Phytase fungicide fertilizer as well as production method and application thereof

Abstract

The invention relates to the technical field of ecological agriculture, and discloses a phytase fungicide fertilizer as well as a production method and application thereof. 1-10 parts of a phytase high expression strain; 1-8 parts of a phytase solution; 1.7 to 9 parts of an embedding material; 3-8 parts of pH buffer particles; 10-30 parts of a micro-carbon carrier; 0.02 to 0.10 part of an indoleacetic acid microcapsule; and 30-60 parts of humus powder. The invention further provides a production method of the phytase fungicide fertilizer. The method comprises the following steps: S1, performing liquid fermentation on a phytase high-expression strain; s2, mixing the thalli with an enzyme solution; s3, adding pH buffer particles into the embedded particles; s4, drying the granules in a spray drying manner; and S5, mixing the dried product with humus powder, and granulating. By constructing a microbial inoculum system containing a phytase high-expression strain, efficient activation and conversion of organic phosphorus resources are realized, and the release efficiency of the fertilizer to phosphorus resources is improved.

Description

Technical Field

[0001] The present invention relates to the technical field of ecological agriculture, and specifically to a phytase bacterial agent fertilizer, its production method and application. Background Art

[0002] At present, traditional chemical fertilizers are widely used in agricultural production, but the utilization efficiency of their phosphate resources is low, especially the poor bioavailability of organic phosphorus, resulting in a large amount of phosphorus in the soil that cannot be effectively absorbed by crops. This not only causes waste of resources, but also exacerbates environmental pollution and affects the sustainable development of agriculture.

[0003] In view of the above related technologies, some studies have attempted to improve the utilization efficiency of phosphorus by adding microbial agents. However, the effects of these agents are not stable and are often limited by environmental factors such as soil pH and temperature. Many microbial agents have too fast or too slow activity in complex soil environments, resulting in insufficiently lasting effects and unable to form a long-term effective phosphorus desorption mechanism.

[0004] In addition, traditional phytase preparations also have certain limitations when applied in the soil. Although phytase can degrade organic phosphorus, its activity varies greatly under different soil conditions, especially in acidic or alkaline environments, it is prone to inactivation. Most of the enzyme fertilizers in the prior art lack a regulation mechanism for soil pH changes, resulting in the degradation ability of the enzyme not being fully exerted in some cases, and the fertilizer effect being difficult to be stable and continuous.

[0005] Currently, in the design of most phytase fertilizers, although the micro-carbon carrier improves the slow-release property of the fertilizer, due to its surface not being specially treated, it may not be able to fully and effectively combine with the active ingredients in the fertilizer. This lack of refined design of the micro-carbon carrier leads to inaccurate release control of the fertilizer and a short fertilizer efficiency cycle, affecting the overall use effect of the fertilizer.

[0006] Finally, the application of biostimulants in traditional fertilizers is mostly limited to single additives. Traditional plant growth regulators often decompose easily in the soil and have a short release time, resulting in their effects not being sustainable. Moreover, the release control of hormones in the prior art is mostly not precise enough to fully match the growth needs of crops and cannot effectively regulate the roots. Summary of the Invention

[0007] In view of the deficiencies of the prior art, the present invention provides a phytase bacterial agent fertilizer, its production method and application, which solve the problems of low utilization efficiency of phosphorus resources, unstable phytase activity and poor persistence of fertilizer effects in existing fertilizers.

[0008] To achieve the above objectives, the present invention is realized through the following technical solutions: A phytase bacterial agent fertilizer, in parts by mass, includes the following components; 1 - 10 parts of phytase highly-expressing strain; 1 - 8 parts of phytase solution; 1.7 - 9 parts of embedding material; 3 - 8 parts of pH buffer granules; 10 - 30 parts of micro-carbon carrier; 0.02 - 0.10 part of indoleacetic acid microcapsule; 30 - 60 parts of humus powder.

[0009] Preferably, the phytase highly-expressing strain is Bacillus subtilis.

[0010] Furthermore, Bacillus subtilis can efficiently decompose phytate in the soil and release phosphorus sources available for plants.

