Gamma-polyglutamic acid-modified nitrification inhibitors, methods of making and using the same

Abstract

The application provides a gamma-polyglutamic acid modified nitrification inhibitor and a preparation method and application thereof. A natural high molecular material gamma-polyglutamic acid (gamma-PGA) is used for structural modification of 3,4-dimethylpyrazole phosphate (DMPP), so that a gamma-polyglutamic acid modified nitrification inhibitor (DMPP-gamma-PGA conjugate) is obtained, and the thermal stability and storage stability of the nitrification inhibitor are significantly improved. The modified product can be applied to stable nitrogen fertilizer. Through soil culture experiments, it is shown that the modification of gamma-PGA not only enhances the nitrification inhibition effect of DMPP, but also significantly reduces the N2O emission flux and cumulative emission amount. In addition, the slow-release effect of gamma-PGA effectively prolongs the persistence period of DMPP, improves the nitrogen regulation capacity of DMPP, and provides a more environmentally friendly and efficient nitrification inhibition scheme for agricultural production.

Description

Technical Field

[0001] This invention belongs to the field of agricultural chemistry and fertilizer enhancement technology, specifically relating to a high-temperature resistant nitrification inhibitor DMPP-γ-PGA based on γ-polyglutamic acid modification, its preparation method, and its application. Background Technology

[0002] Nitrogen fertilizer is a key agricultural input for ensuring high crop yields; however, its utilization rate is generally low (the nitrogen fertilizer utilization rate for grain crops in my country is about 42.6%, significantly lower than the 60%–70% level in developed countries). This results in a large amount of nitrogen entering the environment through ammonia volatilization, nitrate leaching, and greenhouse gas N2O emissions, causing serious environmental pollution and resource waste. Nitrification inhibitors, as an effective means of enhancing nitrogen fertilizer efficiency, can delay nitrogen release, improve nitrogen fertilizer utilization, and reduce N2O emissions by inhibiting the conversion of ammonium nitrogen to nitrate nitrogen in the soil.

[0003] 3,4-Dimethylpyrazole phosphate (DMPP), as a highly efficient and low-toxicity nitrification inhibitor, has been widely used in fertilizers such as urea and ammonium nitrogen fertilizers. However, DMPP still has the following significant drawbacks in practical applications: (1) Poor thermal stability: DMPP is extremely sensitive to high temperatures. During fertilizer granulation (such as high-tower granulation where the temperature often reaches 80-120℃), the activity loss rate of DMPP is as high as 40-60%. Studies have shown that when the temperature exceeds 60℃, its decomposition rate increases exponentially, which seriously restricts its application in the industrial production of fertilizers; (2) Short duration of effect and easy decomposition: In the soil environment, the degradation rate of DMPP is relatively fast, especially under high temperature and high humidity conditions, its nitrification inhibition effect is difficult to last, and it cannot meet the demand for stable nitrogen supply during the entire growth period of crops; (3) Insufficient storage stability: During the mixed storage with nitrogen fertilizers such as urea, ammonium chloride, and ammonium sulfate, DMPP will slowly decompose, especially in the high temperature environment of summer, the content of its effective components will decrease significantly with the extension of storage time, resulting in short product shelf life and unstable fertilizer effect; (4) Limitations of existing modification technologies: At present, the main methods to improve the stability of DMPP include microencapsulation (such as liposome technology) and the addition of inorganic stabilizers (such as diatomaceous earth loading). Although these methods have certain effects, they have problems such as complex preparation process, high cost, possible introduction of secondary pollution or poor compatibility with fertilizers, making it difficult to promote and apply on a large scale. For example, patent CN109665927A discloses a stable nitration inhibitor, its preparation method, and its application. The preparation method involves mixing the nitration inhibitor DMPP with a protective agent (a mixture of protective colloid and soluble starch) into a solution, followed by spray drying to obtain a stable nitration inhibitor. This method requires a high-temperature spray drying process (inlet air temperature 140-160℃, outlet temperature 70-80℃), which leads to DMPP volatilization loss. Furthermore, this method has high energy consumption and requires significant equipment investment. CN113717014A discloses a liquid nitration inhibitor and its preparation method, which involves mixing a protective agent, dispersant, DMPP, and a solvent containing polymerizable monomers. After adding an initiator, polymerization is performed to obtain a liquid nitration inhibitor. This preparation process is complex and costly.

[0004] Therefore, developing a high-efficiency, environmentally friendly, low-cost modification technology that can significantly improve the thermal and storage stability of DMPP is of great practical significance and application demand for promoting the development of the stabilized fertilizer industry, improving nitrogen fertilizer utilization, and reducing agricultural non-point source pollution. Summary of the Invention

[0005] The technical problem to be solved by the present invention is to provide a γ-polyglutamic acid modified nitrification inhibitor, its preparation method and application, in order to address the shortcomings of the prior art. The invention modifies the structure of 3,4-dimethylpyrazole phosphate (DMPP) with natural polymer material γ-polyglutamic acid (γ-PGA), which significantly improves its thermal stability and storage stability. The modified product can be applied to stable nitrogen fertilizers.

[0006] To solve the above-mentioned technical problems, the technical solution adopted by the present invention is: a method for preparing a γ-polyglutamic acid modified nitration inhibitor, comprising the following steps:

[0007] S1. Prepare a γ-polyglutamic acid (γ-PGA) solution using deionized water and adjust the pH to 7.0;

[0008] S2. Prepare a 3,4-dimethylpyrazole phosphate (DMPP) solution using sodium chloride solution, and adjust the pH to 3.0-5.0;

[0009] S3. Add the 3,4-dimethylpyrazole phosphate (DMPP) solution obtained in S2 to the γ-polyglutamic acid (γ-PGA) solution obtained in S1, adjust the pH of the mixture to 5.8, and then carry out the coupling reaction by stirring at 500~800 r / min for 20~30 h at room temperature to obtain the reaction solution.

