Preparation method of drought-resistant polyglycoside synergistic fertilizer

Abstract

The application discloses a preparation method of drought-resistant polyglycoside synergistic fertilizer, and belongs to the technical field of agricultural fertilizer.The gamma-polyglutamic acid from microbial fermentation and the iridoid glycosides from plant extraction are chemically bonded at the molecular level by two-stage gradient induction self-assembly technology to prepare polyglycoside compounds with a three-dimensional network structure, and then the polyglycoside compounds are compounded with fertilizer base materials and granulated by a high tower melting to obtain the drought-resistant polyglycoside synergistic fertilizer.The polyglycoside compounds prepared by the application form a stable three-dimensional network structure through ester bonds and hydrogen bonds, have physical water retention and biological induction dual functions, and the water retention rate in soil is increased by 30%-50% compared with common fertilizer, the corn yield is increased by 20%-30%, and the fertilizer utilization rate can be increased by 18.8%, so that the drought-resistant synergistic effect is remarkable and the industrial application value is high.

Description

Technical Field

[0001] This invention belongs to the field of agricultural fertilizer technology, and specifically discloses a method for preparing a drought-resistant polyglycoside-enhanced fertilizer. Background Technology

[0002] Fertilizer is a crucial material foundation for agricultural production. With the intensification of global climate change, drought has become one of the major abiotic stress factors restricting crop yield and quality. Statistics show that crop yield reduction due to drought exceeds the total reduction caused by other natural disasters. Traditional drought-resistant fertilizers mainly enhance soil water-holding capacity by adding water-retaining agents such as superabsorbent polymers. However, these products often suffer from short-lived water retention, soil compaction, and incoordination with fertilizer nutrient release. In recent years, researchers have begun to focus on fertilizer enhancement technologies that utilize bioactive substances to improve crops' drought resistance. For example, Chinese patent application CN112745163A discloses a humic acid water-soluble fertilizer with drought-resistant properties. By adding drought-resistant agents such as polyglutamic acid and alginic acid, and synergists such as sodium naphthaleneacetate, it achieves a certain drought-resistant effect. However, the components are simply mixed physically, resulting in limited synergistic effects. Chinese patent application CN102167642A discloses a novel drought-resistant and efficiency-enhancing water and fertilizer conditioner, mainly using agricultural organic waste and natural minerals as raw materials. However, the product's function is relatively simple, and its induction effect on the crop's own drought resistance mechanism is insufficient. Chinese patent application CN116693350A discloses a drought-resistant and efficiency-enhancing fertilizer that uses humic acid and compound microbial agents, but does not involve the application of plant glycosides. Chinese patent application CN201410564344.5 discloses a fertilizer synergist additive that uses a combination of polyglutamic acid with urease inhibitors and nitrification inhibitors, but similarly does not involve the introduction of plant glycosides or their synergistic mechanism with polyglutamic acid.

[0003] Plant-derived bioactive substances have attracted widespread attention in the agricultural field due to their natural, environmentally friendly, and multifunctional characteristics. Plant glycosides (cyclohexene glycosides) are a class of natural products with good biological activity; studies have shown that they can regulate plant hormone balance, induce stress-resistance gene expression, and promote root development and nutrient absorption. However, directly applying plant glycosides to fertilizers suffers from problems such as poor stability, easy degradation, and weak binding with fertilizer nutrients, limiting their application in the fertilizer field. To address the shortcomings of existing technologies, this invention develops a method for preparing a drought-resistant polyglycoside-enhanced fertilizer. Through gradient-induced self-assembly technology, microbial-derived γ-polyglutamic acid and plant-derived cyclohexene glycosides are chemically bonded and self-assembled at the molecular level to form a polyglycoside complex with a three-dimensional network structure, which is then further compounded and granulated with fertilizer base materials. Compared with existing technologies, the polyglycoside complex prepared by this invention has both the water-retaining and complexing function of polyglutamic acid and the bio-inducing function of plant glycosides. Through the dual synergistic mechanism of "physical water retention + bio-induction", it significantly improves the drought resistance and nutrient utilization of fertilizers. Moreover, the product has good stability and excellent slow-release performance, achieving a synergistic effect of 1+1>2. Summary of the Invention

[0004] Purpose of this invention: The purpose of this invention is to overcome the shortcomings of the prior art and provide a method for preparing a drought-resistant polyglycoside synergistic fertilizer. By using gradient-induced self-assembly technology, γ-polyglutamic acid and plant glycosides are chemically bonded at the molecular level to form a polyglycoside complex with a three-dimensional network structure. This complex is then used as a synergist to prepare a drought-resistant synergistic fertilizer that has both physical water retention and biological induction functions, thus solving the problems of single function, weak synergistic effect and poor stability of existing drought-resistant fertilizers.

[0005] Technical solution of the present invention: To achieve the above objectives, the present invention provides a method for preparing a drought-resistant polyglycoside-enhanced fertilizer, comprising the following steps: S1. Preparation of polyglycoside synergists: S11. Mix and stir the γ-polyglutamic acid fermentation broth with the plant glycoside extract to obtain a mixture; S12. First induction reaction: Adjust the pH of the mixture with inorganic acid, then stir to allow the carboxyl group of γ-polyglutamic acid to undergo hydrogen bonding and partial esterification with the hydroxyl group of plant glycosides, forming a primary complex; S13. Second stage of self-assembly reaction: Add a cross-linking inducer to the reaction system. The amount added is 0.5% to 2% of the mass of the γ-polyglutamic acid fermentation broth in step S11. Continue to adjust the pH with alkaline solution and then continue stirring to allow the primary complex to further cross-link and self-assemble under the action of the cross-linking inducer to form a polyglycoside complex with a three-dimensional network structure. S14. Purification and drying: The reaction solution was purified by cross-flow filtration using a polyethersulfone membrane, the retentate was collected and spray-dried to obtain polyglycoside synergist powder; S2. Preparation of fertilizer granules: The polyglycoside synergist obtained in step S1 is mixed and stirred with the fertilizer base material. The mixture is then heated to form a molten liquid and pumped to the nozzle at the top of the granulation tower through a high-pressure pump. In the granulation tower, the mixture is cooled and solidified by contact with rising cold air to form granules, thus obtaining drought-resistant polyglycoside synergist fertilizer.

[0006] Further, step S1, preparing the polyglycoside synergist, specifically involves: S11. Mix γ-polyglutamic acid fermentation broth with a molecular weight of 800,000 to 1,500,000 Da and a mass concentration of 3% to 8% with plant glycoside extract at a mass ratio of 1:0.1 to 0.5, and stir evenly at a stirring speed of 100 to 150 rpm to obtain a mixture. S12. First stage of induction reaction: Adjust the pH of the mixture to 3.0-4.5 with an inorganic acid of 0.5-2 mol / L, and stir the mixture at 25℃-35℃ for 1-3 hours to allow the carboxyl group of γ-polyglutamic acid to undergo hydrogen bonding and partial esterification with the hydroxyl group of the plant glycoside to form a primary complex. S13. Second stage self-assembly reaction: Add a cross-linking inducer to the reaction system. The cross-linking inducer is at least one of citric acid, tartaric acid, or malic acid. The amount added is 0.5% to 2% of the mass of the γ-polyglutamic acid fermentation broth in step S11. Adjust the pH to 6.0 to 7.5 with alkali at a concentration of 0.5 to 2 mol / L. Stir the reaction at 40℃ to 50℃ for 2 to 4 hours to allow the primary complex to further cross-link and self-assemble under the action of the cross-linking inducer, forming a polyglycoside complex with a three-dimensional network structure. S14. Purification and Drying: The reaction solution was purified by cross-flow filtration using a polyethersulfone membrane with a molecular weight cutoff of 100,000 to 300,000 Da. The operating pressure was 0.1 to 0.3 MPa and the temperature was 25°C to 35°C. When the volume of the retentate was 1 / 3 to 1 / 2 of the initial volume, an equal volume of deionized water was added and filtration was continued. This process was repeated 2 to 3 times. The retentate was collected and spray-dried to obtain polyglycoside synergist powder with a particle size D50 of 10 to 50 μm and a moisture content of ≤5%.

[0007] Further, step S2, preparing fertilizer granules, specifically involves: placing the polyglycoside synergist obtained in step S1 and the fertilizer base material in a mixer at a mass ratio of 1:50~200, mixing at a speed of 30~60 rpm for 15~30 minutes, then heating the mixture to 110℃~130℃ to form a molten liquid, which is then pumped to the nozzle at the top of the granulation tower through a high-pressure pump. In the granulation tower at a height of 30m~50m, the mixture is cooled and solidified by contact with rising cold air to form granules with a particle size of 1mm~4mm, thus obtaining drought-resistant polyglycoside synergistic fertilizer.

[0008] Further, the γ-polyglutamic acid fermentation broth described in step S11 is prepared by fermentation with Bacillus subtilis. The fermentation medium consists of: glucose 35-45 g / L, yeast powder 4-6 g / L, peptone 8-12 g / L, sodium glutamate 18-22 g / L, NaCl 4-6 g / L, MgSO4·7H2O 0.4-0.6 g / L, K2HPO4 1.5-2.5 g / L, pH 7.0-7.2; the fermentation conditions are 35℃-37℃, 180-220 rpm, and fermentation time is 44-52 h; after fermentation, the bacterial cells are removed by centrifugation at 4℃ and 8000 rpm for 20 min, and the supernatant is filtered through a 0.45 μm microporous membrane to obtain the γ-polyglutamic acid fermentation broth.