[0011] Preferably, the embedding material includes chitosan, sodium alginate and calcium chloride. The embedding material has a pH-responsive bilayer structure, which is formed by mixing chitosan and calcium chloride and then dropping sodium alginate. The mixing ratio of chitosan, sodium alginate and calcium chloride is 1:2 - 4:0.4 - 1.

[0012] Furthermore, chitosan, sodium alginate and calcium chloride form a pH-responsive bilayer structure. In this structure, after chitosan and calcium chloride are mixed, the embedding outer shell is formed by dropping sodium alginate. The function of this outer shell is to protect phytase from being stably present in the fertilizer for a long time and to be able to dynamically release phytase according to the change of pH value in the soil. This responsive structure ensures the high efficiency of the fertilizer in different pH environments. Especially in acidic soils, it can improve the activity of phytase and the release rate of phosphorus.

[0013] Preferably, the micro-carbon carrier is surface-modified bamboo charcoal powder, and the modification method is to coat it with 0.5 - 1.5% chitosan solution.

[0014] Furthermore, bamboo charcoal powder, as a material with a high specific surface area, plays a role in enhancing the stability of the fertilizer and improving the water retention capacity of the soil in the fertilizer. Through the coating modification of chitosan, not only the biocompatibility of bamboo charcoal powder is improved, but also its adhesion to other components in the fertilizer and the ability of sustained release are increased, further enhancing the slow-release effect of the fertilizer.

[0015] Preferably, the pH buffer granules are a complex of sodium citrate and calcium carbonate. Dissolve sodium citrate in an appropriate amount of deionized water to form an aqueous solution with a concentration of 10 - 30%, and slowly add fine calcium carbonate powder under stirring conditions to make it react evenly and form microparticles.

[0016] Furthermore, the pH buffer granules can play a role in stabilizing the pH in the soil and preventing the activity of phytase from being affected due to excessive changes in soil acidity and alkalinity.

[0017] Preferably, the indoleacetic acid microcapsules are prepared by the following steps; Mix indoleacetic acid with vegetable oil to form an indoleacetic acid oil phase; Add gelatin to deionized water, heat to 40 - 50 °C and stir to dissolve; Slowly drop the indoleacetic acid oil phase into it and emulsify to form a water-in-oil emulsion; Obtain indoleacetic acid microcapsules by freeze-drying and sieving to a particle size range of 50 - 100 μm.

[0018] Furthermore, the indoleacetic acid microcapsules play the role of plant hormones in fertilizers. By controlling their release in the soil, they promote the growth of plant roots. The design of the microcapsules effectively reduces the rapid volatilization and excessive release of hormones, while prolonging their effective time and providing continuous growth stimulation for plants.

[0019] A production method of a phytase bacterial fertilizer includes the following steps; S1. Carry out liquid fermentation on phytase highly expressed strains to obtain bacterial cells and enzyme solution; S2. Mix the bacterial cells and enzyme solution and add them to a sodium alginate solution, then drop them into a solution containing chitosan and calcium chloride to form nano-embedded particles; S3. Add pH buffer particles, indoleacetic acid microcapsules and surface-modified bamboo charcoal powder to the embedded particles and mix evenly; S4. Dry the particles by spray drying, with an inlet air temperature of 35 - 45 °C, an outlet air temperature of 28 - 35 °C, a spray pressure of 0.3 - 0.6 MPa, a drying time of 10 - 25 seconds, and a spray method of dual-channel spray drying; S5. Mix and granulate the dried product with humus powder at a mass ratio of 1:1 - 3:1, sieve and package.

[0020] Preferably, in step S1, the fermentation conditions of the strain are a temperature of 30 - 37 °C, a pH of 6.5 - 7.2, a fermentation time of 24 - 48 hours, and a shaker speed of 180 - 220 rpm.

[0021] Preferably, in step S2, the dropping rate is 1 - 5 mL / min, the reaction time is 20 - 40 minutes, and the reaction temperature is 20 - 25 °C.

