Biostimulant composition containing polyglutamic acid sugar ester as well as preparation method and application of biostimulant composition

Abstract

The invention relates to a biostimulant composition containing polymerized glutamate sugar ester as well as a preparation method and application thereof, and belongs to the technical field of fertilizer synergists. According to the scheme, the full-water-soluble composite biostimulant is prepared by taking polymerized glutamate sugar ester and algal polysaccharides as main functional components and compounding active substances such as gamma-aminobutyric acid. The product can achieve the functions of promoting crop rooting, improving nutrient absorption and utilization efficiency, improving crop stress resistance, improving crop quality, improving soil, preserving water and fertilizer and the like according to different composition components.

Description

Technical Field

[0001] This application relates to a biostimulant composition containing polyglutamic acid glycoesters, its preparation method, and its application, belonging to the field of fertilizer synergist technology. Background Technology

[0002] With the continued growth of the global population, the increasing scarcity of arable land resources, and the intensification of climate change, modern agriculture faces enormous pressure to increase crop yields, enhance resilience, and ensure food security. Traditional production models that overly rely on chemical fertilizers and pesticides have led to a series of problems, including soil degradation, environmental pollution, and agricultural product safety. Therefore, developing environmentally friendly and sustainable green agricultural inputs has become an urgent need and an important trend in the development of agriculture worldwide.

[0003] Against this backdrop, biostimulants, as natural substances that can significantly promote crop growth by enhancing nutrient absorption efficiency, improving resistance to abiotic stresses (such as drought, salinity, and extreme temperatures), and improving quality traits, have received widespread attention from industry and academia in recent years. Unlike fertilizers and pesticides, biostimulants function primarily through "regulation" rather than "nutrients" or "killing," achieving healthy growth by stimulating the plant's own potential. Common biostimulants include seaweed extracts, humic acid, amino acids, microbial inoculants, and chitosan.

[0004] Currently, most biostimulant products on the market are single-type or have a prominent main ingredient, such as single alginic acid products, single amino acid mixtures, or single humic acid solutions. These products have significant drawbacks in practical applications:

[0005] 1. Limited Mechanism of Action and Limited Effects: Different types of biostimulants have different mechanisms of action. For example, humic acid mainly improves soil structure and chelates nutrients; amino acids can directly provide plants with organic nitrogen sources and alleviate stress; seaweed extracts are rich in seaweed polysaccharides, which can improve fertilizer utilization and enhance plant resistance. However, single components often only address one aspect of crop needs and are difficult to meet the diverse physiological needs of crops in complex field environments.

[0006] 2. Unstable and unpredictable effects: The effects of a single biostimulant are greatly affected by crop type, growth stage, soil type and climate conditions, resulting in large fluctuations in performance. Farmers face high risks when using it, and it is difficult to establish trust.

[0007] 3. There may be a "ceiling effect": once the application of a single ingredient reaches a certain threshold, further increasing the dosage will not only fail to improve the effect, but may even inhibit or waste the effect.

[0008] In theory, the scientific combination of biostimulants with different functions and mechanisms holds promise for achieving complementary advantages and synergistic effects, thereby overcoming the shortcomings of single formulations. Compound biostimulants can comprehensively regulate plant physiology through multiple pathways, such as simultaneously stimulating root development, improving soil microecology, activating antioxidant systems, and enhancing photosynthesis, thus providing crops with more comprehensive, stable, and efficient growth and stress resistance support.

[0009] With the popularization of integrated water and fertilizer technology, the market demand for efficient, fully water-soluble, and multifunctional biostimulants is becoming increasingly urgent. Summary of the Invention

[0010] To address the aforementioned issues, this application provides a biostimulant composition containing polyglutamic acid glycoesters, its preparation method, and its application. In this application, polyglutamic acid glycoesters are compounded with seaweed polysaccharides at the molecular level, and γ-aminobutyric acid is introduced. This composition works synergistically through multiple pathways and has broad application prospects as a biostimulant.

[0011] The purpose of this application is to overcome the technical defects of existing single-component biostimulants, such as limited function, unstable effects, and immature compound product formulation technology, and to provide a fully water-soluble compound biostimulant with multi-component synergy, clear mechanism, and high stability. Its core objectives are reflected in the following three aspects: 1) By innovatively combining polyglutamic acid esters, seaweed extracts, and GABA at the molecular level, it solves the problems of poor compatibility and insufficient physical stability of multiple active ingredients. By utilizing the functional complementarity of each component, it achieves multi-path synergistic effects of "soil improvement - nutrient synergy - stress resistance signal transduction", overcoming the drawbacks of limited action mechanisms and the existence of effect ceilings of single components; 2) For compound abiotic stresses such as salinity and drought, it not only alleviates soil moisture and ion toxicity through the salt tolerance and water retention and ion adsorption functions of polyglutamic acid esters, but also activates the systemic stress resistance mechanism of crops from multiple dimensions of physical, chemical and molecular biology by utilizing the immune exciton function of seaweed polysaccharides and the signal regulation function of GABA, thereby improving the physiological adaptability and yield stability of crops under adverse environments; 3) It adopts fully bio-based and completely degradable components to ensure that the product improves the soil micro-ecology while effectively exerting its biostimulatory function, without chemical residues and environmental risks, and is perfectly compatible with modern agronomic measures such as drip irrigation and aerial spraying, providing a highly efficient and environmentally friendly synergist for fertigation.

