Preparation of vegetable protection preparation and application of vegetable protection preparation in polycyclic aromatic hydrocarbon polluted soil

Abstract

The invention provides a preparation method of a vegetable protection preparation and application of the vegetable protection preparation in polycyclic aromatic hydrocarbon contaminated soil. The preparation method sequentially comprises the steps of preparation of a carbon-based nano colloidal material preparation, loading of carotenoid, storage of the vegetable protection preparation and application of the vegetable protection preparation in the polycyclic aromatic hydrocarbon contaminated soil. The carotenoid is loaded through the carbon-based nano-colloid material, the PAHs pollution stress resistance of plants is improved, after the carbon-based nano-colloid material is applied, crops have the advantages of being efficient and rapid in plant pollution stress resistance response, and the carbon-based nano-colloid material plays a positive role in improving utilization of PAHs polluted soil in agricultural production in China.

Description

Technical Field

[0001] The invention relates to the technical field of plant protection agents, and in particular to a preparation method of a vegetable protection preparation and application thereof in soil contaminated by polycyclic aromatic hydrocarbons. Background Art

[0002] Polycyclic aromatic hydrocarbons (PAHs) refer to aromatic hydrocarbon compounds containing two or more benzene rings. PAHs are identified as major organic pollutants that affect human health due to their carcinogenic, teratogenic and mutagenic biological toxicity. At the same time, PAHs can also endanger human health through the food chain. At present, there is also a potential risk of PAHs pollution in vegetable fields in Jiangsu Province. According to reports, the ∑PAHs content in the main farmland soils in Jiangsu Province ranges from 45.6 to 2286.8 ppb. The soils in Xinghua City and Wujiang City have reached a severe pollution level; Yixing City and Nanjing City are slightly polluted. Soil contaminated by PAHs will also affect the yield and quality of vegetables. Therefore, there is an urgent need for an agricultural technology that can effectively prevent and control soil PAHs pollution. It is environmentally friendly and highly replicable, and can be promoted and applied to vegetable soils where PAHs pollution occurs across the country.

[0003] At present, the methods for treating PAHs-contaminated soil are mainly on-site chemical oxidation and microbial degradation, but these methods often take a long time to process and cause great damage to the environment; therefore, it is very important to find a carrier to specifically supplement the plant's protective agent against environmental stress.

[0004] The technology of nanomaterials in pollutant control is relatively mature. 1% magnetite nanoparticles applied to soil can effectively reduce 42.5% of As and 49% of PAHs accumulation in corn leaves; Cai et al. found that foliar spraying of SiO2 and ZnO nanoparticles can effectively reduce 20.4%-54.9% of PAHs accumulation in amaranth leaves; and exogenous application of silver nanoparticles can effectively reduce the stress of PAHs on Moringa. Based on the application background of nanomaterials, it is very possible to develop a nanomaterial protective agent that can improve the resistance of plants to environmental stress.

[0005] Carotenoids are reported to be an effective bio-environmental material for improving plant stress resistance, but due to their high price and low direct utilization rate in plants, they have not been effectively used in agricultural production. So is it possible to load carotenoids through nanomaterials, make chitosan nanomaterials loaded with carotenoids, and then apply them in a targeted and quantitative manner to target plants to make them resistant to PAHs stress? Summary of the invention

[0006] In view of the above-mentioned problems, the present invention aims to provide a vegetable protection preparation for treating soil contaminated by polycyclic aromatic hydrocarbons: a method for preparing a carotenoid-loaded carbon-based nanocolloid material preparation.