[0010] S4. The reaction solution obtained in S3 is placed in a dialysis bag and dialyzed for 40-60 hours to remove free 3,4-dimethylpyrazole phosphate (DMPP). Then, it is freeze-dried at -50°C for 40-60 hours to obtain γ-polyglutamic acid modified nitration inhibitor, denoted as DMPP-γ-PGA conjugate.

[0011] Preferably, the concentration of the γ-polyglutamic acid solution in S1 is 8~12 mg / mL.

[0012] Preferably, the mass concentration of the sodium chloride solution in S2 is 0.9% (w / v).

[0013] Preferably, the concentration of the 3,4-dimethylpyrazole phosphate solution in S2 is 1.5~2.5 mg / mL.

[0014] Preferably, the mass ratio of the 3,4-dimethylpyrazole phosphate solution and the γ-polyglutamic acid solution in S3 is 1:1 to 1:10.

[0015] Preferably, the pH adjustment in S1, S2, and S3 uses a sodium hydroxide solution or a hydrochloric acid solution, wherein the mass concentration of the sodium hydroxide solution is 6-10% (w / v) and the mass concentration of the hydrochloric acid solution is 4-8% (w / v).

[0016] Preferably, the molecular weight cutoff range of the dialysis bag in S4 is 8-14 kDa.

[0017] The present invention also provides a γ-polyglutamic acid-modified nitration inhibitor prepared by the above method.

[0018] The present invention also provides an application of the γ-polyglutamic acid modified nitrification inhibitor, which is used in stable nitrogen fertilizer to improve nitrogen fertilizer utilization.

[0019] Compared with the prior art, the present invention has the following significant technical effects:

[0020] 1. Traditional nitrification inhibitors, such as 3,4-dimethylpyrazole phosphate (DMPP), suffer from poor heat resistance, easy decomposition, and short residual effect in practical applications, limiting their stability and long-term efficacy when mixed with fertilizers under high-temperature conditions. To address this issue, this invention modifies the structure of DMPP using the natural polymer γ-polyglutamic acid (γ-PGA) to obtain a γ-polyglutamic acid-modified nitrification inhibitor (DMPP-γ-PGA conjugate), significantly improving its thermal and storage stability. This modified product can be used in stable nitrogen fertilizers.

[0021] 2. Soil incubation experiments showed that γ-PGA modification not only enhanced the nitrification inhibition effect of DMPP but also significantly reduced N2O emission flux and cumulative emissions. Furthermore, the slow-release effect of γ-PGA effectively prolonged the residual effect of DMPP, improving its nitrogen regulation capacity. The γ-polyglutamic acid-modified nitrification inhibitor (DMPP-γ-PGA conjugate) of this invention significantly improves nitrogen fertilizer utilization and nitrogen uptake efficiency, providing a more environmentally friendly and efficient nitrification inhibition solution for agricultural production. These research results not only provide a theoretical basis for the development of novel long-acting nitrification inhibitors but also offer technical support for the efficient utilization of nitrogen fertilizer and environmental protection in agricultural production.

[0022] The present invention will now be described in further detail with reference to the accompanying drawings and embodiments. Attached Figure Description

[0023] Figure 1 The Fourier transform infrared spectra of DMPP, γ-PGA and DMPP-γ-PGA conjugates of the present invention are shown, wherein a: material ratio is 1:1, b: material ratio is 1:5 and c: material ratio is 1:10.

[0024] Figure 2 This is the 1H NMR spectrum of the DMPP-γ-PGA conjugate of the present invention with a material ratio of 1:10.

[0025] Figure 3These are scanning electron microscope (SEM) images of DMPP-γ-PGA conjugates with different material ratios according to the present invention. Figures a and b are SEM images of the DMPP-γ-PGA conjugate with a material ratio of 1:1 at 500 μm and 100 μm, respectively. Figures c and d are SEM images of the DMPP-γ-PGA conjugate with a material ratio of 1:5 at 500 μm and 30 μm, respectively. Figures e and f are SEM images of the DMPP-γ-PGA conjugate with a material ratio of 1:10 at 500 μm and 50 μm, respectively.

[0026] Figure 4 The figures show thermogravimetric analysis (TGA) and differential thermogravimetric analysis (DGA) diagrams of DMPP-γ-PGA couplings with different material ratios according to the present invention. Figures a and b are the TGA and DGA diagrams of the DMPP-γ-PGA coupling with a material ratio of 1:1, respectively; Figures c and d are the TGA and DGA diagrams of the DMPP-γ-PGA coupling with a material ratio of 1:5, respectively; and Figures e and f are the TGA and DGA diagrams of the DMPP-γ-PGA coupling with a material ratio of 1:10, respectively.

[0027] Figure 5 The values ​​represent the DMPP loss rate in DMPP-γ-PGA conjugates under three different heating times and temperatures in Example 4 of this invention: a: heating time 5 min, b: heating time 15 min, c: heating time 30 min, CP1 represents DMPP, CP2 represents 1:1 DMPP-γ-PGA conjugate, CP3 represents 1:5 DMPP-γ-PGA conjugate, and CP4 represents 1:10 DMPP-γ-PGA conjugate.

[0028] Figure 6 The values ​​represent the DMPP loss rate in the DMPP-γ-PGA conjugate under three different heating temperatures and heating times in Example 4 of this invention: a: heating temperature is 70℃, b: heating temperature is 125℃, c: heating temperature is 200℃, CP1 represents DMPP, CP2 represents 1:1 DMPP-γ-PGA conjugate, CP3 represents 1:5 DMPP-γ-PGA conjugate, and CP4 represents 1:10 DMPP-γ-PGA conjugate.

[0029] Figure 7 The values ​​represent the residual DMPP content of the DMPP-γ-PGA conjugate at 25°C after storage in different nitrogen fertilizers in Example 5 of this invention. a: urea as nitrogen source, b: ammonium chloride as nitrogen source, c: ammonium sulfate as nitrogen source, CK1 represents the blank control, CP1 represents the 1:1 DMPP-γ-PGA conjugate, CP2 represents the 1:5 DMPP-γ-PGA conjugate, and CP3 represents the 1:10 DMPP-γ-PGA conjugate.