[0012] This application provides a biostimulant composition containing polyglutamic acid esters, comprising the following components in parts by weight:

[0013] 5-20 parts of polyglutamic acid ester, 3-15 parts of seaweed polysaccharide or seaweed oligosaccharide, 0.1-5 parts of γ-aminobutyric acid, and 1-10 parts of organic acid;

[0014] The polymeric glutamic acid glycoester has the following general formula:

[0015]

[0016] R is a substituent for the functional oligosaccharide source, wherein the functional oligosaccharide is selected from one or more of the following: plant, animal, microbial oligosaccharides or their derivatives, or oligosaccharides or their derivatives obtained from the degradation of plant, animal, or microbial polysaccharides.

[0017] Optionally, the functional oligosaccharide is selected from one or more of seaweed oligosaccharides;

[0018] Optionally, the seaweed oligosaccharide is selected from one or more of green algae oligosaccharides, brown algae oligosaccharides, and red algae oligosaccharides;

[0019] Optionally, the seaweed oligosaccharide is selected from one or more of rhamnosaccharide, alginate oligosaccharide, fucoidan oligosaccharide, carrageenan oligosaccharide, and agar oligosaccharide;

[0020] Optionally, the rhamnooligosaccharide is derived from the enzymatic or chemical hydrolysis products of green algae polysaccharides;

[0021] Optionally, the alginate oligosaccharide is derived from the enzymatic or chemical hydrolysis products of alginate in brown algae;

[0022] Optionally, the rhamnooligosaccharide is an oligosaccharide compound formed by linear linkage of α-L-rhamnosaccharide monomers through 1,3 and / or 1,4 glycosidic bonds as the main constituent units;

[0023] Optionally, the alginate oligosaccharide is a linear oligosaccharide formed by random or block copolymerization of β-D-mannuronic acid and α-L-guluronic acid through 1,4 glycosidic bonds;

[0024] Optionally, the polymeric glutamate ester has a molecular weight of 10–5000 kDa and a molecular weight distribution of ≤1.5.

[0025] The molecular weight distribution of the polymeric glutamic acid glycoester is ≤1.3;

[0026] The molecular weight distribution of the polymeric glutamic acid glycoester is ≤1.2;

[0027] The molecular weight distribution of the polymeric glutamate ester is ≤1.1;

[0028] Optionally, the organic acid is selected from one or more of citric acid, amino acids, humic acid, acetic acid, pyruvic acid, malic acid, α-ketoglutarate, fumaric acid, succinic acid, oxalic acid, and lactic acid.

[0029] Optionally, the amino acid is selected from one or more of glycine, proline, glutamic acid, methionine, phenylalanine, tryptophan, arginine, lysine, threonine, and valine.

[0030] Optionally, the seaweed polysaccharide is selected from one or more of green algae polysaccharides, brown algae polysaccharides, and red algae polysaccharides;

[0031] Optionally, surfactants may also be included;

[0032] Optionally, it may also include 0.5 to 3 parts of surfactant;

[0033] Optionally, the surfactant is selected from one or more of alkyl glycosides and polyglycerol fatty acid esters.

[0034] This application provides the use of the above-mentioned biostimulant composition containing polyglutamic acid esters in the preparation of biostimulant products.

[0035] This application provides a biostimulant product, including the above-mentioned biostimulant composition containing polyglutamic acid glycoesters.

[0036] Optionally, the biostimulant product is in the form of a powder or solution.

[0037] This application provides a method for preparing a biostimulant product, the method comprising the following steps:

[0038] 1) Add the specified amount of water to the reaction vessel, heat to 40-50°C, and slowly add seaweed polysaccharide and / or seaweed oligosaccharide while stirring until dissolved;

[0039] 2) After the solution has cooled to room temperature, add polyglutamic acid ester, γ-aminobutyric acid, and organic acid and stir until homogeneous;

[0040] 3) Add the surfactant and stir until well combined;

[0041] 4) Add organic acid or weak base to adjust the pH of the solution to 5.0–6.5;

[0042] 5) Filter the obtained solution through a filter screen and fill it to obtain the liquid biostimulant product.

[0043] Optionally, the weak base is selected from one or more of carbonates, bicarbonates, phosphates, hydrogen phosphates, and ammonia.

[0044] This application provides a method for preparing a biostimulant product, the method comprising the following steps:

[0045] 1) Add the specified amount of water to the reaction vessel, heat to 40-50°C, and slowly add seaweed polysaccharide and / or seaweed oligosaccharide while stirring until dissolved;

[0046] 2) After the solution has cooled to room temperature, add polyglutamic acid ester, γ-aminobutyric acid, and organic acid and stir until homogeneous;

[0047] 3) After spray drying, the powdered biostimulant product is obtained.

[0048] Optionally, the spray drying conditions include: inlet temperature 180–220°C and outlet temperature 90–120°C;

[0049] This application provides the application of the above-mentioned biostimulant composition containing polyglutamic acid ester, or the above-mentioned biostimulant product, or the biostimulant product prepared by the above-mentioned method of preparing the biostimulant product, in agriculture.

