Soil organic matter improver based on complex microbial inoculant and method for preparing the same

Abstract

The application belongs to the technical field of soil organic matter improver, and discloses a soil organic matter improver based on a composite microbial agent and a preparation method thereof, which comprises components: a composite enzyme, a modified chitosan microsphere complex, a modified polyelectrolyte, an inhibitor L-methionine sulfoximine, a substrate, an auxiliary factor and a composite microbial agent.Taking the composite enzyme system as the core, the soil ammonium ion is efficiently assimilated through the glutamine synthetase / glutamate synthetase cycle and is converted into organic nitrogen, and the energy guarantee is provided by the help of polyphosphate kinase.The modified chitosan microsphere complex protects and releases the enzyme and the substrate, and at the same time, the controlled release of the inhibitor L-methionine sulfoximine is realized through the modified polyelectrolyte, so as to regulate the soil carbon-nitrogen balance.The system and the composite microbial agent synergistically act, effectively solve the problem of soil organic matter decline caused by excessive nitrogen application, significantly increase the crop root biomass and improve the soil organic matter content.

Description

Technical Field

[0001] This invention relates to the field of soil enhancement agent technology, and in particular to a soil organic matter enhancement agent based on a compound microbial agent and its preparation method. Background Technology

[0002] Soil organic matter is the general term for soil components derived from plant and animal remains, microorganisms, and their decomposition and transformation products. It is the core material basis for improving soil fertility and structure. In recent years, due to excessive application of nitrogen fertilizer, the rate of organic matter consumption has far exceeded the rate of organic matter accumulation. At the same time, high concentrations of nitrogen can also interfere with microbial communities, inhibit the activity of microorganisms involved in the synthesis and transformation of organic matter, reduce the amount of organic matter generated, and ultimately lead to a decline in the organic matter content in the soil. Summary of the Invention

[0003] The purpose of this invention is to provide a soil organic matter enhancer based on a compound microbial agent and its preparation method, so as to solve the problem of organic matter decline caused by excessive application of nitrogen fertilizer, while significantly increasing crop root biomass and thus increasing soil organic matter content.

[0004] Mechanism explanation:

[0005] This invention provides a systematic solution to the fundamental problem of excessive nitrogen fertilizer application leading to a rate of soil organic matter consumption exceeding its accumulation rate. In high-nitrogen environments, the large accumulation of inorganic nitrogen ions activates decomposing bacteria such as nitrifying bacteria, consuming existing soil organic matter as a carbon source while simultaneously inhibiting the activity of beneficial microorganisms involved in organic matter synthesis, thus creating a vicious cycle of continuous organic matter depletion.

[0006] Therefore, this invention introduces a complex enzyme, primarily based on the glutamine synthase / glutamate synthase cycle, which is the most important ammonia assimilation pathway in plants and microorganisms. Glutamine synthase is responsible for the assimilation of NH4+. + With extremely high affinity, this enzyme can efficiently capture ammonium ions in the soil. It does so by forming a tetrahedral transition state intermediate, transferring NH4+. + Acid covalently binds to the γ-carboxyl group of glutamate, forming glutamine. Glutamate synthase catalyzes the transfer of the amide group of glutamine to α-ketoglutarate, producing two molecules of glutamate, thus completing the organic fixation of nitrogen. Polyphosphokinase utilizes long-chain polyphosphates as phosphate donors to phosphorylate ADP into ATP, providing a continuous energy supply for the glutamine synthase reaction. This nitrogen assimilation process fixes inorganic nitrogen into organic nitrogen such as proteins, directly increasing soil organic matter reserves.

[0007] As the complex enzyme system continues to assimilate, inorganic nitrogen levels will decrease to the point of disrupting the carbon-nitrogen balance. This invention introduces an L-methionine sulfoxide imine inhibitor, which is a specific and irreversible inhibitor of glutamine synthase. By competitively binding to the glutamate binding site of the active site of glutamine synthase, it forms a stable covalent complex, leading to enzyme inactivation and thus interfering with the nitrogen assimilation process of plants.

[0008] The complex enzymes and inhibitors are originally produced through metabolism within microorganisms and are protected by the cell membrane inside the microbial cell. However, as exogenous additives, these complex enzymes and inhibitors are easily inactivated by environmental factors in the soil environment. Therefore, modified chitosan microspheres and modified polyelectrolytes are added. The modified chitosan microspheres are carboxymethylated to provide a negative charge for electrostatic interaction with the enzyme protein; polyethylene glycol grafting forms a protective molecular brush to block protease degradation; and alginate-Ca... 2+ Cross-linking constructs a mechanically stable three-dimensional network; β-cyclodextrin regulates the surface microenvironment, providing binding sites for subsequent molecular recognition; glutathione scavenge free radicals and maintains enzyme activity. The polyethyleneimine-polyacrylic acid polyelectrolyte complex in the modified polyelectrolyte exhibits the ionic strength sensitivity characteristic of polyelectrolytes. In high ionic strength environments, the carrier is in a contracted state; as the soil ionic strength decreases, the electrostatic shielding effect inside the carrier weakens, leading to swelling and release of L-methionine sulfoxide imine. The adamantyl group binds to the β-cyclodextrin in the modified chitosan microsphere complex, ensuring the precise release of L-methionine sulfoxide imine.

[0009] Soil organic matter enhancer based on compound microbial inoculant includes the following components by weight: 0.3-0.5 parts compound enzyme, 45-60 parts modified chitosan microsphere complex, 10-15 parts modified polyelectrolyte, 2-8 parts inhibitor, 28-56 parts substrate, 5-12 parts cofactor, and 8-15 parts compound microbial inoculant.

