Enzyme-containing liquid fertilizer containing nereis and its preparation method

Abstract

The application belongs to the technical field of agricultural fertilizer, and particularly relates to a high-efficiency yield-increasing liquid fertilizer containing enzymatic nereid and a preparation method thereof, which comprises the following steps: washing and crushing nereid, and then performing freeze-thaw cycle pretreatment; under weak alkaline conditions, a complex protease preparation containing protease and chitinase is used for enzymolysis, and an enzymatic nereid liquid is obtained through solid-liquid separation; urea, phosphate-potassium fertilizer, trace elements, potassium fulvate and polyaspartic acid are mixed in a preparation tank, and then N-carbamoyl-L-glutamic acid-chitosan graft copolymer and seleno-cysteine-alginic acid nanometer microspheres are added to obtain a nutrient liquid; finally, the enzymatic nereid liquid, the nutrient liquid and a plant growth regulator are mixed to obtain a finished product. Through the biological enzymolysis technology, the active substances in the nereid are fully released, the prepared liquid fertilizer has the functions of nutrient supply, physiological regulation and soil improvement, can significantly promote crop growth and increase yield, and realizes high-value utilization of marine biological resources.

Description

TECHNICAL FIELD

[0001] The application belongs to the technical field of agricultural fertilizers, and particularly relates to a high-efficiency yield-increasing liquid fertilizer containing enzymatic sandworm and a preparation method thereof. BACKGROUND

[0002] In agricultural production, fertilizers are the core elements for ensuring crop yield and quality. Although traditional chemical fertilizers can quickly supplement nutrients, long-term overuse can lead to a series of ecological environmental problems such as soil compaction, acidification, organic matter decline, and water eutrophication. At the same time, pure inorganic nutrition cannot fully meet the internal needs of crops for physiological regulation and resistance enhancement at different growth stages. To address these challenges, new liquid fertilizers with both nutrient supply and biological stimulation functions have become a research and development hotspot. Such fertilizers aim to provide a full nutrition package for crops through the scientific combination of organic and inorganic substances, and to improve soil microecology and stimulate the potential of crops using organic active ingredients, thereby achieving the goals of reducing fertilizer use and promoting efficiency and green and sustainable development. However, many existing liquid fertilizers still face problems such as single source of raw materials, low content of active ingredients, limited functions, or rough preparation process, and their stability and universality need to be further improved.

[0003] Exploring efficient, inexpensive, and environmentally friendly sources of organic matter is the key to developing biologically active liquid fertilizers. Currently, commonly used organic raw materials are mainly concentrated in industrial and agricultural by-products, such as monosodium glutamate waste liquid, molasses, soybean meal, and livestock and poultry manure. These raw materials either have large fluctuations in composition, require long fermentation periods, or have single functional substances, limiting the development of high-end fertilizers. Sandworm, as a rich marine biological resource, is rich in protein, amino acids, lipids, and unique chitin and other organic components, making it a potentially high-quality organic fertilizer raw material. Chitooligosaccharides, which are produced by the degradation of chitin, are recognized as a biological stimulant that can stimulate plant immune responses. However, current utilization of sandworm is mainly limited to the feed industry, with little exploration in agricultural fertilizers. This is mainly due to the lack of efficient and targeted conversion technology, making it difficult to effectively degrade complex organic macromolecules into small molecule active substances that can be easily absorbed and utilized by crops, resulting in the long-term neglect of its great value in the fertilizer field.

[0004] In the prior art, similar organic raw materials are usually treated by strong acid, strong base chemical hydrolysis or high temperature and high pressure cooking physical method. Although the chemical method can completely decompose, the condition is severe, and the biological active substances (such as some amino acids and vitamins) sensitive to heat and acid and alkali are easily damaged, and harmful chemical residues may be introduced; the physical method has high energy consumption, and mainly destroys the physical structure, and the biological effectiveness of nutrients is limited. The biological enzyme hydrolysis technology is considered as an ideal treatment method due to its mild condition, strong specificity, high efficiency and better preservation of active ingredients. However, for the special raw material of the sandworm, if the enzyme hydrolysis process for plant fibers (such as using cellulase and pectinase) is simply used, the animal tissue mainly composed of protein and chitin cannot be effectively decomposed, resulting in insufficient enzyme hydrolysis and low product fertilizer efficiency. Therefore, developing a specific biological enzyme hydrolysis process which can efficiently break down the sandworm tissue and maximize the release and preservation of its functional substances is the core prerequisite and technical bottleneck for successfully applying the sandworm to high-end liquid fertilizer. On this basis, how to scientifically compound the enzyme hydrolysis product with inorganic nutrients and other functional additives (such as water retaining agent, chelating agent and growth regulating substance) to build a synergistic composite system is another important problem to be solved for realizing the high-efficiency yield-increasing goal of the fertilizer. SUMMARY

[0005] In view of the deficiencies of the prior art, the purpose of the present application is to provide a high-efficiency yield-increasing liquid fertilizer containing enzyme-hydrolyzed sandworm and a preparation method thereof

[0006] In the first aspect of the present application, a preparation method of a high-efficiency yield-increasing liquid fertilizer containing enzyme-hydrolyzed sandworm is provided, and the steps include:

[0007] S1, collect the sandworm, wash and crush, and perform freeze-thaw cycle; adjust the pH value to 7.5-8.5; deliver the treated sandworm slurry to an enzyme hydrolysis tank, add a compound protease preparation, stir, maintain the temperature at 48-52℃ and the pH at 7.0-8.0, and hydrolyze to obtain an enzyme-hydrolyzed sandworm mixture; perform solid-liquid separation on the enzyme-hydrolyzed sandworm mixture by a plate-and-frame filter press, and collect the filtrate; add water to the solid part obtained by the solid-liquid separation, perform solid-liquid separation again by the plate-and-frame filter press, and combine the filtrates of the two times to obtain an enzyme-hydrolyzed sandworm liquid;

[0008] S2, add water in a preparation tank, stir, and sequentially add urea, potassium dihydrogen phosphate, potassium sulfate and a trace element mixture, and stir until completely dissolved; add potassium fulvate and polyaspartic acid, and continue to stir and mix; add N-carbamoyl-L-glutamic acid-chitosan graft copolymer, and stir; then add seleno-cysteine-alginate nanometer microspheres to obtain a nutrient solution containing modified compounds;

[0009] S3, add the enzyme-hydrolyzed sandworm liquid into the nutrient solution containing modified compounds, stir and mix; and add a plant growth regulator, and continue to stir and mix.

