Preparation and application of water-soluble fertilizer containing polyglutamic acid and alginate polysaccharide synergistic macroelement

Abstract

This invention discloses the preparation and application of an aqueous solution fertilizer containing polyglutamic acid and seaweed polysaccharides to enhance macronutrients, belonging to the field of liquid fertilizer preparation technology. The method includes the following steps: S1, preparation of a linear γ-polyglutamic acid precursor; S2, construction of a cyclic γ-polyglutamic acid backbone; S3, functionalization modification of the cyclic γ-polyglutamic acid; S4, preparation of a brown algae oligosaccharide-calcium complex; and S5, compounding of the liquid fertilizer. This invention systematically solves a series of technical problems existing in the compounding of polyglutamic acid and seaweed polysaccharides in liquid fertilizers, such as high viscosity, easy gelation, and instability. The prepared aqueous solution fertilizer containing polyglutamic acid and seaweed polysaccharides to enhance macronutrients has excellent physicochemical properties, good storage stability, significant application effects, and environmentally friendly characteristics, and has broad application prospects in facility agriculture, fertigation, and other fields.

Description

Technical Field

[0001] This invention belongs to the field of liquid fertilizer preparation technology, and relates to the preparation and application of aqueous liquid fertilizer containing polyglutamic acid and seaweed polysaccharides to enhance the effects of macronutrients. Background Technology

[0002] γ-Polyglutamic acid (γ-PGA) is a water-soluble biopolymer prepared by microbial fermentation. Its molecular chain consists of glutamic acid units linked by γ-amide bonds, and its molecular weight ranges from 50,000 to 10 million Daltons. Due to the large number of active carboxyl groups in its molecular chain, γ-PGA possesses superior water retention capacity and metal ion chelating properties. Its degradation product is pollution-free glutamic acid, which is widely used in the preparation and production of fertilizer synergists. Seaweed polysaccharides, derived from natural seaweed, can induce plant stress resistance, promote seed germination and seedling growth, and improve crop quality; they are also important components of fertilizer synergists.

[0003] With the increasing demands for fertilizer functionality in modern high-efficiency agriculture, the combined use of γ-PGA and seaweed polysaccharides has become an important research direction for developing multifunctional and synergistic liquid fertilizers. Existing technologies have reported simple physical mixing of γ-PGA and seaweed extracts. For example, Chinese patent CN112759459A discloses a liquid fertilizer containing polyglutamic acid, seaweed polysaccharides, and S-inducer, prepared by directly mixing the components. However, in the actual preparation and application of liquid fertilizers, the combination of γ-PGA and seaweed polysaccharides faces complex compatibility issues. Studies have shown that the molecular weight of γ-PGA is closely related to fertilizer stability; high molecular weight γ-PGA of 1000 kDa and above, while exhibiting higher activity, also possesses extremely strong thickening properties. In high-salt systems with macroelements, this can easily lead to a sharp increase in system viscosity, resulting in difficulties in pipeline transportation and low-temperature gelation. Simultaneously, the alginate component in seaweed polysaccharides can react with divalent cations in the system (such as Ca²⁺, ... 2+ Mg 2+ Ionic crosslinking occurs, forming insoluble alginate gels, leading to excessive water-insoluble matter in the product and failing to meet national standards. Furthermore, both synergists are anionic polymers, which are prone to synergistic flocculation in high-concentration nutrient systems and are highly susceptible to stratification and precipitation during storage.

[0004] To address the aforementioned issues, existing technologies have attempted to improve the compatibility and stability of γ-PGA with seaweed polysaccharides in liquid systems through chemical modification or structural regulation. For example, Chinese patent CN121449468A discloses an environmentally friendly water-soluble fertilizer containing seaweed extract and polyglutamic acid. This is achieved by simultaneously grafting 2-hydroxymethyl-5-aminophenylboronic acid and octadecylamine onto sodium poly-L-γ-glutamate to prepare double-grafted modified polyglutamic acid, which is then complexed with a seaweed oligosaccharide solution. Utilizing the dynamic reversibility of borate ester bonds and the self-assembly characteristics of hydrophobic chains, controlled nutrient release is achieved. While this method improves the fertilizer's storage stability and slow-release performance to some extent, its technical solution primarily focuses on regulating the nutrient release rate, offering limited improvement to the compatibility issues of γ-PGA and seaweed polysaccharides—two anionic polymers—in high-salt systems containing macroelements. The thickening problem of high molecular weight γ-PGA, the gel precipitation problem of seaweed polysaccharides in the presence of divalent cations, and the synergistic flocculation problem when the two coexist have not yet been effectively solved. In addition, the double grafting modification process of this method involves multiple steps and the use of organic solvents, making the process complex. Furthermore, some carboxyl groups of γ-PGA are consumed after modification, which may affect its original metal ion chelating ability.

[0005] Therefore, how to effectively reduce the viscosity of γ-PGA in liquid fertilizer while maintaining its high activity, and at the same time suppress the gelation and precipitation tendency of seaweed polysaccharides, so that the two synergists can coexist stably and work synergistically in a macro-element aqueous solution system, has become a technical problem that urgently needs to be solved in this field. Summary of the Invention

[0006] The purpose of this invention is to provide the preparation and application of a liquid fertilizer containing polyglutamic acid and seaweed polysaccharides to enhance the effects of macronutrients. By constructing a complex system consisting of a cyclic γ-PGA backbone, functionalized 5-aminolevulinic acid, and a brown algae oligosaccharide-calcium ion trap, γ-PGA and seaweed polysaccharides can coexist stably and synergistically exert their effects in a high-salt macronutrient system. This solves the core problems in the prior art where the combination of γ-PGA and seaweed polysaccharides leads to excessively high system viscosity, gel precipitation and flocculation upon encountering divalent cations, thereby improving the storage stability and synergistic effect of the liquid fertilizer.

[0007] The technical solution adopted in this invention is a method for preparing an aqueous fertilizer containing polyglutamic acid and seaweed polysaccharides to enhance the effects of macronutrients. The key aspect is that it includes the following steps: S1. Preparation of linear γ-polyglutamic acid precursor: γ-polyglutamic acid was subjected to self-degradation reaction under acidic conditions. Lysine was introduced simultaneously during the reaction to modify its end groups. After purification, a linear γ-PGA solution with lysine end groups was obtained. S2. Construction of the cyclic γ-polyglutamic acid backbone: The linear γ-PGA solution was successively modified with alkyne and cysteine ​​to obtain a bifunctionalized linear γ-PGA containing an alkyne group at one end and a thiol group at the other end. Then, intramolecular cyclization was performed to obtain a cyclic γ-PGA solution. S3. Functional modification of cyclic γ-polyglutamic acid: The cyclic γ-PGA solution was subjected to an amidation grafting reaction with 5-aminolevulinic acid, and after purification, a 5-ALA-grafted cyclic γ-PGA solution was obtained. S4. Preparation of the alginate-calcium complex: The alginate-calcium complex concentrate was obtained by complexing the alginate-calcium complex solution with the calcium nitrate solution and then concentrating it. S5. Compounding of liquid fertilizer: 5-ALA grafted cyclic γ-PGA solution, concentrated brown algae oligosaccharide-calcium complex solution, seaweed polysaccharide and N, P and K elements are compounded to obtain an aqueous liquid fertilizer containing polyglutamic acid and seaweed polysaccharide-enhanced macronutrients.

[0008] Furthermore, in step S1, the above self-degradation reaction is carried out at 65℃~75℃ and pH 3.0~3.5 for a reaction time of 5h~7h.

[0009] Furthermore, in step S1, the above-mentioned γ-polyglutamic acid is prepared into a γ-PGA base solution with a mass concentration of 50 g / L, and the above-mentioned lysine is L-lysine prepared into a lysine solution with a mass concentration of 80 g / L to 120 g / L; the mass ratio of the above-mentioned γ-PGA base solution to the above-mentioned lysine solution is 100:(0.6 to 1.8).

[0010] Furthermore, in step S2, the above-mentioned alkynylation modification is carried out under pH 7.5–8.5 conditions, using alkynyl-succinimide ester as alkynyl donor and reacting with linear γ-PGA solution for 1 h–2 h; the above-mentioned cysteine ​​modification is carried out under pH 5.3–5.7 conditions, using N-hydroxysuccinimide and 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride to activate the alkynyl-modified linear γ-PGA solution for 20 min–40 min, and then reacting with L-cysteine ​​for 3.5 h–4.5 h to obtain a bifunctionalized linear γ-PGA solution.

[0011] Furthermore, in step S2, the intramolecular cyclization is initiated by a mercapto-alkyne photoclick reaction of bifunctionalized linear γ-PGA under ultraviolet light irradiation to obtain a cyclic γ-PGA solution linked by thioether bonds; the intensity of the ultraviolet light irradiation is 5 mW / cm². 2 ~10mW / cm 2 The irradiation reaction time is 2h to 4h.

[0012] Furthermore, in step S3, the above-mentioned amidation grafting reaction is carried out under pH 5.3-5.7 conditions, by activating the cyclic γ-PGA solution with N-hydroxysuccinimide and 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride for 20 min-40 min, and then reacting it with 5-aminolevulinic acid for 5 h-7 h to obtain a 5-ALA-grafted cyclic γ-PGA solution; the mass ratio of the cyclic γ-PGA solution to 5-aminolevulinic acid in the above-mentioned amidation grafting reaction is 1:(0.001-0.02).

[0013] Specifically, in step S4, the mass concentration of the above-mentioned alginate oligosaccharide solution is 100 g / L, the mass concentration of the above-mentioned calcium nitrate solution is 20 g / L, and the volume ratio of the alginate oligosaccharide solution to the calcium nitrate solution is (0.5~1.0):1.

[0014] More specifically, in step S4, the complexation reaction is carried out at 43°C to 47°C and pH 6.3 to 6.7 for 60 min to 90 min.

[0015] It should be noted that in step S5, the above-mentioned mixture also includes the addition of trace elements, betaine, ascorbic acid, β-carotene emulsion, and a concentrated solution rich in lysine, citric acid and γ-PGA oligopeptides obtained by concentrating the ultrafiltration permeate collected during the purification process in step S1.

[0016] Application of fertilizers prepared using the above method in crop cultivation.