[0009] Further, the preparation method of the plant glycoside extract in step S11 includes: drying the plant raw material at 40℃~50℃ to constant weight by forced air, pulverizing it and passing it through a 40~60 mesh sieve, adding 5~10 times its mass of a 60%~80% ethanol aqueous solution, reflux extraction at 60℃~80℃ for 2~4 hours, extracting 2~3 times, combining the extracts, filtering while hot through a 200 mesh filter cloth, and concentrating the filtrate under reduced pressure at 50℃~70℃ and -0.06~-0.09MPa to 1 / 5~1 / 3 of the original volume to obtain the plant glycoside extract; the plant raw material is selected from one or more of Gardenia, Eucommia, Rehmannia, Sophora japonica, Aloe, Safflower, Scrophularia, Chrysanthemum indicum, Citrus peel, Plantago asiatica, Patrinia scabiosaefolia, and Lonicera japonica; the plant glycoside is an iridoid glycoside compound with a molecular weight of 300~1000 Da.

[0010] Further, the inorganic acid in step S12 is one or more of hydrochloric acid, sulfuric acid, or phosphoric acid; the alkaline solution in step S13 is an aqueous solution of sodium hydroxide, an aqueous solution of potassium hydroxide, or ammonia; the crosslinking inducing agent is at least one of citric acid, tartaric acid, or malic acid; and the inlet air temperature of the spray drying in step S14 is 160℃~200℃, and the outlet air temperature is 80℃~100℃.

[0011] Further, at least one of magnesium stearate, silica, or talc powder (0.1% to 0.5% by mass) is added to the polyglycoside synergist powder obtained in step S14 as an anti-caking agent, and the mixture is mixed in a three-dimensional mixer at a speed of 20 to 30 rpm for 10 to 15 minutes to prevent powder agglomeration.

[0012] Further, the fertilizer base material in step S2 includes at least two of nitrogen fertilizer, phosphorus fertilizer, and potassium fertilizer, wherein the nitrogen source is selected from at least one of urea, ammonium nitrate, and ammonium sulfate; the phosphorus source is selected from at least one of monoammonium phosphate, potassium dihydrogen phosphate, and superphosphate; and the potassium source is selected from at least one of potassium sulfate, potassium chloride, and potassium nitrate; the total mass fraction of N, P2O5, and K2O in the fertilizer base material is 30% to 45%.

[0013] Furthermore, in step S2, the inlet air temperature of the granulation tower is 15℃~25℃, the outlet air temperature is 40℃~60℃, and the negative pressure inside the tower is -50~-100Pa; the fertilizer granules have a particle strength ≥15N, a disintegration time in water ≤5min, and a particle uniformity ≥90%.

[0014] The following is the core writing idea (creative concept) of this invention: Innovation Point 1: A dual-source synergistic polyglycoside complex system of "microbial origin & plant origin" was constructed.

[0015] This invention is the first to chemically bond γ-polyglutamic acid derived from microbial fermentation with iridoid glycosides extracted from plants, forming a novel "polyglycoside" complex. γ-polyglutamic acid is a high-molecular-weight polymer produced by Bacillus subtilis fermentation, containing numerous carboxyl groups in its molecular chain, exhibiting excellent water absorption and retention properties and metal ion complexing ability. Plant glycosides are iridoid glycosides extracted from medicinal plants such as gardenia and eucommia, containing multiple hydroxyl groups and glycosidic bonds, possessing biological activities such as regulating plant hormone balance and inducing the expression of stress-resistance genes. The two compounds, originating from different sources and with complementary functions, are chemically bonded at the molecular level using the gradient-induced self-assembly technology of this invention, forming a stable three-dimensional network structure. This invention differs from simple physical mixing, pure chemical synthesis, and the application of a single component; rather, it establishes a dual-source synergistic system of "microbial origin & plant origin," solving the technical challenges of poor stability and easy degradation of plant glycosides.

[0016] Innovation Point 2: Design of a preparation process for "gradient-induced self-assembly".

[0017] This invention utilizes a two-stage gradient-induced self-assembly process tailored to the molecular structural characteristics of γ-polyglutamic acid and phytoglycosides. In the first stage, under acidic conditions (pH 3.0–4.5), the carboxyl groups of γ-polyglutamic acid are protonated, forming strong hydrogen bonds with the hydroxyl groups of the phytoglycosides, while partial esterification occurs, forming a primary complex. In the second stage, after adding a multi-carboxyl crosslinking inducer such as citric acid, the conditions are adjusted to neutral (pH 6.0–7.5). The crosslinking inducer acts as a "molecular bridge," interacting with the remaining carboxyl and hydroxyl groups on the primary complex to form multiple crosslinking points, inducing the folding and aggregation of the complex molecular chains, ultimately leading to self-assembly into a stable three-dimensional network structure. This gradient-induced strategy precisely controls the reaction process, avoiding localized aggregation or incomplete reactions caused by excessively rapid reactions, resulting in a homogeneous and stable polyglycoside complex. This process differs fundamentally from the simple mixing or one-step chemical synthesis methods described in prior art, demonstrating the innovation and complexity of the preparation method, which is difficult for those skilled in the art to easily conceive of from existing technologies.

[0018] Innovation Point 3: Establishing a dual-effect synergistic mechanism of "structural water retention & biological induction".

[0019] The polyglycoside complex prepared in this invention possesses a unique three-dimensional network structure. When applied to the soil, this network structure can adsorb tens of times its own weight in water, which is then slowly released under drought conditions, forming a "miniature reservoir" to achieve physical water retention. Simultaneously, the plant glycoside molecules within the network are slowly released under the influence of water and soil microorganisms. After being absorbed by crop roots, they can regulate the balance of endogenous hormones in plants, reduce the activity of indoleacetic acid oxidase and cytokinin oxidase, activate drought-related signal transduction pathways, and induce the accumulation of osmotic regulatory substances such as proline and soluble sugars, thereby enhancing the crop's intrinsic drought resistance. This dual synergistic mechanism of "physical water retention & biological induction" enables the fertilizer of this invention to exhibit excellent drought resistance and yield-increasing effects under drought stress, with a synergistic effect far exceeding the simple sum of the individual components.

[0020] The relevant theoretical description of this invention: The core mechanism of this invention lies in the intermolecular interaction and synergistic effect between γ-polyglutamic acid and plant glycosides. γ-polyglutamic acid is an anionic polypeptide composed of glutamic acid monomers linked by γ-amide bonds. Its molecular chain contains a large number of free carboxyl groups (-COOH), and in aqueous solution, it exhibits an extended random coil conformation due to electrostatic repulsion, resulting in a large free volume between the molecular chains. Plant glycosides (taking geniposide as an example) are iridoid glycosides, whose molecular structure consists of an iridoid aglycone and glucose. The aglycone contains multiple hydroxyl groups (-OH) and double bonds, while the sugar contains multiple hydroxyl groups. The overall molecule has numerous hydrogen bond donor and acceptor sites.

[0021] In the first induction reaction (pH 3.0–4.5), acidic conditions promote the protonation of γ-polyglutamic acid carboxyl groups, reducing electrostatic repulsion between molecular chains and causing the chains to contract. Simultaneously, the protonated carboxyl groups form strong hydrogen bonds with the hydroxyl groups of the plant glycosides, and some carboxyl groups undergo esterification to form ester bonds, resulting in a primary γ-PGA-glycoside complex. This process can be represented by the following reaction equations: γ-PGA-COOH + HO-glycoside ⇌ γ-PGA-COO-glycoside + H₂O (esterification reaction) and γ-PGA-COOH···HO-glycoside (hydrogen bonding).

[0022] In the second stage of the self-assembly reaction, the added citric acid and other multi-carboxyl crosslinking inducers simultaneously interact with the remaining carboxyl and hydroxyl groups on the primary γ-PGA-glycoside complex, forming multiple crosslinking sites. Citric acid molecules contain three carboxyl groups, which can act as "molecular bridges" connecting different γ-PGA segments or γ-PGA with plant glycoside molecules, inducing the complex molecular chains to fold and aggregate. Simultaneously, under neutral conditions (pH 6.0–7.5), partial deprotonation of carboxyl groups increases intermolecular electrostatic interactions, promoting further aggregation and networking of molecular chains, ultimately leading to the self-assembly of a polyglycoside complex with a three-dimensional network structure. In this network structure, γ-polyglutamic acid segments form a hydrophilic, water-retaining backbone, plant glycoside molecules are uniformly distributed as functional units within the network, and the crosslinking inducers stabilize the network structure.

[0023] When the polyglycoside complex is applied to the soil, the hydrophilic groups (carboxyl groups, hydroxyl groups, etc.) in its three-dimensional network structure bind to water molecules through hydrogen bonds, enabling it to adsorb 30 to 50 times its own weight in water, forming a hydration layer. Under drought conditions, it slowly releases water, achieving a physical water retention function. At the same time, the plant glycoside molecules in the network are gradually hydrolyzed under the action of soil moisture and microorganisms, slowly releasing free plant glycosides. These glycosides are absorbed by the crop roots and transported to the aboveground parts, regulating the balance of endogenous hormones in plants through signal transduction pathways. This reduces the activity of indoleacetic acid oxidase and cytokinin oxidase, increases the levels of growth hormones such as IAA and CTK, activates the expression of drought-resistant genes, induces the accumulation of osmotic regulatory substances such as proline, soluble sugars, and betaine, enhances the osmotic regulation capacity of cells, protects membrane structures and protein functions, thereby improving the crop's intrinsic drought resistance.