[0010] As a preferred embodiment of the present invention, the compound protease preparation is composed of alkaline protease, neutral protease, and chitinase, with a weight ratio of 40-50% alkaline protease, 30-40% neutral protease, and 15-20% chitinase. This compound protease preparation works synergistically during the enzymatic hydrolysis of *Nematocystis jirovecii* slurry. The alkaline and neutral proteases efficiently hydrolyze *Nematocystis jirovecii* muscle and tissue proteins in a weakly alkaline environment, breaking them down into small peptides and amino acids. Simultaneously, the chitinase specifically degrades the chitinous components abundant in the *Nematocystis jirovecii* body surface, generating bioactive chitin oligosaccharides. The combined ratio of the three enzymes fully leverages their synergistic effect, ensuring the full conversion of *Nematocystis jirovecii* organic matter and the maximum release of high-value active ingredients, providing the core functional material basis for the final liquid fertilizer.

[0011] As a preferred embodiment of the present invention, the trace element mixture is composed of sodium iron ethylenediaminetetraacetate, sodium manganese ethylenediaminetetraacetate, sodium zinc ethylenediaminetetraacetate, sodium copper ethylenediaminetetraacetate, boric acid, and ammonium molybdate. The weight ratio of sodium iron ethylenediaminetetraacetate is 35-45%, sodium manganese ethylenediaminetetraacetate is 10-15%, sodium zinc ethylenediaminetetraacetate is 20-25%, sodium copper ethylenediaminetetraacetate is 3-5%, boric acid is 15-20%, and ammonium molybdate is 2-4%. This composition and ratio ensure that iron, manganese, zinc, and copper elements exist in a highly stable chelated form, effectively preventing precipitation and failure in the liquid phase. At the same time, it provides essential boron and molybdenum elements for crops to meet the balanced needs of crops for various trace elements throughout the entire growth cycle, prevent nutrient deficiencies, and participate in various enzymatic reactions and metabolic processes.

[0012] As a preferred embodiment of the present invention, the plant growth regulator is composed of sodium naphthaleneacetate, 6-benzylaminopurine, paclobutrazol, and brassinolide, with the following weight ratio: sodium naphthaleneacetate 40-50%, 6-benzylaminopurine 20-30%, paclobutrazol 15-25%, and brassinolide 0.1-0.5%. This compound utilizes the initiation role of sodium naphthaleneacetate as an auxin analog to promote root development and organ formation, the synergistic effect of 6-benzylaminopurine as a cytokinin to promote cell division and delay senescence, the regulatory role of paclobutrazol as a gibberellin synthesis inhibitor to control vegetative growth, promote reproductive growth, and enhance stress resistance, and the synergistic effect of brassinolide as a highly efficient and broad-spectrum plant hormone that can exert comprehensive regulation, enhance photosynthesis, and alleviate abiotic stress even at extremely low concentrations. The four regulators work synergistically in a scientific ratio to jointly regulate the growth and development process of crops, enabling them to develop towards high yield, high quality, and stress resistance.

[0013] The reaction mechanism of this invention for preparing a highly efficient yield-increasing liquid fertilizer containing enzymatically hydrolyzed *Nematocystis jirovecii* is mainly based on the effects of biological enzymatic hydrolysis, chemical grafting modification, microsphere encapsulation, and multi-component synergistic effects. First, the cell structure of *Nematocystis jirovecii* tissues is physically disrupted through freeze-thaw cycles, creating favorable conditions for subsequent enzymatic hydrolysis. Then, under weakly alkaline and mild conditions, a complex protease preparation precisely and efficiently catalyzes the hydrolysis of proteins within the *Nematocystis jirovecii* into easily absorbed small-molecule peptides and free amino acids. Simultaneously, chitinase degrades chitin in the *Nematocystis jirovecii* body wall into chitin oligosaccharides with plant immune-inducing activity, thereby fully converting the organic matter of the *Nematocystis jirovecii* into an enzymatic hydrolysate with both nutritional and biostimulatory functions. Secondly, the chitosan graft copolymer prepared through chemical modification has active groups on its molecular chain that can integrate nutrients and form a protective film, achieving slow release and stable supply of nutrients, while also stimulating crop stress resistance. Meanwhile, selenocysteine ​​microspheres constructed using microsphere encapsulation technology utilize the cross-linking reaction of sodium alginate and calcium ions to form a stable three-dimensional network structure, effectively protecting selenium from soil fixation and controlling its release rate, significantly improving selenium utilization efficiency. Finally, in the finished product compounding stage, the organic active substances provided by enzymatic hydrolysis of *Nematocystis jirovecii* extract, the fast-acting nutrients provided by inorganic fertilizers, the slow-release and stress-resistance functions provided by the graft copolymer, the precise supply of trace elements provided by nanospheres, and the physiological regulatory effects of plant growth regulators together constitute a complete synergistic system, ultimately achieving the comprehensive goal of promoting crop growth and development, and improving yield and quality.

[0014] As a preferred embodiment of the present invention, in step S1, the enzymatic hydrolysis time is 18-24 h; the number of freeze-thaw cycles is 1-3 times; the conditions for the freeze-thaw cycle are: freezing at -20~-18℃ for 12-16 h and thawing at 24-26℃; the compound protease preparation contains protease and chitinase.

[0015] As a preferred embodiment of the present invention, in step S2, the stirring time is 20-30 minutes.