[0017] Compared with the prior art, the present invention has the following advantages: First, this invention addresses the core technical problems of excessively high viscosity caused by high molecular weight polyglutamic acid in the compounding of polyglutamic acid and seaweed polysaccharides, gel precipitation of seaweed polysaccharides upon encountering divalent cations, and synergistic flocculation when the two coexist. It innovatively constructs a synergistic system combining a cyclic γ-PGA backbone with a brown algae oligosaccharide-calcium ion trap. Unlike existing technologies that use polyglutamic acid in hydrogels, drug carriers, etc., this invention introduces a cyclic structure into liquid fertilizer systems for the first time and provides a systematic design for fertilizer applications. By designing a cyclic structure for polyglutamic acid at the molecular level, the entanglement effect of linear polymer chains is fundamentally eliminated, significantly reducing the viscosity of the fertilizer system. It is important to emphasize that this invention does not simply apply known methods for synthesizing cyclic polyamino acids. Instead, it addresses the high salt and high concentration characteristics of fertilizer systems by combining simultaneous lysine end-group modification with a thiol-acetylene photoclick reaction process, achieving efficient construction and stability control of the cyclic structure. This unique preparation route allows cyclic γ-PGA to maintain its structural integrity even in the presence of a large number of elements, solving the industry problem of the difficulty in stably adding high concentrations of polyglutamic acid to liquid fertilizers. Simultaneously, this invention utilizes the pre-complexation of fucoidan with calcium ions to form a calcium ion trap, effectively shielding the cross-linking effect of free calcium ions on seaweed polysaccharides. Compared to the conventional approach of simply adding chelating agents, this invention converts calcium ions into stable oligosaccharide-calcium complexes through pre-complexation, avoiding the attack of calcium ions on seaweed polysaccharides and making the complexed product itself a bioactive synergistic component.

[0018] Secondly, this invention, through the design of a cyclic γ-PGA framework linked by thioether bonds, endows the fertilizer system with excellent storage stability. Compared with the cyclic structure constructed using disulfide bonds, thioether bonds do not have a dynamic exchange equilibrium in solution, exhibiting higher chemical stability and ensuring the integrity of the cyclic structure during long-term storage. This characteristic stems from the photoclick chemistry strategy employed in the molecular ring-closure step of this invention, namely, initiating a thiol-alkyne addition reaction under 365nm ultraviolet light to form stable thioether bonds under mild conditions, avoiding reversible exchange and side reactions that may occur during the formation of traditional disulfide bonds. Research in this invention shows that the control standard using disulfide bond ring closure exhibits significant flocculation and stratification during storage, while the thioether-linked cyclic structure of this invention remains transparent and free of precipitation even after 180 days of storage at 4°C and 14 days of accelerated storage at 54°C, with a polyglutamic acid retention rate exceeding 92%. The stability of the cyclic structure provides a reliable molecular basis for the functionalization modification of 5-aminolevulinic acid and the effective utilization of calcium ion traps, achieving performance maintenance of the fertilizer product throughout its entire lifecycle from preparation to application.

[0019] Third, this invention covalently grafts 5-aminolevulinic acid (5-aminolevulinic acid) onto the cyclic γ-PGA backbone, supplemented by a synergistic photostable system of ascorbic acid and β-carotene, thus overcoming the inherent defect of 5-aminolevulinic acid's susceptibility to photodegradation in liquid fertilizers as a photosynthesis promoter. Unlike existing technologies that rely on simple physical mixing or encapsulation, this invention utilizes an EDC / NHS-mediated amidation reaction to covalently graft 5-aminolevulinic acid onto the side-chain carboxyl groups of the cyclic γ-PGA, forming a stable chemical link. This design not only avoids precipitation and degradation due to poor compatibility in physical mixing systems, but more importantly, the cyclic backbone provides steric hindrance protection for 5-aminolevulinic acid, enabling it to resist adverse environmental influences while promoting photosynthesis. Building upon this foundation, this invention further introduces ascorbic acid and β-carotene to construct a synergistic photostable system. Ascorbic acid's antioxidant effect scavenge photoinduced free radicals, while β-carotene's light-shielding effect absorbs and attenuates incident light energy. Together, they provide multiple layers of protection for 5-aminolevulinic acid. Photostable tests of this invention show that fertilizer samples combining covalent grafting and photostable systems retain over 90% of 5-aminolevulinic acid after 7 days of natural light exposure, while control samples lacking only one of these protections show significant degradation. This demonstrates that both covalent grafting and the photostable agent are indispensable. This technical solution enables 5-aminolevulinic acid to remain stably present in fertilizer products and continuously promote photosynthesis.

[0020] Fourth, this invention utilizes fucoidan derived from natural seaweed to construct a calcium ion trap. While achieving highly efficient resistance to calcium ion precipitation, it avoids the drawbacks of traditional synthetic chelating agents (such as EDTA) such as poor biodegradability and high environmental residue risks. Unlike existing technologies that simply add chelating agents as additives, this invention achieves controllable pre-complexation of the fucoidan-calcium complex by precisely controlling the molar ratio of fucoidan to calcium ions and the reaction conditions. This preparation process ensures efficient calcium ion complexation while preserving the active structure of the fucoidan. The fucoidan-calcium complex not only effectively complexes free calcium ions in the system, protecting seaweed polysaccharides from cross-linking precipitation, but also possesses the bioactivity of oligosaccharides, serving as a synergistic component in crop growth regulation. Anti-calcium ion precipitation tests of this invention show that fertilizer samples using fucoidan-calcium exhibit a turbidity increase of no more than 26 NTU after the addition of calcium ions, demonstrating calcium resistance comparable to EDTA, but with significant advantages in environmental friendliness and functional versatility, reflecting the design philosophy of green chemistry. In addition, as a natural product, the degradation products of fucoidan are environmentally friendly and meet the requirements of modern agriculture for green and sustainable development.

[0021] Fifth, this invention achieves resource utilization of by-products through complete process design, forming a green closed loop from raw materials to products. In the preparation of the linear γ-PGA precursor in step S1, the permeate collected by ultrafiltration is rich in lysine, citric acid, and γ-PGA oligopeptides. Unlike conventional processes that treat the permeate as waste, this invention uses vacuum concentration to convert these by-products into a synergistic concentrate rich in amino acids, organic acids, and small molecule peptides, which is then reused in the fertilizer compounding process in step S5. This design not only reduces waste emissions and environmental impact but also increases the product's amino acid biostimulant content. Lysine, as an amino acid biostimulant, chelates trace elements and promotes protein synthesis; citric acid, as a natural chelating agent, enhances the stability of trace elements; and γ-PGA oligopeptides, as small molecule peptides, can be directly absorbed and utilized by crops. Fertilizer samples using this invention's technical solution are significantly superior to conventional fertilizers and control products in promoting the growth of Chinese cabbage, increasing chlorophyll content, and improving vitamin C and soluble sugar content. Under drought stress, the plants treated with the fertilizer of this invention were still able to maintain a high relative leaf water content and growth potential, and the accumulation of free proline was significantly increased, proving that the water-retention and efficiency-enhancing effects of the cyclic γ-PGA skeleton and the photosynthetic-promoting function of 5-aminolevulinic acid were fully utilized under adverse conditions.

[0022] In summary, this invention systematically solves a series of technical problems, such as high viscosity, easy gelation, and instability, that exist when polyglutamic acid and seaweed polysaccharides are compounded in liquid fertilizers through a multi-synergistic design involving the construction of a cyclic γ-PGA framework, covalent grafting of 5-aminolevulinic acid, a brown algae oligosaccharide-calcium ion trap, and a photostable protection system. The prepared aqueous liquid fertilizer containing polyglutamic acid and seaweed polysaccharides to enhance macronutrients exhibits excellent physicochemical properties, good storage stability, significant application effects, and environmentally friendly characteristics, showing broad application prospects in facility agriculture and fertigation. Detailed Implementation

[0023] Various exemplary embodiments of the present invention will now be described in detail. This detailed description should not be considered as a limitation of the present invention, but rather as a more detailed description of certain aspects, features, and embodiments of the present invention.

[0024] It should be understood that the terminology used in this invention is merely for describing particular embodiments and is not intended to limit the invention. Furthermore, with respect to numerical ranges in this invention, it should be understood that each intermediate value between the upper and lower limits of the range is also specifically disclosed. Any stated value or intermediate value within a stated range, as well as each smaller range between any other stated value or intermediate value within said range, is also included in this invention. The upper and lower limits of these smaller ranges may be independently included or excluded from the range.

[0025] Unless otherwise stated, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art. While only preferred methods and materials have been described herein, any methods and materials similar or equivalent to those described herein may be used in the implementation or testing of this invention. All references to this specification are incorporated by way of citation to disclose and describe methods and / or materials associated with those references. In the event of any conflict with any incorporated reference, the content of this specification shall prevail.

[0026] Various modifications and variations can be made to the specific embodiments described in this specification without departing from the scope or spirit of the invention, as will be apparent to those skilled in the art. Other embodiments derived from this specification will also be apparent to those skilled in the art. This specification and embodiments are merely exemplary.

[0027] The terms “include,” “including,” “have,” “contain,” etc., used in this article are all open-ended terms, meaning that they include but are not limited to.

[0028] Unless otherwise specified in the examples, conventional conditions can be followed. Reagents or instruments whose manufacturers are not specified or otherwise described are all commercially available products. Specifically, γ-PGA has a weight-average molecular weight of 1000 kDa and a purity of 99%; the fucoidan content in the seaweed polysaccharide is 50%; the water-soluble fertilizer for trace elements contains the following mass ratios: Fe 4%, Zn 5%, Cu 0.5%, Mn 2%, B 2%, and Mo 0.1%; the β-carotene emulsion is a water-dispersible emulsion, with the active ingredient β-carotene accounting for 1% of the total emulsion mass.

[0029] It should be noted that although the alkynyl-succinimide ester used is a commercially available fine chemical, its dosage is extremely low, based on a stoichiometric design for end-group modification. After the reaction, it is completely removed by ultrafiltration, leaving no residue in the final product. During the research and development phase, the cost of this reagent is controllable; in industrial production, its cost can be controlled through bulk purchasing, domestic substitution, and process optimization. Compared with reagents that achieve equivalent functions, alkynyl-succinimide ester has advantages such as convenient procurement, rapid reaction, and simple post-processing, with controllable overall cost, making it suitable for industrial applications.

[0030] N-hydroxysuccinimide is abbreviated as NHS; 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride is abbreviated as EDC; 5-aminolevulinic acid is abbreviated as 5-ALA.