[0024] Beneficial technical effects I. Significant Drought Resistance and Water Retention Effects: The polyglycoside-enhanced fertilizer prepared in this invention can adsorb 30-50 times its own weight in water in the soil, forming a stable water-retaining layer and effectively reducing soil moisture evaporation. Soil water retention rate tests show that soils treated with this invention's fertilizer can still retain more than 32% moisture on the 10th day, which is 30%-50% higher than ordinary fertilizers. Under drought stress, the relative water content of maize leaves treated with this invention's fertilizer reaches more than 75%, which is 30%-40% higher than the drought control; the proline content reaches more than 500 μg / gFW, which is 40%-60% higher than the drought control; and the root activity reaches more than 150 μgTTC / g·h, which is 50%-70% higher than the drought control, demonstrating excellent drought resistance.

[0025] II. Synergistic Nutrient Utilization: The three-dimensional network structure of the polyglycoside complex effectively chelates fertilizer nutrients, reducing nitrogen volatilization and leaching losses. Simultaneously, the plant glycoside components promote root development and nutrient absorption. Field trials show that the fertilizer of this invention can achieve nitrogen fertilizer utilization rates of 44.8%–49.5%, an increase of 9.5–14.2 percentage points compared to conventional fertilization; phosphorus fertilizer utilization rates of 23.5%–27.2%, an increase of 8.7–12.4 percentage points compared to conventional fertilization; and potassium fertilizer utilization rates of 45.7%–50.3%, an increase of 14.2–18.8 percentage points compared to conventional fertilization, achieving long-term and efficient nutrient utilization.

[0026] III. Significant Yield and Quality Improvement Effects: Through the dual effects of physical water retention and biological induction, this invention's fertilizer promotes robust crop growth, increases root quantity and vigor, and enhances photosynthetic efficiency. Field trials on corn show that applying this invention's fertilizer results in a yield of 660-700 kg / mu, an increase of 20%-30% compared to conventional fertilization and 15%-20% compared to commercially available drought-resistant fertilizers; ear length increases by 10%-15%, ear grain number increases by 12%-18%, and thousand-grain weight increases by 8%-12%. Vegetable crops (such as cucumbers and tomatoes) show a 20%-35% increase in yield, and quality indicators such as vitamin C and soluble sugars improve by 10%-20%.

[0027] IV. Excellent product stability and process adaptability: The polyglycoside synergist prepared by this invention is a powdered solid with uniform particle size (D50 10~50μm) and bulk density of 0.3~0.6g / cm³. 3 With an angle of repose ≤35°, good fluidity, and high mixing uniformity with fertilizer base materials, the resulting fertilizer granules have a compressive strength ≥18N, are not easily broken during storage and transportation, have a particle uniformity ≥90%, and a disintegration time in water ≤5min, ensuring rapid dispersion and effective release of the fertilizer after application to the soil. This product is suitable for various industrial production processes such as high-tower melt granulation, rotary drum granulation, and extrusion granulation, and has good prospects for industrial application.

[0028] Powder particle size (D50) determination: The particle size was determined by dry dispersion using a laser particle size analyzer with a dispersion pressure of 0.2 MPa, a sample injection rate of 50%, and a light shading of 0.5% to 5%. The volume average particle size D50 was recorded. Determination of powder bulk density: Refer to the method in GB / T 16913-2008. Allow the thoroughly mixed powder sample to fall freely through a funnel into a 100 mL graduated cylinder of known volume (V) until the cylinder is completely filled and overflows. Level the powder with a ruler and weigh the remaining powder (m). Bulk density (g / cm³) 3 = m / V. Perform three parallel measurements and take the average value. Determination of the angle of repose of powder: The fixed funnel method is used. The funnel is fixed at a certain height (h) above the graph paper, allowing the powder to flow slowly down the funnel wall until the top of the resulting powder cone touches the bottom of the funnel. The diameter of the cone's base (2r) is measured, and the radius (r) is calculated. The angle of repose θ = arctan(h / r). Three parallel measurements are performed, and the average value is taken.

[0029] Particle compressive strength test: Randomly select 20 fertilizer particles with uniform particle size, and use an intelligent particle strength tester to measure the maximum force (N) when each particle is radially crushed. Record the average value, which is the particle strength.

[0030] Determination of disintegration time of granules in water: 500 mL of deionized water was placed in a constant temperature water bath at 25℃±2℃. Ten fertilizer granules were randomly selected and simultaneously placed into the water. Timing was started, and the time required for all granules to completely disintegrate and for no visible hard core to be recorded. The average value was calculated.

[0031] Particle uniformity determination: Take approximately 500g of fertilizer granule sample and place it on a standard vibrating sieve (aperture diameter ±0.5mm of the nominal particle size). After shaking for 5 minutes, weigh the mass (m1) of the granules remaining on the nominal particle size sieve. Particle uniformity (%) = (m1 / 500) × 100%. Attached Figure Description

[0032] Figure 1 This is a schematic diagram of the preparation process of the drought-resistant polyglycoside-enhanced fertilizer of the present invention.

[0033] Figure 2 The graphs show the changes in soil water retention rate over time for the fertilizers prepared in Examples 1-6 and Comparative Examples 1-8 of this invention.

[0034] Figure 3 The bar chart shows the effect of the fertilizers prepared in Examples 1-6 and Comparative Examples 1-8 of this invention on corn yield. Detailed Implementation

[0035] Before further describing specific embodiments of the present invention, it should be understood that the scope of protection of the present invention is not limited to the specific embodiments described below; it should also be understood that the terminology used in the embodiments of the present invention is for describing specific embodiments and not for limiting the scope of protection of the present invention. Test methods in the following embodiments that do not specify specific conditions are generally performed under conventional conditions or as recommended by the respective manufacturers.

[0036] When numerical ranges are given in the embodiments, it should be understood that, unless otherwise stated in this invention, both endpoints of each numerical range and any value between the two endpoints may be selected. Unless otherwise defined, all technical and scientific terms used in this invention have the same meaning as commonly understood by those skilled in the art. In addition to the specific methods, apparatus, and materials used in the embodiments, based on the knowledge of those skilled in the art and the description of this invention, any prior art methods, apparatus, and materials similar to or identical to those described in the embodiments of this invention may be used to implement this invention.

[0037] Unless otherwise stated, the test methods, detection methods and preparation methods disclosed in this invention all adopt conventional techniques in this technical field.

[0038] I. Source of Reagents, Raw Materials and Equipment 1.1 Microbial strains and fermentation reagents The Bacillus subtilis used in the following embodiments of the present invention ( Bacillus subtilis The CICC10732 strain was purchased from the China Industrial Microbial Culture Collection Center (CICC). The activation medium for the strain was beef extract peptone agar, with the following formula: 3g beef extract, 10g peptone, 5g NaCl, 15-25g agar, pH 7.4-7.6, 1000mL water, sterilized at 121℃ for 20min (autoclave).

[0039] The fermentation medium contains glucose, yeast extract, peptone, monosodium glutamate, NaCl, MgSO4·7H2O, and K2HPO4. 4均为 Common raw materials and reagents in this technical field.

[0040] 1.2 Plant raw materials and extraction reagents The plant-based materials are all common items in this field; The ethanol used for extraction was food-grade, 95% by volume, purchased from Sinopharm Chemical Reagent Co., Ltd., and diluted with deionized water to the required concentration before use. Gardenoside standard (purity ≥98%, catalog number: CFN90237) was purchased from Wuhan Tianzhi Biotechnology Co., Ltd.

[0041] 1.3 Chemical reagents and excipients Citric acid, tartaric acid, malic acid, hydrochloric acid, sulfuric acid, phosphoric acid, sodium hydroxide, potassium hydroxide, and ammonia were all analytical grade and purchased from Sinopharm Chemical Reagent Co., Ltd. Magnesium stearate, silica, and talc were pharmaceutical excipient grade and purchased from Wuhan Jixin Yibang Biotechnology Co., Ltd.