[0007] In order to achieve the above object, the technical solution adopted by the present invention is as follows: a method for preparing a vegetable protection preparation, comprising the following steps: 1) Take an appropriate amount of chitosan and dissolve it in 1% glacial acetic acid solution, stir and dissolve it into chitosan acid solution; take an appropriate amount of thiamine pyrophosphate-TPP and dissolve it in ultrapure water to make a 10-20 mg / mL TPP solution; 2) Mix a certain amount of TPP solution and chitosan acid solution, with the mass ratio of chitosan to TPP being 2 to 6:1, and stir and react at 20°C to 40°C for a certain time; 3) After the mixed reaction, the pH of the mixed solution is adjusted to 2-7 with a NaOH solution while stirring, and a carbon-based nanocolloid solution is obtained by cross-linking reaction; 4) Take a certain amount of carbon-based nano-colloidal material and dissolve it in water, add a certain amount of carotenoid solution into the aqueous solution of the carbon-based nano-colloidal material, slowly add it and keep stirring; 5) Add 1% by volume of Triton-100 to blend with the carbon-based nano-colloidal material, and stir to react for 1 hour; obtain a vegetable protection preparation with uniform turbidity: a carotenoid-loaded carbon-based nano-colloidal material preparation.

[0008] In step 1) of the present invention, the mass concentration of chitosan in the chitosan acid solution is 1%, and the concentration of the TPP solution is 10 mg / mL.

[0009] In step 2) of the present invention, the mass ratio of chitosan to TPP is 5:1; as the mass ratio of chitosan CS to TPP increases, the number of protonated amino groups on the surface of nanoparticles in the system gradually increases, and the size of the nanoparticles decreases, but when the ratio is 6:1, the particle size suddenly increases, so the optimal solution is 5:1.

[0010] In step 3) of the present invention, the cross-linking reaction temperature is 20° C., the reaction time is 60 min, and the pH of the mixed solution is adjusted to 5-6 after the reaction. As the pH increases, after exceeding about 6.5, the clear solution begins to become turbid. When the pH reaches 7, chitosan has already undergone obvious aggregation and agglomeration, and subsequent carotenoid loading cannot be performed.

[0011] In step 4) of the present invention, the mass ratio of carotenoid to carbon-based nano-colloidal material is 1:2.

[0012] In step 5) of the present invention, the mass ratio of carotenoids to chitosan and TPP in the carotenoid-loaded carbon-based nano-colloid material preparation is 2.5:5:1, the pH thereof is 6, and the concentration is 100 ppm.

[0013] The carotenoid-loaded carbon-based nano-colloid material preparation of the present invention is dialyzed for 24 hours through a dialysis bag and stored at 4° C. after the dialysis is completed.

[0014] The present invention provides an application of a vegetable protection preparation in soil contaminated by polycyclic aromatic hydrocarbons, comprising the following steps: 1) Place the vegetable protective preparation into a centrifuge tube and ultrasonically treat for 30 minutes before use; 2) Use a syringe to slowly inject a certain amount of vegetable protection agent into the reserved soil hole and let it stand for a certain period of time; 3) Transfer the vegetable seedlings and cover them with uncontaminated soil; complete the root application process of the protective agent.

[0015] The dosage of the vegetable protection preparation of the present invention is 5 ml, and the corresponding soil hole size is 8 cm×8 cm×8 cm.

[0016] The advantages of the present invention are as follows: the present invention is based on environmentally friendly plants with resistance to PAHs pollution stress, carotenoids are loaded on carbon-based nano-colloidal materials, and the resistance of plants to PAHs pollution stress is improved. After the carbon-based nano-colloidal materials are applied, the crop has the characteristics of high efficiency and rapid response to plant pollution stress, which has a positive effect on improving the utilization of PAHs-contaminated soil in my country's agricultural production, and can also play a leading role in improving the resistance of plants to environmental stress with nanomaterials. BRIEF DESCRIPTION OF THE DRAWINGS

[0017] Figure 1 This is a schematic diagram of the carotenoid carbon-based nanocolloid material of the present invention; Figure 2 This is a TEM photo of the carbon-based nano-colloidal material in Example 2; Figure 3 This is a TEM photo of the carbon-based nanocolloid material loaded with carotenoids in Example 2; Figure 4 This is a comparative experimental result diagram of chitosan and TPP in Example 9; Figure 5 The chitosan nanocolloid solutions with different pH values ​​in Example 10; Figure 6 This is a diagram showing the results of a chitosan nanopharmaceutical potted plant experiment at different pH values ​​in Example 10; Figure 7 This is a graph showing the effect of nanoprotectants on vegetable biomass; Figure 8 This is a diagram showing the PAHs resistance effect of nano-protectants on vegetables.