[0022] An application of a phytase bacterial fertilizer in crop cultivation.

[0023] Furthermore, the fertilizer can effectively improve the availability of phosphorus in the soil, promote the root development of crops, and enhance the stress resistance of crops, especially in soils poor in phosphorus resources, showing significant effects.

[0024] The present invention provides a phytase bacterial fertilizer and its production method and application. It has the following beneficial effects: 1. The present invention realizes the efficient activation and transformation of organic phosphorus resources by constructing a microbial agent system containing phytase highly-expressing strains. The strains can quickly establish an enzymatic reaction system in the soil environment, significantly enhance the bioavailability of organic phosphorus in the soil, improve the release efficiency of phosphorus resources in fertilizers, and solve the problem of low phosphorus utilization rate in traditional fertilizers.

[0025] 2. The present invention significantly enhances the stability and activity maintenance ability of phytase under different soil pH values by adopting a pH-responsive double-layer embedding structure formed by chitosan, sodium alginate and calcium chloride. This structure can not only protect the core enzyme components from inactivation, but also regulate the release rhythm, improving the adaptability of the product under various environmental conditions.

[0026] 3. The present invention improves the synergy performance of each functional component in the fertilizer system by introducing surface-modified micro-carbon carriers as functional fillers. The high specific surface area and surface modification structure of the micro-carbon materials enhance the nutrient adsorption and fixation ability, contribute to the formation of a slow-release interface, extend the fertilizer efficiency period and improve the physical and chemical properties of the soil.

[0027] 4. The present invention realizes the coordinated regulation of the slow-release of biostimulants and root development through indole acetic acid microcapsules. This design can effectively extend the hormone action time window, avoid the problems of easy degradation and short effect of traditional hormones in the soil, thereby promoting the healthy development of crop roots and improving the nutrient absorption ability. BRIEF DESCRIPTION OF THE DRAWINGS

[0028] Figure 1 is the flowchart of the method of the present invention. DETAILED DESCRIPTION OF THE EMBODIMENTS

[0029] The technical solutions in the embodiments of the present invention will be clearly and completely described below with reference to the accompanying drawings of the present invention. Obviously, the described embodiments are only a part of the embodiments of the present invention, rather than all the embodiments. All other embodiments obtained by those of ordinary skill in the art based on the embodiments of the present invention without creative efforts shall fall within the protection scope of the present invention.

[0030] Please refer to the attached Figure 1 ; Example 1: Component ratio: Phytase highly-expressing strains: 5 parts Phytase solution: 4 parts Embedding material: 5 parts Chitosan: 1 part Sodium alginate: 2.5 parts Calcium chloride: 1.5 parts pH buffer granules: 5 parts Sodium citrate: 3 parts Calcium carbonate: 2 parts Micro-carbon carrier: 20 parts Surface-modified bamboo charcoal powder: 20 parts Indoleacetic acid microcapsule: 0.05 part Humus powder: 50 parts Strain fermentation: Inoculate the phytase highly expressed strain Bacillus subtilis into an appropriate amount of fermentation medium.

[0031] Fermentation conditions: temperature 37°C, pH 6.8, fermentation time 36 hours, shaker speed 200 rpm, cell concentration reaching 1×10 8 CFU / mL.

[0032] Collect the cells and obtain phytase solution.

[0033] Dissolve chitosan in deionized water at a concentration of 3% (3 parts of chitosan solution).

[0034] Add calcium chloride to the dissolved chitosan solution at a concentration of 0.5% (1.5 parts).

[0035] Gradually add sodium alginate to the solution to form a viscous liquid.

[0036] Drop the obtained solution into the sodium alginate solution and mix to form pH-responsive double-layer embedding particles.

[0037] Microencapsulated plant hormone: Mix indoleacetic acid with vegetable oil to form an indoleacetic acid oil phase.

[0038] Add gelatin to deionized water and heat to 45°C, stir until completely dissolved.

[0039] Slowly drop the oil phase into the gelatin solution to form a water-in-oil emulsion.