[0010] The complex enzyme is prepared by mixing glutamine synthase, glutamate synthase and polyphosphokinase in a mass ratio of 3:2:4.

[0011] The modified chitosan microsphere composite is a chitosan modified with carboxymethyl, grafted with polyethylene glycol, cross-linked with sodium alginate, and surface-coupled with β-cyclodextrin.

[0012] The modified polyelectrolyte carrier is a polyacrylic acid-polyethyleneimine composite polyelectrolyte with adamantane surface modification;

[0013] The inhibitor is L-methionine sulfoxide imine.

[0014] Furthermore, the substrate is prepared by mixing long-chain polyphosphate, glutamic acid, α-ketoglutarate, and adenosine diphosphate in a mass ratio of 8:3:2:1;

[0015] The auxiliary factor was prepared by mixing magnesium chloride and potassium chloride in a mass ratio of 5:3.

[0016] The compound microbial agent is prepared by mixing actinomycetes, lactic acid bacteria, yeast and Bacillus in a volume ratio of 5:5:8:20.

[0017] Furthermore, the modified chitosan microsphere composite was prepared by the following method:

[0018] S11. Add chitosan to deionized water, add 30% NaOH solution, stir at room temperature for 30 min, filter to obtain alkalized chitosan solution; add chloroacetic acid to isopropanol, add it dropwise to alkalized chitosan solution at 60°C, keep the pH at 8-9, react for 2 h, precipitate with acetone, filter to obtain carboxymethyl chitosan.

[0019] S12. Add carboxymethyl chitosan to PBS buffer, then add 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride and N-hydroxysuccinimide, and activate at room temperature for 30 min to obtain an activated carboxymethyl chitosan solution; add polyethylene glycol to deionized water to obtain a polyethylene glycol solution; then add the polyethylene glycol solution dropwise to the activated carboxymethyl chitosan solution, react at 4°C for 4 h with continuous stirring, and finally dialyze for 48 h to obtain a carboxymethyl chitosan-polyethylene glycol solution;

[0020] S13. Add glutamine synthase, glutamate synthase, and polyphosphokinase to Tris-HCl buffer, respectively, and then premix the enzyme solutions at 4°C for 30 min to obtain a composite enzyme premix. Mix long-chain polyphosphate, glutamate, α-ketoglutarate, adenosine diphosphate, magnesium chloride, and potassium chloride, add to deionized water, and adjust the pH to 7.2 to obtain a substrate premix.

[0021] S14. Add sodium alginate to deionized water, then add polyvinyl alcohol, heat until completely dissolved, cool to room temperature, and finally add carboxymethyl chitosan-polyethylene glycol solution to obtain alginate-carboxymethyl chitosan-polyethylene glycol mixture; mix the complex enzyme premix and substrate premix at 4°C, then add glutathione to obtain mixture A; finally, slowly add mixture A to the alginate-carboxymethyl chitosan-polyethylene glycol mixture to obtain mixture B; use a syringe pump to drop mixture B into CaCl2 solution, crosslink at room temperature for 2 hours to obtain gel microspheres;

[0022] S15. Add the gel microspheres to deionized water, disperse by sonication, add 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride and activate at room temperature for 30 min. Wash three times with deionized water, centrifuging each time to obtain an activated gel microsphere suspension. Add β-cyclodextrin to deionized water and heat to 60°C to dissolve completely. Add N-hydroxysuccinimide and stir to activate for 15 min. Adjust the pH to 8.0-8.5 with NaOH solution to obtain a β-cyclodextrin activated solution. Slowly add the activated gel microsphere suspension to the β-cyclodextrin activated solution and react at 4°C for 6-8 h, stirring every 30 min. Add Tris-HCl buffer and stir at room temperature for 30 min. Then centrifuge and wash the precipitate five times with PBS buffer, dispersing by sonication for 5 min each time. After centrifugation, freeze-dry under vacuum to obtain the modified chitosan microsphere complex.

[0023] Furthermore, the modified polyelectrolyte is prepared by the following method:

[0024] S21. Add polyacrylic acid to deionized water, adjust the pH to 9.0 with NaOH, and stir at room temperature for 45 minutes until completely dissolved to obtain an anionic polyacrylic acid solution; add polyethyleneimine to deionized water, adjust the pH to 3.0 with HCl, heat in a 60°C water bath for 30 minutes, and stir until completely dissolved to obtain a cationic polyethyleneimine solution; under vigorous stirring, slowly add the anionic polyacrylic acid solution dropwise to the cationic polyethyleneimine solution, continue stirring for 60 minutes, and adjust the pH to 7.0 to obtain a polyacrylic acid-polyethyleneimine composite suspension;

[0025] S22. The polyacrylic acid-polyethyleneimine composite suspension was transferred to a high-speed homogenizer and homogenized for 15 min. It was then filtered through a 0.22 μm filter membrane, and the filter residue microspheres were collected. The microspheres were washed four times with deionized water and centrifuged each time to obtain polyelectrolyte microspheres. The pH of L-methionine sulfoxide imide was adjusted to 10.0 in NaOH solution to obtain an L-methionine sulfoxide imide anionic solution. The polyelectrolyte microspheres were added to the L-methionine sulfoxide imide anionic solution and adsorbed by shaking at 4°C for 24 h. After centrifugation and washing, drug-loaded microspheres were obtained.