[0016] As a preferred embodiment of the present invention, the preparation method of the N-carbamoyl-L-glutamic acid-chitosan graft copolymer includes: A1, weighing chitosan and adding it to a three-necked flask, adding acetic acid solution, and mechanically stirring under nitrogen protection until completely dissolved; subsequently, adding sodium hydroxide solution dropwise while stirring to adjust the pH of the chitosan solution to 6.0-6.5; dissolving N-carbamoyl-L-glutamic acid in deionized water, adjusting the pH to 6.0-6.5 with sodium hydroxide solution, and then adding 1 -(3-Dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride and N-hydroxysuccinimide were activated at 24-26℃; A2, the activated N-carbamoyl-L-glutamic acid solution was added dropwise to the chitosan solution and reacted at 25-30℃; after the reaction was completed, sodium hydroxide solution was added to adjust the pH to 7.0 to obtain the reaction mixture; the reaction mixture was poured into anhydrous ethanol to precipitate, the precipitate was collected by filtration, washed with ethanol aqueous solution, and dried in a vacuum drying oven at 38-42℃.

[0017] In this invention, the preparation of N-carbamoyl-L-glutamic acid-chitosan graft copolymer is based on a carbodiimide-mediated amidation coupling reaction mechanism, a process that is precise and efficient. First, in a weakly alkaline environment, the carboxyl group of the side chain in the N-carbamoyl-L-glutamic acid molecule undergoes an activation reaction with the added hydroxysuccinimide mediated by carbodiimide hydrochloride. The carbodiimide reagent first condenses with the carboxyl group to form a highly reactive O-acylisourea intermediate. This intermediate is extremely unstable and rapidly undergoes an exchange reaction with the hydroxyl group in the hydroxysuccinimide molecule, generating a more chemically stable but still highly reactive N-hydroxysuccinimide ester. This activation step is crucial, as it transforms a relatively inert carboxyl group into an active ester easily attacked by nucleophiles, laying a solid foundation for the subsequent grafting reaction. Subsequently, this activated solution is added dropwise to a pre-neutralized chitosan solution with the pH adjusted to a weakly acidic state. A large number of free amino groups are distributed on the chitosan molecular chain. These amino groups act as nucleophiles, efficiently attacking the carbonyl carbon atoms of the active ester generated in the aforementioned reaction. Through a single nucleophilic addition-elimination process, a stable amide bond is ultimately formed, thus firmly grafting the N-carbamoyl-L-glutamic acid molecule onto the chitosan molecular backbone in the form of a covalent bond. The entire reaction process needs to be carried out under strictly controlled temperature and pH conditions to ensure that the hydrolysis side reaction of the activated ester is suppressed to a minimum, thereby maximizing the efficiency of the grafting reaction and the uniformity of the product. The resulting graft copolymer inherits the biocompatibility, film-forming properties, and positively charged amino groups of chitosan, as well as the growth-promoting, chelating, and biostimulating activities of N-carbamoyl-L-glutamic acid, achieving synergistic and enhanced functional effects.

[0018] As a preferred embodiment of the present invention, in step A1, the activation time at 24-26°C is 30-40 min.

[0019] As a preferred embodiment of the present invention, in step A2, the reaction time is 6-8 hours at 25-30°C.

[0020] As a preferred technical solution of the present invention, the preparation method of the selenocysteine-alginic acid nanospheres includes: B1, dissolving sodium alginate in deionized water and mechanically stirring in a water bath at 38-42℃ to form an aqueous phase; dissolving selenocysteine ​​in deionized water and adding hydrochloric acid to adjust the pH to 3.8-4.2 to form an inner aqueous phase; B2, adding the inner aqueous phase dropwise to the aqueous phase and homogenizing to form a primary emulsion; adding the primary emulsion dropwise to liquid paraffin containing calcium chloride and sorbitan monooleate and mechanically stirring to crosslink and solidify; adding n-hexane, allowing it to stand and separate into layers, discarding the supernatant, collecting the lower precipitate, washing it alternately with acetone and ethanol, filtering it through a microporous membrane, and drying it in a vacuum drying oven at 28-32℃.

[0021] In this invention, the preparation of selenocysteine-alginic acid nanospheres is based on a precise double emulsion template and ion-transient crosslinking technology. The process begins with the formation of the colostrum: an acidic aqueous inner phase containing selenocysteine ​​is dropwise added to a sodium alginate aqueous phase under high-speed shear force. Sodium alginate is a natural linear polysaccharide with a large number of regularly arranged sodium carboxylate groups on its molecular chains, giving it good water solubility. Under vigorous mechanical stirring or homogenization, the aqueous inner phase is dispersed into countless micron- or even nano-sized droplets, which are then encapsulated by the sodium alginate aqueous phase, forming a water-in-water colostrum system, in which selenocysteine ​​is effectively encapsulated within the inner phase droplets. The next crucial step is crosslinking and curing: this colostrum system is slowly dropwise added to a liquid paraffin outer oil phase containing calcium ions and sorbitan monooleate. Sorbitan monooleate, as a lipophilic nonionic surfactant, rapidly adsorbs onto the oil-water interface of the primary emulsion droplets, significantly reducing interfacial tension and aiding in the dispersion of the droplets in the oil phase, forming a more stable water / oil double emulsion system. At this point, calcium ions in the oil phase, as divalent metal cations, diffuse inwards into the aqueous phase through the interface. Once these calcium ions enter the phase containing sodium alginate, they immediately bind specifically to the guluronic acid units on the sodium alginate molecular chain. Each calcium ion can coordinate with the carboxyl groups of two guluronic acid units like an "egg carton," instantly forming a three-dimensional ionic cross-linking network. This process transforms sodium alginate from a soluble state into an insoluble hydrogel, firmly locking the selenocysteine ​​within the nanosphere network composed of calcium alginate. Subsequent washing and drying steps remove the oil phase, unreacted reagents, and impurities, ultimately yielding well-structured, uniformly sized dried nanosphere powder, achieving effective encapsulation and protection of selenocysteine.

[0022] As a preferred embodiment of the present invention, in step B1, the mechanical stirring time in the water bath at 38-42℃ is 20-30 minutes.

[0023] As a preferred embodiment of the present invention, in step B2, the homogenization time is 5-10 min; the drying time in a vacuum drying oven at 28-32℃ is 6-8 h.