[0042] Example 1 This embodiment provides a method for preparing a drought-resistant polyglycoside-enhanced fertilizer, the specific steps of which are as follows: Preparation of S1.γ-polyglutamic acid fermentation broth: Bacillus subtilis CICC 10732 strain was inoculated onto beef extract peptone agar slant medium and activated by incubation at 37°C for 24 h. The activated bacterial colony was picked and inoculated into a 500 mL Erlenmeyer flask containing 100 mL of seed medium (same as the activation medium, but without agar) and cultured at 37°C with shaking at 200 rpm for 16 h to obtain the seed culture. Prepare the fermentation medium according to the following formula: 40 g / L glucose, 5 g / L yeast extract, 10 g / L peptone, 20 g / L monosodium glutamate, 5 g / L NaCl, 0.5 g / L MgSO4·7H2O, and 2 g / L K2HPO4. Adjust the pH to 7.2 with 1 mol / L NaOH. Dispense the medium into 5 L Erlenmeyer flasks, with each flask containing 1.5 L of liquid. Sterilize at 121 °C for 20 min. After cooling, inoculate the seed culture at a 10% (v / v) inoculation rate and incubate at 37 °C with shaking at 200 rpm for 48 h. After fermentation, the fermentation broth was centrifuged at 4℃ and 8000 rpm for 20 min. The supernatant was collected and filtered through a 0.45 μm microporous membrane to obtain approximately 7.2 L of γ-polyglutamic acid fermentation broth. Molecular weight was determined using gel permeation chromatography (GPC): 1 mL of the fermentation broth was diluted 10-fold with the mobile phase and injected. The chromatographic conditions were: Shodex SB-806MHQ column (8.0 × 300 mm), mobile phase 0.1 mol / L. NaNO3 was used at a flow rate of 0.5 mL / min and a column temperature of 40 °C. A differential refractive index detector was used, with polyethylene glycol as the standard. The results showed that the weight-average molecular weight (Mw) of γ-polyglutamic acid was 1.08 million Da, the number-average molecular weight (Mn) was 830,000 Da, and the polydispersity index (PDI) was 1.30. The content of γ-polyglutamic acid was determined by high performance liquid chromatography: 1 mL of fermentation broth was added to 4 mL of anhydrous ethanol to precipitate the precipitate, which was allowed to stand overnight at 4 °C. The precipitate was centrifuged at 8000 rpm for 10 min, washed three times with anhydrous ethanol, dried at 60 °C to constant weight, weighed, and the content was calculated. The results were repeated in triplicate, and the average value was 6.2% (w / v). S2. Preparation of plant glycoside extract: Take 5.0 kg of dried gardenia fruit and dry it in a 45℃ forced-air drying oven for 24 hours until constant weight. Grind it with a universal pulverizer and pass it through a 50-mesh sieve to obtain about 4.8 kg of gardenia powder. Weigh 4.0 kg of gardenia powder and place it in a 100L stainless steel extraction tank. Add 32.0 kg of 70% ethanol aqueous solution (material-liquid ratio 1:8), turn on stirring and heating, raise the temperature to 70℃, and reflux for 3 hours. After extraction, open the bottom valve of the tank and filter the extract through a 200-mesh filter cloth into a storage tank. Add another 32.0 kg of 70% ethanol aqueous solution to the filter residue and extract under the same conditions for 3 hours. Filter and combine the two extracts. The combined extracts were pumped into a rotary evaporator and concentrated under reduced pressure at 60℃ and -0.08MPa. Ethanol was recovered, and the concentration was stopped when the volume was reduced to 1 / 4 of the original volume, yielding approximately 2.5 kg of phytoglycoside extract. The geniposide content was determined using high-performance liquid chromatography (HPLC): 1 mL of the extract was diluted 10-fold with methanol, filtered through a 0.45 μm filter membrane, and injected. The chromatographic conditions were: Agilent ZORBAX SB-C18 column (4.6 × 250 mm, 5 μm), mobile phase methanol-0.1% phosphoric acid aqueous solution (30:70), flow rate 1.0 mL / min, column temperature 30℃, detection wavelength 238 nm, and injection volume 10 μL. A standard curve was plotted using a series of concentration solutions of geniposide standard, and quantification was performed using the external standard method. The results showed that the geniposide content in the extract was 4.2% (w / v), and the total phytoglycoside content (calculated as geniposide) was 4.2%. S3. Preparation of polyglycoside synergists: Take 10.0 kg of γ-polyglutamic acid fermentation broth prepared in step S1 (γ-polyglutamic acid content 6.2%, molecular weight 1.08 million Da) and 3.0 kg of plant glycoside extract prepared in step S2 (geniposide content 4.2%), with a mass ratio of 1:0.3, add them to a 50L stainless steel reactor, turn on the stirrer, set the speed to 120 rpm, and stir and mix at room temperature for 15 min to obtain a uniform mixture; A 1.0 mol / L hydrochloric acid solution was slowly added dropwise to the mixture while monitoring the pH value. The pH was adjusted to 3.8, and the temperature of the circulating water in the reactor jacket was controlled at 30°C. The reaction was continued with stirring for 2 hours to carry out the first stage of induction reaction. During the reaction, the solution was observed to gradually become viscous and the color changed from light yellow to light brown. After the reaction was completed, 0.10 kg of citric acid (1.0% of the mass of γ-polyglutamic acid fermentation broth) was added to the reaction system and stirred to dissolve for 10 min. Then, 1.0 mol / L sodium hydroxide solution was slowly added dropwise to adjust the pH to 6.8. The temperature of the reactor was raised to 45℃ and the reaction was stirred for 3 h to carry out the second stage of self-assembly reaction. During the reaction, the viscosity of the solution further increased and it became colloidal. After the reaction is complete, the reaction solution is pumped into a membrane separation device, using a polyethersulfone flat sheet membrane with a molecular weight cutoff of 200,000 Da and a membrane area of ​​0.5 m². 2 Set the operating pressure to 0.2 MPa and the temperature to 30 °C, and perform cross-flow filtration. When the volume of the retentate is reduced to 1 / 3 of the initial volume (approximately 4.3 L), add an equal volume of 4.3 L of deionized water and continue filtration until the volume of the retentate is reduced to 1 / 3 again. Repeat this operation 3 times and collect the final retentate, which has a volume of approximately 4.0 L. The retentate was fed into a spray dryer with an inlet air temperature of 180℃, an outlet air temperature of 90℃, an atomizer speed of 20,000 rpm, and a peristaltic pump speed of 15 rpm for spray drying. The dried powder was collected, yielding 1.82 kg of polyglycoside synergist powder. A laser particle size analyzer determined the powder's D50 to be 28 μm and D90 to be 65 μm. A Karl Fischer moisture analyzer determined the moisture content to be 3.8%. The bulk density, determined using the graduated cylinder method, was 0.42 g / cm³. 3 The angle of repose was determined to be 32° using the fixed funnel method. S4. Fertilizer granulation: Take 35.0 kg of urea (N≥46%), 30.0 kg of monoammonium phosphate (N≥11%, P2O5≥44%), and 25.0 kg of potassium sulfate (K2O≥50%), place them in a three-dimensional mixer, set the speed to 40 rpm, and mix for 15 min to obtain 90.0 kg of fertilizer base material; take a sample of this base material for analysis, and the total mass fraction of N+P2O5+K2O is 42.1%; Take 0.90 kg of the polyglycoside synergist powder obtained in step S3 and add it to 90.0 kg of the above fertilizer base material. Continue mixing in a three-dimensional mixer at 40 rpm for 15 min to ensure that the polyglycoside synergist and fertilizer base material are fully and evenly mixed. The mass ratio of polyglycoside synergist to fertilizer base material is 1:100. The mixture was fed into a melting tank and heated to 120°C to completely melt it, obtaining a molten liquid. The molten liquid was then pumped through a high-pressure pump to a nozzle at the top of a granulation tower. The nozzle orifice diameter was 1.5 mm, the nozzle speed was 300 rpm, the granulation tower height was 45 m, the inlet air temperature was 20°C, the outlet air temperature was 50°C, and the negative pressure inside the tower was -80 Pa. The molten liquid droplets cooled and solidified upon contact with rising cold air during their 45 m fall, forming granules. The granules were collected from the bottom of the tower and classified by a vibrating screen. Particles with a diameter of 3 mm were collected, yielding approximately 90.5 kg of drought-resistant polyglycoside-enhanced fertilizer. Twenty granules were randomly tested using a particle strength tester, with an average compressive strength of 18.6 N. Ten granules were then placed in deionized water at 25°C, and the complete disintegration time was recorded, with an average disintegration time of 4.2 min. The proportion of particles with a diameter of 3 mm was determined by sieving, and 94.5% of the particles were found to be 3 mm.

[0043] Example 2 This embodiment is basically the same as Example 1, except that the mass ratio of γ-polyglutamic acid fermentation broth to plant glycoside extract is adjusted to 1:0.1. The specific steps are as follows: Take 10.0 kg of γ-polyglutamic acid fermentation broth (same as in Example 1), 1.0 kg of plant glycoside extract (4.2% geniposide content), mix them, adjust the pH to 3.0 with 1.0 mol / L hydrochloric acid, and stir at 25°C for 1 h; then add 0.05 kg of tartaric acid (0.5% of the mass of γ-polyglutamic acid fermentation broth), adjust the pH to 6.0 with 1.0 mol / L sodium hydroxide, and stir at 40°C for 2 h; subsequent purification uses a membrane with a molecular weight cutoff of 100,000 Da, operating pressure of 0.1 MPa, other parameters are the same as in Example 1; spray drying inlet air temperature is 160°C, outlet air temperature is 80°C, atomizer speed is 15000 rpm, to obtain 1.65 kg of polyglycoside synergist powder, D50=35 μm, moisture content 4.2%, bulk density 0.45 g / cm³. 3 The angle of repose is 34°. When granulating fertilizer, 0.90 kg of polyglycoside synergist is mixed with 45.0 kg of fertilizer base material (mass ratio 1:50), heated to 110℃ for melting and granulation, and the finished product is about 45.4 kg with a compressive strength of 17.2 N, a disintegration time of 3.8 min, and a particle uniformity of 92.8%.