[0050] The beneficial effects of this application include, but are not limited to:

[0051] 1) The active ingredients provided in this application are polyglutamic acid ester and seaweed polysaccharide, and are compounded with γ-aminobutyric acid. They have a good synergistic effect. Depending on the combination of ingredients, they can promote crop rooting, improve nutrient absorption and utilization efficiency, improve crop stress resistance, improve crop quality, improve soil, and retain water and fertilizer.

[0052] 2) Polyglutamic acid glycoesters work synergistically with other components, resulting in a significant improvement in efficacy compared to using unesterified polyglutamic acid and seaweed oligosaccharides alone.

[0053] 3) It has overcome the salt-drought coupling barrier and solved the technical problem of the inactivation of traditional water-retaining agents in high-salt environments, which can simultaneously alleviate water stress and ion poisoning.

[0054] 4) It uses fully bio-based, completely biodegradable components, which can perform their functions without leaving environmental residues and maintain soil health.

[0055] 5) Polyglutamic acid glycoesters work synergistically with other components to enhance the crop's resistance to adverse environments such as low temperature, drought, and waterlogging by inducing the expression of key stress-resistance genes such as LEA and SOS1 in the crop.

[0056] 6) Polyglutamic acid sugar esters work synergistically with other components to promote the formation of soil aggregates, thereby enhancing soil water retention and permeability, and achieving the goal of soil improvement with long-term use.

[0057] 7) Field trial results show that polyglutamic acid esters, in synergy with other components, can increase nitrogen fertilizer utilization by 20%–30%, phosphorus fertilizer utilization by 10%–30%, and potassium fertilizer utilization by 10%–15%.

[0058] 8) Polyglutamic acid sugar esters have good compatibility and a wide range of applications. They can be added to products as bio-enhancing agents for nitrogen fertilizers, phosphate fertilizers, compound fertilizers, water-soluble fertilizers containing macro-elements, liquid fertilizers, pesticides, etc. Attached Figure Description

[0059] The accompanying drawings, which are included to provide a further understanding of this application and form part of this application, illustrate exemplary embodiments and are used to explain this application, but do not constitute an undue limitation of this application. In the drawings:

[0060] Figure 1 This is a schematic diagram of the structure of the polymeric glutamic acid glycoester involved in this application;

[0061] Figure 2 This is a schematic diagram of a specific structure of the polymeric glutamic acid glycoester involved in this application;

[0062] Figure 3 This application pertains to the biosynthetic pathway of the polyglutamic acid glycoester involved in the present application.

[0063] Figure 4 This is a graph showing the yield results of shake-flask fermentation of polyglutamic acid rhamnosyl ester by different engineered strains involved in Example 1 of this application;

[0064] Figure 5 This is a liquid chromatogram of the molecular weight of the product of the biosynthetic route of polyglutamic acid glycoester involved in Example 1 of this application;

[0065] Figure 6 The infrared spectrum of the product of the biosynthetic route of polyglutamic acid glycoester involved in Example 1 of this application;

[0066] Figure 7 This is a liquid chromatogram of the molecular weight of the product of the biosynthetic route of polyglutamic acid glycoester involved in Example 1 of this application;

[0067] Figure 8 The infrared spectrum of the product of the biosynthetic route of polyglutamic acid glycoester involved in Example 1 of this application;

[0068] Figure 9 This is a graph showing the difference in growth at harvest time for different treatments involved in Example 3 of this application;

[0069] Figure 10 This is a field growth diagram of romaine lettuce under different treatments as described in Embodiment 4 of this application;

[0070] Figure 11 This is a graph showing the differences in plant growth among different treatments involved in Example 4 of this application;

[0071] Figure 12 This is a diagram showing the field performance results of maize under different treatments involved in Example 4 of this application;

[0072] Figure 13 This is a graph showing the differences in maize plant growth among different treatments involved in Example 4 of this application;

[0073] Figure 14This is a graph showing the effects of different treatments on various indicators of the aboveground parts of maize in Example 4 of this application;

[0074] Figure 15 This is a difference image of maize root system scans during the seedling stage under different treatments involved in Example 4 of this application;

[0075] Figure 16 This is a graph showing the effects of different treatments on various indicators of the seedling root system in Example 4 of this application;

[0076] Figure 17 This is a graph showing the effects of different treatments on the corn harvest period in Example 4 of this application;

[0077] Figure 18 This is a graph showing the effects of different treatments on the length and diameter of maize ears in Example 4 of this application.

[0078] Figure 19 This refers to the experimental apparatus described in Embodiment 6 of this application;

[0079] Figure 20 The following graphs show the effects of different treatments on the content of available nitrogen, phosphorus, and potassium in Example 6 of this application (the top three graphs represent the top layer of soil (0-20cm), and the bottom three graphs represent the bottom layer (20-40cm)).

[0080] Figure 21 This is a graph showing the effect of different treatments involved in Example 6 of this application on the soil moisture content of the topsoil layer (0-20cm);

[0081] Figure 22 This is a comparison chart of wheat growth under different treatments 18 days after the jointing stage treatment involved in Example 7 of this application. Detailed Implementation

[0082] The present application is described in detail below with reference to the embodiments, but the present application is not limited to these embodiments. Unless otherwise specified, the raw materials and reagents in the embodiments of the present application are all purchased through commercial channels.