[0024] In a second aspect, the present invention provides a method for preparing a highly efficient yield-increasing liquid fertilizer containing enzymatically hydrolyzed sandworms. The liquid fertilizer comprises the following raw materials in parts by weight: 50-70 parts fresh sandworms; 3-8 parts N-carbamoyl-L-glutamic acid-chitosan graft copolymer; 2-5 parts selenocysteine-alginic acid nanospheres; 5-10 parts urea; 3-6 parts potassium dihydrogen phosphate; 2-4 parts potassium sulfate; 1-3 parts a mixture of trace elements; 0.5-2 parts polyaspartic acid; 1-3 parts potassium humate; and 0.1-0.5 parts plant growth regulator.

[0025] Compared with the prior art, the present invention has the following beneficial effects:

[0026] (1) The high-yield liquid fertilizer containing enzymatically hydrolyzed sandworms provided by this invention exhibits significant comprehensive technical effects through innovative preparation processes and scientific component design. First, in terms of resource utilization and process innovation, this invention successfully realizes the high-value transformation and utilization of the marine biological resource sandworm. By using a compound enzyme preparation containing protease and chitinase, and in conjunction with a precisely controlled temperature and pH environment, mild, efficient, and targeted enzymatic hydrolysis of sandworm tissue is achieved, completely changing the drawbacks of traditional chemical or physical methods for treating organic raw materials, such as destruction of active ingredients, high energy consumption, and low efficiency. In particular, the innovative freeze-thaw cycle pretreatment process effectively destroys the cell structure of sandworms, creating extremely favorable conditions for enzymatic hydrolysis, and significantly improving the efficiency of enzymatic hydrolysis and the yield of the target product. The entire preparation process is clear, the conditions are mild and controllable, suitable for large-scale industrial production, and provides a new raw material path and technical paradigm for the development of new functional fertilizers.

[0027] (2) In terms of product functionality and effects, this fertilizer achieves a perfect unity of nutrient supply, physiological regulation, and soil improvement. Enzymatic hydrolysis of *Nematocystis jirovecii* extract not only provides abundant direct nitrogen sources such as small-molecule amino acids and peptides, but its chitosan oligosaccharide content is also a highly efficient biostimulant that can stimulate systemic resistance in crops and improve their resistance to abiotic stress. N-carbamoyl-L-glutamic acid-chitosan graft copolymer has both nutrient slow-release and immune-inducing functions, forming a protective layer around the roots to reduce nutrient loss and continuously stimulate crop growth. Selenocysteine-alginic acid nanospheres achieve precise controlled release of the trace element selenium, effectively preventing selenium fixation and inactivation in the soil, greatly improving the bioavailability of selenium, and laying a solid foundation for the production of selenium-rich agricultural products. The addition of polyaspartic acid and potassium humate further enhances the fertilizer's chelating ability and the formation of soil aggregates, improving the rhizosphere microenvironment.

[0028] (3) In terms of agricultural application and ecological benefits, the liquid fertilizer of this invention exhibits excellent yield-increasing and quality-improving effects as well as environmentally friendly characteristics. Field trials show that crops treated with this fertilizer have more developed root systems, stronger plants, darker green leaves, and significantly improved photosynthetic efficiency. In grain crops, this is reflected in increased ear grain number and thousand-grain weight, while in fruit and vegetable crops, it is reflected in improved fruit setting rate and fruit quality. The synergistic effect of various active substances in the product effectively reduces the amount of traditional chemical fertilizers used, which is in line with the green development concept of reducing fertilizer use and increasing efficiency in agriculture. At the same time, all components of this fertilizer are biodegradable, with no harmful residues, and do not pose a pollution risk to soil and groundwater. In summary, this invention not only provides a new type of efficient and environmentally friendly fertilizer product for agricultural production, but also opens up broad prospects for the innovative application of marine biological resources in the agricultural field, and has extremely high promotion and application value. Detailed Implementation

[0029] To facilitate understanding of the present invention, the following embodiments are provided. Those skilled in the art should understand that these embodiments are merely illustrative and should not be construed as limiting the scope of the invention.

[0030] The sources of some components in the examples and comparative examples are as follows:

[0031] The plate and frame filter press was purchased from Jingjin Equipment Co., Ltd.

[0032] The urea was purchased from Hubei Yihua Group Co., Ltd.

[0033] The potassium dihydrogen phosphate was purchased from Sichuan Lanjian Yingdun Technology Co., Ltd.

[0034] The potassium sulfate was purchased from SDIC Xinjiang Lop Nur Potash Co., Ltd.

[0035] The potassium humate was purchased from Beijing Aerospace Hengfeng Technology Co., Ltd.

[0036] The polyaspartic acid was purchased from Shandong Taihe Water Treatment Technology Co., Ltd.

[0037] The curing tank was purchased from Jiangsu Leke Energy Saving Technology Co., Ltd.

[0038] The chitosan was purchased from Zhejiang Jinke Biochemical Co., Ltd.

[0039] The acetic acid was purchased from Jiangsu Suopu (Group) Co., Ltd.

[0040] The N-carbamoyl-L-glutamic acid was purchased from Shanghai Yuanye Biotechnology Co., Ltd.

[0041] The 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride was purchased from Shanghai Maclean Biochemical Technology Co., Ltd.

[0042] The N-hydroxysuccinimide was purchased from Aladdin Reagent (Shanghai) Co., Ltd.

[0043] The sodium alginate was purchased from Qingdao Mingyue Seaweed Group Co., Ltd.

[0044] The selenocysteine ​​was purchased from Xi'an Ruixi Biotechnology Co., Ltd.

[0045] The calcium chloride was purchased from Inner Mongolia Lantai Industrial Co., Ltd.

[0046] The sorbitan monooleate was purchased from Liaoyang Huaxing Chemical Co., Ltd.