[0040] Through freeze-drying technology, dry the emulsion into microcapsules and finally screen to a particle size range of 50–100 μm.

[0041] Mix the phytase cells, embedding material, micro-carbon carrier, pH buffer particles, indoleacetic acid microcapsules and humus powder evenly.

[0042] Use spray-drying technology, inlet air temperature 40°C, outlet air temperature 30°C, spray pressure 0.4 MPa, drying time 15 seconds.

[0043] Mix the dried product with humus powder at a mass ratio of 2:1, granulate and screen to ensure uniform particle size.

[0044] Packaging is completed, and the final product is granular fertilizer.

[0045] Example 2: Component ratio: High-expression strain of phytase: 1 part Phytase solution: 1 part Embedding material: 1.7 parts Chitosan: 0.5 part Sodium alginate: 1 part Calcium chloride: 0.2 part pH buffer granules: 3 parts Sodium citrate: 1.5 parts Calcium carbonate: 1.5 parts Micro-carbon carrier: 10 parts Surface-modified bamboo charcoal powder: 10 parts Indoleacetic acid microcapsule: 0.02 part Humus powder: 30 parts Production steps: Ferment using a medium with a temperature of 30 °C and a pH value of 6.5.

[0046] The fermentation time is 24 hours, and the shaker speed is 180 rpm.

[0047] Obtain the bacterial cells by filtration and extract the phytase solution.

[0048] Mix chitosan and calcium chloride, then drip sodium alginate to form double-layer embedded granules.

[0049] Mixing ratio: chitosan:sodium alginate:calcium chloride is 1:2:0.4.

[0050] After preparing the solution, perform embedding at low temperature to ensure that the phytase is fully encapsulated.

[0051] According to the oil-in-water principle, mix the indoleacetic acid oil phase and the gelatin solution.

[0052] Use the freeze-drying technique to prepare microcapsules with a particle size of 50–100 μm.

[0053] Mix all the above components such as the embedded granules, micro-carbon carrier, and pH buffer granules according to the given ratio.

[0054] Use the spray-drying technique, with an inlet temperature of 35 °C, an outlet temperature of 28 °C, a drying time of 20 seconds, and a spray pressure of 0.3 MPa.

[0055] Mix the dried granules with the humus powder and granulate them at a mass ratio of 1:1 to 3:1.

[0056] Perform screening to finally obtain fertilizers with uniform particle size.

[0057] Example 3: Group allocation ratio: Phytase highly-expressing strain: 10 parts Phytase solution: 8 parts Embedding material: 9 parts Chitosan: 3 parts Sodium alginate: 4 parts Calcium chloride: 2 parts pH buffer granules: 8 parts Sodium citrate: 5 parts Calcium carbonate: 3 parts Micro-carbon carrier: 30 parts Surface-modified bamboo charcoal powder: 30 parts Indoleacetic acid microcapsule: 0.10 part Humus powder: 60 parts Production steps: Set the temperature at 37°C, pH value at 6.9, and shaker speed at 220 rpm.

[0058] Fermentation time is 48 hours, and finally a phytase solution with a relatively high concentration is obtained.

[0059] After dissolving chitosan and calcium chloride, add sodium alginate dropwise to form embedding particles, and the mixing ratio is 1:2.5:0.67.

[0060] After mixing evenly, add it dropwise to the calcium chloride solution, and a double-layer embedding material is formed after the reaction.

[0061] Mix indoleacetic acid and vegetable oil in proportion to prepare an oil phase; Emulsify the gelatin solution and the oil phase to form microcapsules.

[0062] Use the freeze-drying process to process the microcapsules and screen them to 50–100 μm.

[0063] Mix components such as phytase bacteria, embedding material, and micro-carbon carrier in proportion.

[0064] Use spray-drying technology, with an inlet air temperature of 45°C, an outlet air temperature of 35°C, a spray pressure of 0.5 MPa, and a drying time of 20 seconds.

[0065] Mix the dried product and humus powder in a ratio of 1:1 to 3:1, and then perform granulation and screening to obtain uniform granular fertilizer.