[0083] Example 1 Polymeric glutamic acid glycoester and its preparation method

[0084] Construction of engineered strains for the production of polyglutamic acid sugar esters

[0085] The construction of engineered strains for producing polyglutamic acid glycoesters mainly consists of two modules. First, using glutamic acid or its salts as substrates, the polyglutamic acid (γ-PGA) synthase complex is overexpressed in Bacillus subtilis to catalyze the polymerization of glutamic acid into γ-PGA. When γ-PGA has polymerized to a certain extent, the expression of the second module, namely acyltransferase and glycosyltransferase, is initiated. The production and secretion of polyglutamic acid glycoesters are achieved through the addition of functional oligosaccharides.

[0086] The steps for constructing engineered strains and screening key enzymes are as follows:

[0087] 1) Source of the γ-PGA synthase complex in the first module

[0088] Table 1. Sources of γ-PGA synthase complex

[0089]

[0090] 2) First module promoter strength gradient design

[0091] Table 2 Promoter strength and regulation characteristics

[0092]

[0093] Table 3 Design of different promoter strength combinations in Module 1

[0094] promoter combination Promoter strength combination 1 P43-pgsA-xylA-pgsB-veg-pgsC 2 P43-pgsA-xylA-pgsC-veg-pgsB 3 P43-pgsC-xylA-pgsA-veg-pgsB 4 P43-pgsC-xylA-pgsB-veg-pgsA 5 P43-pgsB-xylA-pgsC-veg-pgsA 6 P43-pgsB-xylA-pgsA-veg-pgsC

[0095] 3) First module: Genome integration (CRISPR-Cas9 system)

[0096] 3.1) Integration site design:

[0097] Target: amyE gene locus (non-essential region, does not affect growth);

[0098] Donor plasmids: containing expression cassettes (e.g., promoter combination 1: P43-pgsA-xylA-pgsB-veg-pgsC), and screening for different donor plasmids constructed from those in Table 3;

[0099] 3.2) Electroconversion and Screening:

[0100] Competent cells of Bacillus subtilis were prepared (pretreated with 0.1M CaCl2) and then co-electroporated with the following: donor plasmid to be screened (10 μg) and CRISPR plasmid pJOE8999 (5 μg, containing Cas9 and sgRNA). Electroporation conditions: 2.5 kV, 5 ms, 4℃. Recovery: SMM liquid medium, 37℃ shaker for 1 h. PCR verification and screening were performed.

[0101] Expression cassettes with different promoter combinations were integrated into the amyE site of the Bacillus subtilis genome to obtain six engineered strains: B-1, B-2, B-3, B-4, B-5, and B-6 (corresponding to promoter combinations 1 to 6 in Table 3).

[0102] 4) The second module heterologously expresses acyltransferases and glycosyltransferases

[0103] The RhlA (acyltransferase) and RhlB (glycosyltransferase) genes from Pseudomonas aeruginosa were linked by a DNA sequence encoding a (Gly4Ser)3 flexible linker to form a fusion gene rhlA-linker-rhlB. This fusion gene was cloned downstream of the xylose-inducible promoter xylA in the Bacillus subtilis expression vector pAX01 to construct the recombinant plasmid pAX01-rhlAB. The pAX01-rhlAB plasmid was introduced into the six strains constructed in step 3) by electroporation (2.5 kV, 5 ms) to obtain engineered strains BS-1, BS-2, BS-3, BS-4, BS-5, and BS-6, which were equipped with two modules for the production of polyglutamic acid glycol esters.

[0104] Preparation of Polyglutamic Acid Rhamnosyl Ester

[0105] 1) Screening and validation of engineered strains for high-yield production of polyglutamic acid esters

[0106] Table 4 Screening and Validation of High-Yielding Polyglutamic Acid Rhamnosyl Ester Strains

[0107]

[0108] The results are as follows Figure 4 As shown, the engineered strain BS-6 produced the highest yield of polyglutamic acid rhamnosyl ester, reaching 25.8 g / L. Therefore, the obtained engineered strain BS-6 was used for subsequent process optimization.

[0109] 2) Fermentation and Induced Expression

[0110] The engineered strain BS-6 was inoculated into LB medium containing 5 μg / mL erythromycin and activated at 37°C for 12 hours. The culture was then transferred to a 5L fermenter (working volume 3L) at an 8% inoculation rate. The initial culture medium consisted of: 60 g / L sodium glutamate, 30 g / L glucose, 5 g / L yeast extract, and 0.1 mM MnCl2. Control parameters were: temperature 37°C, pH 7.0, dissolved oxygen >30% (stirring speed 300 rpm). Key induction procedures (performed simultaneously at 36 hours of fermentation): added sterile xylose to a final concentration of 5 g / L (to activate the xylA promoter, initiating the expression of RhlA (acyltransferase), RhlB (glycosyltransferase), and pgsA genes); adjusted the fermentation temperature to 30°C; reduced the stirring speed to 160 rpm to alleviate shear stress; and continued induction for 8 hours.