[0030] Figure 8 This refers to the DMPP residual rate of the DMPP-γ-PGA conjugate stored in different nitrogen fertilizers at 45°C in Example 5 of this invention. a: urea as nitrogen source, b: ammonium chloride as nitrogen source, c: ammonium sulfate as nitrogen source, CK1 represents blank control, CP1 represents 1:1 DMPP-γ-PGA conjugate, CP2 represents 1:5 DMPP-γ-PGA conjugate, and CP3 represents 1:10 DMPP-γ-PGA conjugate.

[0031] Figure 9 The different treatments in Example 6 of this invention affect ammonium nitrogen (NH4) + The effects of γ-N were investigated. CK1 was the control group with only urea added, CP1 was urea with 0.8% DMPP added, CP2 was urea with 1.2% DMPP added, CP3 was urea with 0.8% DMPP-γ-PGA added, and CP4 was urea with 1.2% DMPP-γ-PGA added.

[0032] Figure 10 The different treatments in Example 6 of this invention affect nitrate nitrogen (NO3). - The effects of γ-N were investigated. CK1 was the control group with only urea added, CP1 was urea with 0.8% DMPP added, CP2 was urea with 1.2% DMPP added, CP3 was urea with 0.8% DMPP-γ-PGA added, and CP4 was urea with 1.2% DMPP-γ-PGA added.

[0033] Figure 11 Figure 6 shows the N2O emission flux and cumulative emission amount of different treatments in Example 6 of this invention. Figure a shows the N2O emission flux, Figure b shows the cumulative N2O emission amount, CK1 is the control group with only urea added, CP1 is urea with 0.8% DMPP added, CP2 is urea with 1.2% DMPP added, CP3 is urea with 0.8% DMPP-γ-PGA added, and CP4 is urea with 1.2% DMPP-γ-PGA added. Detailed Implementation

[0034] The nitration inhibitor DMPP used in this invention was provided by Shanghai Kingbase Biotechnology Co., Ltd., and was of analytical grade (AR); γ-polyglutamic acid (γ-PGA) was provided by Shandong Xinhua Medical Instrument Co., Ltd., and was a biological reagent (BR). Dialysis bags with a molecular weight cutoff range of 8-14 kDa were provided by Beijing Lvbaicao Technology Development Co., Ltd. Example 1

[0035] This embodiment describes a method for preparing a γ-polyglutamic acid-modified nitration inhibitor, comprising the following steps:

[0036] S1. Prepare a γ-polyglutamic acid (γ-PGA) solution with a concentration of 10 mg / mL using deionized water, and adjust the pH to 7.0 using 8% (w / v) sodium hydroxide solution or 6% (w / v) hydrochloric acid solution.

[0037] S2. Prepare a 2 mg / mL solution of 3,4-dimethylpyrazole phosphate (DMPP) using 0.9% (w / v) sodium chloride solution. Adjust the pH using 8% (w / v) sodium hydroxide solution or 6% (w / v) hydrochloric acid solution, and finally maintain the pH at 4.0.

[0038] S3. Add the DMPP solution obtained in S2 to the γ-PGA solution obtained in S1 at a mass ratio of 1:1, adjust the pH of the mixture to 5.8, and then carry out the coupling reaction by stirring at 600 r / min for 24 h at room temperature to obtain the reaction solution.

[0039] S4. The reaction solution obtained in S3 was placed in a dialysis bag with a molecular weight cutoff range of 8-14 kDa and dialyzed for 48 hours to remove free DMPP. Then, it was freeze-dried at -50°C for 48 hours to obtain γ-polyglutamic acid modified nitration inhibitor, denoted as 1:1 DMPP-γ-PGA conjugate. Example 2

[0040] This embodiment describes a method for preparing a γ-polyglutamic acid-modified nitration inhibitor, comprising the following steps:

[0041] S1. Prepare a γ-polyglutamic acid (γ-PGA) solution with a concentration of 8 mg / mL using deionized water, and adjust the pH to 7.0 using a 6% (w / v) sodium hydroxide solution or a 4% (w / v) hydrochloric acid solution.

[0042] S2. Prepare a 1.5 mg / mL solution of 3,4-dimethylpyrazole phosphate (DMPP) using a 0.9% (w / v) sodium chloride solution. Adjust the pH using a 6% (w / v) sodium hydroxide solution or a 4% (w / v) hydrochloric acid solution until the final pH is maintained at 3.0.

[0043] S3. Add the DMPP solution obtained in S2 to the γ-PGA solution obtained in S1 at a mass ratio of 1:5, adjust the pH of the mixture to 5.8, and then carry out the coupling reaction by stirring at 500 r / min for 30 h at room temperature to obtain the reaction solution.

[0044] S4. The reaction solution obtained in S3 was placed in a dialysis bag with a molecular weight cutoff range of 8-14 kDa and dialyzed for 40 h to remove free DMPP. Then, it was freeze-dried at -50℃ for 40 h to obtain γ-polyglutamic acid modified nitration inhibitor, denoted as 1:5 DMPP-γ-PGA conjugate.

[0045] Example 3

[0046] This embodiment describes a method for preparing a γ-polyglutamic acid-modified nitration inhibitor, comprising the following steps:

[0047] S1. Prepare a γ-polyglutamic acid (γ-PGA) solution with a concentration of 12 mg / mL using deionized water, and adjust the pH to 7.0 using a 10% (w / v) sodium hydroxide solution or an 8% (w / v) hydrochloric acid solution.

[0048] S2. Prepare a 2.5 mg / mL solution of 3,4-dimethylpyrazole phosphate (DMPP) using a 0.9% (w / v) sodium chloride solution. Adjust the pH using a 10% (w / v) sodium hydroxide solution or an 8% (w / v) hydrochloric acid solution until the final pH is maintained at 5.0.