[0044] Example 3 This embodiment is basically the same as Example 1, except that the mass ratio of γ-polyglutamic acid fermentation broth to plant glycoside extract is adjusted to 1:0.5. The specific steps are as follows: 10.0 kg of γ-polyglutamic acid fermentation broth and 5.0 kg of plant glycoside extract were mixed and the pH was adjusted to 4.5 with 1.0 mol / L hydrochloric acid. The mixture was stirred at 35°C for 3 h. Then, 0.20 kg of malic acid (2.0% of the mass of the γ-polyglutamic acid fermentation broth) was added, and the pH was adjusted to 7.5 with 1.0 mol / L sodium hydroxide. The mixture was stirred at 50°C for 4 h. Subsequent purification was performed using a membrane with a molecular weight cutoff of 300,000 Da and an operating pressure of 0.3 MPa. Spray drying was carried out at an inlet air temperature of 200°C, an outlet air temperature of 100°C, and an atomizer speed of 25,000 rpm to obtain 2.05 kg of polyglycoside synergist powder with a D50 of 22 μm, a moisture content of 3.2%, and a bulk density of 0.38 g / cm³. 3 The angle of repose is 30°. When granulating fertilizer, 0.90 kg of polyglycoside synergist is mixed with 180.0 kg of fertilizer base material (mass ratio 1:200), heated to 130℃ for melting and granulation, and the finished product is about 180.4 kg with a compressive strength of 19.5 N, a disintegration time of 4.5 min, and a particle uniformity of 95.2%.

[0045] Example 4 This embodiment is basically the same as Example 1, except that the plant glycoside extract uses a compound plant raw material of gardenia, eucommia, and honeysuckle. The specific steps are as follows: Take 2.0 kg of dried gardenia fruit, 1.0 kg of dried eucommia bark, and 1.0 kg of dried honeysuckle buds (mass ratio 2:1:1), mix them, and pulverize them through a 50-mesh sieve to obtain approximately 3.8 kg of compound plant powder. Take 4.0 kg of the compound plant powder (actually weigh 4.0 kg, with the shortfall made up with gardenia powder), add 32.0 kg of 60% ethanol aqueous solution, reflux at 60℃ for 3 hours, filter, and add another 32.0 kg of ethanol aqueous solution to the residue. Extracted with 60% ethanol under reflux at 60°C for 3 hours, then filtered; the filtrates were combined and concentrated under reduced pressure at 60°C and -0.08 MPa to 1 / 4 of the original volume, yielding 2.6 kg of plant glycoside extract; HPLC analysis showed that the content of geniposide was 2.8%, the content of genipin was 1.5%, and the total plant glycoside content was 4.3%; subsequent steps were the same as in Example 1, yielding 1.85 kg of polyglycoside synergist powder with a D50 of 27 μm, a moisture content of 3.7%, and a bulk density of 0.41 g / cm³. 3 The fertilizer granulation was carried out in the same manner as in Example 1, yielding approximately 90.6 kg of finished product with a granule compressive strength of 18.2 N.

[0046] Example 5 This embodiment is basically the same as Example 1, except that the plant glycoside extract uses a compound plant raw material of Sophora japonica, aloe vera, and wild chrysanthemum. The specific steps are as follows: Take 2.0 kg of dried Sophora japonica flower buds, 1.0 kg of dried Aloe vera leaves, and 1.0 kg of dried Chrysanthemum capitula (mass ratio 2:1:1), mix them, and pulverize them through a 50-mesh sieve to obtain approximately 3.7 kg of compound plant powder. Take 4.0 kg of the compound plant powder (the insufficient part is supplemented with Sophora japonica flower powder), add 32.0 kg of 80% ethanol aqueous solution, and reflux extract at 80℃ for 2 hours. Extract three times, combine the filtrates, and concentrate to obtain 2.4 kg of plant glycoside extract. HPLC determination shows that the content of aloin is 2.1%, the content of sucralose is 1.8%, and the total plant glycoside content is 3.9%. The subsequent steps are the same as in Example 1 to obtain 1.78 kg of polyglycoside synergist powder with D50=29μm and moisture content of 4.0%. The fertilizer granulation is the same as in Example 1 to obtain approximately 90.4 kg of finished product.

[0047] Example 6 This embodiment is basically the same as Example 1, except that an anti-caking agent is added to the polyglycoside synergist powder. The specific steps are as follows: 1.82 kg of polyglycoside synergist powder was prepared according to the method in Example 1. 1.82 g of magnesium stearate (0.1% of the powder mass) was added and mixed in a three-dimensional mixer at 30 rpm for 10 min to obtain modified polyglycoside synergist powder. During fertilizer granulation, 0.90 kg of modified polyglycoside synergist was mixed with 90.0 kg of fertilizer base material, and the rest was the same as in Example 1, to obtain approximately 90.5 kg of finished product. The powder did not clump after being placed for 30 days under a relative humidity of 75%.

[0048] Comparative Example 1 (Physical Mixed Group) This comparative example does not involve gradient-induced self-assembly reaction; instead, the γ-polyglutamic acid fermentation broth and plant glycoside extract are physically mixed. 10.0 kg of γ-polyglutamic acid fermentation broth (same as in Example 1) and 3.0 kg of plant glycoside extract (4.2% geniposide content) are mixed in a reactor for 30 min (120 rpm). The pH is not adjusted, and no cross-linking inducer is added. The mixture is directly purified by membrane separation (using a 200,000 Da molecular weight cutoff membrane, same as in Example 1), and spray-dried (under the same conditions as in Example 1) to obtain 1.78 kg of physically mixed powder. 0.90 kg of this powder is mixed with 90.0 kg of fertilizer base material and granulated according to the method in Example 1 to obtain the finished fertilizer product.

[0049] Comparative Example 2 (One-step reaction group) This comparative example does not involve segmented pH control and adopts a one-step reaction. 10.0 kg of γ-polyglutamic acid fermentation broth and 3.0 kg of plant glycoside extract were mixed, and 0.10 kg of citric acid was added directly. The pH was adjusted to 6.8 with 1.0 mol / L sodium hydroxide, and the mixture was stirred at 45°C for 5 h (the total reaction time was the same as the total reaction time of the two stages in Example 1). Then, membrane separation purification was performed (same as Example 1), and spray drying was carried out to obtain 1.80 kg of powder. 0.90 kg of this powder was mixed with 90.0 kg of fertilizer base material and granulated.

[0050] Comparative Example 3 (γ-polyglutamic acid alone) This comparative example does not add plant glycoside extract, but only uses γ-polyglutamic acid fermentation broth; 10.0 kg of γ-polyglutamic acid fermentation broth was taken and directly purified by membrane separation (membrane with molecular weight cutoff of 200,000 Da), and spray dried (under the same conditions as in Example 1) to obtain 1.65 kg of γ-polyglutamic acid powder. 0.90 kg of this powder was mixed with 90.0 kg of fertilizer base material and granulated.

[0051] Comparative Example 4 (Plant glycosides alone) This comparative example does not add γ-polyglutamic acid fermentation broth, but only uses plant glycoside extract; take 3.0 kg of plant glycoside extract (gardenioside content 4.2%), add 0.3 kg of β-cyclodextrin as a spray drying aid, and spray dry directly (under the same conditions as in Example 1) to obtain 0.58 kg of plant glycoside powder (containing β-cyclodextrin); take 0.90 kg of this powder (equivalent to about 0.6 kg of pure plant glycoside) and mix it with 90.0 kg of fertilizer base material for granulation.

[0052] Comparative Example 5 (Sodium Alginate Substitution Group) In this comparative example, sodium alginate (molecular weight approximately 100,000 Da) was used to replace γ-polyglutamic acid. 0.62 kg of sodium alginate (equivalent to 0.62 kg of γ-polyglutamic acid in 10.0 kg of γ-polyglutamic acid fermentation broth) was dissolved in 9.38 kg of deionized water to prepare 10.0 kg of sodium alginate solution with a mass concentration of 6.2%. This solution was mixed with 3.0 kg of plant glycoside extract and processed according to the gradient-induced self-assembly process in Example 1 to obtain 1.75 kg of sodium alginate-plant glycoside composite powder. 0.90 kg of this powder was mixed with 90.0 kg of fertilizer base material and granulated.

[0053] Comparative Example 6 (Saponin Substitution Group) In this comparative example, soybean saponins (80% purity) were used to replace plant glycosides. 0.126 kg of soybean saponins (equivalent to 0.126 kg of plant glycosides in 3.0 kg of plant glycoside extract) were dissolved in 2.874 kg of deionized water to prepare 3.0 kg of saponin solution with a mass concentration of 4.2%. This solution was mixed with 10.0 kg of γ-polyglutamic acid fermentation broth and operated according to the gradient-induced self-assembly process in Example 1 to obtain 1.80 kg of polyglutamic acid-saponin composite powder. 0.90 kg of this powder was mixed with 90.0 kg of fertilizer base material and granulated.

[0054] Comparative Example 7 (Example 1 of Chinese Invention Patent CN112745163A) Referring to Example 1 of Chinese Invention Patent CN112745163A, a humic acid water-soluble fertilizer with drought-resistant function was prepared: by weight percentage, urea 15%, industrial monoammonium phosphate 15%, potassium sulfate 15%, mineral humic acid 5%, small molecule organic carbon 5%, polyglutamic acid 0.6%, alginic acid 0.6%, polyaspartic acid 0.6%, sodium naphthaleneacetate 0.3%, photosynthetic acid 0.3%, salicylic acid 0.3%, with the balance being magnesium sulfate; all raw materials were mixed in proportion and mixed at 70 rpm for 30 minutes to obtain a fertilizer sample; the fertilizer sample was converted according to the total mass fraction of N+P2O5+K2O to make it equivalent to the nutrients in Example 1 for application.