[0066] Comparative Example 1: Compared with Example 1, the difference is that the dosage of the phytase highly-expressing strain is reduced, and the rest are the same.

[0067] Comparative Example 2: Compared with Example 1, the difference is that a conventional organic carrier is used instead of the micro-carbon carrier, and the rest are the same.

[0068] Comparative Example 3: Compared with Example 1, the difference lies in that indoleacetic acid microcapsules were not added, and the rest were the same. Comparative Example 4: Compared with Example 1, the difference lies in that calcium chloride was not used in the embedding material, and the rest were the same.

[0069] Experiment name: Phytate degradation rate test Experiment purpose: To verify the influence of different dosages of phytase highly expressed strains on the phytate degradation ability, so as to compare the differences in functional performance between Example 1 and Comparative Example 1.

[0070] Experiment materials and reagents: Sodium phytate solution (initial concentration 5 mg / mL) pH 6.8 buffer solution Each treatment sample (Example 1, Comparative Example 1) Distilled water Colorimetric tubes, microtiter plates, spectrophotometer (700 nm) Preparation of treatment solution: Add equal amounts (0.5 g by dry weight) of Example 1 and Comparative Example 1 into two independent beakers respectively, add 10 mL of buffer solution, and ultrasonically vibrate for 30 min to form a treatment solution.

[0071] Add 0.5 mL of sodium phytate solution + 0.5 mL of treatment solution into a group of colorimetric tubes; Set 3 replicates for each group (n = 3), and set a blank control (without treatment solution, only add buffer solution); The reaction is left standing in a 37 °C water bath for 48 hours.

[0072] Add 1 mL of trichloroacetic acid solution to each sample to terminate the reaction; Centrifuge for 10 minutes (8000 rpm), and take the supernatant for determination.

[0073] Determination of phosphorus release amount: Take 0.5 mL of the supernatant and add 1 mL of ammonium molybdate color reagent; After standing for 30 minutes, measure the absorbance at 700 nm; Calculate the phosphorus concentration released by phytate degradation through the standard curve, and calculate the degradation rate (degradation rate = measured phosphorus release amount / theoretical phytate phosphorus content × 100%).

[0074] Table name: Table 1 Comparative experimental data of phytate degradation rate: Summary: Phytic acid exists in the soil in the form of organic phosphorus. Its phosphorus bond structure is stable, and it is difficult to effectively release the absorbable phosphorus in it through conventional fertilization. The results of this experiment show that when the addition amount of the high-expressing phytase strain decreases, the degradation rate of phytic acid significantly decreases, indicating that the accumulation level of phytase activity has a decisive influence on the conversion efficiency of organic phosphorus. By constructing a high-expressing phytase microbial community system, the enzymatic phosphorus-solubilizing reaction can be quickly initiated in the soil, significantly improving the effective conversion of insoluble phosphorus resources, and thus increasing the utilization rate of soil phosphorus sources by crops.

[0075] From the perspective of enzymatic kinetics, sufficient enzyme sources are the prerequisite for maintaining the reaction rate and stable release. The use of high-expressing strains not only increases the total enzyme activity level in the unit mass of the sample, but also cooperates with the stable embedding process during preparation to keep the phytase in a relatively slow-release state in the soil, thus achieving the goal of continuous phosphorus solubilization. The decrease in enzyme concentration caused by insufficient enzyme sources in the control group limits the rate of the enzymatic phosphorus-solubilizing reaction in the soil, reflecting the positive correlation between the microbial load and its phosphorus-solubilizing ability.

[0076] The solution of the present invention realizes an efficient and continuous phytic acid degradation reaction in the soil environment through the combined design of the regulation of the strain expression level and the high-activity enzyme release carrier.

[0077] Experiment name: Determination of the ability to increase available phosphorus in soil Experiment purpose: Under the same fertilization conditions, compare the effects of different carrier systems on the content of available phosphorus (quick-acting phosphorus) in the soil, so as to evaluate the effect of the micro-carbon carrier in promoting the stable release of nutrients after the action of phytase.