[0049] S3. Add the DMPP solution obtained in S2 to the γ-PGA solution obtained in S1 at a mass ratio of 1:10, adjust the pH of the mixture to 5.8, and then carry out the coupling reaction by stirring at 800 r / min for 20 h at room temperature to obtain the reaction solution.

[0050] S4. The reaction solution obtained in S3 was placed in a dialysis bag with a molecular weight cutoff range of 8-14 kDa and dialyzed for 60 h to remove free DMPP. Then, it was freeze-dried at -50℃ for 60 h to obtain γ-polyglutamic acid modified nitration inhibitor, denoted as 1:10 DMPP-γ-PGA conjugate.

[0051] Figure 1 The figures show the Fourier transform infrared spectra of DMPP, γ-PGA, and DMPP-γ-PGA conjugates, where a represents a material ratio of 1:1 (corresponding to Example 1), b represents a material ratio of 1:5 (corresponding to Example 2), and c represents a material ratio of 1:10 (corresponding to Example 3). As can be seen from the figures, the characteristic peak positions of the infrared spectra of the DMPP-γ-PGA conjugates with the three different material ratios are generally consistent, but the differences in peak shape and intensity reveal variations in their structural interactions and the degree of reaction. Specifically: 3269 cm⁻¹ -1 The decrease in intensity of the broad peak of the NH amino group indicates that the amino group in γ-PGA interacts with other groups; 1390 cm⁻¹ -1 and 1579 cm -1 The characteristic peak contraction at the 3,4-dimethylpyrazole ring may be related to the formation of hydrogen bonds between γ-PGA molecules; 1729 cm⁻¹ -1 The newly emerging carbonyl peak did not interact with the carboxyl group, confirming the stable existence of γ-PGA; 2935 cm⁻¹ -1The intensity of the broad methyl peak in DMPP decreases, reflecting the bonding of the methyl group with other groups; 1210 cm⁻¹ -1 (CN key) and 983 cm -1 The peak of (PO bond) further confirms the retention of the DMPP structure. By comparing samples with different material ratios, it was found that the vibration intensity was strongest at a ratio of 1:10 and weakest at a ratio of 1:1, indicating that the reaction between DMPP and γ-PGA is more complete and the binding between the two is more thorough when the material ratio is 1:10, while the coupling efficiency is relatively limited under low material ratio conditions.

[0052] Figure 2 The image shows the 1H NMR spectrum of the DMPP-γ-PGA conjugate with a material ratio of 1:10. Analysis reveals that the main characteristic peak of the synthesized conjugate occurs at a chemical shift of 4.66 ppm, with a peak area integral of 1.00, which highly matches the reported characteristic peak position of γ-PGA in the literature, confirming that the structural integrity of the carboxylic acid group in the γ-PGA backbone is maintained after coupling. Simultaneously, the characteristic proton signal of DMPP is distributed in the 1-2 ppm range, with the characteristic peaks at 1.93 ppm (integral 0.06) and 1.80 ppm (integral 0.09) indicating that the methyl group of DMPP exists in a specific chemical environment within the conjugate. Based on the synthetic ratio of DMPP to γ-PGA (1:10) and quantitative analysis of the integrated area, the binding concentration of DMPP in the conjugate is calculated to be 88 μg / mg γ-PGA, with a binding efficiency of 88%, demonstrating a significant stoichiometric relationship between the two.

[0053] Figure 3These are scanning electron microscope (SEM) images of DMPP-γ-PGA conjugates with different material ratios. Figures a and b show the SEM images of the DMPP-γ-PGA conjugate with a material ratio of 1:1 at 500 μm and 100 μm, respectively; figures c and d show the SEM images of the DMPP-γ-PGA conjugate with a material ratio of 1:5 at 500 μm and 30 μm, respectively; and figures e and f show the SEM images of the DMPP-γ-PGA conjugate with a material ratio of 1:10 at 500 μm and 50 μm, respectively. Analysis shows that the microstructure of the DMPP-γ-PGA conjugates differs significantly under different material ratios, exhibiting a trend of evolution from a loose and heterogeneous structure to a dense and stable structure. At a material ratio of 1:1 (Figures a and b), the samples mainly exhibit porous and network structures, but the overall morphology is relatively irregular, the pore size distribution is uneven, and no obvious DMPP particles are observed adhering, or the amount of adhering particles is extremely small. This indicates that the interaction between γ-PGA and DMPP is weak at this ratio, resulting in a loose composite structure that is difficult to effectively load DMPP. As the material ratio is adjusted to 1:5 (Figures c and d), the sample gradually exhibits a certain layered structure, but the interlayer gaps are small and the arrangement is not regular, which may limit the effective embedding and adsorption of DMPP. Although a small number of DMPP particles can be observed distributed on the surface of the structure in Figure d, the overall loading is still limited, which is speculated to be related to the low DMPP ratio and the influence of reaction conditions on the binding efficiency. In contrast, when the material ratio is 1:10 (Figures e and f), the sample forms a more uniform layered-network composite structure with a uniform distribution of interlayer pores, providing a more ideal loading space for DMPP particles. Especially in Figure f, it can be clearly observed that DMPP particles are uniformly distributed in the pores of the layered structure, and the loading is significantly higher than that under the 1:1 and 1:5 ratio conditions.