[0018] Wherein, TK is a blank control group, and T1 to T8 correspond to the experimental results of Examples 1 to 8 respectively; DETAILED DESCRIPTION

[0019] The present invention is further described in detail below in conjunction with the accompanying drawings and specific implementation methods.

[0020] Example 1: A vegetable protection preparation: A method for preparing a carotenoid-loaded carbon-based nanocolloid material preparation, taking spinach, a common green leafy vegetable, as an example, specifically comprising: 1) Preparation of carbon-based nano-colloidal materials: Chitosan was dissolved in 1% (%, w / v) glacial acetic acid solution to make the concentration of the chitosan solution 10 mg / mL, and 10 mg / mL TPP solution was added dropwise to the chitosan solution. The mass ratio of chitosan to TPP was 5:1. The addition was accompanied by magnetic stirring until the mixed solution was added. Stirring was continued at 20°C and 1000 r / min for 60 min. The pH value of the mixed solution was adjusted to 5 to obtain a carbon-based nano-colloidal solution.

[0021] 2) Carotenoid loading: Carotenoids and carbon-based nano-colloids were mixed in a mass ratio of 1:2, and the reacted substances were dissolved in deionized water. The complex was dialyzed for 24 h using a dialysis bag with a molecular weight cutoff of 20,000 Da to remove free carbon-based nano-colloidal materials and carotenoids. The obtained carotenoid-loaded carbon-based nano-colloidal materials were as follows: Figure 1 As shown in ①; 3) Preservation of the protective preparation: the carotenoid-loaded carbon-based nano-colloidal material preparation is transferred into a dialysis bag, and then vacuum-evacuated for vacuum treatment; the treated carotenoid-loaded carbon-based nano-colloidal material preparation is stored at a low temperature of 4 degrees Celsius; 4) Application of nanomaterials: Transfer the carotenoid-loaded carbon-based nano-colloidal material preparation into a 50 mL centrifuge tube and ultrasonicate for 30 min for use; take a 10 mL syringe, transfer 5 mL of the carotenoid-loaded carbon-based nano-colloidal material preparation, slowly inject it into the reserved soil hole (8×8×8 cm), and let it stand for 5 min; transfer the vegetable seedlings into the hole, cover with soil (use uncontaminated soil), and complete the root application process of the plant protectant.

[0022] Example 2: A vegetable protection preparation: A method for preparing a carotenoid-loaded carbon-based nanocolloid material preparation, taking spinach, a common green leafy vegetable, as an example, specifically comprising: 1) Preparation of carbon-based nano-colloidal materials: Chitosan was dissolved in 1% (%, w / v) glacial acetic acid solution to make the concentration of the chitosan solution 10 mg / mL, and 10 mg / mL TPP solution was added dropwise to the chitosan solution. The mass ratio of chitosan to TPP was 5:1. The addition was accompanied by magnetic stirring until the mixed solution was added. Stirring was continued at 20°C and 1000 r / min for 60 min. The pH value of the mixed solution was adjusted to 6 to obtain a carbon-based nano-colloidal solution.

[0023] The subsequent preparation steps are the same as those in Example 1, and the obtained carotenoid-loaded carbon-based nanocolloid material preparation is as follows: Figure 1 As shown in ②.

[0024] Example 3: A vegetable protection preparation: A method for preparing a carotenoid-loaded carbon-based nanocolloid material preparation, taking spinach, a common green leafy vegetable, as an example, specifically comprising: 1) Preparation of carbon-based nano-colloidal materials: Chitosan was dissolved in 1% (%, w / v) glacial acetic acid solution to make the concentration of the chitosan solution 10 mg / mL, and 20 mg / mL TPP solution was added dropwise to the chitosan solution. The mass ratio of chitosan to TPP was 5:1. The addition was accompanied by magnetic stirring until the mixed solution was added. Stirring was continued at 20°C and 1000 r / min for 60 min. The pH value of the mixed solution was adjusted to 5 to obtain a carbon-based nano-colloidal solution.

[0025] The subsequent preparation steps are the same as those in Example 1, and the obtained carotenoid-loaded carbon-based nanocolloid material preparation is as follows: Figure 1 As shown in ③.