[0111] 3) Intracellular enzymes catalyze glycoesterification reactions

[0112] After induction, a sterile rhamnosaccharide solution (200 g / L, dissolved in 50 mM Tris-HCl buffer, pH 7.5) was added at a flow rate of 5 g / L / h. The reaction conditions were maintained at 30 °C, pH 7.0, and stirring speed of 160 rpm. Samples were taken every 2 hours during the addition process to monitor substrate consumption.

[0113] 4) Self-assembly of biosynthetic products

[0114] Twelve hours after the addition of feedstock, the viscosity of the fermentation broth increased significantly (>5000 cP), indicating that the self-assembly of polyglutamic acid rhamnosyl ester was complete. The reaction was terminated by centrifugation (8000 rpm, 10 min), collection of the supernatant, and HPLC analysis of the product. The results are as follows: Figure 5 As shown, the product has a weight-average molecular weight of 12.82 kDa and a PDI of 1.09. Infrared detection results are as follows... Figure 6 As shown, at 1725cm -1 The presence of a characteristic absorption peak for the carbonyl group (OC=O) indicates that the esterification of the product was successful.

[0115] Preparation of Polyglutamic Acid Fucoidoester

[0116] The preparation process is basically the same as that of polyglutamic acid rhamnosyl ester, except that the flow-added sterile rhamnosyl oligosaccharide solution is replaced with the flow-added sterile alginate oligosaccharide solution (prepared by enzymatic hydrolysis and purification of alginate extracted from kelp).

[0117] The product was detected by HPLC, and the results are as follows: Figure 7 As shown, the product has a weight-average molecular weight of 12.75 kDa and a PDI of 1.10. Infrared detection results are as follows... Figure 8 As shown, at 1745cm -1 The presence of a characteristic absorption peak for the carbonyl group (OC=O) indicates that the esterification of the product was successful.

[0118] Example 2 Biostimulant and its preparation method

[0119] Preparation of 1000 kg of liquid compound biostimulant

[0120] Materials include: Polyglutamic acid rhamnosyl ester (molecular weight 10kDa): 400kg, alginate oligosaccharide (molecular weight 2000Da): 200kg, γ-aminobutyric acid (GABA): 100kg, citric acid: 20kg, alkyl glycoside (APG-0810): 5kg, tap water: 275kg.

[0121] The preparation method includes the following steps:

[0122] 1) Dissolving: Add the specified amount of water to the reaction vessel, heat to 40-50℃, and slowly add seaweed polysaccharide while stirring until completely dissolved;

[0123] 2) Compound preparation: After the solution cools to room temperature (25-30℃), add polyglutamic acid ester, γ-aminobutyric acid and organic acid in sequence, and continue stirring until a homogeneous and transparent solution is formed;

[0124] 3) Conditioning: Add surfactant and stir until homogeneous. Adjust the pH of the solution to between 5.0 and 6.5 using an organic acid or weak base (such as potassium hydroxide) to maintain the stability and activity of all components;

[0125] 4) Filtration and packaging: The obtained solution is filtered through a 200-mesh filter and then bottled to obtain the finished liquid biostimulant.

[0126] Prepared using the above method, a pale yellow to amber transparent liquid is obtained, with a pH of approximately 5.5, a specific gravity of 1.15, and no precipitate.

[0127] Preparation of 400 kg of compound biostimulant powder

[0128] Materials include: Polyglutamic acid alginate (molecular weight 15kDa): 300kg, Ulva prolifera polysaccharide (molecular weight 2000-15000Da): 50kg, γ-aminobutyric acid (GABA): 50kg, glycine: 20kg.

[0129] The preparation method includes the following steps:

[0130] 1) Dissolving: Add the specified amount of water to the reaction vessel, heat to 40-50℃, and slowly add seaweed polysaccharide while stirring until completely dissolved;

[0131] 2) Compound preparation: After the solution cools to room temperature (25-30℃), add polyglutamic acid ester, γ-aminobutyric acid and organic acid in sequence, and continue stirring until a homogeneous and transparent solution is formed;

[0132] 3) Dissolve the above active ingredients in water and spray dry to obtain a light yellow to light brown powder with a pH value (diluted 250 times) of about 6.0. The inlet temperature during spray drying is 180-200℃ and the outlet temperature is 90-100℃. Finally, mix it evenly with the drying filler to obtain the final product.

[0133] Preparation of 100kg of compound biostimulant powder

[0134] Materials include: Polyglutamic acid alginate (molecular weight 15kDa): 50kg, Ulva prolifera polysaccharide (molecular weight 2000-15000Da): 30kg, γ-aminobutyric acid (GABA): 10kg, glycine: 10kg.

[0135] The preparation method is the same as above.

[0136] Preparation of 500 kg of compound biostimulant powder

[0137] Materials include: Polyglutamic acid alginate (molecular weight 15kDa): 200kg, Ulva prolifera polysaccharide (molecular weight 2000-15000Da): 150kg, γ-aminobutyric acid (GABA): 50kg, glycine: 100kg.

[0138] The preparation method is the same as above.