[0054] Figure 4Figures show the thermogravimetric analysis (TGA) and differential thermogravimetric analysis (DGA) plots of DMPP-γ-PGA couplings with different material ratios. Figures a and b show the TGA and DGA plots for a DMPP-γ-PGA coupling with a material ratio of 1:1, figures c and d show the TGA and DGA plots for a DMPP-γ-PGA coupling with a material ratio of 1:5, and figures e and f show the TGA and DGA plots for a DMPP-γ-PGA coupling with a material ratio of 1:10. As can be seen from the figures, the thermal stability of the DMPP-γ-PGA couplings exhibits significant differences under different material ratios. This is mainly reflected in the shift of the DMPP decomposition temperature towards a higher temperature range, the improved thermal stability due to coupling, and the decrease in high-temperature residual mass as the γ-PGA ratio decreases. At a material ratio of 1:1, thermogravimetric analysis (TGA) showed an overall mass loss of 6.41% in the range of 100°C to 200°C, indicating that γ-PGA contributes significantly to the thermal stability of the system at this ratio. Differential thermogravimetric analysis (DTGA) further revealed that the maximum weight loss rate temperature occurred at 319.46°C, much higher than the decomposition temperature of DMPP, indicating that the coupling effect of γ-PGA can effectively improve the thermal stability of DMPP. In addition, no obvious decomposition peak was observed near 178°C, suggesting that the thermal decomposition characteristics of DMPP have changed due to the coupling effect. As the material ratio increased to 1:5, the overall mass loss was 4.13% in the range of 100°C to 200°C. The DTGA curve showed that the maximum loss rate temperature further increased to 349.66°C, indicating that coupling enhanced the heat resistance of DMPP. At the same time, a relatively gentle shoulder peak appeared near 200°C, which may correspond to the partial decomposition signal of DMPP, but compared with free DMPP, its thermal decomposition path has changed significantly. When the material ratio increases to 1:10, the overall mass loss is 4.75% in the range of 100°C to 200°C. The DTGA curve shows that the maximum loss rate temperature rises to 353.06°C, indicating that the overall thermal decomposition stability of the system is further improved. At the same time, the shoulder peak signal near 200°C is slightly enhanced, suggesting that the high proportion of DMPP still partially retains its thermal decomposition characteristics.

[0055] The above results demonstrate the successful preparation of DMPP-γ-PGA conjugates. In its unmodified state, DMPP decomposes at approximately 178°C, exhibiting low heat resistance and making it prone to loss during fertilizer granulation or high-temperature storage. However, in the DMPP-γ-PGA conjugates prepared in this invention, the thermal stability of DMPP significantly improves with increasing γ-PGA proportion. The maximum weight loss rate temperature of the 1:10 conjugate reaches 353.06°C, significantly higher than the decomposition temperature of DMPP. This phenomenon may stem from the high heat resistance of γ-PGA and its physical coating and chemical bonding effect on DMPP, which not only enhances the heat resistance of DMPP but also positively impacts its storage stability.

[0056] Example 4

[0057] This embodiment is a thermal stability test of DMPP-γ-PGA coupling.

[0058] The DMPP-γ-PGA conjugate was heated at 70℃, 125℃, and 200℃ for 5-30 min, respectively. The DMPP content was detected by HPLC, and the DMPP loss rate was calculated.

[0059] Figure 5 These represent the DMPP loss rate in DMPP-γ-PGA conjugates at three different heating times and temperatures: a: heating time 5 min, b: heating time 15 min, c: heating time 30 min. CP1 represents DMPP, CP2 represents a 1:1 DMPP-γ-PGA conjugate, CP3 represents a 1:5 DMPP-γ-PGA conjugate, and CP4 represents a 1:10 DMPP-γ-PGA conjugate. Figure 5 It was found that the loss rate of DMPP exhibited a significant temperature-time dependence (70℃ to 200℃, 5-30 min) under different temperature and heating time conditions, while the introduction of γ-PGA significantly improved its thermal stability through a molecular encapsulation mechanism. Experimental data showed that the loss rate of pure DMPP (CP1) reached 20.34% after heating at 70℃ for 5 min, while the loss rate surged to 64.32% under the extreme condition of heating at 200℃ for 30 min, indicating that high temperature accelerated its degradation kinetics. When DMPP formed a 1:10 coupling compound (CP4) with γ-PGA, its thermal stability was significantly improved, with a loss rate of 2.97% under the condition of heating at 70℃ for 5 min, and a loss rate of 17.63% under the extreme condition of heating at 200℃ for 30 min.

[0060] Figure 6 The figures show the DMPP loss rate in DMPP-γ-PGA conjugates under three different heating temperatures and times: a: heating temperature 70℃, b: heating temperature 125℃, c: heating temperature 200℃. CP1 represents DMPP, CP2 represents a 1:1 DMPP-γ-PGA conjugate, CP3 represents a 1:5 DMPP-γ-PGA conjugate, and CP4 represents a 1:10 DMPP-γ-PGA conjugate. Figure 6It was found that the thermal degradation behavior of DMPP and the stability of its coupling with γ-PGA differed significantly under different temperature gradients (70℃, 125℃, and 200℃). Overall, the loss rate of DMPP increased exponentially with increasing temperature and heating time, while the introduction of γ-PGA effectively suppressed its degradation kinetics through a proportion-dependent mechanism. Specifically, at 70℃, the loss rate of pure DMPP (CP1) increased from 20.34% to 26.74% with heating time (5-30 min), while the loss rate of DMPP in the 1:10 DMPP-γ-PGA coupling (CP4) only slightly increased from 2.79% to 3.47%. At 125℃, the loss rate of pure DMPP (CP1) increased from 21.35% to 32.46% with heating time (5-30 min), while the loss rate of the 1:10 DMPP-γ-PGA coupling (CP4) only slightly increased from 7.95% to 9.26%. When the temperature rises to 200℃, the thermal degradation of DMPP accelerates significantly: the loss rate of pure DMPP (CP1) increases from 43.52% to 64.32% with heating time (5-30 min), while the loss rate of 1:10 DMPP-γ-PGA conjugate (CP4) only increases slightly from 9.85% to 18.14%.

[0061] In summary, this invention successfully improved the thermal stability of DMPP by introducing γ-PGA, with the 1:10 DMPP-γ-PGA coupling exhibiting the best thermal stability.

[0062] Example 5

[0063] This embodiment is a storage resistance test of DMPP-γ-PGA conjugate.

