A complex bio-stimulating hormone based on organic polypeptide enzyme class, and a preparation method and application thereof

Abstract

The application provides a kind of complex biological stimulants based on organic polypeptide enzyme and its preparation method and application, the complex biological stimulants are prepared by precise process with the core effective components of complex organic polypeptide liquid, complex enzyme active substance, amino acid component, trace element chelate, plant source bioactive substance and natural plant growth regulating substance, and auxiliary agent is matched.The synergistic effect of each component is realized by innovative design of polypeptide molecular weight gradient ratio, enzyme activity synergistic system and multi-component targeting ratio;The application of the complex biological stimulants is suitable for field crops, economic crops and special crops, and the targeting application scheme is designed according to different needs such as stress resistance, flower protection and yield increase, which can significantly improve the growth performance of crops and the quality of agricultural products, and the whole biological source component has no residue, is environment-friendly, and has wide agricultural application prospect.

Description

Technical Field

[0001] This invention relates to the field of agricultural biostimulant technology, specifically to a composite biostimulant based on organic polypeptide enzymes, its preparation method, and its application. Background Technology

[0002] With the deepening of the concept of green agricultural development, biostimulants have become an important direction for replacing traditional chemical fertilizers due to their ability to enhance crop stress resistance, improve the quality of agricultural products, and be environmentally friendly. However, existing biostimulant products still face many technical bottlenecks, making it difficult to meet the needs of modern precision planting.

[0003] In terms of component design, most products simply mix single functional ingredients such as peptides, amino acids, and trace elements, lacking a scientifically sound synergistic formulation logic. Some products use peptides from a single source with a wide molecular weight distribution, failing to accurately match the physiological needs of crops at different growth stages. Enzyme components often lack a clearly defined ratio of activity units, leading to poor synergistic effects with other components, or even functional antagonism. Trace elements mostly exist in inorganic or single chelated forms, resulting in low crop absorption rates and a tendency to react and precipitate with peptides and enzymes, reducing product effectiveness. The ambiguous proportions of plant growth regulators often cause excessive vegetative growth or poor crop development, further limiting the overall product efficacy.

[0004] At the preparation process level, existing technologies suffer from significant limitations due to their crudeness. The peptide enzymatic hydrolysis process lacks precise molecular weight control, and the purification steps are simplistic, resulting in insufficient purity of active ingredients. Enzyme components lack targeted stabilization and protection technologies, or rely solely on a single encapsulation method, making enzyme activity susceptible to inactivation due to temperature and pH fluctuations during storage, transportation, and field application. Activity loss typically exceeds 30% after six months of storage at room temperature. Key parameters such as the molar ratio, pH, and temperature in the trace element chelation reaction are not clearly defined, leading to low chelation rates. Furthermore, the mixing order and stirring parameters during compound formulation are arbitrary, resulting in uneven dispersion of components, poor product uniformity and repeatability, and significant quality fluctuations during mass production.

[0005] In terms of application scenarios, existing products mostly provide general application solutions, without designing targeted solutions for different crop types, growth stages, and functional needs (such as stress resistance, flower and fruit promotion, and quality and yield improvement). They only provide general dilution ratios without adjusting the application timing and complementary materials based on component characteristics and crop physiological rhythms. This results in the active ingredients not being able to fully exert their effects, leading to prominent problems such as unstable stress resistance, limited yield improvement, and insignificant quality improvement.

[0006] Furthermore, some products pose a risk of ingredient residues or lead to resource waste due to a lack of synergistic mechanisms, which does not meet the requirements of sustainable agricultural development. Therefore, developing a compound biostimulant with precise component ratios, stable active ingredients, controllable preparation processes, and targeted application schemes to address the pain points of existing products such as poor synergy, unstable effects, and limited applicability is of significant practical importance and market value for promoting agricultural quality and efficiency improvement and achieving green and low-carbon development. Summary of the Invention

[0007] Purpose of the invention: The purpose of this invention is to overcome the shortcomings of the prior art and provide a compound biostimulant based on organic polypeptide enzymes, its preparation method and application. Through precise component ratio design and systematic preparation process, the synergistic effect of each active ingredient is achieved, thereby improving product stability and application effect.