[0026] S23. Add 1-adamantane carboxylic acid to N,N-dimethylformamide, add 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride and N-hydroxysuccinimide to activate for 30 min, adjust the pH to 8.5 with triethylamine, disperse the drug-loaded microspheres in PBS buffer, slowly add adamantane activation solution, react under N2 protection for 4 h, wash 6 times with dimethyl sulfoxide:H2O (1:4) mixture, wash 3 times with deionized water, dialyze in a dialysis bag for 48 h, freeze dry for 72 h to obtain modified polyelectrolyte.

[0027] It should be noted that in step S21, the polyacrylic acid has a molecular weight of 100,000 and the polyethyleneimine has a molecular weight of 25,000; the mass ratio of polyethyleneimine to polyacrylic acid is 5:4. In step S23, the adamantane modification rate is ≥15%.

[0028] The preparation method of soil organic matter enhancer based on compound microbial inoculant includes the following steps:

[0029] S1. Preparation of modified chitosan microsphere composites;

[0030] S2, Preparation of modified polyelectrolytes;

[0031] S3. Preparation of compound microbial inoculant: Actinomycetes, lactic acid bacteria, yeast, and Bacillus were activated and propagated separately. The effective viable count of actinomycetes was 0.1 × 10⁻⁶. 9 ~1.0×10 9 CFU / ml, effective viable count of lactic acid bacteria 0.5×10⁻⁶ 9 ~5.0×10 9 CFU / ml, effective viable yeast count 0.5×10⁻⁶ 9 ~5.0×10 9 CFU / ml, effective viable Bacillus count 5×10⁻⁶ 9 ~20.0×10 9 The concentration of CFU / ml was then mixed at a volume ratio of 5:5:8:20 to prepare a compound microbial agent, wherein the effective viable count of the compound microbial agent is greater than 0.5 × 10⁻⁶. 9 CFU / m;

[0032] S4. Modified chitosan microsphere composite, modified polyelectrolyte, and composite microbial agent were sheared and mixed at 80 rpm for 40 min, and then freeze-dried under vacuum for 24 h to obtain a soil organic matter enhancer based on composite microbial agent.

[0033] Compared with the prior art, the present invention has the following beneficial effects:

[0034] 1. This invention achieves regulation of soil carbon-nitrogen ratio through the synergistic effect of a complex enzyme and an inhibitor. The complex enzyme first efficiently assimilates excess ammonium ions, converting them into organic nitrogen and directly increasing soil organic matter reserves. Before inorganic nitrogen is consumed to the point of causing carbon-nitrogen imbalance, the inhibitor L-methionine sulfoxide imine is released, specifically and irreversibly inhibiting glutamine synthase activity, thus terminating the nitrogen assimilation process in a timely manner, thereby avoiding excessive consumption of organic matter and maintaining a reasonable soil carbon-nitrogen balance.

[0035] 2. The modified chitosan microsphere complex of this invention effectively prevents the inactivation of the complex enzyme and substrate by encapsulating and protecting them, ensuring the continuous and efficient enzymatic reaction in the soil environment. The modified polyelectrolyte loads the inhibitor, and its unique ionic strength response characteristics cause it to swell and release the inhibitor when the soil ion concentration decreases. The two components combine through the molecular recognition of β-cyclodextrin and adamantane to form a delivery and regulation system.

[0036] 3. The synergistic effect of this compound enzyme system and compound microbial inoculants significantly increases crop root biomass and, consequently, soil organic matter content. Attached Figure Description

[0037] To more clearly illustrate the technical solutions of the embodiments of the present invention, the accompanying drawings used in the embodiments will be briefly described below. It should be understood that the following drawings only show some embodiments of the present invention and should not be regarded as a limitation of the scope. For those skilled in the art, other related drawings can be obtained based on these drawings without creative effort, wherein:

[0038] Figure 1 This is a process flow diagram of the preparation process of the soil organic matter enhancer based on the compound microbial agent of the present invention. Detailed Implementation

[0039] The technical solutions in the embodiments of the present invention are described below. The described embodiments are only some embodiments of the present invention, and not all embodiments. The specific parameters given in the following embodiments, such as temperature, speed, concentration, etc., are all exemplary illustrations. Those skilled in the art can adjust them within a reasonable range according to actual conditions, and such adjustments should be included within the protection scope of the present invention.

[0040] Examples 1-7: Soil organic matter enhancers based on composite microbial agents in Examples 1-7 of this invention comprise the following components by weight: a composite enzyme, a modified chitosan microsphere composite, a modified polyelectrolyte, an inhibitor, a substrate, a cofactor, and a composite microbial agent; the composite enzyme is prepared by mixing glutamine synthase, glutamate synthase, and polyphosphokinase in a mass ratio of 3:2:4; the modified chitosan microsphere composite is chitosan modified with carboxymethyl groups, grafted with polyethylene glycol, cross-linked with sodium alginate, and surface-coupled with β-cyclodextrin; the modified polyelectrolyte carrier is a polyelectrolyte with polyacrylic acid-polyethyleneimine composite and surface-modified with adamantane; the inhibitor is L-methionine sulfoxide imide.

[0041] The substrate is prepared by mixing long-chain polyphosphate, glutamic acid, α-ketoglutarate, and adenosine diphosphate in a mass ratio of 8:3:2:1.

[0042] The auxiliary factor was prepared by mixing magnesium chloride and potassium chloride in a mass ratio of 5:3.