[0055] Comparative Example 8 (Example 1 of Chinese Invention Patent CN105585371A) Referring to Example 1 of Chinese Invention Patent CN105585371A, a fertilizer synergist was prepared: Polyglutamic acid fermentation broth (polyglutamic acid content 10%) was mixed with diatomaceous earth at a weight ratio of 1:1, stirred for 10 min, and dried at 80℃ for 5 h until the moisture content was <10%; 80 kg of the mixture was mixed with 15 kg of pulverized cyanamide (80 mesh) for 10 min, and then 1 kg of pulverized thiourea (80 mesh) and 4 kg of n-butylthiophosphoric triamine were added and stirred for 30 min to obtain a solid fertilizer synergist; this synergist was mixed with corn-specific fertilizer (urea, diammonium phosphate, and potassium chloride were formulated to form N-P2O5-K2O=25-12-8) at a weight ratio of 8%:1 to obtain a synergistic fertilizer; a sample of this fertilizer was taken and converted according to the total mass fraction of N-P2O5-K2O to ensure that it was applied with the same nutrients as in Example 1.

[0056] Performance testing The fertilizers prepared in Examples 1-6 and Comparative Examples 1-8 of this application were subjected to performance tests. The test methods for each indicator are as follows: 1. Structural characterization of the polyglycoside complex 1.1 Determination of molecular weight distribution: The change in molecular weight before and after the reaction was determined by gel permeation chromatography (GPC). 0.1 g of each of the polyglycoside synergist powders prepared in Examples 1-6 and Comparative Examples 1-2 and 5-6 was dissolved in 10 mL of mobile phase (0.1 mol / L NaNO3), filtered through a 0.45 μm filter membrane, and injected. The chromatographic conditions were the same as in step S1. The degree of polymerization was characterized by the change in weight-average molecular weight (Mw) (Mw product / Mw raw material). 1.2 Rheological property determination: The above samples were prepared into a 5% (w / v) aqueous solution, and the storage modulus (G') and loss modulus (G'') were determined using a rheometer; Test conditions: conical rotor (diameter 40 mm, cone angle 1°), temperature 25℃, strain 1%, frequency scan range 0.1~100Hz; G'>G'' and G' not significantly increasing with frequency were used as the criterion for the formation of the three-dimensional network structure; 1.3 Scanning electron microscopy observation: Take the above sample powder, adhere it to the conductive adhesive, spray it with gold, and observe it under a scanning electron microscope with an accelerating voltage of 5kV to observe the microstructure of the powder.

[0057] 2. Soil water retention rate test The tested soil was collected from alluvial soil in Xinxiang, Henan Province. Soil from the 0-20cm topsoil layer was taken, air-dried, and sieved through a 2mm sieve. Basic physicochemical properties: organic matter 12.5g / kg, available nitrogen 75mg / kg, available phosphorus 18mg / kg, available potassium 95mg / kg, pH 7.8, field water holding capacity 28.5%. Weigh 100.0g of air-dried soil (accurate to 0.1g), mix it evenly with 2.0g of the fertilizer to be tested, place it in a PVC pipe with a diameter of 5cm and a height of 10cm, seal the bottom with 200-mesh nylon mesh, and gently compact it to a bulk density of 1.2g / cm³. 3 Place the PVC pipe in a water tank, add water until the soil surface is just moistened, soak for 2 hours, then remove it and let it drain in a ventilated place for 30 minutes. Weigh it to obtain the initial wet weight W0. Then place the sample in a constant temperature incubator at 25℃ and relative humidity at 60%. Weigh it every 2 days and record the weight Wt. Water retention rate (%) = (Wt / W0) × 100%. Each group is repeated 3 times and the average value is taken. 3. Potted corn drought resistance experiment The maize variety used in the test was Zhengdan 958. Plastic pots (25cm in diameter and 20cm in height) were used, and each pot contained 5.0kg of air-dried soil. The fertilizer application rate for each treatment was calculated based on N 0.36g / kg soil, P2O5 0.18g / kg soil, and K2O 0.18g / kg soil. The amount applied per pot was calculated based on the nutrient content of each fertilizer sample. Any deficiencies were supplemented with urea, monoammonium phosphate, and potassium sulfate. The fertilizer was mixed evenly with the soil before being placed in the pots. Sow 5 corn seeds per pot, and after emergence, thin to 3 seedlings per pot. Before sowing, water to 80% of field capacity. Begin drought stress treatment at the three-leaf stage, controlling soil moisture content to 40%–45% of field capacity, maintaining this by daily weighing and watering for 15 days. After the stress treatment ends, take samples from each pot to measure the following indicators, while simultaneously setting up a normal watering control group (70%–75% of field capacity): Relative moisture content of leaves: Take the functional leaves of corn (the third leaf from the bottom), cut them into 2cm sections, weigh them fresh (Wf), soak them in deionized water for 4 hours, take them out, use filter paper to absorb the surface moisture, weigh them saturated (Wt), and dry them at 105℃ to constant weight, weigh them dry (Wd); Relative moisture content of leaves (%) = (Wf-Wd) / (Wt-Wd)×100%.

[0058] Proline content: Take 0.5g of fresh leaves, cut them into small pieces, add 5mL of 3% sulfosalicylic acid solution, extract in a boiling water bath for 10min, cool and filter; take 2mL of filtrate, add 2mL of glacial acetic acid and 2mL of acidic ninhydrin reagent, develop color in a boiling water bath for 30min, cool and extract with 4mL of toluene, allow to stand for layering, take the upper toluene phase, and measure the absorbance at 520nm. Prepare a series of concentration solutions of proline standard to plot a standard curve, and calculate the proline content (μg / gFW) in the sample.

[0059] Root activity: Corn roots were collected, rinsed thoroughly with deionized water, and dried. 0.5 g of root tip was placed in a 10 mL centrifuge tube, and 5 mL of 0.4% TTC solution and 5 mL of phosphate buffer (0.1 mol / L, pH 7.0) were added. The mixture was incubated at 37°C in the dark for 2 h. The reaction was terminated by adding 1 mL of 1 mol / L sulfuric acid. The roots were removed, dried with filter paper, and 5 mL of ethyl acetate was added to grind and extract triphenylhydrazone. The extract was transferred to a 10 mL volumetric flask and diluted to volume. The absorbance was measured at 485 nm. A standard curve was plotted using TTC standards, and root activity (μg TTC / g·h) was calculated.

[0060] 4. Maize field yield trial The experiment was conducted at the Xinxiang Academy of Agricultural Sciences experimental base in Henan Province. The soil was alluvial soil, and the basic fertility was the same as in the pot experiment. The experiment included 16 treatments: Examples 1-6, Comparative Examples 1-8, a conventional fertilization control (without synergist), and a blank control (no fertilization). Each treatment was replicated three times, arranged in a randomized block design, with each plot area of ​​30 m². 2 .

[0061] The fertilizer application rate for each treatment was 180 kg / hm² N. 2 P2O5 90kg / hm 2 K2O 90kg / hm 2 The application rate for each plot was calculated based on the nutrient content of each fertilizer sample, with any shortfall supplemented by urea, monoammonium phosphate, and potassium sulfate. All fertilizers were applied as base fertilizer in a single application before sowing, with no top dressing during the growing season. The corn variety was Zhengdan 958, with a planting density of 60,000 plants / hm². 2 The row spacing is 60cm and the plant spacing is 27.8cm. The sowing date and the harvest date are 4 months apart.

[0062] During the mature stage, take the middle two rows (10m) of each plot. 2 Actual yield was measured and the yield (kg / mu) was calculated. At the same time, 10 representative ears were randomly selected from each plot to measure yield components such as ear length, number of grains per ear, and thousand-grain weight.

[0063] 5. Fertilizer utilization rate determination In a pot experiment on maize, a blank control (CK) without fertilizer was included. The above-ground parts of maize plants in each treatment were harvested, blanched at 105℃ for 30 min, dried at 75℃ to constant weight, weighed, and then pulverized through a 0.5 mm sieve. 0.5 g of sample (accurate to 0.0001 g) was weighed, placed in a digestion tube, and 5 mL of concentrated H₂SO₄ was added. The mixture was shaken well and left overnight. The next day, the solution was digested in a digestion furnace, gradually heated to 300℃, and maintained until the solution turned brownish-black. After slightly cooling, H₂O₂ was added dropwise, and the solution was heated again until clear and colorless. Heating continued for 30 min to remove acid. After cooling, the solution was transferred to a 100 mL volumetric flask and diluted to volume.

[0064] Total nitrogen determination: Take 5 mL of the digestion solution and determine it using a Kjeldahl nitrogen analyzer. Total phosphorus determination: Take 5 mL of the digestion solution, add 2 drops of dinitrophenol indicator, adjust it to yellow with 6 mol / L NaOH, then adjust it to colorless with 2 mol / L H2SO4, add 5 mL of ammonium vanadomolybdate color reagent, make up the volume to 25 mL, and measure the absorbance at 440 nm after 15 min, and quantify it with the standard curve of potassium dihydrogen phosphate. Total potassium determination: Take 5 mL of the digestion solution and determine it using a flame photometer, and quantify it with the standard curve of potassium chloride.