[0078] Experiment materials and apparatus: Samples: Fertilizers treated in Example 1 and Comparative Example 2 Acidic sandy soil (low-phosphorus background soil, available phosphorus < 5mg / kg) The fertilizer application rate is uniformly 1g / kg soil Potted plastic containers (12 cm in diameter) Deionized water Extractant: 0.5mol / L NaHCO3 (Olsen method) Spectrophotometer (700nm) Filters, centrifuge tubes, balances, measuring cylinders.

[0079] Experiment steps: Divide the air-dried and sieved acidic soil equally into each potted container, adding 1 kg to each pot; Set 3 replicates for each group, with a total of 6 groups of potted plants being treated (Example 1 × 3, Comparative Example 2 × 3); Add the samples at 1g / kg, mix well and add water to 60% of the field water holding capacity.

[0080] Incubate at a constant temperature at room temperature while keeping the humidity constant; Set the curing time to 14 days. During this period, replenish water without disturbing the soil structure.

[0081] Take 10 g of representative soil samples from the middle of each pot; Add 100 mL of 0.5 mol / L NaHCO3 solution and shake on a shaker for 30 minutes; Determine the phosphorus content of the filtrate using a spectrophotometer and calculate the available phosphorus (unit: mg / kg) Table name: Table 2 Comparison experimental data of soil available phosphorus content: Summary: It can be seen from the results of this experiment that under the same application dose, the treatment group using the micro-carbon carrier system can significantly improve the release level of available phosphorus in the soil. This result reflects that the micro-carbon carrier not only provides a physical support structure but also plays an interfacial synergistic role in enzymatic reactions. Its surface oxygen-containing functional groups and porous structure provide stable adsorption sites for phytase, which helps to enhance the directional action of the enzyme and the contact efficiency with the substrate, thereby promoting the continuous decomposition and transformation of organic phosphorus.

[0082] The high specific surface area and loose porous structure of the micro-carbon carrier can also adsorb and slowly release the released phosphorus to a certain extent, reducing the rapid inactivation problem caused by the poor mobility of phosphate and the strong soil fixation ability. This structure helps to form a microenvironment buffer zone, keeping the released phosphorus in a state that can be absorbed by crops for a longer time, thus enhancing the persistence of fertilizer efficiency. In contrast, conventional organic carriers lack this structural characteristic, resulting in a limited action area of phytase and unstable release of phosphorus, ultimately affecting the cumulative effect of available phosphorus.

[0083] Experiment name: Comparison of crop root length and root system quantity Experiment purpose: Evaluate the promoting effect of indole-3-acetic acid microcapsule treatment on plant root development and reflect the release efficiency of its biological activity in the system through actual root length and lateral root quantity.

[0084] Experiment materials and instruments: Samples: Example 1, Comparative Example 3 (both contain phytase and carrier system, the difference is whether indole-3-acetic acid microcapsules are added) Test plants: Chinese cabbage (or other model plants with short growth cycles) Seedling raising substrate (peat soil and vermiculite 1:1) Seedling trays, forceps, rulers, scalpels Deionized water, spray bottles Incubators (or natural light environments) Sowing and treatment: Evenly spread the seedling-raising substrate into the seedling tray, sow the pakchoi seeds, and cover them with a shallow layer of soil; Each treatment group has 30 plants. There are two groups in total, namely Example 1 and Comparative Example 3, with 3 replicates in each group (180 plants in total); Start the treatment on the 3rd day after emergence, and add 2 mL of the corresponding sample suspension to the rhizosphere of each plant.

[0085] Place it in a light incubator at a temperature of 25±2°C and a photoperiod of 12 h; Keep the substrate moist and replenish the liquid once every 2 days (the sample is not applied repeatedly); The cultivation period is 14 days in total.

[0086] Take samples on the 14th day, and select 10 plants with uniform growth in each group; Wash the roots, measure the length of the main root (cm) with a ruler, and count the number of lateral roots at the same time; Record the data of each plant and take the average value for statistical analysis.