[0008] The technical solution of this invention: A complex biostimulant based on organic polypeptide enzymes, Based on 100% by weight, this compound biostimulant based on organic polypeptide enzymes contains the following components: 20-40% compound organic polypeptide liquid, 8-15% compound enzyme active ingredients, 15-25% amino acid components, 5-12% trace element chelates, 8-15% plant-derived bioactive substances, 0.1-0.5% natural plant growth regulators, and the balance being adjuvants and deionized water.

[0009] Furthermore, the preparation of the composite organic polypeptide liquid includes the following steps: Raw material pretreatment: Crush the plant protein raw material to 60-80 mesh, defatt the animal protein raw material at 60-70℃ for 2-3 hours, and mix them in proportion to obtain mixed protein raw material; Compound enzymatic hydrolysis: Add deionized water to the mixed protein raw materials, adjust the pH to 6.5-7.5, and the temperature to 45-55℃. Then add 1.5-2.5% of the weight of the mixed protein raw materials with compound protease. Hydrolyze for 6-8 hours, adjusting the pH to 6.5-7.5 every 1-2 hours. Control the molecular weight distribution of plant-derived hydrolyzed peptides, animal-derived hydrolyzed peptides, and microbial fermentation peptides during the hydrolysis process. Enzyme inactivation and purification: Heat to 85-90℃ and maintain for 15 minutes to inactivate enzymes. Purify by filtration through an ultrafiltration membrane with a molecular weight cutoff of 3000 Da. Collect the retentate containing plant-derived enzymatically hydrolyzed peptides, animal-derived enzymatically hydrolyzed peptides, and the permeate containing microbial fermentation peptides. Concentrate them separately under vacuum to a solid content of 35-40%, and then mix them in proportion to obtain a composite organic peptide solution.

[0010] Furthermore, the weight ratio of the plant-derived enzymatically hydrolyzed polypeptide, the animal-derived enzymatically hydrolyzed polypeptide, and the microbial fermentation polypeptide is (2-4):(1-3):(1-2).

[0011] Furthermore, the preparation method of the complex enzyme active substance includes the following steps: Enzyme activity protection: Protease, cellulase, superoxide dismutase, peroxidase, and chitinase were dissolved separately in buffer solutions containing 0.1-0.3 wt% trehalose and 0.05-0.1 wt% ascorbic acid to prepare protease buffer, cellulase buffer, superoxide dismutase buffer, peroxidase buffer, and chitinase buffer, respectively. The pH of the protease buffer was adjusted to 7.0-7.5, the cellulase buffer to 4.8-5.5, the superoxide dismutase buffer to 7.8-8.2, the peroxidase buffer to 6.0-6.5, and the chitinase buffer to 5.0-5.5 for later use. Microcapsule encapsulation: Using a chitosan-sodium alginate mixed solution as the wall material solution, the above-mentioned protease buffer, cellulase buffer, superoxide dismutase buffer, peroxidase buffer, and chitinase buffer were mixed with the wall material solution, and enzyme microcapsules were prepared by spray drying. The spray drying inlet temperature was 160-180℃ and the outlet temperature was 80-90℃. The encapsulation rate was ≥85%, the enzyme activity retention rate was ≥90%, and the microcapsule particle size was 1-10μm, thus obtaining a composite enzyme active product.

[0012] Furthermore, the weight ratio of the protease, cellulase, superoxide dismutase, peroxidase, and chitinase is (2-4):(1-3):(0.5-1.5):(1-2):(0.5-1), and the protease activity is ≥50000 U / g, cellulase ≥30000 U / g, superoxide dismutase ≥20000 U / g, peroxidase ≥25000 U / g, and chitinase ≥15000 U / g.

[0013] Furthermore, the amino acid component is a mixture of proline, glutamic acid, and complex amino acids; the weight ratio of proline, glutamic acid, and complex amino acids is (2-3):(3-4.5):(5-10).

[0014] Furthermore, the trace element chelate is one or a mixture of zinc-ethylenediaminetetraacetic acid / amino acid chelate, boron-plant polysaccharide chelate, calcium-polysaccharide alcohol chelate, iron-citrate chelate, and manganese-ethylenediaminetetraacetic acid chelate.

[0015] Furthermore, the plant-derived bioactive substance is a mixture of seaweed extract, humic acid / fulvic acid, and chitosan / chitooligosaccharide, with a weight ratio of (1.5-2):(1.5-1.8):1; the natural plant growth regulator is a mixture of brassinolide, methyl jasmonic acid, and salicylic acid, with a weight ratio of (1-1.5):(2-2.5):(3-3.5).