[0043] The compound microbial agent is prepared by mixing actinomycetes, lactic acid bacteria, yeast and Bacillus in a volume ratio of 5:5:8:20.

[0044] The modified chitosan microsphere composite was prepared by the following method:

[0045] S11. Add chitosan to deionized water at a solid-liquid ratio of 1:25, and add 30% NaOH solution at a chitosan:NaOH solution ratio of 1:2 (g / mL). Stir at room temperature for 30 min, and filter through a 0.22 μm filter membrane to obtain an alkalized chitosan solution. Add chloroacetic acid to isopropanol at a solid-liquid ratio of 3:20, and add it dropwise to the alkalized chitosan solution at 60°C at a rate of 1-2 drops / s. Adjust the pH to 8-9 with 0.1 mol / L HCl or NaOH. After reacting for 2 h, precipitate with acetone (3 times the volume of the reaction solution), and filter to obtain carboxymethyl chitosan.

[0046] S12. Carboxymethyl chitosan was added to PBS buffer (pH 7.4) at a solid-liquid ratio of 1:40. 1-Ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride and N-hydroxysuccinimide were added to the solution at a mass ratio of 3:2. The mixture was activated at room temperature for 30 min to obtain an activated carboxymethyl chitosan solution. Polyethylene glycol was added to deionized water at a solid-liquid ratio of 4:30 to obtain a polyethylene glycol solution. The polyethylene glycol solution was then added dropwise to the activated carboxymethyl chitosan solution at a rate of 1-2 drops / s. The reaction was carried out at 4°C for 4 h with continuous stirring at 150 rpm. Finally, the mixture was dialyzed through a dialysis bag (molecular cutoff of 12000-14000 Da) for 48 h to obtain a carboxymethyl chitosan-polyethylene glycol solution.

[0047] S13. Glutamine synthase, glutamate synthase, and polyphosphokinase were added to 50 mmol / L Tris-HCl buffer (pH 7.4) at a mass ratio of 3:2:4 to prepare a solution with a total enzyme concentration of 5 mg / mL. The enzyme solution was then premixed at 4°C for 30 min to obtain a composite enzyme premix. Long-chain polyphosphate, glutamate, α-ketoglutarate, and adenosine diphosphate were mixed with magnesium chloride and potassium chloride at a mass ratio of 5:3 at a mass ratio of 8:3:2:1 and added to deionized water to prepare a solution with a total concentration of 100 mg / mL. The pH was adjusted to 7.2 with 0.1 mol / L HCl or NaOH to obtain a substrate premix.

[0048] S14. Add sodium alginate to deionized water at a solid-liquid ratio of 1:32, then add polyvinyl alcohol at a mass ratio of sodium alginate:polyvinyl alcohol = 5:2. Heat to 80°C until completely dissolved, then cool to room temperature. Finally, add carboxymethyl chitosan-polyethylene glycol solution at a volume ratio of 1:1 to obtain alginate-carboxymethyl chitosan-polyethylene glycol mixture. Mix the complex enzyme premix and substrate premix at a volume ratio of 1:1 at 4°C, then add glutathione at a mass ratio of sodium alginate:glutathione = 5:1 to obtain mixture A. Finally, slowly add mixture A to the alginate-carboxymethyl chitosan-polyethylene glycol mixture at a rate of 1-2 drops / s to obtain mixture B. Use a syringe pump (flow rate 0.5 mL / min) to drop mixture B into a 2% CaCl2 solution and crosslink at room temperature for 2 hours to obtain gel microspheres.

[0049] S15. Add gel microspheres to deionized water at a solid-liquid ratio of 1:15, disperse using ultrasonic power at 300W for 5 min, then add 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride at 0.5 times the mass of the gel microspheres, activate at room temperature for 30 min, wash three times with deionized water, centrifuge at 4000 rpm for 10 min each time, to obtain an activated gel microsphere suspension; add β-cyclodextrin to deionized water at a solid-liquid ratio of 1:20, heat to 60°C to completely dissolve, add N-hydroxysuccinimide at 0.3 times the mass of β-cyclodextrin, stir and activate for 15 min, then use 0.1 mol / L... The pH was adjusted to 8.0-8.5 with NaOH solution to obtain a β-cyclodextrin activation solution. The activated gel microsphere suspension was slowly added to the β-cyclodextrin activation solution at a volume ratio of 1:3. The reaction was carried out at 4°C for 6-8 hours, with stirring every 30 minutes (150 rpm). After the reaction, 50 mmol / L Tris-HCl buffer (pH 7.4) was added at twice the mass of the microspheres. The mixture was stirred at room temperature for 30 minutes, then centrifuged at 4000 rpm for 15 minutes. The precipitate was washed 5 times with PBS buffer (pH 7.4), and each time it was dispersed by sonication at 200 W for 5 minutes and then centrifuged at 4000 rpm for 10 minutes. Finally, the mixture was freeze-dried at -30°C and vacuum degree ≤10 Pa for 24 hours to obtain the modified chitosan microsphere complex.

[0050] The modified polyelectrolyte is prepared by the following method:

[0051] S21. Polyacrylic acid is added to deionized water at a solid-liquid ratio of 1:37.5. The pH is adjusted to 9.0 with 1 mol / L NaOH, and stirred at room temperature for 45 min until completely dissolved to obtain an anionic polyacrylic acid solution. Polyethylene imine is added to deionized water at a solid-liquid ratio of 1:20. The pH is adjusted to 3.0 with 1 mol / L HCl, and heated in a 60°C water bath for 30 min. The mixture is stirred until completely dissolved to obtain a cationic polyethylene imine solution. Under vigorous stirring (500 rpm), the anionic polyacrylic acid solution is slowly added dropwise to the cationic PEI solution at a rate of 1-2 drops / s. The mixture is stirred for another 60 min, and the pH is adjusted to 7.0 with 1 mol / L HCl or NaOH to obtain a polyacrylic acid-polyethylene imine composite suspension.