[0047] The liquid paraffin was purchased from China Petroleum & Chemical Corporation (Sinopec). Example 1

[0048] This embodiment provides a method for preparing a highly efficient liquid fertilizer containing enzymatically hydrolyzed *Nematocystis jirovecii* to increase yield. The method involves preparing an N-carbamoyl-L-glutamic acid-chitosan graft copolymer: 10g of chitosan is weighed and added to a three-necked flask, followed by 200mL of a 2% (w / w) acetic acid solution. The solution is mechanically stirred under nitrogen protection until completely dissolved. Then, a 1mol / L sodium hydroxide solution is added dropwise while stirring to adjust the pH of the chitosan solution to 6.2. 15g of N-carbamoyl-L-glutamic acid is dissolved in 100mL of deionized water, and the pH is adjusted to 6.2 with a 1mol / L sodium hydroxide solution. Then, 1.5g of 1-3-dimethylaminopropyl-3-ethylcarbodiimide hydrochloride and 0.75g of [unspecified ingredient] are added. N-hydroxysuccinimide was activated at 25°C for 35 minutes. The activated solution was slowly added dropwise to the chitosan solution, and the reaction was carried out at 28°C for 7 hours. After the reaction was completed, 1 mol / L sodium hydroxide solution was added to adjust the pH to 7.0. The reaction mixture was poured into 500 mL of anhydrous ethanol to precipitate the product. The precipitate was collected by filtration, washed three times with 80% ethanol aqueous solution, and dried to constant weight in a vacuum drying oven at 40°C to obtain the product.

[0049] Preparation of selenocysteine-alginic acid nanospheres: 5g of sodium alginate was dissolved in 200mL of deionized water and mechanically stirred in a 40℃ water bath for 25 minutes to form an aqueous phase; 3g of selenocysteine ​​was dissolved in 50mL of deionized water, and 0.1mol / L hydrochloric acid was added to adjust the pH to 4.0 to form an inner aqueous phase; the inner aqueous phase was added dropwise to the aqueous phase and homogenized at 9000 rpm for 4 minutes to form a pre-emulsion; the pre-emulsion was added dropwise to 400mL of liquid paraffin containing 4% calcium chloride and 2% sorbitan monooleate and mechanically stirred at 500 rpm for 30 minutes to crosslink and solidify; 400mL of n-hexane was added, and after standing and separation, the supernatant was discarded, the lower precipitate was collected, and washed three times alternately with acetone and anhydrous ethanol. The precipitate was filtered through a 0.45μm microporous membrane and dried in a vacuum drying oven at 30℃ for 7 hours to obtain the product.

[0050] Preparation of liquid fertilizer: Weigh 600g of fresh *Nepenthes*, wash and mechanically crush them, and perform two freeze-thaw cycles: freeze at -19℃ for 14h, then thaw at 25℃. Add deionized water to adjust the total volume of the *Nepenthes* slurry to 2000mL, and adjust the pH to 8.0 with sodium hydroxide solution. Transfer the slurry to an enzymatic hydrolysis tank, add 18g of a compound protease preparation, which consists of 8g of alkaline protease, 7g of neutral protease, and 3g of chitinase. Maintain the temperature at 50℃ and pH at 7.5, and mechanically stir for 20h for enzymatic hydrolysis. Separate the enzymatically hydrolyzed *Nepenthes* mixture into solid and liquid phases using a plate and frame filter press, and collect the filtrate. Add 1000mL of deionized water to the solid portion obtained by pressing, filter again, and combine the two filtrates to obtain approximately 1500mL of enzymatically hydrolyzed *Nepenthes* liquid for later use. Add 500 mL of deionized water to a 2000 mL preparation tank. While mechanically stirring, add 80 g of urea, 45 g of potassium dihydrogen phosphate, 30 g of potassium sulfate, and 20 g of a trace element mixture, stirring until completely dissolved. Add 20 g of potassium humate and 10 g of polyaspartic acid, and continue stirring for 25 min. Add 50 g of N-carbamoyl-L-glutamic acid-chitosan graft copolymer, and stir for 30 min to ensure uniform dispersion. Then add 30 g of selenocysteine-alginic acid nanospheres, and stir slowly for 10 min to obtain a nutrient solution containing the modified compound. Add all the enzymatically hydrolyzed *Nematocystis jirovecii* extract to the above nutrient solution and stir to mix for 15 min. Finally, add 3 g of plant growth regulator, and continue stirring for 20 min. After mixing evenly, the high-efficiency yield-increasing liquid fertilizer containing enzymatically hydrolyzed *Nematocystis jirovecii* is obtained. Example 2

[0051] This embodiment provides a method for preparing a highly efficient liquid fertilizer containing enzymatically hydrolyzed *Nematocystis jirovecii* to increase yield. The method involves preparing an N-carbamoyl-L-glutamic acid-chitosan graft copolymer: 10g of chitosan is weighed and added to a three-necked flask, followed by 200mL of a 2% (w / w) acetic acid solution. The solution is mechanically stirred under nitrogen protection until completely dissolved. Then, a 1mol / L sodium hydroxide solution is added dropwise while stirring to adjust the pH of the chitosan solution to 6.0. 12g of N-carbamoyl-L-glutamic acid is dissolved in 100mL of deionized water, and the pH is adjusted to 6.0 with a 1mol / L sodium hydroxide solution. Then, 1.2g of 1,3-dimethylaminopropyl-3-ethylcarbodiimide hydrochloride and 0.6g of... N-hydroxysuccinimide was activated at 24°C for 30 minutes. The activated solution was slowly added dropwise to the chitosan solution, and the reaction was carried out at 25°C for 6 hours. After the reaction was completed, 1 mol / L sodium hydroxide solution was added to adjust the pH to 7.0. The reaction mixture was poured into 500 mL of anhydrous ethanol to precipitate the product. The precipitate was collected by filtration, washed three times with 80% ethanol aqueous solution, and dried to constant weight in a vacuum drying oven at 38°C to obtain the product.

[0052] Preparation of selenocysteine-alginic acid nanospheres: 5g of sodium alginate was dissolved in 200mL of deionized water and mechanically stirred in a 38℃ water bath for 20 minutes to form an aqueous phase; 2g of selenocysteine ​​was dissolved in 50mL of deionized water, and 0.1mol / L hydrochloric acid was added to adjust the pH to 3.8 to form an inner aqueous phase; the inner aqueous phase was added dropwise to the aqueous phase and homogenized at 8000 rpm for 5 minutes to form a pre-emulsion; the pre-emulsion was added dropwise to 400mL of liquid paraffin containing 3% calcium chloride and 1% sorbitan monooleate and mechanically stirred at 400 rpm for 20 minutes to crosslink and solidify; 400mL of n-hexane was added, and after standing and separation, the supernatant was discarded, the lower precipitate was collected, and washed three times alternately with acetone and anhydrous ethanol. The precipitate was filtered through a 0.45μm microporous membrane and dried in a vacuum drying oven at 28℃ for 6 hours to obtain the product.