[0031] Example 1 In this embodiment, an aqueous solution fertilizer containing polyglutamic acid and seaweed polysaccharides to enhance the effects of macronutrients was prepared. The specific preparation process is as follows: S1. Preparation of linear γ-PGA precursor: Dissolve γ-PGA in deionized water to prepare a basic γ-PGA solution with a mass concentration of 50 g / L. Dissolve citric acid monohydrate in deionized water to prepare a citric acid solution with a mass concentration of 200 g / L. L-lysine was dissolved in deionized water to prepare a lysine solution with a mass concentration of 100 g / L. At 70℃, the mixture was stirred at 100r / min, and 18 parts of citric acid solution were added to 100 parts of γ-PGA basic solution while stirring. The pH of the system was controlled at 3.2. After the citric acid solution was added, the temperature and stirring speed were maintained to carry out the reaction. The 1.2 parts of lysine solution were divided into three equal parts, which were added at the beginning of the reaction after the citric acid solution was added, at 2 hours of the reaction, and at 4 hours of the reaction, respectively, in order to maintain the free lysine in the system in an excess state at all times. The total reaction time was controlled at 6 hours. The intrinsic viscosity of the solution was measured every 30 minutes at the start of the reaction and during the reaction. When the intrinsic viscosity dropped to 45.6% of the initial value, the reaction system was immediately cooled to 4°C in an ice bath, and the pH was adjusted to 7.0 with a 10% sodium hydroxide solution to terminate the reaction. After the reaction was completed, the reaction solution was transferred to an ultrafiltration system with a molecular weight cutoff of 3 kDa and concentrated by ultrafiltration at a pressure of 0.15 MPa. At the same time, deionized water was used as the replacement fluid for washing until no lysine and citric acid were detected in the permeate. The retentate was a purified lysine end-modified linear γ-PGA solution, which is the linear precursor solution. The permeate was concentrated to 1 / 7 of its original volume under reduced pressure to obtain a concentrate rich in lysine, citric acid, and γ-PGA oligopeptides.

[0032] S2, Construction of the cyclic γ-PGA framework: S2.1, Terminal functionalization of linear precursors (introduction of an alkynyl group at one end) Take 10 portions of the linear precursor solution prepared by S1, add 0.1 mol / L, pH 8.0 phosphate buffer to a total mass of 20 portions, and prepare the reaction solution. Under conditions of 25℃, protection from light, and stirring at 100 r / min, 0.2 parts of alkynyl-succinimide ester were dissolved in 2 parts of dimethyl sulfoxide to obtain an alkynyl ester solution. The acetylacetic acid ester solution was slowly added to the reaction solution, the pH was adjusted to 8.0, and the reaction was stirred continuously at 25°C for 1.5 h. After the reaction was completed, the reaction solution was transferred to a tangential flow ultrafiltration system with a molecular weight cutoff of 3 kDa. The system was washed with deionized water as the replacement fluid at a pressure of 0.15 MPa. The washing volume was 7 times the initial volume of the reaction solution until no alkynyl-NHS ester and its hydrolysis products were detected in the permeate. After ultrafiltration, the retentate is an alkyne-modified linear γ-PGA solution (one end retains the lysine terminal group, and the other end introduces the alkyne group through alkyne-succinimide ester).

[0033] S2.2, Terminal functionalization of linear precursors (introduction of cysteine ​​to the lysine terminal) Take 10 portions of alkyne-modified linear γ-PGA solution and add 0.1 mol / L, pH 5.8 MES buffer to a total mass of 20 portions to prepare the reaction solution; Under conditions of 25°C, protection from light, and stirring at 100 r / min, 0.2 parts of NHS and 0.3 parts of EDC were added to the above reaction solution while stirring, and the activation reaction was carried out for 30 min. Take 0.1 to 0.3 parts of L-cysteine, dissolve it in 2 parts of the above MES buffer, and adjust the pH to 5.5 to obtain a cysteine ​​solution. The cysteine ​​solution was slowly added to the activated reaction system, and the reaction was stirred continuously for 4.0 h. After the reaction was completed, the reaction solution was transferred to an ultrafiltration system with a molecular weight cutoff of 3 kDa. The system was washed with deionized water as the replacement fluid at a pressure of 0.15 MPa. The washing volume was 6 times the initial volume of the reaction solution until no cysteine ​​was detected in the permeate. After ultrafiltration, the retentate is a bifunctional linear γ-PGA solution with an alkyne group at one end and a mercapto group at the other end.

[0034] S2.3, Intramolecular cyclization (thiol-acetylene photoclick reaction): Take 10 parts of the bifunctionalized linear γ-PGA solution and dilute it with 6500 parts of deionized water to obtain the cyclization reaction solution; Under conditions of 25°C and protection from light, the ring-closing reaction solution was slowly added to an empty ring-closing reaction vessel. When the liquid level covered the stirring shaft, stirring was started and maintained at 100 r / min. The reaction solution was then irradiated with a 365 nm ultraviolet lamp with a light intensity of 8 mW / cm². 2 Continue stirring and irradiating the reaction for 3 hours; After the reaction was completed, the reaction solution was transferred to an ultrafiltration system with a molecular weight cutoff of 3 kDa and concentrated to 1 / 80 of the original reaction solution volume under a pressure of 0.15 MPa. At the same time, deionized water was used as the replacement fluid for washing and filtration. The volume of deionized water used for washing and filtration was 7 times the volume of the concentrated solution to remove any possible photoinitiated byproducts. After ultrafiltration, the retentate is a cyclic γ-PGA solution linked by thioether bonds.

[0035] S3, functionalization of cyclic γ-PGA: Take 50 portions of cyclic γ-PGA solution and add MES buffer to a total mass of 100 portions to prepare the functionalized modification reaction solution; Under light-protected conditions at 25°C, the mixture was stirred at 100 rpm while adding 1.0 part NHS and 0.15 part EDC, and the activation reaction was carried out for 30 min. Take 0.08 parts of 5-ALA, dissolve it in 18 parts of the above MES buffer, adjust the pH to 5.5, and obtain a 5-ALA solution; Under light-protected conditions at 25°C, the 5-ALA solution was slowly added to the activated reaction system, and the mixture was stirred at 100 r / min for 6 h. After the reaction was completed, the reaction solution was transferred to an ultrafiltration system with a molecular weight cutoff of 3 kDa and concentrated by ultrafiltration at a pressure of 0.15 MPa. At the same time, deionized water was used as the replacement fluid for washing. The volume of deionized water used for washing was 7 times the volume of the concentrated solution to remove unreacted 5-ALA, activator and byproducts. After ultrafiltration, the retentate is a 5-ALA-grafted cyclic γ-PGA solution, and the concentration is adjusted to 40 g / L.

[0036] S4. Preparation of the brown algae oligosaccharide-calcium complex: Take the alginate oligosaccharide and prepare a 100 g / L alginate oligosaccharide solution with deionized water; Take calcium nitrate tetrahydrate and prepare a calcium nitrate solution with a mass concentration of 20 g / L using deionized water; At 45℃, the mixture was stirred at 100 r / min, and the calcium nitrate solution was slowly added to the alginate oligosaccharide solution while stirring. The volume ratio of calcium nitrate solution to alginate oligosaccharide solution was 0.8:1. The mixture was stirred and reacted for 75 min, and the pH of the system was maintained at 6.5 during the reaction. After the reaction was completed, the concentration of free calcium was determined by selective electrode method to confirm that the free calcium was not higher than 5% of the initial total calcium, that is, the complexation efficiency should be higher than 95%. In this embodiment, the complexation efficiency was 97.3%. The reaction solution was transferred to a rotary evaporator and concentrated under reduced pressure at 55°C to obtain a brown algae oligosaccharide-calcium complex with a mass concentration of 150 g / L. The complex was then sealed and stored at 4°C for later use.

[0037] S5, Compound formulation of liquid fertilizer: S5.1, Addition of synergistic components: Add 100 parts of deionized water to a compounding vessel, control the rotation speed at 100 r / min and the temperature at 28℃, add 7.5 parts of betaine and 0.75 parts of ascorbic acid, stir until completely dissolved, adjust the pH of the solution to 6.5, and obtain the betaine basic solution.

[0038] Take 200 parts of 5-ALA-grafted cyclic γ-PGA solution and slowly add it to the above compounding vessel while stirring. After the addition is complete, continue stirring for 10 minutes. Take 7.5 parts of seaweed polysaccharide, pre-dissolve it in 40℃ warm water to prepare a seaweed polysaccharide solution with a mass concentration of 100g / L, and slowly add the seaweed polysaccharide solution into the above compounding kettle while stirring. After the addition is complete, continue stirring for 10 minutes. Take 105 parts of the brown algae oligosaccharide-calcium complex and slowly add it to the compounding vessel while stirring. After the addition is complete, continue stirring for 10 minutes. Take 7.5 portions of the concentrated solution rich in lysine, citric acid and γ-PGA oligopeptides prepared in step S1, and slowly add it to the compounding vessel while stirring. After the addition is complete, continue stirring for 10 minutes.

[0039] S5.2 Preparation and addition of basic nutrient concentrate solution: Add 225 parts of deionized water to the mixing tank and heat to 45°C; Weigh the solid raw materials according to the following formula: 190 parts urea, 230 parts potassium dihydrogen phosphate, 150 parts potassium nitrate, and 3.5 parts water-soluble fertilizer for trace elements. Under stirring at 100 r / min, the above solid raw materials were added sequentially, the pH was adjusted to 6.5, and stirring was continued for 15 min to obtain a basic nutrient concentrated solution. The above-mentioned basic nutrient concentrated solution was slowly added to the compounding vessel at 100 r / min, and stirring was continued for 15 min after the addition was completed.

[0040] S5.3 Homogenization and Volume Adjustment: Turn on the homogenizer and homogenize at 2250 r / min for 7.5 min. Add 0.2 parts of β-carotene emulsion and continue homogenizing for 2 min to ensure that the components are fully dispersed and the system is homogeneous and stable. Add deionized water to bring the total volume to 1000 parts, and continue stirring at 100 r / min for 15 min; The sample was passed through a 200-mesh sieve to remove any possible undissolved particles. It was then dispensed, sealed, and used to obtain an aqueous fertilizer containing polyglutamic acid and seaweed polysaccharide-enhanced macronutrients. This fertilizer was designated as fertilizer sample 1 and stored in a cool, dark place.