[0052] S22. The polyacrylic acid-polyethyleneimine composite suspension was transferred to a high-speed homogenizer and homogenized at 12000 rpm for 15 min. The mixture was then filtered through a 0.22 μm filter membrane, and the filter residue microspheres were collected. The microspheres were washed four times with deionized water, and centrifuged at 4000 rpm for 10 min each time to obtain polyelectrolyte microspheres. L-methionine sulfoxide imide was added to 0.1 mol / L NaOH solution to adjust the pH to 10.0 to obtain an L-methionine sulfoxide imide anionic solution. The polyelectrolyte microspheres were added to the solution at a ratio of polyelectrolyte microspheres:L-methionine sulfoxide imide anionic solution = 1:10 (w / v). The mixture was shaken at 120 rpm at 4°C for 24 h to adsorb the microspheres. The microspheres were washed three times with PBS buffer (pH 7.4), and centrifuged at 4000 rpm for 10 min each time to obtain drug-loaded microspheres.

[0053] S23. Add 1-adamantane carboxylic acid to N,N-dimethylformamide at a solid-liquid ratio of 1:25. Activate the mixture for 30 min with an 1:3:1.5 mass ratio of adamantane carboxylic acid: 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride: N-hydroxysuccinimide. Adjust the pH to 8.5 with 0.1 mol / L triethylamine to obtain the adamantane activation solution. Disperse the drug-loaded microspheres in PBS buffer (pH 8.0) at a solid-liquid ratio of 1:20. Slowly add the adamantane activation solution at a volume ratio of 1:2 (activation solution to microsphere suspension). React at 25°C for 4 h under N2 protection, stirring every 30 min. After the reaction is complete, use dimethyl sulfoxide:H2O (1:4). The mixture was washed 6 times, centrifuged at 4000 rpm for 10 min each time, then washed 3 times with deionized water, and finally dialyzed for 48 h with a dialysis bag (molecular weight cutoff 12,000-14,000 Da). It was then freeze-dried for 72 h at -30°C and vacuum degree ≤10 Pa to obtain the modified polyelectrolyte.

[0054] The soil organic matter enhancers based on composite microbial agents in Examples 1-7 of this invention were prepared by the following methods:

[0055] S1. Preparation of modified chitosan microsphere composites;

[0056] S2, Preparation of modified polyelectrolytes;

[0057] S3. Preparation of compound microbial inoculant: Actinomycetes, lactic acid bacteria, yeast, and Bacillus were activated and propagated separately. The effective viable count of actinomycetes was 0.1 × 10⁻⁶. 9 ~1.0×10 9 CFU / ml, effective viable count of lactic acid bacteria 0.5×10⁻⁶ 9 ~5.0×10 9 CFU / ml, effective viable yeast count 0.5×10⁻⁶ 9 ~5.0×10 9 CFU / ml, effective viable Bacillus count 5×10⁻⁶ 9 ~20.0×10 9 The concentration of CFU / ml was then mixed at a volume ratio of 5:5:8:20 to prepare a compound microbial agent, wherein the effective viable count of the compound microbial agent is greater than 0.5 × 10⁻⁶. 9 CFU / m;

[0058] S4. Modified chitosan microsphere composite, modified polyelectrolyte, and composite microbial agent were sheared and mixed at 80 rpm for 40 min, then freeze-dried at -30℃ and vacuum degree ≤10 Pa for 24 h to obtain a soil organic matter enhancer based on composite microbial agent. The preparation process is as follows: Figure 1 As shown.

[0059] The formulations of the composite enzyme, modified chitosan microsphere composite, modified polyelectrolyte, inhibitor, substrate, cofactor, and composite microbial agent in Examples 1-7 of this invention are shown in Table 1.

[0060] Table 1 Formulations of Examples 1-7

[0061]

[0062] Comparative Example 1: Based on Example 3, but differing from Example 3 in that no complex enzyme was added in this comparative example. Specifically, no complex enzyme was added during mixing in step S4. Accordingly, in preparing the modified chitosan microsphere complex, no complex enzyme premix was added in steps S13 and S14. Instead, the substrate premix was mixed with glutathione, and then further processed with an alginate-carboxymethyl chitosan-polyethylene glycol mixture to finally obtain blank chitosan microspheres containing only the substrate and cofactor. The remaining components and preparation methods were consistent with those in Example 3.

[0063] Comparative Example 2: Based on Example 3, but differing from Example 3 in that no modified chitosan microsphere complex was added in this comparative example. Specifically, during mixing in step S4, an equal amount of blank chitosan microspheres, unencapsulated with enzymes and substrate, was used instead. The preparation of these blank chitosan microspheres only included steps S11, S12, S14 (without adding any mixture other than the carboxymethyl chitosan-polyethylene glycol solution) and S15, i.e., only the carrier framework was constructed. The remaining components and preparation methods were consistent with Example 3.