[0053] Preparation of liquid fertilizer: Weigh 500g of fresh *Nepenthes*, wash and mechanically crush, and perform one freeze-thaw cycle: freeze at -20℃ for 12h, then thaw at 24℃. Add deionized water to adjust the total volume of the *Nepenthes* slurry to 1800mL, and adjust the pH to 7.5 with sodium hydroxide solution. Transfer the slurry to an enzymatic hydrolysis tank, add 15g of a compound protease preparation, which consists of 7g of alkaline protease, 6g of neutral protease, and 2g of chitinase. Maintain the temperature at 48℃ and pH at 7.0, and mechanically stir for 18h for enzymatic hydrolysis. Subsequent solid-liquid separation is the same as in Example 1, yielding approximately 1300mL of enzymatically hydrolyzed *Nepenthes* liquid for later use. Add 400mL of deionized water to a preparation tank, then add 50g of urea, 30g of potassium dihydrogen phosphate, 20g of potassium sulfate, and 10g of a trace element mixture, stirring to dissolve. Add 10g of potassium humate and 5g of polyaspartic acid, and continue stirring for 20min. Add 30g of N-carbamoyl-L-glutamic acid-chitosan graft copolymer and stir for 25 minutes. Then add 20g of selenocysteine-alginic acid nanospheres and stir slowly for 8 minutes to obtain a nutrient solution. Add the enzymatically hydrolyzed nervone extract to the nutrient solution and stir to mix for 12 minutes. Add 1g of plant growth regulator and continue stirring for 15 minutes until well mixed to obtain the final product. Example 3

[0054] This embodiment provides a method for preparing a highly efficient liquid fertilizer containing enzymatically hydrolyzed *Nematocystis jirovecii* to increase yield. The method involves preparing an N-carbamoyl-L-glutamic acid-chitosan graft copolymer: 10g of chitosan is weighed and added to a three-necked flask, followed by 200mL of a 2% (w / w) acetic acid solution. The solution is mechanically stirred under nitrogen protection until completely dissolved. Then, a 1mol / L sodium hydroxide solution is added dropwise while stirring to adjust the pH of the chitosan solution to 6.5. 18g of N-carbamoyl-L-glutamic acid is dissolved in 100mL of deionized water, and the pH is adjusted to 6.5 with a 1mol / L sodium hydroxide solution. Then, 1.8g of 1,3-dimethylaminopropyl-3-ethylcarbodiimide hydrochloride and 0.9g of... N-hydroxysuccinimide was activated at 26°C for 40 minutes. The activated solution was slowly added dropwise to the chitosan solution, and the reaction was carried out at 30°C for 8 hours. After the reaction was completed, 1 mol / L sodium hydroxide solution was added to adjust the pH to 7.0. The reaction mixture was poured into 500 mL of anhydrous ethanol to precipitate the product. The precipitate was collected by filtration, washed three times with 80% ethanol aqueous solution, and dried to constant weight in a vacuum drying oven at 42°C to obtain the product.

[0055] Preparation of selenocysteine-alginic acid nanospheres: 5g of sodium alginate was dissolved in 200mL of deionized water and mechanically stirred in a 42℃ water bath for 30 minutes to form an aqueous phase; 4g of selenocysteine ​​was dissolved in 50mL of deionized water, and 0.1mol / L hydrochloric acid was added to adjust the pH to 4.2 to form an inner aqueous phase; the inner aqueous phase was added dropwise to the aqueous phase and homogenized at 10000 rpm for 3 minutes to form a primary emulsion; the primary emulsion was added dropwise to 400mL of liquid paraffin containing 5% calcium chloride and 2% sorbitan monooleate and mechanically stirred at 600 rpm for 40 minutes for crosslinking and curing; 400mL of n-hexane was added, and after standing and separation, the supernatant was discarded, the lower precipitate was collected, and washed three times alternately with acetone and anhydrous ethanol. The precipitate was filtered through a 0.45μm microporous membrane and dried in a vacuum drying oven at 32℃ for 8 hours to obtain the product.

[0056] Preparation of liquid fertilizer: Weigh 700g of fresh *Nepenthes*, wash and mechanically crush, and perform three freeze-thaw cycles: freeze at -18℃ for 16h, then thaw at 26℃. Add deionized water to adjust the total volume of the *Nepenthes* slurry to 2200mL, and adjust the pH to 8.5 with sodium hydroxide solution. Transfer the slurry to an enzymatic hydrolysis tank, add 21g of a compound protease preparation, which consists of 9g of alkaline protease, 8g of neutral protease, and 4g of chitinase. Maintain the temperature at 52℃ and pH at 8.0, and mechanically stir for 24h for enzymatic hydrolysis. Subsequent solid-liquid separation is the same as in Example 1, yielding approximately 1600mL of enzymatically hydrolyzed *Nepenthes* liquid for later use. Add 600mL of deionized water to a preparation tank, then add 100g of urea, 60g of potassium dihydrogen phosphate, 40g of potassium sulfate, and 30g of a trace element mixture, stirring to dissolve. Add 30g of potassium humate and 20g of polyaspartic acid, and continue stirring for 30min. Add 80g of N-carbamoyl-L-glutamic acid-chitosan graft copolymer and stir for 35 minutes. Then add 50g of selenocysteine-alginic acid nanospheres and stir slowly for 12 minutes to obtain a nutrient solution. Add the enzymatically hydrolyzed nervone extract to the nutrient solution and stir to mix for 18 minutes. Add 5g of plant growth regulator and continue stirring for 25 minutes until evenly mixed to obtain the final product.