[0139] The compound biostimulant product provided in this application achieves dual regulation of the "soil-crop" system through the synergistic effect of the following multiple mechanisms:

[0140] Table 5 Synergistic Regulation of Each Component in the Scheme of this Application

[0141]

[0142]

[0143] Example 3: Effect Test of Polyglutamic Acid Rhamnose Ester on Rapeseed Drenching

[0144] Experimental location: Vegetable greenhouse in Qingdao High-tech Zone, Shandong Province; Experimental period: October 2023 - December 2023; Application method: Root application (3 weeks after transplanting); Experimental design: Treatment 1: 10% γ-PGA + 5% Chlorella oligosaccharide + 2.5‰ GABA; Treatment 2: 15% Polyglutamic acid rhamnosyl ester + 2.5‰ GABA; Treatment 3: 15% Polyglutamic acid rhamnosyl ester; Climate environment: Daytime 18-20℃, Nighttime 12-14℃, Soil EC value 1.32mS / cm, pH=6.2 (Winter low temperature + mild salt stress).

[0145] Test results: Passed Figure 9 It can be seen that rapeseed treated with polyglutamic acid rhamnosyl ester (treatments 2 and 3) exhibited vigorous growth and uniform plant size, showing significantly better growth than γ-PGA + chlorophyll oligosaccharide + GABA (treatment 1). Compared to rapeseed treated with polyglutamic acid rhamnosyl ester alone (treatment 3), rapeseed treated with GABA in combination (treatment 2) had darker green leaves.

[0146] Table 6. Differences in yield and economic benefits among different treatments

[0147]

[0148]

[0149] Experimental conclusions: Drainage application of polyglutamic acid rhamnosyl ester (treatments 2 and 3) can improve the stress resistance of rapeseed in low temperature, low light, and saline-alkali soils, ensuring growth rate under adverse conditions and thus increasing yield. Compared with the polyglutamic acid + chlorophyll oligosaccharide compound product (treatment 1), the rapeseed treated with polyglutamic acid rhamnosyl ester (treatments 2 and 3) showed a yield increase of 13.34%–10.18%, and a net profit increase of 248–328 yuan / mu. Compared with drainage application of polyglutamic acid rhamnosyl ester alone (treatment 3), the combined use of polyglutamic acid rhamnosyl ester and GABA (treatment 2) further enhanced the functional effect, resulting in higher rapeseed yield, greener leaves, and improved marketability.

[0150] Example 4: Effect of Polyglutamic Acid Rhamnose Ester Combined with Urea on Lettuce

[0151] Experimental location: Chengyang District, Qingdao City, Shandong Province; Experimental period: June 2024 - July 2024; Application method: Base fertilizer application; Experimental design: CK: 5‰ polyglutamic acid + 5‰ chlorophyll oligosaccharide + 2.5‰ GABA + urea (20 kg / mu), T1: 7.5‰ polyglutamic acid rhamnosyl ester + 2.5‰ GABA + urea (20 kg / mu), T2: 10‰ polyglutamic acid rhamnosyl ester + 2.5‰ GABA + urea (20 kg / mu); Climate environment: High summer temperatures affected the emergence and growth rate of lettuce.

[0152] Experimental results: Figure 10 and Figure 11 The differences in overall and individual plant growth of romaine lettuce among different treatments are shown in Tables 7 and 8. As can be seen from the data, the nutrient content and various morphological indicators of plants treated with polyglutamic acid rhamnosyl ester (T1 and T2) are better than those of the control (polyglutamic acid + chlorophyll oligosaccharide).

[0153] Table 7 Nutrient content in plants under different treatments

[0154]

[0155] Table 8. Effects of different treatments on various morphological indicators of romaine lettuce.

[0156]

[0157] Experimental conclusion: Adding polyglutamic acid rhamnosyl ester (T1 and T2) to urea can improve nitrogen fertilizer utilization and promote crop growth compared to polyglutamic acid + chlorophyll oligosaccharide (CK). Adding 7.5‰ and 10‰ of polyglutamic acid rhamnosyl ester increased N, P, and K utilization by 9.7%, 2.3%, and 3.6% and 10.5%, 2.1%, and 3.5%, respectively, compared to the control.

[0158] Example 5: Effect test of polyglutamic acid fucoidan ester combined with diammonium phosphate on maize.

[0159] Experimental location: Laixi City, Qingdao, Shandong Province; Experimental period: June 2024 - October 2024; Application method: Base fertilizer application; Experimental design: CK: 2.5‰ polyglutamic acid + 2.5‰ fucoidan + 2.5‰ GABA + diammonium phosphate (55 kg / mu), T1: 5‰ polyglutamic acid fucoidan + diammonium phosphate (55 kg / mu), T2: 5‰ polyglutamic acid fucoidan + 2.5‰ GABA + diammonium phosphate (55 kg / mu).

[0160] 1) Effects of different fertilization treatments on maize seedling growth

[0161] Figure 12 and Figure 13 The effects of different treatments on the overall and individual growth of maize in the field were demonstrated. Overall, the maize treated with polyglutamic acid fucoidan + GABA + diammonium phosphate (T2) showed the best growth. Figure 14 As can be seen, the maize treated with polyglutamic acid fucoidan + GABA + diammonium phosphate (T2) showed the best performance in the above-ground indicators across all regions. Plant height, stem diameter, SPAD value, and above-ground fresh weight increased by 32.9% and 10.9%, 33.3% and 18.4%, 3.27 and 3.59%, and 70.5% and 20.0% respectively compared with CK and T1.