[0064] 0.5 g of DMPP-γ-PGA conjugate was mixed with 100 g of nitrogen fertilizer (urea, ammonium chloride, or ammonium sulfate). After thorough mixing, the mixture was divided into 30 equal portions and stored in sealed small sample bottles. The sample bottles were placed in a constant temperature incubator at two temperatures: 25°C and 45°C. Samples were taken on days 1, 7, 21, 35, and 50 of storage. Immediately after sampling, the DMPP content was quantitatively analyzed to evaluate the effects of different nitrogen fertilizer types, storage temperatures, and storage times on the residual rate of the nitrification inhibitor DMPP.

[0065] Figure 7The values ​​represent the residual DMPP content of the DMPP-γ-PGA conjugate after storage in different nitrogen fertilizers at 25℃. a: urea as nitrogen source, b: ammonium chloride as nitrogen source, c: ammonium sulfate as nitrogen source, CK1 represents the blank control, CP1 represents the 1:1 DMPP-γ-PGA conjugate, CP2 represents the 1:5 DMPP-γ-PGA conjugate, and CP3 represents the 1:10 DMPP-γ-PGA conjugate. As shown in the figure, under constant temperature of 25℃, the DMPP content of the three nitrogen source systems (urea, ammonium chloride, and ammonium sulfate) decreased with prolonged storage time. However, γ-PGA modification significantly slowed down the degradation process: In the urea system, the 50-day residual rates of the three DMPP-γ-PGA couplings (1:1, 1:5, and 1:10) reached 88.67%, 88.94%, and 89.39%, respectively, which were 0.91%, 1.18%, and 1.63% higher than the control group (87.76%). When the nitrogen source was replaced with ammonium chloride, the modification effect was the most prominent, with the 50-day residual rate of the couplings increasing by 1.46%, 1.97%, and 3.53% (87.95% in the control group and 91.48% in the modified group). In the ammonium sulfate system, the 50-day residual rate of the 1:10 coupling increased by 2.15% (90.14% in the control group and 92.29% in the modified group).

[0066] Figure 8 The figure shows the residual DMPP content of DMPP-γ-PGA conjugates after storage in different nitrogen fertilizers at 45℃. a: urea as nitrogen source, b: ammonium chloride as nitrogen source, c: ammonium sulfate as nitrogen source, CK1 represents the blank control, CP1 represents the 1:1 DMPP-γ-PGA conjugate, CP2 represents the 1:5 DMPP-γ-PGA conjugate, and CP3 represents the 1:10 DMPP-γ-PGA conjugate. As shown in the figure, under 45℃ storage conditions, the degradation process of DMPP in the three nitrogen source systems (urea, ammonium chloride, and ammonium sulfate) all exhibited time-dependent characteristics, while γ-PGA modification treatment showed a certain improvement effect on its long-term storage (50 days) stability. In the urea system, the DMPP residue rates of DMPP-γ-PGA couplings with different material ratios (1:1, 1:5, 1:10) after 50 days were 88.44%, 88.95%, and 89.12%, respectively. Compared with the control group (CK1, 87.73%), the residue rates increased by 0.71%, 1.22%, and 1.39%, respectively. In the ammonium chloride system, the effect of modification treatment was more significant: after 50 days of storage, the coupling residue rates were 88.99%, 90.09%, and 91.58% (compared to 87.56% in the control group), with residue rate increases of 1.43%, 2.53%, and 4.02%, respectively.

[0067] Experimental results show that the introduction of γ-PGA significantly slowed down the degradation rate of DMPP, and its protective effect was closely related to the type of nitrogen source, storage temperature, and the proportion of additive (γ-PGA). For different nitrogen source types, the stability of DMPP was ammonium chloride > ammonium sulfate > urea, and the stability was significantly improved at high temperature (45℃). The stabilizing effect of γ-PGA may stem from its physicochemical synergistic effect with DMPP: on the one hand, the polymer chain of γ-PGA can form a complex with DMPP through hydrophobic interactions or hydrogen bonds, reducing its direct contact with the environmental medium, thereby inhibiting hydrolysis and volatilization; on the other hand, the steric hindrance effect of γ-PGA may enhance the encapsulation efficiency of DMPP.

[0068] Example 6

[0069] This embodiment verifies the agronomic effects of DMPP-γ-PGA conjugates.

[0070] The effects of DMPP-γ-PGA conjugates on nitrogen transformation were systematically studied through soil culture experiments. Soil samples were collected from the topsoil layer (0-20 cm) of the Quzhou Experimental Station in Hebei Province (36°51'50"N, 115°0'58"E), and the soil type was saline-alkaline soil. A five-point sampling method was used. Fresh soil samples were brought back to the laboratory, mixed thoroughly, and after manual sieving to remove impurities (such as stones, plants, and roots), the samples were air-dried, ground, and sieved through a 2 mm sieve. A portion of the samples was used for soil physicochemical property testing, and the remainder was used for soil culture experiments. The basic physicochemical properties of the soil are shown in Table 1.

[0071] Table 1 Soil physicochemical properties

[0072]

[0073] An indoor soil culture method was used. 50.00 g of air-dried soil was weighed into a culture bottle (250 mL), and the soil moisture content was adjusted to 60% WFPS (water-filled pore space). After pre-culturing at room temperature for 4 days, fertilizer treatment was applied, with a fertilizer addition amount of 0.1 g N / kg soil. The fertilizer preparation method was as follows: DMPP and DMPP-γ-PGA conjugate (1:10) were added to urea, with the addition amounts of DMPP and DMPP-γ-PGA conjugate set at 0.80% and 1.20% of the total amide nitrogen and ammonium nitrogen in urea, respectively. Each treatment was repeated in triplicate. Soil culture was maintained at room temperature for 21 days. During the culture period, water was added every 2 days using a weighing method to maintain the soil moisture content at 60% WFPS. Destructive sampling was performed on days 1, 3, 7, 14, and 21 after culture to determine the contents of N2O, ammonium nitrogen, and nitrate nitrogen.