[0057] Comparative Example 1

[0058] The difference between this comparative example and Example 1 is that this comparative example does not contain N-carbamoyl-L-glutamic acid-chitosan graft copolymer and selenocysteine-alginic acid nanospheres.

[0059] Comparative Example 2

[0060] The difference between this comparative example and Example 1 is that this comparative example only adds N-carbamoyl-L-glutamic acid-chitosan graft copolymer, and does not add selenocysteine-alginic acid nanospheres.

[0061] Comparative Example 3

[0062] The difference between this comparative example and Example 1 is that this comparative example uses ordinary chitosan and sodium selenite instead of N-carbamoyl-L-glutamic acid-chitosan graft copolymer and selenocysteine-alginic acid nanospheres.

[0063] The performance of the high-yield-increasing liquid fertilizer containing enzymatically hydrolyzed nervone provided in the above embodiments and comparative examples was tested using the following methods:

[0064] In accordance with national and industry standard testing specifications, the high-yield-increasing liquid fertilizer samples containing enzymatically hydrolyzed sandworms obtained in Examples 1-3 and Comparative Examples 1-3 were systematically evaluated for performance. This performance test employed a pot experiment method. Uniformly growing tomato varieties were selected as the test crop and sown in standardized seedling trays. When the seedlings reached the 4-leaf stage, robust and uniformly sized seedlings were transplanted into plastic pots containing an equal volume of mixed substrate nutrient soil (25 cm in diameter and 20 cm in height), with one seedling per pot. The experiment included seven treatments: the fertilizer prepared in Example 1, Example 2, Example 3, Comparative Example 1, Comparative Example 2, and Comparative Example 3, and a blank control group irrigated with an equal volume of water. Each treatment was replicated 10 times, for a total of 70 pots. All groups of plants were cultivated under the same environmental conditions. During cultivation, the temperature was controlled at 25-28℃ during the day and 15-18℃ at night, and the relative humidity was maintained at 60%-70%. Natural light was supplemented with artificial light to maintain a photocycle of 12 hours of light / 12 hours of darkness. Fertilization began after the seedlings had recovered from transplanting. Each fertilizer sample was diluted 500 times with deionized water and then slowly poured along the pot wall, 200 mL per pot each time. The blank control group was given an equal amount of water. A total of 3 top-dressings were applied throughout the growing season, with an interval of 15 days between each application. On the 60th day after the first fertilization, the height of the plant from the substrate surface to the growing point was measured using a measuring tape. The stem diameter of the third node above the base of the plant was measured using a digital vernier caliper. The relative chlorophyll content (SPAD value) of the fully expanded functional leaves at the top of the plant was measured using a portable chlorophyll meter. Each indicator was measured 3 times per plant, and the average value was taken. After the fruit entered the ripening stage, the number of fruits with a diameter greater than 2 cm on each plant was counted as the fruit set per plant. All ripe fruits were harvested, and the weight of each fruit was measured using a 0.1 g precision electronic balance to calculate the total yield per plant. After harvesting the fruit, samples were collected from the above-ground parts of the plant. After washing, blanching at 105℃, drying at 80℃, and pulverizing, the total nitrogen content was determined using the Kjeldahl method, the total phosphorus content using the vanadium-molybdenum yellow colorimetric method, and the total potassium content using flame photometry. The nitrogen utilization rate of the high-yield-increasing liquid fertilizer containing enzymatically hydrolyzed sandworms was calculated based on the nutrient input. Simultaneously, samples of the edible fruit parts were microwave-digested, and the selenium content was determined using atomic fluorescence spectrometry. All experimental data were recorded and imported into SPSS statistical software. One-way ANOVA was used for data processing, and Duncan's new multiple range test was used to test the significance of differences, with a significance level set at p < 0.05.

[0065] The performance test data above are shown in Table 1.

[0066] Table 1 Performance Test Results:

[0067] .

[0068] As can be seen from the above, the enzymatically hydrolyzed nervus liquid fertilizers prepared in Examples 1-3 exhibit significant advantages in promoting crop growth, increasing yield and quality, and effectively solving the technical problems of traditional fertilizers such as single function, low nutrient utilization rate, and lack of biological activity. Specifically, the plant height, stem diameter, and chlorophyll content of Examples 1-3 are significantly better than those of Comparative Examples 1-3 and the blank control. Among them, the plant height of Example 1 reached 125.6 cm and the stem diameter reached 11.5 mm, which were 27.3% and 29.2% higher than those of Comparative Example 1, respectively, indicating that it fully activated the organic active substances in the nervus. In terms of yield composition, the number of fruits per plant in Example 1 reached 25.3, and the yield per plant reached 3.85 kg, which were 39.0% and 49.2% higher than those of Comparative Example 1, proving that it achieved the yield increase target through the synergistic effect of enzymatic hydrolysis technology and functional components. In particular, regarding nutrient utilization efficiency, the nitrogen fertilizer utilization rate of Example 1 reached 58.4%, which was 38.1% higher than that of Comparative Example 1. This was attributed to the slow-release fertilizer retention function of the N-carbamoyl-L-glutamic acid-chitosan graft copolymer in Example 1 and the biostimulatory effect of the enzymatic hydrolysis of *Nematocystis caryophylla* extract. Comparative Example 1, lacking the two modified compounds, showed significantly lower performance across all indicators, confirming the core role of the functional components in the system. Although Comparative Example 2 retained the graft copolymer, the lack of selenocysteine-alginic acid nanospheres resulted in undetectable selenium content in its fruit and a lower yield than Example 1, indicating that nanospheres are indispensable for the effective supply of trace elements. Comparative Example 3 used ordinary chitosan and sodium selenite to replace the two modified compounds. While its growth and yield indicators were better than those of Comparative Example 1, which completely lacked the functional components, they were lower than those of Comparative Example 2, which contained the N-carbamoyl-L-glutamic acid-chitosan graft copolymer, and all other examples. This confirms that ordinary chitosan has limited slow-release and synergistic effects due to the lack of graft modification. Meanwhile, sodium selenite, lacking the protection of nanospheres, is easily fixed or transformed in the soil, resulting in a significantly lower bioavailability than in Examples 1-3, with the fruit selenium content only 20.4% of that in Example 1. Examples 1-3, through compound enzymatic hydrolysis technology, fully released amino acids and chitosan oligosaccharides from *Nematocystis jirovecii*, combined with the slow-release synergistic effect of graft copolymers and the precise selenium supply from nanospheres, constructing a complete nutrient-stimulation-regulation system. This successfully achieved high-efficiency yield increase and functional enhancement of the fertilizer, completely solving the technical bottlenecks mentioned in the background technology, such as low active ingredients, easy degradation of trace elements, and uncoordinated nutrient release in traditional organic fertilizers.