[0026] Example 4: A vegetable protection preparation: A method for preparing a carotenoid-loaded carbon-based nanocolloid material preparation, taking spinach, a common green leafy vegetable, as an example, specifically comprising: 1) Preparation of carbon-based nano-colloidal materials: Chitosan was dissolved in 1% (%, w / v) glacial acetic acid solution to make the concentration of the chitosan solution 10 mg / mL, and 20 mg / mL TPP solution was added dropwise to the chitosan solution. The mass ratio of chitosan to TPP was 5:1. The addition was accompanied by magnetic stirring until the mixed solution was added. Stirring was continued at 20°C and 1000 r / min for 60 min. The pH value of the mixed solution was adjusted to 6 to obtain a carbon-based nano-colloidal solution.

[0027] The subsequent preparation steps are the same as those in Example 1, and the obtained carotenoid-loaded carbon-based nanocolloid material preparation is as follows: Figure 1 As shown in ④.

[0028] Example 5: A vegetable protection preparation: A method for preparing a carotenoid-loaded carbon-based nanocolloid material preparation, taking spinach, a common green leafy vegetable, as an example, specifically comprising: 1) Preparation of carbon-based nano-colloidal materials: Chitosan was dissolved in 1% (%, w / v) glacial acetic acid solution to make the concentration of the chitosan solution 10 mg / mL, and 10 mg / mL TPP solution was added dropwise to the chitosan solution. The mass ratio of chitosan to TPP was 5:1. The addition was accompanied by magnetic stirring until the mixed solution was added. Stirring was continued at 40°C and 1000 r / min for 60 min. The pH value of the mixed solution was adjusted to 5 to obtain a carbon-based nano-colloidal solution.

[0029] The subsequent preparation steps are the same as those in Example 1, and the obtained carotenoid-loaded carbon-based nanocolloid material preparation is as follows: Figure 1 As shown in ⑤.

[0030] Example 6: A vegetable protection preparation: A method for preparing a carotenoid-loaded carbon-based nanocolloid material preparation, taking spinach, a common green leafy vegetable, as an example, specifically comprising: 1) Preparation of carbon-based nano-colloidal materials: Chitosan was dissolved in 1% (%, w / v) glacial acetic acid solution to make the concentration of the chitosan solution 10 mg / mL, and 10 mg / mL TPP solution was added dropwise to the chitosan solution. The mass ratio of chitosan to TPP was 5:1. The addition was accompanied by magnetic stirring until the mixed solution was added. Stirring was continued at 40°C and 1000 r / min for 60 min. The pH value of the mixed solution was adjusted to 6 to obtain a carbon-based nano-colloidal solution.

[0031] The subsequent preparation steps are the same as those in Example 1, and the obtained carotenoid-loaded carbon-based nanocolloid material preparation is as follows: Figure 1 As shown in ⑥.

[0032] Example 7: A vegetable protection preparation: A method for preparing a carotenoid-loaded carbon-based nanocolloid material preparation, taking spinach, a common green leafy vegetable, as an example, specifically comprising: 1) Preparation of carbon-based nano-colloidal materials: Chitosan was dissolved in 1% (%, w / v) glacial acetic acid solution to make the concentration of the chitosan solution 10 mg / mL, and 20 mg / mL TPP solution was added dropwise to the chitosan solution. The mass ratio of chitosan to TPP was 5:1. The addition was accompanied by magnetic stirring until the mixed solution was added. Stirring was continued at 40°C and 1000 r / min for 60 min. The pH value of the mixed solution was adjusted to 5 to obtain a carbon-based nano-colloidal solution.

[0033] The subsequent preparation steps are the same as those in Example 1, and the obtained carotenoid-loaded carbon-based nanocolloid material preparation is as follows: Figure 1 As shown in ⑦.