[0162] Figure 15 The image shows root scans of maize from different treatments. It can be seen that the maize treated with polyglutamic acid fucoidan + GABA + diammonium phosphate (T2) had a more developed root system and a greater number of capillary roots. Figure 16 The results show that the root fresh weight of the polyglutamic acid fucoidan + GABA + diammonium phosphate (T2) treatment was the highest, at 5.0 g, which was 51.5% and 6.4% higher than that of the control (CK) and T1 treatments, respectively. The number of primary roots in the T2 treatment was the same as that in the T1 treatment, both higher than that in the CK treatment. The total number of roots and the total root surface area in the T2 treatment were the highest, at 3596 and 12188 mm², respectively. 2 .

[0163] 2) Effects of different fertilization treatments on maize harvest time

[0164] Figure 17 This demonstrates the effects of different treatments on maize growth and ears at harvest, combined with... Figure 18It can be seen that the ear length and ear diameter of the polyglutamic acid fucoidan + GABA + diammonium phosphate (T2) treatment reached 19.8 cm and 47.0 mm, respectively, both superior to CK and T1. Table 9 shows that at harvest, the stem diameter, 100-kernel dry weight, leaf area, and leaf dry weight of the polyglutamic acid fucoidan + GABA + diammonium phosphate (T2) treatment were all superior to those of CK and T1 treatments. Finally, yield measurements revealed that the maize yield of the polyglutamic acid fucoidan + GABA + diammonium phosphate (T2) treatment reached 940 kg / mu, an increase of 11.1% and 9.2% compared to CK and T1, respectively; after using polyglutamic acid fucoidan, the net income per mu increased by 184 yuan and 151.1 yuan compared to CK and T1, respectively (Table 10).

[0165] Table 9. Effects of different treatments on relevant indicators at maize harvest time.

[0166]

[0167] Table 10. Effects of different treatments on maize yield and income.

[0168]

[0169] Experimental Conclusion: The synergistic effect of adding polyglutamic acid fucoidan and GABA to diammonium phosphate significantly promotes maize growth. Overall, the effects are mainly reflected in plant height and the number of fine roots during the seedling stage, and in ear size and yield during the harvest stage. Field trials have verified that polyglutamic acid fucoidan + GABA has a significant advantage over polyglutamic acid + fucoidan + GABA in synergistic effects with phosphate fertilizer, achieving the effect of increasing yield and income.

[0170] Example 6: Nutrient Leaching Effect Test of Polyglutamic Acid Rhamnose Ester

[0171] Test location: Haida Bio-industry Park, High-tech Zone, Qingdao City, Shandong Province; Test time: May 2024 - June 2024; Test method: Product leaching test was carried out with reference to the equipment and method mentioned in patent CN219328791U; Test design: CK: macro-elements (18-18-18) + water; T1: macro-elements (18-18-18) + 10‰ polyglutamic acid rhamnosyl ester.

[0172] Test results: The test setup is as follows Figure 19 As shown, the test results are available in [reference needed]. Figure 20 and Figure 21 As shown in the figure, the application of polyglutamic acid rhamnosyl ester increased the nitrogen, phosphorus and potassium content in the topsoil layer (0-20cm) and decreased the nitrogen, phosphorus and potassium content in the non-topsoil layer (20-40cm); the soil moisture content increased by 23.4% compared with the control.

[0173] Experimental conclusion: Polyglutamic acid rhamnosyl ester, as a nutrient synergist for macronutrients, can improve the utilization rate of nutrients in the topsoil and enhance soil water retention. The nitrogen, phosphorus, and potassium contents in the topsoil increased by 22.4%, 19.9%, and 11.1% respectively compared with the control.

[0174] Example 7: Application effect of polyglutamic acid glycoester as an aerial spraying companion in semi-arid regions

[0175] Experimental crop and time period: wheat, jointing stage and early flowering stage.

[0176] The experimental protocols are shown in Tables 11 and 12.

[0177] Table 11 Experimental Program for Wheat "Aerial Spraying Companion" at the Wheat Jointing Stage

[0178]

[0179]

[0180] Table 12 Experimental Program for Wheat "Aerial Spray Companion" in the Early Flowering Stage

[0181]

[0182] The experimental results are shown in Tables 13 and 14.

[0183] Table 13 Survey data on different treatments at the wheat jointing stage

[0184] serial number Plant height / cm SPAD CK 62.99±3.77a 50.87±4.88c T1 62.63±3.04a 53.88±2.47a T2 63.82±2.74a 54.00±1.84a T3 64.46±2.35a 53.07±3.03ab T4 64.395±3.79a 54.67±3.24a T5 64.26±2.56a 54.42±1.83a

[0185] Table 14 Survey data on different treatments during the early flowering stage of wheat

[0186]

[0187]

[0188] According to Tables 13 and 14 and Figure 22 The following experimental conclusions can be drawn:

[0189] In semi-arid regions, polyglutamic acid glycolipids (PGD) were used as a companion to aerial spraying, promoting the growth of wheat during both the jointing and booting stages. During the jointing stage, γ-polyglutamic acid, PGA rhamnosyl ester, and PGA fucoidan showed the best performance on the flag leaf SPAD. Among different drought-resistant agents, PGA D performed well. There were no significant differences in plant height and flag leaf area among different treatments. During the booting stage, 8% chlorella oligosaccharide and PGA fucoidan showed good performance on the flag leaf SPAD. 8% chlorella oligosaccharide, 3.5% polyglutamic acid, PGA rhamnosyl ester, and PGA fucoidan showed good performance on the second-to-last leaf SPAD. PGA rhamnosyl ester and PGA fucoidan showed the best spike length. Overall, the two PGA D application groups showed the best effects. There were no significant differences in flag leaf area among different treatments.