[0041] Example 2 In this embodiment, an aqueous solution fertilizer containing polyglutamic acid and seaweed polysaccharides to enhance the effects of macronutrients was prepared. The specific preparation process is as follows: S1. Preparation of linear γ-PGA precursor: Dissolve γ-PGA in deionized water to prepare a basic γ-PGA solution with a mass concentration of 50 g / L. Dissolve citric acid monohydrate in deionized water to prepare a citric acid solution with a mass concentration of 250 g / L. L-lysine was dissolved in deionized water to prepare a lysine solution with a mass concentration of 80 g / L. At 75℃, the mixture was stirred at 120 r / min, and 25 parts of citric acid solution were added to 100 parts of γ-PGA basic solution while stirring. The pH of the system was controlled at 3.5. After the citric acid solution was added, the temperature and stirring speed were maintained to carry out the reaction. Divide 1.8 parts of lysine solution into three equal parts, and add them respectively at the beginning of the reaction after the citric acid solution is added, at 2 hours of reaction, and at 4 hours of reaction. The total reaction time was controlled at 5 hours. During the reaction, the intrinsic viscosity of the solution was measured every 30 minutes. When the intrinsic viscosity dropped to 40.2% of the initial value, the reaction system was immediately cooled to 4°C in an ice bath, and the pH was adjusted to 6.8 with a 10% sodium hydroxide solution to terminate the reaction. After the reaction was completed, the reaction solution was transferred to an ultrafiltration system with a molecular weight cutoff of 3 kDa and concentrated by ultrafiltration at a pressure of 0.2 MPa. At the same time, deionized water was used as the replacement fluid for washing until no lysine and citric acid were detected in the permeate. The retentate was a purified lysine end-modified linear γ-PGA solution, which is the linear precursor solution. The permeate was concentrated to 1 / 5 of its original volume under reduced pressure to obtain a concentrated solution rich in lysine, citric acid, and γ-PGA oligopeptides.

[0042] S2, Construction of the cyclic γ-PGA framework: S2.1, Terminal functionalization of linear precursors (introduction of an alkynyl group at one end): Take 10 portions of the linear precursor solution prepared by S1, add 0.1 mol / L, pH 8.5 phosphate buffer to a total mass of 20 portions, and prepare the reaction solution. Under conditions of 25℃, protection from light, and stirring at 120 r / min, 0.3 parts of alkynyl-succinimide ester were dissolved in 3 parts of dimethyl sulfoxide to obtain an alkynyl ester solution. The acetylacetic acid ester solution was slowly added to the reaction solution, the pH was adjusted to 8.5, and the reaction was stirred continuously at 25°C for 1 hour. After the reaction was completed, the reaction solution was transferred to a tangential flow ultrafiltration system with a molecular weight cutoff of 3 kDa. The system was washed with deionized water as the replacement fluid at a pressure of 0.2 MPa. The washing volume was 8 times the initial volume of the reaction solution until no alkynyl-NHS ester and its hydrolysis products could be detected in the permeate. After ultrafiltration, the retentate is an alkyne-modified linear γ-PGA solution.

[0043] S2.2, Terminal functionalization of linear precursors (introduction of cysteine ​​to the lysine terminal): Take 10 portions of alkyne-modified linear γ-PGA solution and add 0.1 mol / L, pH 6.0 MES buffer to a total mass of 20 portions to prepare the reaction solution; Under conditions of 25°C, in the dark, and with stirring at 120 r / min, 0.3 parts of NHS and 0.4 parts of EDC were added to the above reaction solution while stirring, and the activation reaction was carried out for 20 min. Take 0.3 parts of L-cysteine, dissolve it in 3 parts of the above MES buffer, adjust the pH to 5.7, and obtain a cysteine ​​solution; The cysteine ​​solution was slowly added to the activated reaction system, and the reaction was stirred continuously for 3.5 hours. After the reaction was completed, the reaction solution was transferred to an ultrafiltration system with a molecular weight cutoff of 3 kDa. The system was washed with deionized water as the replacement fluid at a pressure of 0.2 MPa. The washing volume was 8 times the initial volume of the reaction solution until no cysteine ​​was detected in the permeate. After ultrafiltration, the retentate is a bifunctional linear γ-PGA solution with an alkyne group at one end and a mercapto group at the other end.

[0044] S2.3, Intramolecular cyclization (thiol-acetylene photoclick reaction): Take 10 parts of the bifunctionalized linear γ-PGA solution and dilute it with 5000 parts of deionized water to obtain the cyclization reaction solution; Under conditions of 25°C and protection from light, the ring-closing reaction solution was slowly added to an empty ring-closing reaction vessel. When the liquid level covered the stirring shaft, stirring was started and maintained at 120 r / min. The reaction solution was then irradiated with a 365 nm ultraviolet lamp with a light intensity of 10 mW / cm². 2 Continue stirring and irradiating the reaction for 2 hours; After the reaction was completed, the reaction solution was transferred to an ultrafiltration system with a molecular weight cutoff of 3 kDa and concentrated to 1 / 50 of the original reaction solution volume under a pressure of 0.2 MPa. At the same time, deionized water was used as the replacement fluid for washing and filtration. The volume of deionized water used for washing and filtration was 8 times the volume of the concentrated solution to remove any possible photoinitiated byproducts. After ultrafiltration, the retentate is a cyclic γ-PGA solution linked by thioether bonds.

[0045] S3, functionalization of cyclic γ-PGA: Take 50 portions of cyclic γ-PGA solution and add MES buffer to a total mass of 100 portions to prepare the functionalized modification reaction solution; Under light-protected conditions at 25°C, the mixture was stirred at 120 rpm while adding 1.5 parts NHS and 0.2 parts EDC, and the activation reaction was carried out for 20 min. Take 0.1 part of 5-ALA, dissolve it in 25 parts of the above MES buffer, adjust the pH to 5.7, and obtain a 5-ALA solution; Under light-protected conditions at 25°C, the 5-ALA solution was slowly added to the activated reaction system, and the mixture was stirred at 120 r / min for 5 h. After the reaction was completed, the reaction solution was transferred to an ultrafiltration system with a molecular weight cutoff of 3 kDa and concentrated by ultrafiltration at a pressure of 0.2 MPa. At the same time, deionized water was used as the replacement fluid for washing. The volume of deionized water used for washing was 8 times the volume of the concentrated solution to remove unreacted 5-ALA, activator and byproducts. After ultrafiltration, the retentate is a 5-ALA-grafted cyclic γ-PGA solution, and the concentration is adjusted to 40 g / L.

[0046] S4. Preparation of the brown algae oligosaccharide-calcium complex: Take the alginate oligosaccharide and prepare a 100 g / L alginate oligosaccharide solution with deionized water; Take calcium nitrate tetrahydrate and prepare a calcium nitrate solution with a mass concentration of 20 g / L using deionized water; At 47℃, the mixture was stirred at 120 r / min, and the calcium nitrate solution was slowly added to the alginate oligosaccharide solution while stirring. The volume ratio of calcium nitrate solution to alginate oligosaccharide solution was 1.0:1. The mixture was stirred and reacted for 60 min, and the pH of the system was maintained at 6.7 during the reaction. After the reaction was completed, the concentration of free calcium was determined by selective electrode method to confirm that the free calcium was not higher than 5% of the initial total calcium, that is, the complexation efficiency should be higher than 95%. In this embodiment, the complexation efficiency was 96.5%. The reaction solution was transferred to a rotary evaporator and concentrated under reduced pressure at 60°C to obtain a brown algae oligosaccharide-calcium complex with a mass concentration of 150 g / L. The complex was then sealed and stored at 4°C for later use.

[0047] S5. Compounding of liquid fertilizer: Same as step S5 in Example 1, to obtain an aqueous liquid fertilizer containing polyglutamic acid and seaweed polysaccharide to enhance macro-elements, denoted as fertilizer sample 2, and stored in a cool, dark place.

[0048] Example 3 In this embodiment, an aqueous solution fertilizer containing polyglutamic acid and seaweed polysaccharides to enhance the effects of macronutrients was prepared. The specific preparation process is as follows: S1. Preparation of linear γ-PGA precursor: Dissolve γ-PGA in deionized water to prepare a basic γ-PGA solution with a mass concentration of 50 g / L. Dissolve citric acid monohydrate in deionized water to prepare a citric acid solution with a mass concentration of 150 g / L. L-lysine was dissolved in deionized water to prepare a lysine solution with a mass concentration of 120 g / L. At 65℃, the mixture was stirred at 80r / min, and 10 parts of citric acid solution were added to 100 parts of γ-PGA basic solution while stirring. The pH of the system was controlled at 3.0. After the citric acid solution was added, the temperature and stirring speed were maintained to carry out the reaction. Divide 0.6 parts of lysine solution into three equal parts, and add them respectively at the beginning of the reaction after the citric acid solution is added, at 2 hours of reaction, and at 4 hours of reaction. The total reaction time was controlled at 7 hours. During the reaction, the intrinsic viscosity of the solution was measured every 30 minutes. When the intrinsic viscosity dropped to 49.6% of the initial value, the reaction system was immediately cooled to 4°C in an ice bath, and the pH was adjusted to 7.2 with a 10% sodium hydroxide solution to terminate the reaction. After the reaction was completed, the reaction solution was transferred to an ultrafiltration system with a molecular weight cutoff of 3 kDa and concentrated by ultrafiltration at a pressure of 0.1 MPa. At the same time, deionized water was used as the replacement fluid for washing until no lysine and citric acid were detected in the permeate. The retentate was a purified lysine end-modified linear γ-PGA solution, which is the linear precursor solution. The permeate was concentrated to 1 / 10 of its original volume under reduced pressure to obtain a concentrate rich in lysine, citric acid, and γ-PGA oligopeptides.

[0049] S2, Construction of the cyclic γ-PGA framework: S2.1, Terminal functionalization of linear precursors (introduction of an alkynyl group at one end): Take 10 portions of the linear precursor solution prepared by S1, add 0.1 mol / L, pH 7.5 phosphate buffer to a total mass of 20 portions, and prepare the reaction solution. Under conditions of 25℃, protection from light, and stirring at 80 r / min, 0.1 part of alkynyl-succinimide ester was dissolved in 1 part of dimethyl sulfoxide to obtain an alkynyl ester solution; The acetylacetic acid ester solution was slowly added to the reaction solution, the pH was adjusted to 7.5, and the reaction was stirred continuously at 25°C for 2 hours. After the reaction was completed, the reaction solution was transferred to a tangential flow ultrafiltration system with a molecular weight cutoff of 3 kDa. The system was washed with deionized water as the replacement fluid at a pressure of 0.1 MPa. The washing volume was 5 times the initial volume of the reaction solution until no alkynyl-NHS ester and its hydrolysis products were detected in the permeate. After ultrafiltration, the retentate is an alkyne-modified linear γ-PGA solution.