[0064] Comparative Example 3: Based on Example 3, but differing from Example 3 in that no modified polyelectrolyte was added in this comparative example. Specifically, during mixing in step S4, equal amounts of blank polyelectrolyte microspheres (prepared solely through step S21) without adamantane modification and inhibitor loading were physically mixed with free L-methionine sulfoxide imine. The remaining components and preparation methods were consistent with those of Example 3.

[0065] Comparative Example 4: Based on Example 3, but differing from Example 3 in that the inhibitor L-methionine sulfoxide imide was not added in this comparative example. Specifically, in the preparation of the modified polyelectrolyte, the drug loading process in step S22 was omitted, and only steps S21 and S23 were performed, ultimately obtaining blank polyelectrolyte microspheres without inhibitor loading and modified only with adamantane. These blank microspheres were used during mixing in step S4. The remaining components and preparation methods were consistent with those of Example 3.

[0066] Comparative Example 5: Based on Example 3, but differing from Example 3 in that no compound microbial agent was added in this comparative example. Specifically, the addition of this component was omitted during mixing in step S4. The remaining components and preparation methods were the same as in Example 3.

[0067] Comparative Example 6: Based on Example 3, but differing from Example 3 in that the composition ratio of the complex enzyme was changed. Specifically, the complex enzyme used was prepared by mixing glutamine synthase, glutamate synthase, and polyphosphokinase in a mass ratio of 1:1:1. The preparation of this complex enzyme and the subsequent process of embedding it into modified chitosan microspheres were the same as in Example 3. The remaining components and preparation methods were consistent with those in Example 3.

[0068] Comparative Example 7: Based on Example 3, but differing from Example 3 in that the composition ratio of the composite microbial agent was changed. Specifically, the composite microbial agent used was prepared by mixing actinomycetes, lactic acid bacteria, yeast, and Bacillus in a volume ratio of 1:1:1:1. The remaining components and preparation methods were the same as in Example 3.

[0069] Comparative Example 8: Based on Example 3, but differing from Example 3 in that unmodified polyelectrolytes were used in this comparative example. Specifically, in preparing the polyelectrolyte carrier, only step S21 was performed to obtain polyacrylic acid-polyethyleneimine composite microspheres, without adamantane modification and inhibitor loading. In step S4, the unmodified microspheres were physically mixed with free L-methionine sulfoxide imine. The remaining components and preparation methods were consistent with Example 3.

[0070] Comparative Example 9: Based on Example 3, but differing from Example 3 in that no substrate was added in this comparative example. Specifically, in preparing the modified chitosan microsphere complex, no substrate premix was added in steps S13 and S14. The complex enzyme premix was simply mixed with glutathione, and then further processed with an alginate-carboxymethyl chitosan-polyethylene glycol mixture to finally obtain chitosan microspheres encapsulated only with the complex enzyme. The remaining components and preparation methods were consistent with Example 3.

[0071] Comparative Example 10: Based on Example 3, but differing from Example 3 in that no auxiliary factors were added in this comparative example. Specifically, when preparing the modified chitosan microsphere composite, magnesium chloride and potassium chloride were not added when preparing the substrate premix in step S13. The remaining components and preparation methods were the same as in Example 3.

[0072] Experimental Example 1: Determination of Soil Physicochemical Indicators.

[0073] I. Experimental Materials:

[0074] Test site soil: Brown soil with organic matter content of 16.8±2.1 g / kg, ammonium ion content of 38.5±5.0 mg / kg, carbon content of 9.75±1.27 g / kg, nitrogen content of 1.43±0.19 g / kg, carbon-nitrogen ratio of 6.8±0.9, electrical conductivity of 88.5±11.5 μS / cm, pH of 6.2±0.3, available phosphorus content of 18.5±2.4 mg / kg, and available potassium content of 125±16 mg / kg was selected.

[0075] Treatment groups T1-T7 correspond to the soil organic matter enhancers formulated in Examples 1-7, control groups C1-C10 correspond to formulations in proportions 1-10, and CK is a blank control, without any formulation applied.

[0076] II. Experimental Methods:

[0077] 1. The experiment involved applying 2 kg of soil conditioner per acre.

[0078] 2. Trial period: 120 days.

[0079] 3. Experimental crop: Peanut (variety: Luhua 11).

[0080] 4. Detection method:

[0081] Soil organic matter content: potassium dichromate titration method - external heating method.

[0082] Ammonium ion content: Indophenol blue colorimetric method.

[0083] Carbon content: elemental analysis method.

[0084] Nitrogen content: Total nitrogen was determined by the Kjeldahl method.

[0085] Carbon-nitrogen ratio: Calculation method (carbon content / nitrogen content).

[0086] Total ion concentration: conductivity method.

[0087] Table 2 Results of Soil Physicochemical Indicators

[0088]

[0089] The soils treated in Examples 1-7 showed significantly higher organic matter content than the control. Simultaneously, the ammonium ion content was effectively reduced. Carbon and nitrogen content increased simultaneously, with the carbon-to-nitrogen ratio remaining stable within an optimal range. The decrease in total ion concentration indicates an improvement in soil salinity. These data collectively demonstrate that this enhancer effectively promotes the conversion of inorganic nitrogen to organic matter, increases soil organic matter reserves, and optimizes the soil chemical environment.