[0087] Table name: Table 3 Comparison data of crop root growth indicators: Summary: The experimental results show that the group treated with indoleacetic acid microcapsules shows a more significant promoting effect on both the main root growth and the number of lateral roots, indicating that the slow-release plant hormone has good functions in promoting root differentiation and growth. As a classic endogenous auxin, indoleacetic acid can stimulate cell elongation and division, especially at the root tip. However, its chemical instability and easy degradation characteristics make its active release period short in the unencapsulated state, and it is difficult to maintain a stable concentration in the rhizosphere, resulting in limited biological effects.

[0088] In this system, indoleacetic acid is designed to be in a microencapsulated state, forming a compatible system with other components, and gradually releasing active substances in the rhizosphere. Through the controlled release mechanism, low-concentration and long-term acting auxin can be continuously provided at the root, effectively simulating the natural secretion rhythm of plants and avoiding the excessive or inhibitory effects caused by the sudden release of hormones. The obvious differentiation advantage shown by the plant roots in the experiment reflects the practical application value of this release strategy in regulating the development of root structure.

[0089] In addition, this system combines the growth-promoting mechanism based on phytic acid degradation, and constructs a dual-regulation model of nutrient release and root growth promotion. The indoleacetic acid slow-release structure not only improves the response ability of the roots to available phosphorus, but also helps the plants quickly establish a rhizosphere absorption system.

[0090] Experiment name: Enzyme activity stability test (pH buffer system) Experiment purpose: Compare the stability of phytase in different embedding systems under multi-pH environments, evaluate the role of calcium chloride in enzyme protection and buffer regulation, and verify its impact on maintaining enzyme activity.

[0091] Experimental materials and equipment: Samples: Example 1 (containing calcium chloride) and Comparative Example 4 (without calcium chloride) Buffer systems: pH 5.5, 6.0, 6.5, 7.0 (each prepared with citrate-phosphate buffer) Sodium phytate substrate solution (5 mg / mL) Water bath (constant temperature 37 °C) Centrifuge tubes, pipettes Ammonium molybdate color reagent, spectrophotometer (700 nm) Experimental procedures: Enzyme extraction: Add equal amounts of Example 1 and Comparative Example 4 to 10 mL of distilled water respectively, and ultrasonically vibrate for 30 min; After centrifugation, take the supernatant as the enzyme solution to be measured.

[0092] In 4 groups of buffers with different pH values, set the reaction system respectively: 0.5 mL of enzyme solution + 0.5 mL of sodium phytate substrate; Set 3 replicates for each group.

[0093] After reacting in a water bath at 37 °C for 60 minutes, add 1 mL of trichloroacetic acid to terminate the reaction; Centrifuge and take the supernatant, measure the absorbance at 700 nm after color development; Take the absorbance of Example 1 at pH 6.5 as the 100% enzyme activity standard, and calculate the relative enzyme activity percentage of other treatments.

[0094] Table name: Table 4 Comparison of enzyme activity stability under different pH conditions: Summary; The experimental results show that under different pH conditions, the embedding system constructed with the participation of calcium chloride can significantly improve the activity retention rate of phytase, showing stronger pH adaptability and structural stability. Calcium chloride forms a cross-linked network with sodium alginate during the embedding process, making the microcapsule structure denser, which can provide an effective physical barrier to enzyme molecules in a changing environment and slow down the direct impact of external pH fluctuations on the enzyme active center. This steady-state structure is of great significance for the continuous exertion of enzyme activity under weak acid to neutral conditions.

[0095] Meanwhile, the presence of calcium chloride helps to form a strong local buffering system in the microenvironment. In cooperation with the embedding matrix, it can maintain a relatively stable micro-pH environment around the enzyme molecules, avoiding irreversible denaturation of the enzyme conformation caused by changes in the external acidity and alkalinity. This mechanism of action is particularly crucial in the process of enzymatic hydrolysis, which helps to improve the maintenance ability of the entire system for enzyme function and enhance the continuity and consistency of phosphorus activation efficiency.