[0050] S2.2, Terminal functionalization of linear precursors (introduction of cysteine ​​to the lysine terminal): Take 10 portions of alkyne-modified linear γ-PGA solution and add 0.1 mol / L, pH 5.5 MES buffer to a total mass of 20 portions to prepare the reaction solution; Under conditions of 25°C, in the dark, and with stirring at 80 r / min, 0.1 parts of NHS and 0.2 parts of EDC were added to the above reaction solution while stirring, and the activation reaction was carried out for 40 min. Take 0.1 part of L-cysteine, dissolve it in 1 part of the above MES buffer, adjust the pH to 5.3, and obtain a cysteine ​​solution; The cysteine ​​solution was slowly added to the activated reaction system, and the reaction was stirred continuously for 4.5 hours. After the reaction was completed, the reaction solution was transferred to an ultrafiltration system with a molecular weight cutoff of 3 kDa. The system was washed with deionized water as the replacement fluid at a pressure of 0.1 MPa. The washing volume was 5 times the initial volume of the reaction solution until no cysteine ​​was detected in the permeate. After ultrafiltration, the retentate is a bifunctional linear γ-PGA solution with an alkyne group at one end and a mercapto group at the other end.

[0051] S2.3, Intramolecular cyclization (thiol-acetylene photoclick reaction): Take 10 parts of the bifunctionalized linear γ-PGA solution and dilute it with 8000 parts of deionized water to obtain the cyclization reaction solution; Under conditions of 25°C and protection from light, the ring-closing reaction solution was slowly added to an empty ring-closing reaction vessel. When the liquid level covered the stirring shaft, stirring was started and maintained at 80 r / min. The reaction solution was then irradiated with a 365 nm ultraviolet lamp with a light intensity of 5 mW / cm². 2 The reaction was stirred and irradiated for 4 hours. After the reaction was completed, the reaction solution was transferred to an ultrafiltration system with a molecular weight cutoff of 3 kDa and concentrated to 1 / 100 of the original reaction solution volume under a pressure of 0.1 MPa. At the same time, deionized water was used as the replacement fluid for washing and filtration. The volume of deionized water used for washing and filtration was 5 times the volume of the concentrated solution to remove any possible photoinitiated byproducts. After ultrafiltration, the retentate is a cyclic γ-PGA solution linked by thioether bonds.

[0052] S3, functionalization of cyclic γ-PGA: Take 50 portions of cyclic γ-PGA solution and add MES buffer to a total mass of 100 portions to prepare the functionalized modification reaction solution; Under light-protected conditions at 25°C, the mixture was stirred at 80 rpm while adding 0.5 parts NHS and 0.1 parts EDC, and the activation reaction was carried out for 40 min. Take 0.05 parts of 5-ALA, dissolve it in 10 parts of the above MES buffer, adjust the pH to 5.3, and obtain a 5-ALA solution; Under light-protected conditions at 25°C, the 5-ALA solution was slowly added to the activated reaction system, and the mixture was stirred at 80 r / min for 7 h. After the reaction was completed, the reaction solution was transferred to an ultrafiltration system with a molecular weight cutoff of 3 kDa and concentrated by ultrafiltration at a pressure of 0.1 MPa. At the same time, deionized water was used as the replacement fluid for washing. The volume of deionized water used for washing was 5 times the volume of the concentrated solution to remove unreacted 5-ALA, activator and byproducts. After ultrafiltration, the retentate is a 5-ALA-grafted cyclic γ-PGA solution, and the concentration is adjusted to 40 g / L.

[0053] S4. Preparation of the brown algae oligosaccharide-calcium complex: Take the alginate oligosaccharide and prepare a 100 g / L alginate oligosaccharide solution with deionized water; Take calcium nitrate tetrahydrate and prepare a calcium nitrate solution with a mass concentration of 20 g / L using deionized water; At 43℃, the mixture was stirred at 80 r / min, and the calcium nitrate solution was slowly added to the alginate oligosaccharide solution while stirring. The volume ratio of calcium nitrate solution to alginate oligosaccharide solution was 0.5:1. The mixture was stirred and reacted for 90 min, and the pH of the system was maintained at 6.3 during the reaction. After the reaction was completed, the concentration of free calcium was determined by selective electrode method, confirming that the free calcium was not higher than 5% of the initial total calcium. The complexation efficiency in this embodiment was 98.4%. The reaction solution was transferred to a rotary evaporator and concentrated under reduced pressure at 50°C to obtain a brown algae oligosaccharide-calcium complex with a mass concentration of 150 g / L. The complex was then sealed and stored at 4°C for later use.

[0054] S5. Compounding of liquid fertilizer: Same as step S5 in Example 1, to obtain an aqueous liquid fertilizer containing polyglutamic acid and seaweed polysaccharide to enhance macro-elements, denoted as fertilizer sample 3, and stored in a cool, dark place.

[0055] Example 4 In this embodiment, an aqueous solution of macro-elements containing polyglutamic acid and seaweed polysaccharides is prepared. The preparation process of S1 to S4 is the same as in Example 1. The compounding process of the liquid fertilizer in S5 is as follows: S5.1, Addition of synergistic components: Add 100 parts of deionized water to a compounding vessel, control the rotation speed at 80 r / min and the temperature at 25℃, add 5 parts of betaine and 0.5 parts of ascorbic acid, stir until completely dissolved, adjust the pH of the solution to 6.3, and obtain the betaine basic solution.

[0056] Take 150 portions of 5-ALA-grafted cyclic γ-PGA solution and slowly add it to the above compounding vessel while stirring. After the addition is complete, continue stirring for 5 minutes. Take 5 portions of seaweed polysaccharide, pre-dissolve them in 40℃ warm water to prepare a seaweed polysaccharide solution with a mass concentration of 100g / L, and slowly add the seaweed polysaccharide solution into the above compounding kettle while stirring. After the addition is complete, continue stirring for 5 minutes. Take 80 parts of the brown algae oligosaccharide-calcium complex and slowly add it to the compounding vessel while stirring. After the addition is complete, continue stirring for 5 minutes. Take 5 portions of the concentrated solution rich in lysine, citric acid and γ-PGA oligopeptides prepared in step S1, add it slowly to the compounding vessel while stirring, and continue stirring for 5 minutes after the addition is complete.

[0057] S5.2 Preparation and addition of basic nutrient concentrate solution: Add 200 parts of deionized water to the mixing tank and heat to 40°C; Weigh the solid raw materials according to the following formula: 180 parts urea, 220 parts potassium dihydrogen phosphate, 140 parts potassium nitrate, and 2 parts water-soluble fertilizer for trace elements. Under stirring at 80 r / min, the above solid raw materials were added sequentially, the pH was adjusted to 6.3, and stirring was continued for 20 min to obtain a basic nutrient concentrated solution; The above-mentioned basic nutrient concentrated solution was slowly added to the compounding vessel at 80 r / min, and stirring was continued for 20 min after the addition was completed.

[0058] S5.3 Homogenization and Volume Adjustment: Turn on the homogenizer and homogenize at 2000 r / min for 10 min. Add 0.1 part of β-carotene emulsion and continue homogenizing for 3 min to ensure that the components are fully dispersed and the system is homogeneous and stable. Add deionized water to bring the total volume to 1000 parts, and continue stirring at 80 r / min for 20 min; The sample was passed through a 200-mesh sieve to remove any possible undissolved particles. It was then packaged, sealed, and a liquid fertilizer containing polyglutamic acid and seaweed polysaccharide-enhanced macronutrients was obtained. This sample was designated as fertilizer sample 4 and stored in a cool, dark place.

[0059] Example 5 In this embodiment, an aqueous solution of macro-elements containing polyglutamic acid and seaweed polysaccharides is prepared. The preparation process of S1 to S4 is the same as in Example 1. The compounding process of the liquid fertilizer in S5 is as follows: S5.1, Addition of synergistic components: Add 100 parts of deionized water to a compounding vessel, control the rotation speed at 120 r / min and the temperature at 30℃, add 10 parts of betaine and 1.0 part of ascorbic acid, stir until completely dissolved, adjust the pH of the solution to 6.7, and obtain the betaine basic solution.

[0060] Take 250 portions of 5-ALA-grafted cyclic γ-PGA solution and slowly add it to the above compounding vessel while stirring. After the addition is complete, continue stirring for 15 minutes. Take 10 parts of seaweed polysaccharide, pre-dissolve it in 40℃ warm water to prepare a seaweed polysaccharide solution with a mass concentration of 100g / L, and slowly add the seaweed polysaccharide solution into the above compounding kettle while stirring. After the addition is complete, continue stirring for 15 minutes. Take 130 parts of the brown algae oligosaccharide-calcium complex and slowly add it to the compounding vessel while stirring. After the addition is complete, continue stirring for 15 minutes. Take 10 portions of the concentrated solution rich in lysine, citric acid and γ-PGA oligopeptides prepared in step S1, and slowly add it to the compounding vessel while stirring. After the addition is complete, continue stirring for 15 minutes.

[0061] S5.2 Preparation and addition of basic nutrient concentrate solution: Add 250 parts of deionized water to the mixing tank and heat to 50°C; Weigh out the solid raw materials according to the following formula: 200 parts urea, 240 parts potassium dihydrogen phosphate, 160 parts potassium nitrate, and 5 parts water-soluble fertilizer for trace elements. Under stirring at 120 r / min, the above solid raw materials were added sequentially, the pH was adjusted to 6.7, and stirring was continued for 10 min to obtain a basic nutrient concentrated solution. The above-mentioned basic nutrient concentrated solution was slowly added to the compounding vessel at 120 r / min, and stirring was continued for 10 min after the addition was completed.

[0062] S5.3 Homogenization and Volume Adjustment: Turn on the homogenizer and homogenize at 2500 r / min for 5 min. Add 0.3 parts of β-carotene emulsion and continue homogenizing for 1 min to ensure that the components are fully dispersed and the system is homogeneous and stable. Add deionized water to bring the total volume to 1000 parts, and continue stirring at 120 r / min for 10 min; The sample was passed through a 200-mesh sieve to remove any possible undissolved particles. It was then packaged, sealed, and a liquid fertilizer containing polyglutamic acid and seaweed polysaccharide-enhanced macronutrients was obtained. This sample was designated as fertilizer sample 5 and stored in a cool, dark place.