[0074] Determination of ammonium nitrogen and nitrate nitrogen content: During sampling, the soil in the bottle was stirred evenly with a glass rod. 10 g of fresh soil sample was weighed into a 100 mL centrifuge tube. 149.84 g of potassium chloride was placed in a 2 L volumetric flask to prepare a 1 mol / L potassium chloride solution. 50 mL of the potassium chloride solution was added to the centrifuge tube for extraction. The tube was placed on a shaker and shaken at 180 r / min for 60 min at 25℃. During shaking, the soil sample was briefly stored in a 4℃ refrigerator. After filtration, the NH4+ content in the filtrate was determined using a flow analyzer. + -N, NO3 - -N content.

[0075] N2O emission determination: A three-way valve compatible with the culture flask was used for sampling. One end was connected to the culture flask, the other end was sealed, and the other end was connected to a medical syringe. Gas samples were collected four times at 0, 45, 90, and 135 minutes, injected into the chromatographic bottle, and analyzed using a gas chromatograph. Measurement conditions: ECD detector temperature 250 ℃, column temperature 50 ℃, carrier gas high-purity argon-methane, gas flow rate 40 mL / min. Sampling time and ambient temperature were strictly recorded.

[0076] The formula for calculating gas emission flux is: F = V × dc / dt × ρ × 273 / (273 + T) / W, where: F is the gas emission flux, mg·kg -1 ·h -1 or μg·kg -1 ·h -1 V represents the volume of the upper space of the culture flask, in L; dc / dt represents the rate of change of gas concentration in the culture flask per unit time, in mg·kg⁻¹. -1 ·h -1 or μg·kg -1 ·h -1 ρ is the density of the gas under standard conditions, in g·L. -1 T represents the average temperature inside the bottle during the evacuation process, in °C; W represents the dry mass of the soil sample, in g.

[0077] The cumulative greenhouse gas emissions during the cultivation period were estimated using linear interpolation. The calculation formula is: TF = ∑(Fi+1+Fi) / 2 × (Ti+1-Ti) × 24, where TF is the cumulative emissions during the cultivation period, in mg·g -1 ;Fi+1 represents the average emission flux of the gas collected in the (i+1)th experiment, in mg·g -1 ·h -1 Fi represents the average emission flux of the gas collected in the i-th sampling, in mg·g⁻¹. -1 ·h -1 ;Ti+1-Ti is the interval between the (i+1)th gas sampling and the ith gas sampling, d.

[0078] Figure 9 Different treatments affect ammonium nitrogen (NH4) + The effect of modified nitrification inhibitor (DMPP-γ-PGA) on ammonium nitrogen (NH4+) at each culture time point is shown in the figure. CK1 is the control group with only urea, CP1 is urea with 0.8% DMPP, CP2 is urea with 1.2% DMPP, CP3 is urea with 0.8% DMPP-γ-PGA, and CP4 is urea with 1.2% DMPP-γ-PGA. + The retention of NH4+ was significantly better in the control group (CK1) than in the unmodified (DMPP) group, with 1.2% DMPP-γ-PGA (CP4) exhibiting the best stability. Specifically, the NH4+ retention in the control group (CK1) was significantly better than that in the unmodified (DMPP) group. + The ammonium nitrogen (NH4+) content decreased sharply from 92.94 mg / kg on D1 to 1.30 mg / kg on D21, indicating that nitrification led to rapid consumption of ammonium nitrogen. In the unmodified inhibitor group, although 0.8% DMPP (CP1) and 1.2% DMPP (CP2) showed short-term ammonium nitrogen accumulation on D3 (reaching 121.79 mg / kg and 103.11 mg / kg, respectively), the NH4+ content decreased significantly on D21. + -N content decreased to 11.20 mg / kg and 19.73 mg / kg, respectively; the inhibitory effect was relatively enhanced in the modified inhibitor group, with 0.8% DMPP-γ-PGA (CP3) showing improved NH4+ levels at D21. + The -N content was 14.93 mg / kg, while 1.2% DMPP-γ-PGA (CP4) maintained the highest level at all time points, reaching 127.64 mg / kg at D3 and 21.76 mg / kg at D21, which was 10.2% higher than that of unmodified CP2 (19.73 mg / kg), and its D21 residual amount was 16.7 times that of CK1.

[0079] Figure 10 Different treatments affect nitrate nitrogen (NO3) - The effect of modified nitrification inhibitor (DMPP-γ-PGA) on nitrate nitrogen (NO3-N) is shown in the figure. CK1 is the control group with only urea, CP1 is urea with 0.8% DMPP, CP2 is urea with 1.2% DMPP, CP3 is urea with 0.8% DMPP-γ-PGA, and CP4 is urea with 1.2% DMPP-γ-PGA. The figure illustrates the effect of modified nitrification inhibitor (DMPP-γ-PGA) on nitrate nitrogen (NO3-N). - The inhibitory effect on NO3- accumulation was superior to that of the unmodified treatment group, with 1.2% DMPP-γ-PGA (CP4) showing the best persistence during long-term culture. Specific results are as follows: NO3- accumulation in the control group (CK1) was significantly lower than that in the control group (CK1). --N content surged from 40.41 mg / kg on D1 to a peak of 128.47 mg / kg on D7, stabilizing at 120.02 mg / kg on D21, indicating that uninhibited nitrification led to rapid accumulation of nitrate nitrogen; in the unmodified inhibitor group, NO3- in 0.8% DMPP (CP1) and 1.2% DMPP (CP2) - Although the -N content was significantly lower than CK1 (reduced by 14.9% and 22.5% respectively at D21), it still showed an increasing trend over time: CP1 was 102.14 mg / kg at D21, and CP2 was 93.68 mg / kg; the inhibitory effect of the modified inhibitor group was more significant and persistent, with 0.8% DMPP-γ-PGA (CP3) showing a decrease in NO3 at D21. - -N content was 99.61 mg / kg; while 1.2% DMPP-γ-PGA (CP4) was 33.86 mg / kg on D7 and 92.91 mg / kg on D21.