[0034] Example 8: A vegetable protection preparation: A method for preparing a carotenoid-loaded carbon-based nanocolloid material preparation, taking spinach, a common green leafy vegetable, as an example, specifically comprising: 1) Preparation of carbon-based nano-colloidal materials: Chitosan was dissolved in 1% (%, w / v) glacial acetic acid solution to make the concentration of the chitosan solution 10 mg / mL, and 20 mg / mL TPP solution was added dropwise to the chitosan solution. The mass ratio of chitosan to TPP was 5:1. The addition was accompanied by magnetic stirring until the mixed solution was added. Stirring was continued at 40°C and 1000 r / min for 60 min. The pH value of the mixed solution was adjusted to 6 to obtain a carbon-based nano-colloidal solution.

[0035] The subsequent preparation steps are the same as those in Example 1, and the obtained carotenoid-loaded carbon-based nanocolloid material preparation is as follows: Figure 1 As shown in ⑧.

[0036] Example 9: Chitosan dosage comparison experiment; 1) Preparation of carbon-based nano-colloidal materials: Chitosan was dissolved in 1% (%, w / v) glacial acetic acid solution to make the concentration of the chitosan solution 10 mg / mL, and 10 mg / mL TPP solution was added dropwise to the chitosan solution. The mass ratios of chitosan and TPP were 2:1, 3:1, 4:1, 5:1, and 6:1, respectively. The addition was accompanied by magnetic stirring until the mixed solution was added. Stirring was continued at 20°C and 1000 r / min for 60 min. The pH of the mixed solution was adjusted to 5 to obtain a carbon-based nano-colloidal solution.

[0037] The subsequent steps are the same as in Example 1, and the results obtained are as follows Figure 4 As shown: from Figure 4 It can be seen that as the mass ratio of chitosan CS to TPP increases, the particle size first decreases from 227.9 nm to 106.7 nm. When the mass ratio reaches 6:1, the particle size suddenly increases to 136.2 nm. This is mainly caused by the cross-linking of a small amount of phosphate groups with a large amount of protonated amino groups. As the mass ratio of CS to TPP increases, the number of protonated amino groups on the surface of nanoparticles in the system gradually increases, and the nanoparticle size decreases. In summary, the mass ratio of CS to TPP of 5:1 is finally selected as the optimal embodiment of the present invention.

[0038] Example 10: Chitosan dosage comparison experiment; 1) Preparation of carbon-based nano-colloidal materials: Chitosan was dissolved in 1% (%, w / v) glacial acetic acid solution to make the concentration of the chitosan solution 10 mg / mL, and 10 mg / mL TPP solution was added dropwise to the chitosan solution. The mass ratio of chitosan to TPP was 5:1, respectively. The addition was accompanied by magnetic stirring until the mixed solution was added. Stirring was continued at 20°C and 1000 r / min for 60 min. The pH value of the mixed solution was adjusted to 2, 3, 4, 5, 6, and 7 to obtain a carbon-based nano-colloidal solution.

[0039] The subsequent steps are the same as in Example 1, and the results obtained are as follows Figure 5 As shown: from Figure 5 It can be seen that after the reaction, the pH of the mixed solution was adjusted. As the pH increased, after exceeding about 6.5, the clear solution began to become turbid. When the pH reached 7, chitosan had already aggregated and lumped significantly, and subsequent carotenoid loading could not be performed. At the same time, chitosan nanocolloid solutions with pH 2-6 were loaded with carotenoids and potted experiments were conducted. It was found that when the pH was 2, the roots were severely damaged when exposed, and the vegetables could not grow after the roots were burned; the growth of vegetables at pH 5 / 6 was obviously better than that at pH 2 / 3 / 4, and the leaves were larger and fuller. Therefore, pH 5-6 was selected for subsequent experiments ( Figure 6 ).

[0040] Example 11: The microscopic morphology of the carotenoid-loaded carbon-based nanocolloid material prepared in Example 2 was observed. The specific results are shown in Figure 2 and 3 shown.

[0041] The carotenoid-loaded carbon-based nanocolloid materials prepared in Examples 1 to 8 were used in a spinach experiment. The specific experimental results are shown in Figure 7 .

[0042] pass Figure 7 It can be seen that the spinach root-exposed to the nanoparticle prepared in Example 2, i.e., 5 mL of 100 ppm carotenoid-loaded carbon-based nano-colloidal material (TPP concentration 10 mg / mL, 20°C, pH=6), increased the spinach biomass by 45.03% compared to the blank treatment group.