[0063] Comparative Example 1 The comparative preparation of the macro-element aqueous solution fertilizer for this example is carried out in the same manner as in Example 1, except that: The intramolecular cyclization step S2.3 is omitted. The bifunctionalized linear γ-PGA solution prepared in S2.2 is directly used in step S3. Subsequent steps are the same as in Example 1, resulting in a 5-ALA-grafted γ-PGA solution with a linear structure. Subsequent steps are the same as in S4 and S5 of Example 1. The resulting fertilizer is designated as fertilizer control 1.

[0064] Comparative Example 2 The comparative example preparation of the macro-element aqueous solution fertilizer is carried out in the same manner as in Example 1, except that in step S2, air oxidation of disulfide bonds is used instead of the ultraviolet light-induced mercapto-alkyne click reaction. The specific process of step S2 is as follows: S2.1, Diterminal functionalization of linear precursors: Take 10 portions of the linear precursor solution prepared by S1, add 0.1 mol / L, pH 5.5 MES buffer to a total mass of 20 portions, and prepare the reaction solution. Under conditions of 25°C, protection from light, and stirring at 100 r / min, 0.4 parts of NHS and 0.6 parts of EDC were added to the above reaction solution while stirring, and the activation reaction was carried out for 30 min. Take 0.4 parts of L-cysteine, dissolve it in 4 parts of the above MES buffer, adjust the pH to 5.5, and obtain a cysteine ​​solution. The cysteine ​​solution was slowly added to the activated reaction system, and the reaction was stirred continuously for 4 hours. After the reaction was completed, the reaction solution was transferred to an ultrafiltration system with a molecular weight cutoff of 3 kDa and washed with deionized water at a pressure of 0.15 MPa until no cysteine ​​was detected in the permeate. After ultrafiltration, the retentate is a linear γ-PGA solution modified with cysteine ​​at both ends.

[0065] S2.2, Intramolecular ring closure (disulfide bond formation): Take 10 parts of the linear γ-PGA solution modified with 10 cysteine ​​ends and dilute it with 6500 parts of deionized water to obtain the cyclization reaction solution. At 25°C and in the dark, the ring-closing reaction solution was slowly added to an empty ring-closing reaction vessel. When the liquid level was above the stirring shaft, the stirring was turned on and the reaction was carried out at 100 r / min for 24 h. After the reaction was completed, the reaction solution was transferred to an ultrafiltration system with a molecular weight cutoff of 3 kDa. The solution was concentrated to 1 / 80 of the original reaction solution volume under a pressure of 0.15 MPa. At the same time, deionized water was used as the replacement fluid for washing and filtration. The volume of deionized water used for washing was 7 times the volume of the concentrated solution to remove any possible oxidation byproducts. After ultrafiltration, the retentate was the control cyclic γ-PGA solution.

[0066] The subsequent steps S3 to S4 are the same as those in Example 1, except that an equal mass of the comparative cyclic γ-PGA solution is used instead of the cyclic γ-PGA solution used in Example 1. The resulting fertilizer is designated as fertilizer control 2.

[0067] Comparative Example 3 The comparative preparation of the macro-element aqueous solution fertilizer for this example is carried out in the same manner as in Example 1, except that: In step S3, the 5-ALA grafting modification process is omitted, and the unmodified cyclic γ-PGA solution is directly used for subsequent compounding. That is, in this comparative example, step S3 only adjusts the concentration of the cyclic γ-PGA solution prepared in step S2 to 40 g / L. Step S4 is the same as S4 in Example 1. In step S5, 200 parts of the cyclic γ-PGA solution with adjusted concentration are used instead of the 5-ALA grafted cyclic γ-PGA solution, and 0.4 parts of 5-ALA are added to make its concentration in the final fertilizer equivalent to that in Example 1. The resulting fertilizer is recorded as fertilizer control 3.

[0068] Comparative Example 4 This comparative example prepares a macro-element aqueous solution fertilizer, and the specific implementation method is the same as in Example 1. The difference is that the preparation process of the brown algae oligosaccharide-calcium complex is omitted in step S4, and instead, an EDTA-calcium solution is prepared. The specific operation is as follows: EDTA-2Na was dissolved in deionized water to prepare an EDTA solution with a mass concentration of 100 g / L. Calcium nitrate tetrahydrate was prepared to prepare a calcium nitrate solution with a mass concentration of 20 g / L. The two solutions were mixed at a molar ratio of 1:1 for EDTA and calcium ions and reacted at 45 °C and pH 6.5 for 60 min. The reaction solution was then transferred to a rotary evaporator and concentrated under reduced pressure at 55 °C to obtain an EDTA-calcium complex solution with a mass concentration of 150 g / L. The subsequent step S5 is the same as in Example 1, except that an equal mass of EDTA-calcium solution is added instead of the concentrated brown algae oligosaccharide-calcium complex solution, and the resulting fertilizer is designated as fertilizer control 4.

[0069] Comparative Example 5 The comparative preparation of the macro-element aqueous solution fertilizer was carried out in the same manner as in Example 1, except that the addition of ascorbic acid and β-carotene emulsion was omitted in step S5 and replaced with an equal amount of deionized water; other operations were the same as in Example 1, and the resulting fertilizer was designated as fertilizer control 5.

[0070] Analysis and Testing To verify the technical effect of the present invention and to compare it with the comparative scheme, the obtained fertilizer samples 1-5 and fertilizer control products 1-5 were systematically analyzed and tested.

[0071] In accordance with the relevant provisions of T / CPFIA 0013-2024 "Water-soluble Fertilizers Containing Polyglutamic Acid", NY / T 3039-2016 "Determination of Polyglutamic Acid Content in Water-soluble Fertilizers" and NY / T 1107-2020 "Water-soluble Fertilizers Containing Macro-elements", fertilizer samples and reference standards were systematically analyzed and tested.

[0072] I. Key Quality Indicator Testing and Analysis Polyglutamic acid is the core synergistic ingredient of this invention, and its content directly affects the fertilizer's effectiveness. Following the method specified in NY / T3039-2016, the polyglutamic acid content of each fertilizer sample and control was determined, and the results are shown in Table 1.

[0073] The water-insoluble matter content of each fertilizer sample and the reference standard was determined according to the method (gravimetric method) specified in Appendix of T / CPFIA 0013-2024; the pH value of each fertilizer sample and the reference standard was directly measured (25℃) using a pH meter, and the results are shown in Table 1.

[0074] Table 1: Test Results of Key Quality Indicators for Fertilizer Samples and Controls As shown in Table 1, the polyglutamic acid content of fertilizer samples 1-5 of this invention is significantly higher than the standard requirements. Specifically, the polyglutamic acid content of fertilizer samples 1-3 is 7.0 g / L to 7.5 g / L. Fertilizer sample 4 has the lowest content (5.2 g / L) due to the lowest amount of 5-ALA-grafted cyclic γ-PGA solution added in step S5, while fertilizer sample 5 has the highest content (8.6 g / L) due to the highest amount added. The measured results are consistent with the feeding design.

[0075] The polyglutamic acid (PCA) content of fertilizer reference standards 1-3 was significantly lower than that of samples 1-3 of the present invention. Among them, fertilizer reference standard 1, lacking the ring-closing step in S2.3 and thus lacking a ring structure, had the lowest PCA content. Fertilizer reference standard 2, using disulfide bond ring closure instead of thioether bond ring closure, had a slightly higher content than reference standard 1, but still lower than the samples of the present invention. Fertilizer reference standard 3, using 5-ALA physical mixing instead of covalent grafting, also had a significantly lower content than the samples of the present invention. These results indicate that the ring structure, thioether bond ring closure, and 5-ALA covalent grafting all play important roles in maintaining the stability and effective content of PCA. Reference standard 4, using EDTA-calcium instead of fucoidan-calcium, had a PCA content comparable to the samples of the present invention, but EDTA has poor biodegradability. Reference standard 5, omitting ascorbic acid and β-carotene emulsion in step S5, had a slightly lower PCA content than the samples of the present invention, indicating that the light stabilizer also has a certain protective effect on PCA.

[0076] Regarding water-insoluble matter, the fertilizer samples 1-5 of this invention all had a content below 1.5 g / L, far below the standard requirement, proving that the filtration process using a 200-mesh sieve employed in this invention effectively removed any potentially undissolved particles, improving the purity of the fertilizer. The causes of water-insoluble matter mainly include trace impurities introduced from the raw materials, byproducts formed during the reaction process, and precipitates that may form during storage. The extremely low water-insoluble matter content in the samples of this invention indicates that the ultrafiltration purification process at each step can effectively remove small molecule impurities and byproducts. Furthermore, the unique ring structure design of this invention fundamentally ensures the stability of the fertilizer samples during production, testing, and storage, preventing precipitation.

[0077] The water-insoluble matter content of fertilizer reference standards 1, 2, and 3 was significantly higher than that of the fertilizer sample of this invention. Specifically, fertilizer reference standard 1, due to the omission of the S2.3 ring-closing step during preparation, lacks a ring structure, making the linear polyglutamic acid molecular chains prone to entanglement and forming microgels or insoluble matter during storage. Fertilizer reference standard 2, prepared using disulfide bond ring closure, is more prone to slow breakage or exchange reactions during storage compared to the thioether bonds used in this invention, leading to partial opening of the ring structure and the formation of soluble or insoluble aggregates. Fertilizer reference standard 3, prepared using physical mixing of 5-ALA, has ungrafted 5-ALA with poor compatibility with the system, potentially precipitating or reacting with other components during storage.

[0078] The water-insoluble matter content of reference standards 4 and 5 was comparable to that of the samples of this invention, indicating that the substitution of EDTA and the omission of ascorbic acid and β-carotene emulsions had no significant effect on water-insoluble matter in the short term. The pH values ​​of all samples remained stable between 6.4 and 6.6, meeting the standard requirements.

[0079] II. Fertilizer viscosity and rheological property testing To address the high viscosity problem of polyglutamic acid that this invention aims to solve, the viscosity of various fertilizer samples and control standards was measured. The dynamic viscosity was determined using a rotational viscometer at 25°C and 60 r / min.

[0080] For comparison, a conventional linear γ-PGA solution was prepared as a control: γ-PGA (weight average molecular weight 1000 kDa) from the same source as in this invention was prepared with deionized water to the same polyglutamic acid concentration (7.2 g / L) as fertilizer sample 1, and after stirring evenly, a conventional linear control was obtained.

[0081] The viscosity reduction rate of each fertilizer sample and the control was calculated according to Equation 1, and the results are shown in Table 2.