[0090] Comparative Example 1 showed no difference in any of its indicators compared to the blank control, demonstrating that the exogenously added complex enzyme (GS / GOGAT cycle) is the core of the entire system initiating inorganic nitrogen assimilation and organic matter synthesis. Comparative Example 2 showed a significantly weakened effect, indicating that the modified chitosan microspheres, acting as a protective and slow-release carrier for the enzyme and substrate, are crucial for maintaining the long-term activity of the complex enzyme in complex soil environments. Comparative Examples 3 and 4 showed high levels of residual ammonium ions, demonstrating that the ionic strength-responsive swelling characteristics of the modified polyelectrolyte enabled intelligent controlled release of L-methionine sulfoxide imine. This inhibitor terminates the assimilation process by precisely inactivating the GS enzyme, preventing excessive nitrogen consumption and disruption of the carbon-nitrogen balance. Comparative Examples 9 and 10 showed poor results, confirming that the substrate and cofactors provide the necessary raw materials and energy for the enzymatic reaction, which is fundamental to maintaining efficient metabolic cycles.

[0091] Experimental Example 2: Soil Microbial Detection.

[0092] Experimental materials:

[0093] Soil and treatment group similar to those in Experiment 1.

[0094] Detected bacterial species: Actinomycetes, lactic acid bacteria, yeast, and Bacillus.

[0095] Test method:

[0096] 1. The viable count of each bacterial species was determined using the dilution plate method.

[0097] 2. Testing time: 30 days, 60 days, 90 days, and 120 days after application.

[0098] 3. Culture media: Actinomycetes: Modified Gao's No. 1 medium; Lactic acid bacteria: MRS medium; Yeast: Martin medium; Bacillus: Nutrient agar medium.

[0099] 4. Culture conditions: 28±2℃, culture for 48-72h.

[0100] Table 3 Soil microbial test results

[0101]

[0102] 120 days after application of the enhancer, the number of the four major functional microorganisms in the soils treated in Examples 1-7 was significantly higher than that in the blank control. This indicates that the enhancer can not only improve the physical and chemical properties of the soil, but also significantly promote the proliferation of beneficial microbial communities in the soil, thus building a healthier micro-ecosystem.

[0103] Although Comparative Example 5 showed a slight increase in the number of each bacterial group compared to the control group, it was still far lower than that of the Example 1. This directly proves that the exogenously added compound microbial agent is the core source for rapidly building a dominant beneficial bacterial community. The number of microorganisms in Comparative Example 1 was not significantly different from that in the control group, indicating that improving the soil microenvironment through enzymatic reactions is an important way to promote the growth of native microorganisms. The effect of Comparative Example 7 was better than the control but less effective than the Example 1 with the optimal ratio, indicating that the specific ratio of Actinomycetes:Lactic Acid Bacteria:Yeast:Bacillus = 5:5:8:20 is an optimized synergistic combination that can most effectively colonize and exert its effects.

[0104] Experimental Example 3: Peanut Root Growth Detection.

[0105] I. Experimental Materials:

[0106] Peanut variety: Same as in Experiment 1.

[0107] Soil and treatment group: Same as Experiment 1.

[0108] Experimental scale: Each treatment group had 3 replicates, and each replicate had 20 plants.

[0109] II. Experimental Methods:

[0110] 1. The reproductive period is 120 days, and samples are taken at maturity.

[0111] 2. Testing indicators: Root morphology (main root length: ruler measurement method).

[0112] Table 4. Results of peanut root growth test (maturity period 120 days)

[0113]

[0114] Peanuts treated in Examples 1-7 showed improved root development. The comparative treatment data were generally inferior to those in Example 3, confirming that improved crop root systems are the ultimate manifestation of the synergistic improvement in soil physicochemical and microbiological indicators.

[0115] The above description is merely a preferred embodiment of the present invention and is not intended to limit the scope of protection of the present invention. Any modifications, equivalent substitutions, and improvements made by those skilled in the art within the spirit and principles of the present invention should be included within the scope of protection of the present invention.

Claims

1. A soil organic matter enhancer based on compound microbial inoculants, characterized in that, It includes the following components by weight: 0.3-0.5 parts of complex enzyme, 45-60 parts of modified chitosan microsphere complex, 10-15 parts of modified polyelectrolyte, 2-8 parts of inhibitor, 28-56 parts of substrate, 5-12 parts of cofactor, and 8-15 parts of complex microbial agent. The complex enzyme is prepared by mixing glutamine synthase, glutamate synthase and polyphosphokinase in a mass ratio of 3:2:

4. The modified chitosan microsphere composite is a chitosan modified with carboxymethyl, grafted with polyethylene glycol, cross-linked with sodium alginate, and surface-coupled with β-cyclodextrin. The modified chitosan microsphere composite was prepared by the following method: S11. Add chitosan to deionized water, add 30% NaOH solution, stir at room temperature for 30 min, filter to obtain alkalized chitosan solution; add chloroacetic acid to isopropanol, add it dropwise to alkalized chitosan solution at 60℃, keep the pH at 8-9, react for 2 h, precipitate with acetone, filter to obtain carboxymethyl chitosan. S12. Add carboxymethyl chitosan to PBS buffer, then add 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride and N-hydroxysuccinimide, and activate at room temperature for 30 min to obtain an activated carboxymethyl chitosan solution; add polyethylene glycol to deionized water to obtain a polyethylene glycol solution; then add the polyethylene glycol solution dropwise to the activated carboxymethyl chitosan solution, react at 4℃ for 4 h with continuous stirring, and finally dialyze for 48 h to obtain a carboxymethyl chitosan-polyethylene glycol solution; S13. Add glutamine synthase, glutamate synthase, and polyphosphokinase to Tris-HCl buffer, respectively, and then premix the enzyme solutions at 4°C for 30 min to obtain a composite enzyme premix. Mix long-chain polyphosphate, glutamate, α-ketoglutarate, adenosine diphosphate, magnesium chloride, and potassium chloride, add to deionized water, and adjust the pH to 7.2 to obtain a substrate premix. S14. Add sodium alginate to deionized water, then add polyvinyl alcohol, heat until completely dissolved, cool to room temperature, and finally add carboxymethyl chitosan-polyethylene glycol solution to obtain a mixture of alginate-carboxymethyl chitosan-polyethylene glycol. The premixed enzyme solution and the premixed substrate solution were mixed at 4°C, and then glutathione was added to obtain mixture A. Finally, mixture A was slowly added to the alginate-carboxymethyl chitosan-polyethylene glycol mixture to obtain mixture B. Mixture B was dropped into CaCl2 solution using a syringe pump and crosslinked at room temperature for 2 hours to obtain gel microspheres. S15. Add the gel microspheres to deionized water, disperse by sonication, add 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride and activate at room temperature for 30 min. Wash three times with deionized water, centrifuging each time to obtain an activated gel microsphere suspension. Add β-cyclodextrin to deionized water and heat to 60℃ to dissolve completely. Add N-hydroxysuccinimide and stir to activate for 15 min. Adjust the pH to 8.0-8.5 with NaOH solution to obtain a β-cyclodextrin activated solution. Slowly add the activated gel microsphere suspension to the β-cyclodextrin activated solution and react at 4℃ for 6-8 h, stirring every 30 min. Add Tris-HCl buffer and stir at room temperature for 30 min. Then centrifuge. Wash the precipitate five times with PBS buffer, dispersing by sonication for 5 min each time and centrifuging. Freeze-dry under vacuum to obtain the modified chitosan microsphere complex. The modified polyelectrolyte carrier is a polyacrylic acid-polyethyleneimine composite polyelectrolyte with adamantane surface modification; The modified polyelectrolyte was prepared by the following method: S21. Add polyacrylic acid to deionized water, adjust the pH to 9.0 with NaOH, stir at room temperature for 45 minutes until completely dissolved to obtain an anionic polyacrylic acid solution; add polyethyleneimine to deionized water, adjust the pH to 3.0 with HCl, heat in a 60°C water bath for 30 minutes, and stir until completely dissolved to obtain a cationic polyethyleneimine solution. Under vigorous stirring, the anionic polyacrylic acid solution was slowly added dropwise to the cationic polyethyleneimine solution, and stirring was continued for 60 min. The pH was then adjusted to 7.0 to obtain a polyacrylic acid-polyethyleneimine complex suspension. S22. The polyacrylic acid-polyethyleneimine composite suspension was transferred to a high-speed homogenizer and homogenized for 15 min. It was then filtered through a 0.22 μm filter membrane, and the filter residue microspheres were collected. The microspheres were washed four times with deionized water and centrifuged each time to obtain polyelectrolyte microspheres. The pH of L-methionine sulfoxide imide was adjusted to 10.0 in NaOH solution to obtain an L-methionine sulfoxide imide anionic solution. The polyelectrolyte microspheres were added to the L-methionine sulfoxide imide anionic solution and adsorbed by shaking at 4 °C for 24 h. After centrifugation and washing, drug-loaded microspheres were obtained. S23. Add 1-adamantane carboxylic acid to N,N-dimethylformamide, add 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride and N-hydroxysuccinimide to activate for 30 min, adjust the pH to 8.5 with triethylamine, disperse the drug-loaded microspheres in PBS buffer, slowly add adamantane activation solution, react under N2 protection for 4 h, wash 6 times with dimethyl sulfoxide:H2O mixture, wash 3 times with deionized water, dialyze through a dialysis bag for 48 h, freeze dry for 72 h to obtain the modified polyelectrolyte; The inhibitor is L-methionine sulfoxide imine; The substrate is prepared by mixing long-chain polyphosphate, glutamic acid, α-ketoglutarate, and adenosine diphosphate in a mass ratio of 8:3:2:1; the cofactor is prepared by mixing magnesium chloride and potassium chloride in a mass ratio of 5:3; and the compound microbial agent is prepared by mixing actinomycetes, lactic acid bacteria, yeast, and Bacillus in a volume ratio of 5:5:8:

20.

2. The method for preparing the soil organic matter enhancer based on the composite microbial agent according to claim 1, characterized in that, Includes the following steps: S1. Preparation of modified chitosan microsphere composites; S2, Preparation of modified polyelectrolytes; S3. Preparation of compound microbial inoculant: Actinomycetes, lactic acid bacteria, yeast, and Bacillus were activated and propagated separately. The effective viable count of actinomycetes was 0.1 × 10⁻⁶. 9~ 1.0×10 9 CFU / ml, effective viable count of lactic acid bacteria 0.5×10⁻⁶ 9 ~5.0×10 9 CFU / ml, effective viable yeast count 0.5×10⁻⁶ 9 ~5.0×10 9 CFU / ml, effective viable Bacillus count 5×10⁻⁶ 9 ~20.0×10 9 The concentration of CFU / ml was then mixed at a volume ratio of 5:5:8:20 to prepare a compound microbial inoculant. The effective viable count of the compound microbial agent is greater than 0.5 × 10⁻⁶. 9 CFU / ml; S4. Modified chitosan microsphere composite, modified polyelectrolyte, and composite microbial agent were sheared and mixed at 80 rpm for 40 min, and then vacuum freeze-dried for 24 h to obtain a soil organic matter enhancer based on composite microbial agent.

Images