Claims

1. A method for preparing a yield-increasing liquid fertilizer containing enzymatically hydrolyzed sandworms, characterized in that the steps include... include: S1. Collect sandworms, wash them, crush them, and then perform freeze-thaw cycles. Adjust the pH to 7.5-8.5; The treated worm slurry was transferred to an enzymatic hydrolysis tank, a compound protease preparation was added, and the mixture was stirred. The temperature was maintained at 48-52℃ and the pH at 7.0-8.0, and enzymatic hydrolysis was performed to obtain an enzymatically hydrolyzed worm mixture. The enzymatically hydrolyzed worm mixture was then subjected to solid-liquid separation using a plate and frame filter press, and the filtrate was collected. The solid portion obtained from the solid-liquid separation was added to water, and the mixture was subjected to solid-liquid separation again using a plate and frame filter press. The two filtrates were combined to obtain the enzymatically hydrolyzed worm solution. S2. Add water to the preparation tank and stir. Then add urea, potassium dihydrogen phosphate, potassium sulfate, and trace element mixture in sequence and stir until completely dissolved. Add potassium humate and polyaspartic acid and continue stirring and mixing. Add N-carbamoyl-L-glutamic acid-chitosan graft copolymer and stir. Then add selenocysteine-alginic acid nanospheres to obtain a nutrient solution containing modified compounds. S3. Add the enzymatically hydrolyzed nervone solution to the nutrient solution containing the modified compound and stir to mix; add the plant growth regulator and continue stirring to mix. The preparation method of the N-carbamoyl-L-glutamic acid-chitosan graft copolymer includes: A1. Weigh chitosan and add it to a three-necked flask. Add acetic acid solution and stir mechanically under nitrogen protection until completely dissolved. Then, add sodium hydroxide solution dropwise while stirring to adjust the pH of the chitosan solution to 6.0-6.

5. Dissolve N-carbamoyl-L-glutamic acid in deionized water and adjust the pH to 6.0-6.5 with sodium hydroxide solution. Then add 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride and N-hydroxysuccinimide and activate at 24-26℃. A2. Add the activated N-carbamoyl-L-glutamic acid solution dropwise to the chitosan solution and react at 25-30℃. After the reaction is complete, add sodium hydroxide solution to adjust the pH to 7.0 to obtain the reaction mixture. Pour the reaction mixture into anhydrous ethanol to precipitate, filter to collect the precipitate, wash with ethanol aqueous solution, and dry in a vacuum drying oven at 38-42℃. The preparation method of the selenocysteine-alginic acid nanospheres includes: B1. Weigh out sodium alginate and dissolve it in deionized water. Stir mechanically in a water bath at 38-42℃ to form an aqueous phase. Dissolve selenocysteine ​​in deionized water and add hydrochloric acid to adjust the pH to 3.8-4.2 to form an inner aqueous phase. B2. Add the inner aqueous phase dropwise to the aqueous phase, homogenize, and form a pre-emulsion; add the pre-emulsion dropwise to liquid paraffin containing calcium chloride and sorbitan monooleate, stir mechanically, and cross-link and solidify; add n-hexane, let stand to separate into layers, discard the supernatant, collect the lower precipitate, wash with acetone and ethanol alternately, filter through a microporous membrane, and dry in a vacuum drying oven at 28-32℃.

2. The method for preparing the yield-increasing liquid fertilizer containing enzymatically hydrolyzed nervous silkworms according to claim 1, characterized in that, In step S1, the enzymatic hydrolysis time is 18-24 hours; the number of freeze-thaw cycles is 1-3 times; the conditions for the freeze-thaw cycle are: freezing at -20~-18℃ for 12-16 hours and thawing at 24-26℃; the complex protease preparation contains protease and chitinase.

3. The method for preparing the yield-increasing liquid fertilizer containing enzymatically hydrolyzed nervous silkworms according to claim 1, characterized in that, In step S2, continue stirring for 20-30 minutes.

4. The method for preparing the yield-increasing liquid fertilizer containing enzymatically hydrolyzed sandworms according to claim 1, characterized in that, In step A1, the activation time is 30-40 minutes at 24-26℃.

5. The method for preparing the yield-increasing liquid fertilizer containing enzymatically hydrolyzed nervous silkworms according to claim 1, characterized in that, In step A2, the reaction time is 6-8 hours at 25-30℃.

6. The method for preparing the yield-increasing liquid fertilizer containing enzymatically hydrolyzed nervous silkworms according to claim 1, characterized in that, In step B1, the mechanical stirring time in a water bath at 38-42℃ is 20-30 minutes.

7. The method for preparing the yield-increasing liquid fertilizer containing enzymatically hydrolyzed nervous silkworms according to claim 1, characterized in that, In step B2, the homogenization time is 5-10 min; the drying time in a vacuum drying oven at 28-32℃ is 6-8 h.

8. A yield-increasing liquid fertilizer containing enzymatically hydrolyzed nervone, prepared by the method for preparing the liquid fertilizer containing enzymatically hydrolyzed nervone according to any one of claims 1-7, characterized in that, The raw materials include the following parts by weight: 50-70 parts fresh sandworms; 3-8 parts N-carbamoyl-L-glutamic acid-chitosan graft copolymer; 2-5 parts selenocysteine-alginic acid nanospheres; 5-10 parts urea; 3-6 parts potassium dihydrogen phosphate; 2-4 parts potassium sulfate; 1-3 parts trace element mixture; 0.5-2 parts polyaspartic acid; 1-3 parts potassium humate; and 0.1-0.5 parts plant growth regulator.