[0082] Viscosity reduction rate (%) = (Viscosity of conventional linear reference standard - Measured viscosity of sample or reference standard) / Viscosity of conventional linear reference standard × 100% Equation 1 Table 2: Viscosity test results of fertilizer samples and control standards As shown in Table 2, the viscosity of fertilizer samples 1-5 of this invention is all below 200 mPa·s, which is more than 86% lower than that of conventional linear PGA at the same concentration. This proves that the high viscosity problem of polyglutamic acid is effectively solved through the ring structure design. Among them, fertilizer sample 3 has the lowest viscosity, with a reduction rate of 89.4%, indicating that fertilizer sample 3 is most effective in reducing the viscosity of the system while ensuring structural stability. Fertilizer samples 4 and 5 have slightly different viscosities due to the different amounts of 5-ALA-grafted ring γ-PGA solution added in step S5, resulting in corresponding differences in polyglutamic acid content, but both are far lower than that of conventional linear PGA.

[0083] The viscosity reduction rates of fertilizer reference standards 1 and 3 were only 58.9% and 62.2%, respectively, significantly higher than those of the samples of this invention. Fertilizer reference standard 1, due to the omission of the S2.3 ring-closing step during preparation, lacked a ring structure, leading to easy entanglement of linear polyglutamic acid molecular chains and resulting in persistently high viscosity. Fertilizer reference standard 3, while possessing a ring structure due to the use of physical mixing of 5-ALA instead of covalent grafting, exhibited poor compatibility between the ungrafted 5-ALA and the system, potentially interfering with the orderly arrangement of molecular chains and significantly reducing the viscosity reduction effect. These results demonstrate that the ring structure itself plays a crucial role in reducing viscosity, while the covalent grafting method of 5-ALA also affects the final rheological properties.

[0084] The viscosity reduction rate of fertilizer reference standard 2 was 73.7%, which was better than that of reference standards 1 and 3, but still significantly higher than that of the sample of this invention. This is because the ring-closing process of reference standard 2 used disulfide bonds instead of thioether bonds for ring closing. Compared with the thioether bonds used in the preparation method of this invention, disulfide bonds have a dynamic exchange equilibrium in solution, resulting in a less stable ring structure than thioether bonds. Some molecular chains may dissociate or rearrange, thereby weakening the viscosity reduction effect.

[0085] The viscosity reduction rates of fertilizer reference standards 4 and 5 were comparable to those of the samples of this invention, indicating that the substitution of EDTA for fucoidan-calcium and the omission of ascorbic acid and β-carotene emulsions had no significant effect on viscosity, which is consistent with expectations, as these components do not participate in the construction of the ring structure.

[0086] III. Fertilizer Stability Analysis (a) Storage stability test Each fertilizer sample and control was stored in a sealed, light-protected container at 4℃ for 180 days and in a constant temperature incubator at 54℃ for 14 days. After storage, the appearance changes were observed and the polyglutamic acid content was determined. The retention rate was calculated according to Formula 2. The results are shown in Table 3.

[0087] Retention rate (%) = Polyglutamic acid content after storage / Initial polyglutamic acid content × 100% Equation 2 Table 3: Storage stability test results of fertilizer samples and control standards As shown in Table 3, fertilizer samples 1-5 of this invention remained transparent and free of precipitation after being stored at 4℃ for 180 days and at 54℃ for 14 days under accelerated storage conditions, exhibiting excellent storage stability. Among them, fertilizer sample 3 showed the highest retention rate, which corroborates its lowest viscosity result in Table 2, indicating that its ring structure was the most stable.

[0088] Reference standards 1 and 3 showed a small amount of precipitation at the bottom after storage at 4°C, with polyglutamic acid retention rates of only 79.6% and 82.3%, respectively. After accelerated storage at 54°C, obvious stratification occurred, and the retention rates dropped to 68.4% and 71.5%, respectively, indicating significantly inferior stability compared to the sample of this invention. Specifically, reference standard 1, lacking a cyclic structure, was prone to entanglement of linear polyglutamic acid molecular chains during storage, forming microgels or insoluble substances, leading to changes in appearance and content loss. Reference standard 3, using physical mixing of 5-ALA instead of covalent grafting, had poor compatibility with the ungrafted 5-ALA, which may precipitate or trigger side reactions during storage, compromising the stability of the system.

[0089] Although the stability of reference standard 2 was better than that of reference standards 1 and 3, it was still significantly lower than that of the sample of this invention, and slight flocculation was observed. This is because reference standard 2 uses disulfide bond ring closure, which, compared with the thioether bond used in this invention, results in a dynamic exchange equilibrium of disulfide bonds in solution. During long-term storage, some ring structures may undergo reversible dissociation or rearrangement, leading to the formation of aggregates in the system.

[0090] The stability of reference standard 4 is comparable to that of the sample of the present invention, indicating that EDTA substitution for fucoidan-calcium has no significant effect on storage stability. The stability of reference standard 5 is slightly lower than that of the sample of the present invention, and its appearance shows a lightening of color, indicating that the lack of ascorbic acid and β-carotene leads to the degradation of 5-ALA. However, the ring structure itself still maintains good physical stability, which corresponds to the result of its lower viscosity in Table 2.

[0091] (ii) Test for resistance to calcium ion precipitation In actual agricultural production, irrigation water and fertilizers often contain a certain amount of calcium ions, especially in hard water areas or when using calcium-containing fertilizers. When these calcium ions encounter components in fertilizers such as seaweed polysaccharides, sulfate, and phosphate, they readily undergo ion cross-linking reactions, forming insoluble calcium salts or gel-like precipitates. This not only leads to problems such as flocculation, stratification, and excessive water-insoluble matter in the fertilizer itself, but more seriously, during drip irrigation, these precipitates gradually accumulate in the dripper channels, causing dripper blockage, severely affecting irrigation uniformity and fertilization efficiency, and even paralyzing the entire drip irrigation system. Therefore, examining the fertilizer system's tolerance to calcium ions is an important indicator for evaluating its practical application performance.

[0092] Add 0.1% (by weight) of calcium chloride (calculated as solid CaCl2) to each fertilizer sample and control, stir, and let stand for 24 hours. Measure the turbidity change using a turbidimeter. The smaller the increase in turbidity, the stronger the system's resistance to calcium ion precipitation. Calculate the increase in turbidity according to Equation 3; the results are shown in Table 4.

[0093] Turbidity increase (NTU) = Turbidity after calcium addition - Initial turbidity (Equation 3) Table 4: Test results of the resistance to calcium ion precipitation of fertilizer samples and control standards As shown in Table 4, the initial turbidity of fertilizer samples 1-5 of this invention was all below 20 NTU. Visually, they were clear, transparent liquids without visible sediment or turbidity, consistent with the low water-insoluble matter content results in Table 1. After the addition of calcium ions, although the turbidity increase did not exceed 26 NTU, the appearance remained transparent, with no visible flocculent matter or sediment, demonstrating excellent resistance to calcium ion precipitation. Among them, fertilizer sample 3 showed the smallest increase in turbidity, which corroborates its best storage stability result in Table 3, indicating that it had the highest calcium ion trapping efficiency.

[0094] The initial turbidity of reference standards 1 and 3 was significantly higher than that of the sample of this invention. After the addition of calcium, the turbidity increased dramatically to over 200 NTU, and severe flocculation and white precipitate were observed. Among them, reference standard 1 lacked a cyclic structure, and the linear polyglutamic acid molecular chains could not effectively bind the seaweed polysaccharide; reference standard 3 was physically mixed with 5-ALA, and the ungrafted 5-ALA may have interfered with the stability of the calcium ion trap.

[0095] The initial turbidity of reference standard 2 was 28.5 NTU, and the turbidity increased by 127.7 NTU after the addition of calcium. Obvious flocculation was observed to the naked eye. Although it was better than reference standards 1 and 3, it was still significantly worse than the sample of this invention. This is because the dynamic exchange characteristics of disulfide bonds cause the ring structure to partially open, weakening the protective effect of the calcium ion trap.

[0096] The initial turbidity and the increase in turbidity after calcium addition of reference standards 4 and 5 were comparable to those of the sample of this invention, and both remained clear and transparent to the naked eye. It is worth noting that reference standard 4 used EDTA-calcium instead of fucoidan-calcium. Although its ability to resist calcium ion precipitation was similar to that of the sample of this invention, EDTA, as a synthetic chelating agent, has problems such as poor biodegradability and high environmental residue risk. In contrast, the fucoidan-calcium used in this invention is derived from natural seaweed and has advantages such as biodegradability and environmental friendliness. The results of reference standard 5 showed that the omission of the light stabilizer had no significant impact on the ability to resist calcium ion precipitation, consistent with the results in Table 1 showing its low water-insoluble content.

[0097] (III) Light stability test 5-Aminolevulinic acid (5-ALA), as a photosynthesis promoter, exhibits photosensitivity as an intrinsic characteristic. This invention addresses the photostability issue of 5-ALA in liquid fertilizers by covalently grafting 5-ALA onto a cyclic γ-PGA framework and supplementing it with a synergistic protective system of ascorbic acid and β-carotene.

[0098] To verify the effectiveness of the protection system, each fertilizer sample and the control were exposed to natural light for 7 days (25℃±2℃). Samples were taken at days 0, 3, and 7 to determine the 5-ALA content and calculate the retention rate. The retention rate was calculated according to Equation 4, and the results are shown in Table 5.

[0099] 5-ALA retention rate (%) = 5-ALA content after light exposure / initial 5-ALA content × 100% (Equation 4) Table 5: Results of photostability tests on fertilizer samples and control standards As shown in Table 5, after 7 days of exposure to natural light, fertilizer samples 1-4 of this invention all exhibited a 5-ALA retention rate of over 91%, demonstrating excellent photostability. Among them, fertilizer sample 3 showed the highest retention rate, reaching 93.8% after 7 days, further confirming that its overall structure was the most stable.

[0100] The 5-ALA retention rates of fertilizer reference standards 1, 2, and 4 were comparable to those of the sample of this invention, with retention rates all exceeding 90% after 7 days. While fertilizer reference standards 1 and 2 exhibited defects in their ring structure, resulting in poorer storage stability and resistance to calcium ion precipitation, the photostability of 5-ALA was not significantly affected because step S3 still covalently grafted 5-ALA onto the polyglutamic acid backbone, and step S5 contained a complete ascorbic acid and β-carotene photostability system. The 5-ALA retention rate of reference standard 4 was comparable to that of the sample of this invention, indicating that EDTA substitution for fucoidan-calcium had no effect on the photostability of 5-ALA.

