of polyoxypropylene glycerol ether and stir for 10 minutes; The pH value of the system is adjusted to 7-7.5 with sodium hydroxide solution, and then nisin is added and stirred evenly to obtain the bio-glue.

8. Use of the bioglue according to any one of claims 1 to 6 for sampling pesticide residues on the surface of fruits and vegetables.

9. A pesticide residue rapid detection card, comprising an immobilized enzyme portion and a color developer portion, characterized in that: The immobilized enzyme portion is obtained by coating the bio-glue as described in any one of claims 1 to 7 on a test strip.

10. The pesticide residue rapid detection card according to claim 8, characterized in that: The color developer part is obtained by coating the chemical color developer solution on the test strip.

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