[0065] Nutrient uptake amount (kg / hm 2 )= Dry weight of plants (kg / hm 2 ) × Nutrient content (%). Fertilizer utilization rate (%) = (Nutrient uptake amount in the fertilized area - Nutrient uptake amount in the blank area) / Fertilizer application amount × 100%.

[0066] Table 1 Results of structural characterization of the polyglycoside complex It can be seen from the data in Table 1 that the Mw of Examples 1 - 6 is significantly higher than that of the raw material γ-polyglutamic acid, and the change rate of Mw exceeds 200%, indicating that polymerization or cross-linking reactions have occurred. Among them, the Mw of Example 3 is the highest (2.85 million Da), and the change rate reaches 264%. Rheological parameters show that the storage modulus G' of Examples 1 - 6 is significantly higher than the loss modulus G'', and the value of G' is much larger than that of the comparative example, indicating that a stable three-dimensional network structure has been formed. The change rate of Mw in Comparative Example 1 (physical mixture) is only 6%, G' < G'', and no network structure is formed; although there is a certain degree of polymerization in Comparative Example 2 (one-step method), G' is significantly lower than that of the examples, and the network structure is incomplete; the Mw and G' of Comparative Example 5 (substituted with sodium alginate) and Comparative Example 6 (substituted with saponin) are lower than those of the examples, indicating the uniqueness of the raw material combination of the present invention. Scanning electron microscopy further confirmed that the samples of the examples showed an obvious three-dimensional network structure, while the samples of the comparative examples were amorphous aggregates or flaky structures.

[0067] Table 2 Dynamic changes in soil water retention rate (%) under different fertilizer treatments As shown in Table 2, the soil water retention rates of Examples 1-6 were all above 32% on day 10, significantly higher than all comparative examples. Example 3 showed the best effect, with a water retention rate of 35.4% on day 10, 2.4 times that of conventional fertilization. Comparative Example 1 (physical mixing) had a water retention rate of 25.3%, and Comparative Example 2 (one-step method) had 26.8%, both significantly lower than the examples, demonstrating that the three-dimensional network structure formed by chemical bonding is crucial for water retention performance. Comparative Example 3 (γ-polyglutamic acid alone) had a water retention rate of 27.6%, and Comparative Example 4 (plant glycosides alone) only 18.2%. The synergistic effect of the combination of the two (34.2% in Example 1) far exceeded the simple summation (27.6 + 18.2 - 14.6 = 31.2%, considering the background of conventional fertilization), proving the existence of synergistic enhancement. Comparative Example 5 (sodium alginate substitution) and Comparative Example 6 (saponin substitution) had water retention rates of 22.8% and 24.5%, respectively, lower than the examples, indicating that the raw material combination of the present invention is irreplaceable. The water retention rates of Comparative Examples 7 and 8 were 20.3% and 22.1%, respectively, which are significantly lower than those of the Example, demonstrating the advanced nature of the technical solution of the present invention. Note: Water retention rate is as follows: Figure 2 As shown.

[0068] Table 3 Physiological indicators of maize seedlings under drought stress As shown in Table 3, under drought stress, the relative leaf water content, proline content, and root activity of maize seedlings treated in Examples 1-6 were significantly higher than those in Comparative Examples 1-8. The relative leaf water content of the treatment in Example 3 reached 80.1%, which is 86.6% of that under normal irrigation; the proline content was 542.5 μg / gFW, 1.52 times that of the drought control, indicating that phytoglycosides effectively induced the accumulation of osmotic regulators; and the root activity was 163.4 μg TTC / g·h, recovering to 88.1% of that under normal irrigation. All indicators of Comparative Examples 7 and 8 were significantly lower than those of the Examples, demonstrating that the polyglycoside complex formed by chemical bonding in this invention has unique advantages in improving crop drought resistance.

[0069] Table 4. Field yield and fertilizer utilization rate of maize As shown in Table 4, the corn yields of Examples 1-6 were significantly higher than those of Comparative Examples 1-8. Example 3 had the highest yield, reaching 698.7 kg / mu, representing a 27.2% increase compared to conventional fertilization, a 19.3% increase compared to Comparative Example 7, and an 18.8% increase compared to Comparative Example 8. Regarding fertilizer utilization, Example 3 achieved nitrogen, phosphorus, and potassium utilization rates of 49.5%, 27.2%, and 50.3%, respectively, representing increases of 19.2, 12.4, and 18.8 percentage points compared to conventional fertilization, 14.2, 10.2, and 14.2 percentage points compared to Comparative Example 7, and 13.3, 9.6, and 13.1 percentage points compared to Comparative Example 8. The yields and fertilizer utilization rates of Comparative Examples 1-8 were significantly lower than those of Examples 1-6, demonstrating that the polyglycoside synergist prepared in this invention has unique advantages in improving fertilizer utilization and crop yield. Note: Yields are as follows... Figure 3 As shown.

[0070] Based on the above test results, the drought-resistant polyglycoside-enhanced fertilizer prepared in this embodiment of the invention is significantly superior to the comparative example in terms of water retention performance, crop drought resistance physiological indicators, yield, and fertilizer utilization rate. The reasons for this difference can be mainly attributed to the following aspects: 1. The three-dimensional network structure formed by chemical bonding is the structural basis for performance improvement.

[0071] GPC and rheological test results (Table 1) show that the gradient-induced self-assembly process of this invention enables effective chemical bonding between γ-polyglutamic acid and plant glycosides, forming a stable three-dimensional network structure. The weight-average molecular weight of the example samples is more than twice that of the raw materials, and the storage modulus G' is much greater than the loss modulus G'', exhibiting typical gel network characteristics. In contrast, the molecular weight of Comparative Example 1 (physical mixing) remains basically unchanged, and no network structure is formed; although Comparative Example 2 (one-step method) shows some degree of polymerization, the network structure is incomplete. This structural difference directly leads to the performance difference: the example samples with complete three-dimensional network structure can form stable "micro-reservoirs" in soil, continuously adsorbing and slowly releasing water, and their water retention rate (Table 2) is significantly higher than that of the comparative examples; at the same time, the network structure's coating and protection of plant glycosides enables them to be released slowly and continuously exert their bio-inducing function.

[0072] 2. The dual synergistic mechanism of "physical water retention & biological induction" is the functional guarantee for performance enhancement.

[0073] Comparative analysis of the data in Tables 2, 3, and 4 reveals that the performance indicators of Examples 1-6 are significantly superior to those of the single components (Comparative Examples 3 and 4) and their simple sum. Comparative Example 3 (γ-polyglutamic acid alone) mainly provides physical water retention, while Comparative Example 4 (plant glycosides alone) mainly provides biological induction, but the simple sum of the two is still lower than that of Example 1 (34.2%). The proline content is 437.9 μg / gFW, lower than that of Example 1 (528.7 μg / gFW); the yield is 589.3 kg / mu, lower than that of Example 1 (685.3 kg / mu). The test results demonstrate that there is indeed a synergistic effect between the two. The mechanism is that the three-dimensional network structure not only provides physical water retention but also protects plant glycosides from rapid degradation, allowing for continuous release. The drought-resistant physiological response induced by plant glycosides in crops, in turn, enhances the crop's water absorption and utilization efficiency, forming a virtuous cycle.

[0074] 3. Gradient-induced self-assembly is the key to achieving chemical bonding.

[0075] Comparing the results of Example 1 with those of Comparative Examples 1-2, it can be seen that the preparation process has a significant impact on product performance. Comparative Example 1 (physical mixing) failed to form a network structure due to the absence of chemical bonding, resulting in poor performance. Comparative Example 2 (one-step method), although exhibiting some reaction, lacked a pre-induction stage under acidic conditions, leading to insufficient reaction, an incomplete network structure, and performance falling between Comparative Example 1 and the Example 2. In contrast, the two-stage gradient-induced self-assembly process employed in Examples 1-6 first promotes carboxyl protonation and hydrogen bond formation under acidic conditions, inducing initial aggregation of molecular chains; then, under crosslinking inducer and neutral conditions, it promotes ester bond formation and network crosslinking, achieving precise reaction control and yielding structurally complete and high-performance polyglycoside complexes.

[0076] 4. The specific selection of raw materials is a material prerequisite for synergistic effects.

[0077] The performance of Comparative Example 5 (sodium alginate replacing γ-polyglutamic acid) and Comparative Example 6 (saponins replacing phytoglycosides) was lower than that of the Examples, indicating that the raw material combination of the present invention is irreplaceable. The large number of carboxyl groups on the γ-polyglutamic acid molecular chain gives it excellent hydrophilicity and reactivity, enabling it to form stable ester and hydrogen bonds with the hydroxyl groups of phytoglycosides. Furthermore, the unique molecular structure of phytoglycosides (cyclohexene ether glycosides) gives them good biological activity, capable of inducing the expression of drought-resistant genes in crops. Although sodium alginate is also a polysaccharide, its molecular structure and reactivity differ from γ-polyglutamic acid, making it difficult to form a network structure of equal stability. Although saponins are also glycoside compounds, their molecular structure and biological activity differ from cyclohexene ether glycosides, resulting in weaker drought-inducing ability.