[0101] The 5-ALA retention rate of fertilizer reference standard 3 was significantly lower than that of samples 1-5 of this invention, decreasing to 84.7% after 7 days. This is because fertilizer reference standard 3 used physical mixing of 5-ALA instead of covalent grafting in step S3 of its preparation. Although step S5 contained a complete photostable system of ascorbic acid and β-carotene, the ungrafted 5-ALA had poor compatibility with the system and was more prone to photolysis under light conditions. This result demonstrates that covalent grafting of 5-ALA is more effective than physical mixing in stabilizing 5-ALA, and even in the presence of a light stabilizer, physically mixed 5-ALA still degrades more rapidly.

[0102] The 5-ALA retention rate of control 5 was the lowest among all samples, at only 72.8% after 3 days and decreasing to 56.3% after 7 days. This is because fertilizer control 5 omitted the addition of ascorbic acid and β-carotene emulsion in step S5 of its preparation. Although its step S3 still involved covalent grafting, the lack of a photostable protection system led to severe degradation of 5-ALA under light conditions. The combined results of the two comparative studies indicate that the photostable stability of 5-ALA requires dual protection from both covalent grafting and photostable agents; both are synergistic and indispensable.

[0103] IV. Fertilizer Application Effect Test To verify the effectiveness of the fertilizer of this invention in practical applications, a pot experiment was designed to investigate the effects of different fertilizer treatments on the growth, physiological characteristics, and quality of Chinese cabbage. Simultaneously, to verify the water-retention and synergistic effect of cyclic γ-PGA, two water treatments—normal irrigation and drought stress—were set up to comprehensively evaluate the application effect of the fertilizer of this invention.

[0104] Test crop: Chinese cabbage ( Brassica chinensis L. The variety is Shanghai Bok Choy.

[0105] Test soil: Garden soil and vermiculite were mixed at a volume ratio of 3:1, air-dried, sieved, and then placed in pots, with 2 kg of soil in each pot.

[0106] Experimental design: A total of 8 treatments were set up, with two water gradients for each treatment: normal irrigation and drought stress. Each treatment was repeated 3 times in a randomized block design.

[0107] The following steps will be taken: T1: Water control (CK); T2: Commercially available conventional water-soluble fertilizer with macro-elements (Teli water-soluble fertilizer with macro-elements 20-20-20+TE); T3: Fertilizer sample 3; T4: Fertilizer reference standard 1; T5: Fertilizer reference standard 2; T6: Fertilizer reference standard 3; T7: Fertilizer reference standard 4; T8: Fertilizer reference standard 5; Fertilization method: All fertilizers were applied at the same nitrogen level (N 0.2g / kg soil), and the base fertilizer was applied all at once.

[0108] Moisture treatment methods: Normal irrigation: Maintain soil moisture content at 70%–80% of field capacity. Drought stress: Irrigate normally for 14 days after sowing. After the seedlings have survived, start controlling the water supply and maintain the field water holding capacity at 40% to 50% until harvest.

[0109] Cultivation and management: Sow 10 seeds per pot, thin out to 3 seedlings after emergence, and the growth cycle is 45 days.

[0110] The measured indicators included growth indicators (fresh weight of aboveground parts), physiological indicators (chlorophyll content, relative water content of leaves), quality indicators (vitamin C content, soluble sugar content) and stress resistance indicators (free proline content). The results are shown in Tables 6 and 7.

[0111] Table 6: Effects of different treatments on relevant indicators of Chinese cabbage under normal irrigation conditions Table 7: Effects of different treatments on relevant indicators of Chinese cabbage under drought stress As shown in Table 6, under normal irrigation conditions, the pakchoi treated with fertilizer sample 3 showed significantly better performance in all indicators compared to other treatments. The fresh weight of the aboveground parts was much higher than that of the water control and the conventional fertilizer control. The chlorophyll content, vitamin C content, and soluble sugar content also showed significant advantages, indicating that the fertilizer of this invention has a significant growth-promoting and quality-improving effect.

[0112] As shown in Table 7, under drought stress, all growth indicators decreased compared to normal irrigation, but fertilizer sample 3 showed the smallest decrease, with significantly higher aboveground fresh weight and relative leaf water content than the other controls. Simultaneously, fertilizer sample 3 accumulated far more free proline than the other treatments, indicating that it effectively enhanced drought resistance through osmotic regulation.

[0113] Fertilizer control 1, lacking a ring structure, showed significantly lower growth indicators than fertilizer sample 3 under both moisture conditions, demonstrating the crucial role of the ring structure in maintaining fertilizer stability and enhancing water retention and efficiency. While fertilizer control 2, employing disulfide bond ring closure, was superior to fertilizer control 1, its effect was still lower than fertilizer sample 3, proving that thioether bonds are more suitable for constructing stable ring structures than disulfide bonds. Fertilizer control 3, using a physical mixture of 5-ALA, showed lower growth-promoting and quality-improving effects than fertilizer sample 3, demonstrating that covalent grafting of 5-ALA provides a more stable photosynthetic-promoting effect than physical mixing.

[0114] Fertilizer control 4, which uses EDTA-calcium as a substitute, is basically equivalent to fertilizer sample 3. However, considering the biodegradability of EDTA, fucoidan-calcium, as a naturally sourced green chelating agent, has a significant environmental advantage. Fertilizer control 5, which omits ascorbic acid and β-carotene, is lower than sample 3 in all indicators, demonstrating the important role of the photostable protection system in maintaining fertilizer activity.

[0115] In summary, fertilizer sample 3 of this invention has shown excellent performance in promoting the growth of Chinese cabbage, improving photosynthesis, enhancing quality, and strengthening drought resistance, which fully demonstrates the effectiveness of the multi-synergistic design of the ring γ-PGA framework, 5-ALA covalent grafting, brown algae oligosaccharide-calcium ion trap, and photostable protection system.

[0116] The embodiments described above are merely preferred embodiments of the present invention and are not intended to limit the scope of the present invention. Various modifications and improvements made by those skilled in the art to the technical solutions of the present invention without departing from the spirit of the present invention should fall within the protection scope defined by the claims of the present invention.

Claims

1. A method for preparing an aqueous solution fertilizer containing polyglutamic acid and seaweed polysaccharides to enhance the effects of macronutrients, characterized in that, Includes the following steps: S1. Preparation of linear γ-polyglutamic acid precursor: γ-polyglutamic acid was subjected to self-degradation reaction under acidic conditions. Lysine was introduced simultaneously during the reaction to modify its end groups. After purification, a linear γ-PGA solution with lysine end groups was obtained. S2. Construction of the cyclic γ-polyglutamic acid backbone: The linear γ-PGA solution was successively modified with alkyne and cysteine ​​to obtain a bifunctionalized linear γ-PGA containing an alkyne group at one end and a thiol group at the other end. Then, intramolecular cyclization was performed to obtain a cyclic γ-PGA solution. S3. Functional modification of cyclic γ-polyglutamic acid: The cyclic γ-PGA solution was subjected to an amidation grafting reaction with 5-aminolevulinic acid, and after purification, a 5-ALA-grafted cyclic γ-PGA solution was obtained. S4. Preparation of the alginate-calcium complex: The alginate-calcium complex concentrate was obtained by complexing the alginate-calcium complex solution with the calcium nitrate solution and then concentrating it. S5. Compounding of liquid fertilizer: 5-ALA grafted cyclic γ-PGA solution, concentrated brown algae oligosaccharide-calcium complex solution, seaweed polysaccharide and N, P and K elements are compounded to obtain an aqueous liquid fertilizer containing polyglutamic acid and seaweed polysaccharide-enhanced macronutrients.

2. The preparation method according to claim 1, characterized in that, In step S1, the self-degradation reaction is carried out at 65℃~75℃ and pH 3.0~3.5 for 5h~7h.

3. The preparation method according to claim 1, characterized in that, In step S1, the γ-polyglutamic acid is prepared into a γ-PGA base solution with a mass concentration of 50 g / L, and the lysine is L-lysine prepared into a lysine solution with a mass concentration of 80 g / L to 120 g / L; the mass ratio of the γ-PGA base solution to the lysine solution is 100:(0.6 to 1.8).

4. The preparation method according to claim 1, characterized in that, In step S2, the alkynylation modification is carried out by reacting an alkynyl-succinimide ester as an alkynyl donor with a linear γ-PGA solution for 1 to 2 hours at pH 7.5 to 8.5; the cysteine ​​modification is carried out by activating the alkynyl-modified linear γ-PGA solution with N-hydroxysuccinimide and 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride for 20 to 40 minutes at pH 5.3 to 5.7, followed by reaction with L-cysteine ​​for 3.5 to 4.5 hours to obtain a bifunctionalized linear γ-PGA solution.

5. The preparation method according to claim 1, characterized in that, In step S2, the intramolecular cyclization is initiated by a mercapto-acetylene photoclick reaction of bifunctionalized linear γ-PGA under ultraviolet light irradiation to obtain a cyclic γ-PGA solution linked by thioether bonds; the intensity of the ultraviolet light irradiation is 5 mW / cm². 2 ~10mW / cm 2 The irradiation reaction time is 2h to 4h.

6. The preparation method according to claim 1, characterized in that, In step S3, the amidation grafting reaction is carried out under pH 5.3-5.7 conditions, by activating the cyclic γ-PGA solution with N-hydroxysuccinimide and 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride for 20-40 min, followed by reaction with 5-aminolevulinic acid for 5-7 h to obtain a 5-ALA-grafted cyclic γ-PGA solution; the mass ratio of the cyclic γ-PGA solution to 5-aminolevulinic acid in the amidation grafting reaction is 1:(0.001-0.02).

7. The preparation method according to claim 1, characterized in that, In step S4, the mass concentration of the alginate oligosaccharide solution is 100 g / L, the mass concentration of the calcium nitrate solution is 20 g / L, and the volume ratio of the alginate oligosaccharide solution to the calcium nitrate solution is (0.5-1.0):

1.

8. The preparation method according to claim 1, characterized in that, In step S4, the complexation reaction is carried out at 43℃~47℃ and pH 6.3~6.7 for 60min~90min.

9. The preparation method according to claim 1, characterized in that, In step S5, the mixed compound also includes the addition of trace elements, betaine, ascorbic acid, β-carotene emulsion, and a concentrated solution rich in lysine, citric acid and γ-PGA oligopeptides obtained by concentrating the ultrafiltration permeate collected during the purification process in step S1.

10. The application of fertilizer prepared by any one of claims 1-9 in crop cultivation.

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