[0190] The above description is merely an embodiment of this application, and the scope of protection of this application is not limited to these specific embodiments, but is determined by the claims of this application. Various modifications and variations can be made to this application by those skilled in the art. Any modifications, equivalent substitutions, improvements, etc., made within the technical concept and principles of this application should be included within the scope of protection of this application.

Claims

1. A biostimulant composition containing polyglutamic acid esters, characterized in that, It comprises the following components by weight: 5-20 parts of polyglutamic acid ester, 3-15 parts of seaweed polysaccharide or seaweed oligosaccharide, 0.1-5 parts of γ-aminobutyric acid, and 1-10 parts of organic acid; The polymeric glutamic acid glycoester has the following general formula: R is a substituent for the functional oligosaccharide source, wherein the functional oligosaccharide is selected from one or more of the following: plant, animal, or microbial oligosaccharides or their derivatives, or oligosaccharides or their derivatives obtained from the degradation of plant, animal, or microbial polysaccharides.

2. The biostimulant composition containing polyglutamic acid esters according to claim 1, characterized in that, Optionally, the functional oligosaccharide is selected from one or more of seaweed oligosaccharides; Optionally, the seaweed oligosaccharide is selected from one or more of green algae oligosaccharides, brown algae oligosaccharides, and red algae oligosaccharides; Optionally, the seaweed oligosaccharide is selected from one or more of rhamnosaccharide, alginate oligosaccharide, fucoidan oligosaccharide, carrageenan oligosaccharide, and agar oligosaccharide; Optionally, the rhamnooligosaccharide is derived from the enzymatic or chemical hydrolysis products of green algae polysaccharides; Optionally, the alginate oligosaccharide is derived from the enzymatic or chemical hydrolysis products of alginate in brown algae; Optionally, the rhamnooligosaccharide is an oligosaccharide compound formed by linear linkage of α-L-rhamnosaccharide monomers through 1,3 and / or 1,4 glycosidic bonds as the main constituent units; Optionally, the alginate oligosaccharide is a linear oligosaccharide formed by random or block copolymerization of β-D-mannuronic acid and α-L-guluronic acid through 1,4 glycosidic bonds; Optionally, the polymeric glutamate ester has a molecular weight of 10–5000 kDa and a molecular weight distribution of ≤1.

5.

3. The biostimulant composition containing polyglutamic acid esters according to claim 1, characterized in that, The seaweed polysaccharide is selected from one or more of green algae polysaccharides, brown algae polysaccharides, and red algae polysaccharides; Optionally, the organic acid is selected from one or more of citric acid, amino acids, humic acid, acetic acid, pyruvic acid, malic acid, α-ketoglutarate, fumaric acid, succinic acid, oxalic acid, and lactic acid. Optionally, the amino acid is selected from one or more of glycine, proline, glutamic acid, methionine, phenylalanine, tryptophan, arginine, lysine, threonine, and valine.

4. The use of the biostimulant composition containing polyglutamic acid glycoesters as described in any one of claims 1 to 3 in the preparation of biostimulant products.

5. A biostimulant product, characterized in that, The biostimulant composition comprising polyglutamic acid glycoesters as described in any one of claims 1 to 3.

6. The biostimulant product according to claim 5, characterized in that, The biostimulant product is in the form of powder or solution.

7. A method for preparing a biostimulant product, characterized in that, The preparation method includes the following steps: 1) Add the specified amount of water to the reaction vessel, heat to 40-50°C, and slowly add seaweed polysaccharide and / or seaweed oligosaccharide while stirring until dissolved; 2) After the solution has cooled to room temperature, add polyglutamic acid ester, γ-aminobutyric acid, and organic acid and stir until homogeneous; 3) Add the surfactant and stir until well combined; 4) Add organic acid or weak base to adjust the pH of the solution to 5.0–6.5; 5) Filter the obtained solution through a filter screen and fill it to obtain the liquid biostimulant product.

8. A method for preparing a biostimulant product, characterized in that, The preparation method includes the following steps: 1) Add the specified amount of water to the reaction vessel, heat to 40-50°C, and slowly add seaweed polysaccharide and / or seaweed oligosaccharide while stirring until dissolved; 2) After the solution has cooled to room temperature, add polyglutamic acid ester, γ-aminobutyric acid, and organic acid and stir until homogeneous; 3) After spray drying, the powdered biostimulant product is obtained.

9. The method for preparing the biostimulant product according to claim 8, characterized in that, The spray drying conditions include: inlet temperature 180–220°C and outlet temperature 90–120°C.

10. The application in agriculture of the biostimulant composition containing polyglutamic acid glycoesters as described in any one of claims 1 to 3, or the biostimulant product as described in claim 5 or 6, or the biostimulant product prepared by the method described in any one of claims 7 to 9.

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