[0078] 5. Comparison with existing technologies verifies the inventiveness of this invention. Comparative Examples 7 and 8 are existing technologies. Test results show that the water retention rate, physiological indicators, yield, and fertilizer utilization rate of both are lower than those of the embodiments of this invention, demonstrating that the technical solution of this invention represents a significant improvement over existing technologies. In particular, Comparative Example 7 used a physical mixture of polyglutamic acid with various drought-resistant agents and synergists, and Comparative Example 8 used a combination of polyglutamic acid with urease inhibitors / nitrification inhibitors; neither achieved chemical bonding and network structure construction, thus limiting their effectiveness. This invention constructs a three-dimensional network structure through chemical bonding, achieving a dual synergy of "physical water retention & biological induction," and its technical effect exceeds the reasonable expectations of those skilled in the art.

[0079] In summary, the polyglycoside synergist prepared by the present invention through gradient-induced self-assembly technology achieves molecular-level chemical bonding and three-dimensional network structure construction of γ-polyglutamic acid and plant glycosides. The prepared drought-resistant synergistic fertilizer has significant drought-resistant and yield-increasing effects and improved fertilizer utilization. Its technical solution has outstanding substantive features and significant progress compared with the prior art, and possesses the inventiveness required by the patent law.

[0080] Finally, it should be noted that the above embodiments are used to illustrate the technical solutions of the present invention and not to limit the scope of protection of the present invention. Although the present invention has been described in detail with reference to preferred embodiments, those skilled in the art should understand that modifications or equivalent substitutions can be made to the technical solutions of the present invention without departing from the essence and scope of the technical solutions of the present invention.

Claims

1. A method for preparing a drought-resistant polyglycoside-enhanced fertilizer, characterized in that, Includes the following steps: S1. Preparation of polyglycoside synergists: S11. Mix and stir the γ-polyglutamic acid fermentation broth with the plant glycoside extract to obtain a mixture; S12. First induction reaction: Adjust the pH of the mixture with inorganic acid, then stir to allow the carboxyl group of γ-polyglutamic acid to undergo hydrogen bonding and partial esterification with the hydroxyl group of plant glycosides, forming a primary complex; S13. Second stage of self-assembly reaction: Add a cross-linking inducer to the reaction system. The amount added is 0.5% to 2% of the mass of the γ-polyglutamic acid fermentation broth in step S11. Continue to adjust the pH to 6.0 to 7.5 with alkaline solution, and then continue stirring to allow the primary complex to further cross-link and self-assemble under the action of the cross-linking inducer to form a polyglycoside complex with a three-dimensional network structure. S14. Purification and drying: The reaction solution was purified by cross-flow filtration using a polyethersulfone membrane, the retentate was collected and spray-dried to obtain polyglycoside synergist powder; S2. Preparation of fertilizer granules: The polyglycoside synergist obtained in step S1 is mixed and stirred with the fertilizer base material. The mixture is then heated to form a molten liquid and pumped to the nozzle at the top of the granulation tower through a high-pressure pump. In the granulation tower, the mixture is cooled and solidified by contact with rising cold air to form granules, thus obtaining drought-resistant polyglycoside synergist fertilizer.

2. The method according to claim 1, characterized in that: Step S1, preparing the polyglycoside synergist, specifically involves: S11. Mix γ-polyglutamic acid fermentation broth with a molecular weight of 800,000 to 1,500,000 Da and a mass concentration of 3% to 8% with plant glycoside extract at a mass ratio of 1:0.1 to 0.5, and stir evenly at a stirring speed of 100 to 150 rpm to obtain a mixture. S12. First stage of induction reaction: Adjust the pH of the mixture to 3.0-4.5 with an inorganic acid of 0.5-2 mol / L, and stir the mixture at 25℃-35℃ for 1-3 hours to allow the carboxyl group of γ-polyglutamic acid to undergo hydrogen bonding and partial esterification with the hydroxyl group of the plant glycoside to form a primary complex. S13. Second stage self-assembly reaction: Add a cross-linking inducer to the reaction system. The cross-linking inducer is at least one of citric acid, tartaric acid, or malic acid. The amount added is 0.5% to 2% of the mass of the γ-polyglutamic acid fermentation broth in step S11. Adjust the pH to 6.0 to 7.5 with alkali at a concentration of 0.5 to 2 mol / L. Stir the reaction at 40℃ to 50℃ for 2 to 4 hours to allow the primary complex to further cross-link and self-assemble under the action of the cross-linking inducer, forming a polyglycoside complex with a three-dimensional network structure. S14. Purification and Drying: The reaction solution was purified by cross-flow filtration using a polyethersulfone membrane with a molecular weight cutoff of 100,000 to 300,000 Da. The operating pressure was 0.1 to 0.3 MPa and the temperature was 25°C to 35°C. When the volume of the retentate was 1 / 3 to 1 / 2 of the initial volume, an equal volume of deionized water was added and filtration was continued. This process was repeated 2 to 3 times. The retentate was collected and spray-dried to obtain polyglycoside synergist powder with a particle size D50 of 10 to 50 μm and a moisture content of ≤5%.

3. The method according to claim 1, characterized in that: S2. The preparation of fertilizer granules specifically involves: placing the polyglycoside synergist obtained in step S1 and the fertilizer base material in a mixer at a mass ratio of 1:50~200, mixing at a speed of 30~60 rpm for 15~30 minutes, then heating the mixture to 110℃~130℃ to form a molten liquid, which is then pumped to the nozzle at the top of the granulation tower through a high-pressure pump. In the granulation tower at a height of 30m~50m, the mixture is cooled and solidified by contact with rising cold air to form granules with a particle size of 1mm~4mm, thus obtaining drought-resistant polyglycoside synergistic fertilizer.

4. The method according to claim 1, characterized in that: The γ-polyglutamic acid fermentation broth described in step S11 was prepared by fermentation with Bacillus subtilis. The fermentation medium consisted of: glucose 35-45 g / L, yeast extract 4-6 g / L, peptone 8-12 g / L, sodium glutamate 18-22 g / L, NaCl 4-6 g / L, MgSO4·7H2O 0.4-0.6 g / L, K2HPO4 1.5-2.5 g / L, and pH 7.0-7.

2. The fermentation conditions were 35℃-37℃, 180-220 rpm, and fermentation time of 44-52 h. After fermentation, the bacterial cells were removed by centrifugation at 4℃ and 8000 rpm for 20 min. The supernatant was filtered through a 0.45 μm microporous membrane to obtain the γ-polyglutamic acid fermentation broth.

5. The method according to claim 1, characterized in that: The preparation method of the plant glycoside extract in step S11 includes: drying the plant raw material at 40℃~50℃ to constant weight by forced air, pulverizing it and passing it through a 40~60 mesh sieve, adding 5~10 times its mass of a 60%~80% ethanol aqueous solution, refluxing at 60℃~80℃ for 2~4 hours, extracting 2~3 times, combining the extracts, filtering while hot through a 200 mesh filter cloth, and concentrating the filtrate under reduced pressure at 50℃~70℃ and -0.06~-0.09MPa to 1 / 5~1 / 3 of the original volume to obtain the plant glycoside extract; the plant raw material is selected from one or more of Gardenia, Eucommia, Rehmannia, Sophora japonica, Aloe, Safflower, Scrophularia, Chrysanthemum indicum, Citrus peel, Plantago asiatica, Patrinia scabiosaefolia, and Lonicera japonica; the plant glycoside is an iridoid glycoside compound with a molecular weight of 300~1000 Da.

6. The method according to claim 1, characterized in that: The inorganic acid in step S12 is one or more of hydrochloric acid, sulfuric acid, or phosphoric acid; the alkaline solution in step S13 is an aqueous solution of sodium hydroxide, an aqueous solution of potassium hydroxide, or ammonia; the crosslinking inducing agent is at least one of citric acid, tartaric acid, or malic acid; the inlet air temperature of the spray drying in step S14 is 160℃~200℃, and the outlet air temperature is 80℃~100℃.

7. The method according to claim 1, characterized in that: Add at least one of magnesium stearate, silica, or talc as an anti-caking agent at 0.1% to 0.5% of its mass to the polyglycoside synergist powder obtained in step S14, and mix in a three-dimensional mixer at a speed of 20 to 30 rpm for 10 to 15 minutes to prevent powder agglomeration.

8. The method according to claim 1, characterized in that: The fertilizer base material mentioned in step S2 includes at least two of nitrogen fertilizer, phosphorus fertilizer, and potassium fertilizer, wherein the nitrogen source is selected from at least one of urea, ammonium nitrate, and ammonium sulfate; the phosphorus source is selected from at least one of monoammonium phosphate, potassium dihydrogen phosphate, and superphosphate; and the potassium source is selected from at least one of potassium sulfate, potassium chloride, and potassium nitrate. The total mass fraction of N, P2O5, and K2O in the fertilizer base material is 30% to 45%.

9. The method according to claim 1, characterized in that: The granulation tower described in step S2 has an inlet air temperature of 15℃~25℃, an outlet air temperature of 40℃~60℃, and a negative pressure of -50~-100Pa inside the tower; the fertilizer granules have a particle strength ≥15N, a disintegration time in water ≤5min, and a particle uniformity ≥90%.

10. A drought-resistant polyglycoside-enhanced fertilizer, characterized in that, It is prepared by the method described in any one of claims 1-9.

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