[0096] In addition, this experiment further verified that the components in the embedding system are not simply physically mixed, but achieve functional coupling through the construction of the spatial structure. Calcium chloride not only exists as a cross-linking agent, but also plays a dual role in terms of stable release and microenvironment regulation in terms of function.

[0097] Although the embodiments of the present invention have been shown and described, those of ordinary skill in the art can understand that various changes, modifications, substitutions and variations can be made to these embodiments without departing from the principle and spirit of the present invention. The scope of the present invention is defined by the appended claims and their equivalents.

Claims

1. A phytase bacterial agent fertilizer, characterized in that, Comprising the following components by mass parts; High phytase-expressing strain 1 - 10 parts; Phytase solution 1 - 8 parts; Embedding material, 1.7 - 9 parts; pH buffer particles 3 - 8 parts; Micro-carbon carrier 10 - 30 parts; Indoleacetic acid microcapsule 0.02 - 0.10 parts; Humus powder 30 - 60 parts.

2. The phytase bacterial agent fertilizer according to claim 1, wherein The high phytase-expressing bacterium is Bacillus subtilis.

3. The phytase bacterial agent fertilizer according to claim 1, wherein The embedding material includes chitosan, sodium alginate and calcium chloride. The embedding material is a pH-responsive bilayer structure formed by mixing chitosan and calcium chloride and then dropping sodium alginate. The mixing ratio of chitosan, sodium alginate and calcium chloride is 1:2 - 4:0.4 - 1.

4. The phytase bacterial agent fertilizer according to claim 1, characterized in that, The micro-carbon carrier is surface-modified bamboo charcoal powder, and the modification method is coating treatment with a 0.5 - 1.5% chitosan solution.

5. The phytase bacterial agent fertilizer according to claim 1, characterized in that, The pH buffer particles are a complex of sodium citrate and calcium carbonate. Dissolve sodium citrate in an appropriate amount of deionized water to form an aqueous solution with a concentration of 10 - 30%, and slowly add fine calcium carbonate powder under stirring conditions to make it react evenly and form micro-particles.

6. The phytase bacterial agent fertilizer according to claim 1, characterized in that, The indoleacetic acid microcapsules are prepared by the following steps; Mix indoleacetic acid with vegetable oil to form an indoleacetic acid oil phase; Add gelatin to deionized water and heat to 40 - 50°C with stirring to dissolve; Slowly drop the indoleacetic acid oil phase into it and emulsify to form a water-in-oil emulsion; Obtain indoleacetic acid microcapsules by freeze-drying and sieving to a particle size range of 50 - 100μm.

7. A production method of a phytase bacterial agent fertilizer for preparing a phytase bacterial agent fertilizer as described in any one of claims 1-6, characterized in that, Including the following steps; S1. Carry out liquid fermentation on the high phytase-expressing strain to obtain thalli and enzyme solution; S2. After mixing the thalli and the enzyme solution, add them to a sodium alginate solution, and drop them into a solution containing chitosan and calcium chloride to form nano-embedded particles; S3. Add pH buffer particles, indoleacetic acid microcapsules and surface-modified bamboo charcoal powder to the embedded particles and mix evenly; S4. Dry the particles by spray drying. The inlet air temperature is 35 - 45°C, the outlet air temperature is 28 - 35°C, the spray pressure is 0.3 - 0.6MPa, the drying time is 10 - 25 seconds, and the spray method is dual-channel spray drying; S5. Mix and granulate the dried product and the humus powder according to a mass ratio of 1:1 to 3:1, sieve and package.

8. The production method of a phytase bacterial agent fertilizer according to claim 7, characterized in that, The fermentation conditions of the strain in step S1 are temperature 30 - 37°C, pH 6.5 - 7.2, fermentation time 24 - 48 hours, and the shaker speed is 180 - 220 rpm.

9. The production method of a phytase bacterial agent fertilizer according to claim 7, characterized in that, The dropping rate in step S2 is 1 - 5 mL / min, the reaction time is 20 - 40 minutes, and the reaction temperature is 20 - 25°C.

10. Application of a phytase bacterial fertilizer as described in claims 1 - 6 in crop cultivation.

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