[0043] The carotenoid-loaded carbon-based nano-colloidal materials prepared in Examples 1 to 8 were subjected to spinach experiment treatment. The specific experimental results are shown in Figure 8 .

[0044] pass Figure 7It can be seen that after 4 weeks of PAHs treatment, the roots are the main site of pyrene accumulation. The spinach treated with T2 in Example 2 has the lowest PYR accumulation, which is 0.98 mg / kg. The spinach treated with T3 in Example 3 and T7 in Example 7 have the highest PYR increases, which are 2.62 and 1.71 mg / kg, respectively.

[0045] Working principle: Through the application of carotenoid-loaded carbon-based nano-colloidal materials to plants, plants can absorb as much exogenous carotenoids as possible, thereby effectively improving the resistance of crops to soil PAHs pollution stress.

[0046] It should be noted that the above are merely preferred embodiments of the present invention and are not intended to limit the protection scope of the present invention. Any combination or equivalent transformation made on the basis of the above embodiments shall fall within the protection scope of the present invention.

Claims

1. A method for preparing a vegetable protective preparation, characterized in that: The preparation method comprises the following steps: 1) Take an appropriate amount of chitosan and dissolve it in 1% glacial acetic acid solution, stir and dissolve it into chitosan acid solution; take an appropriate amount of thiamine pyrophosphate-TPP and dissolve it in ultrapure water to make a 10-20 mg / mL TPP solution; 2) Mix a certain amount of TPP solution and chitosan acid solution, with the mass ratio of chitosan to TPP being 2 to 6:1, and stir and react at 0°C to 40°C for a certain time; 3) After the mixed reaction, the pH of the mixed solution is adjusted to 2-7 with a NaOH solution while stirring, and a carbon-based nanocolloid solution is obtained by cross-linking reaction; 4) Take a certain amount of carbon-based nano-colloidal material and dissolve it in water, add a certain amount of carotenoid solution into the aqueous solution of the carbon-based nano-colloidal material, slowly add it and keep stirring; 5) Add 1% by volume of Triton-100 to blend with the carbon-based nano-colloidal material, and stir to react for 1 hour; obtain a vegetable protection preparation with uniform turbidity: a carotenoid-loaded carbon-based nano-colloidal material preparation.

2. The preparation method according to claim 1, characterized in that In the step 1), the mass concentration of chitosan in the chitosan acid solution is 1%, and the concentration of the TPP solution is 10 mg / mL.

3. The preparation method according to claim 1, characterized in that: In the step 2), the mass ratio of chitosan to TPP is 5:

1.

4. The preparation method according to claim 1, characterized in that: In the cross-linking reaction in step 3), the solution pH value is 5-6, the temperature is 20° C., and the reaction time is 60 min.

5. The preparation method according to claim 1, characterized in that: In the step 4), the mass ratio of carotenoid to carbon-based nano-colloidal material is 1:

2.

6. The preparation method according to claim 1, characterized in that: In the step 5), the mass ratio of carotenoids to chitosan and TPP in the carotenoid-loaded carbon-based nano-colloid material preparation is 2.5:5:1, the pH value is 6, and the concentration is 100 ppm.

7. The preparation method according to claim 1, characterized in that: The carotenoid-loaded carbon-based nano-colloid material preparation is dialyzed for 24 hours through a dialysis bag and stored at 4° C. after the dialysis is completed.

8. Use of the vegetable protection preparation prepared by the method according to claim 1 in soil contaminated by polycyclic aromatic hydrocarbons, characterized in that: The steps include: 1) Place the vegetable protective preparation into a centrifuge tube and ultrasonicate it for later use; 2) Use a syringe to slowly inject a certain amount of vegetable protection agent into the reserved soil hole and let it stand for a certain period of time; 3) Transfer in the vegetable seedlings and cover them with uncontaminated soil.

9. The use according to claim 8, characterized in that The dosage of the vegetable protection preparation is 5 ml, and the specification of the soil hole is 8 cm×8 cm×8 cm.

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