[0080] Table 2 shows the effect of DMPP-γ-PGA coupling on nitrification inhibition rate. It was found that γ-PGA modification significantly improved the inhibition performance of DMPP, and the high-concentration treatment (1.2% DMPP-γ-PGA) showed particularly outstanding performance in long-term inhibition. Data showed that the nitrification inhibition rate of each treatment exhibited a trend of "rapid increase, peak maintenance, and slow decline," reaching its peak on day 7 of cultivation and then gradually decreasing, but the modified group still maintained a significant advantage at day 21. In the short-term effect (1-7 days), 1.2% DMPP-γ-PGA showed the highest inhibition rate on the first day (22.16%±0.0465), which was 42.9% higher than the unmodified group at the same concentration (15.48%±0.0057). In the mid-term (7-14 days), although the difference between the modified group and the unmodified group narrowed, it was still higher than the unmodified group. By the time of long-term culture at 21 days, the inhibition rate of the 1.2% modified group (22.59%±0.0069) was still significantly higher than that of the unmodified group (21.95%±0.0068) and the 0.8% modified group (17.01%±0.0059).

[0081] Table 2 Effects of each treatment on nitrification inhibition rate

[0082]

[0083] Figure 11Figures a and b show the N2O emission flux and cumulative emissions for different treatments. Figure a represents the N2O emission flux, and Figure b represents the cumulative N2O emissions. CK1 is the control group with only urea; CP1 is urea with 0.8% DMPP; CP2 is urea with 1.2% DMPP; CP3 is urea with 0.8% DMPP-γ-PGA; and CP4 is urea with 1.2% DMPP-γ-PGA. The figures demonstrate the crucial role of DMPP and its γ-PGA modification in inhibiting N2O generation and release. As shown in Figures a and b, N2O emissions from each treatment exhibit a typical pattern of "rapid surge - sustained inhibition - slow decline": the control group CK1 (urea only) showed a significant peak (>0.022 mg·m³) on day 3 of culture. -2 ·h -1 Furthermore, the cumulative emissions reached their highest value (>1.35 mg / kg) during the D4-D7 stages, confirming the strong promoting effect of rapid urea nitrification on N2O emissions. In contrast, DMPP treatments (CP1, CP2) inhibited nitrification, preventing a significant peak in emission flux at D3 and reducing cumulative emissions by 1-2 times across the D1-D21 stages. γ-PGA-modified DMPP (CP3, CP4) further enhanced the inhibitory effect, with emission flux consistently lower than the unmodified group, and cumulative emissions reduced by 1-2 times compared to the control group at each stage from D1 to D21 (p < 0.05), demonstrating that the modification treatment prolonged the action period by delaying DMPP release.

[0084] In summary, in soil treated with 1.2% DMPP-γ-PGA + urea, NH4+ + The highest N-N retention was achieved, the lowest cumulative N2O emissions were observed, and the nitrification inhibition rate was significantly improved.

[0085] This invention provides a γ-polyglutamic acid-modified nitrification inhibitor (DMPP-γ-PGA conjugate), which significantly improves its thermal and storage stability. This modified product can be applied to nitrogen fertilizers to improve nitrogen fertilizer utilization and nitrogen absorption efficiency, providing a more environmentally friendly and efficient nitrification inhibition solution for agricultural production. The results of this study not only provide a theoretical basis for the development of novel long-acting nitrification inhibitors but also offer technical support for the efficient utilization of nitrogen fertilizers and environmental protection in agricultural production.

[0086] The above description is merely a preferred embodiment of the present invention and is not intended to limit the invention in any way. Any simple modifications, alterations, and equivalent changes made to the above embodiments based on the inventive essence shall still fall within the protection scope of the present invention.

Claims

1. A method for preparing a γ-polyglutamic acid-modified nitration inhibitor, characterized in that, Includes the following steps: S1. Prepare a γ-polyglutamic acid solution with a concentration of 8~12 mg / mL using deionized water, and adjust the pH to 7.0; S2. Prepare a 3,4-dimethylpyrazole phosphate solution with a concentration of 1.5~2.5 mg / mL using sodium chloride solution, and adjust the pH to 3.0-5.0; S3. Add the 3,4-dimethylpyrazole phosphate solution obtained in S2 to the γ-polyglutamic acid solution obtained in S1, wherein the mass ratio of the 3,4-dimethylpyrazole phosphate solution to the γ-polyglutamic acid solution is 1:1 to 1:

10. Adjust the pH of the mixture to 5.8, and then carry out the coupling reaction by stirring at 500 to 800 r / min at room temperature for 20 to 30 hours to obtain the reaction solution. S4. The reaction solution obtained in S3 is placed in a dialysis bag and dialyzed for 40-60 hours to remove free 3,4-dimethylpyrazole phosphate. Then, it is freeze-dried at -50°C for 40-60 hours to obtain γ-polyglutamic acid modified nitration inhibitor, denoted as DMPP-γ-PGA conjugate.

2. The method according to claim 1, characterized in that, The sodium chloride solution in S2 has a mass concentration of 0.9% w / v.

3. The method according to claim 1, characterized in that, The pH adjustment described in S1, S2, and S3 uses sodium hydroxide solution or hydrochloric acid solution, wherein the mass concentration of the sodium hydroxide solution is 6-10% w / v and the mass concentration of the hydrochloric acid solution is 4-8% w / v.

4. The method according to claim 1, characterized in that, The molecular weight cutoff range of the dialysis bag described in S4 is 8-14 kDa.

5. A γ-polyglutamic acid-modified nitration inhibitor prepared by the method described in any one of claims 1 to 4.

6. The application of the γ-polyglutamic acid-modified nitration inhibitor according to claim 5, characterized in that, The γ-polyglutamic acid-modified nitrification inhibitor is used in stable nitrogen fertilizers to improve nitrogen fertilizer utilization.

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