[0016] Furthermore, the adjuvants include glycerin, propylene glycol, sodium citrate, and potassium sorbate.

[0017] This invention provides a method for preparing a complex biostimulant based on organic polypeptide enzymes, comprising the following steps: (1) Segmented mixing: Add the compound organic polypeptide liquid, amino acid components, and plant-derived bioactive substances to the mixing tank, stir at 200-300 rpm, and stir at 40-45℃ for 30-45 minutes to obtain phase A; dissolve the trace element chelate and natural plant growth regulator in part of the deionized water, and stir until completely dissolved to obtain phase B; disperse the compound enzyme active substances in the adjuvant and the remaining deionized water, and sonicate for 10-15 minutes at 200-300W to obtain phase C; (2) Sequential mixing: Add phase B slowly to phase A at a rate of 5-10 ml / min, while increasing the stirring speed to 300-500 rpm, and mix for 15-25 minutes; then add phase C at a rate of 10-15 ml / min, reduce the stirring speed to 150-200 rpm, and mix for 25-30 minutes; finally add the additives and continue stirring for 10-20 minutes to obtain the mixture. (3) Homogenization and aging: The mixture is homogenized 2-3 times under a pressure of 30-40MPa using a high-pressure homogenizer, with each homogenization time being 5-10 minutes, so that the product particle size is concentrated at 1-5μm; then it is aged for 24-48 hours at 20-25℃ and 50-60% humidity to obtain the compound biostimulant based on organic polypeptide enzymes.

[0018] This invention provides an application of a compound biostimulant based on organic polypeptide enzymes in crops, applicable to field crops, cash crops, and specialty crops. It employs targeted application schemes tailored to the different growth stages and functional needs of various crops, specifically including: (1) Cold resistance / drought resistance / salt resistance: Suitable for field crops. Apply 3-5 days before adverse stress by foliar spraying. Dilute 600-800 times and use 30-40 kg per mu. Use with 50 g of potassium dihydrogen phosphate in 30 kg of water. (2) Promote flowering and fruit setting: Applicable to flowering and fruiting economic crops. Apply once each during the flower bud differentiation period, the initial flowering period, and the young fruit stage. For foliar spraying, dilute 1000-1200 times and use 25-30 kg per mu. Use with 15 g boron fertilizer per 30 kg of water. (3) Improve quality and increase yield: Applicable to grapes, cucumbers and tea. During the fruit enlargement period / vigorous growth period, the foliar spray dilution ratio is 800-1000 times. During the color change period / 20 days before harvest, the foliar spray dilution ratio is 1200-1500 times. The dosage is 20-25 kg per mu. (4) Seed treatment application: Applicable to all crops. Soak seeds in a 200-300 times diluted solution for 2-4 hours. Use 10-20 ml of the stock solution per kilogram of seeds. After soaking, air dry before sowing. Beneficial effects: The compound biostimulant based on organic polypeptide enzymes of the present invention has the following beneficial effects: 1. Significant synergistic effect of components: By combining compound organic polypeptide liquid with five highly active enzymes and chelated trace elements, a core synergistic system is constructed, which effectively improves crop stress resistance, yield and quality, and solves the problem of poor synergistic effect of existing products.

[0019] 2. High enzyme activity stability: The dual stabilization technology of buffer protection and microencapsulation ensures long-term stable enzyme activity and avoids enzyme inactivation during application.

[0020] 3. High efficiency in the absorption of trace elements: The absorption rate of chelated trace elements is improved, and they will not antagonize other components. The fully biodegradable ligands are environmentally friendly and leave no residue.

[0021] 4. High controllability of the preparation process: The combination of processes such as segmented mixing and sequential feeding ensures product uniformity, minimizes quality fluctuations in batch production, and meets the requirements of industrial scale-up.

[0022] 5. Wide range of applications and strong targeting: Different application programs are designed for different crops and different growth stages, which can meet various needs such as stress resistance, flower and fruit promotion, quality improvement and yield increase, and have strong market applicability. Detailed Implementation

[0023] The present invention will be described below with reference to specific embodiments. It should be noted that the following embodiments are examples of the present invention and are used only to illustrate the invention, not to limit it. Other combinations and various modifications within the scope of the present invention can be made without departing from its spirit or scope.

[0024] Alkaline protease, neutral protease, protease, and cellulase were purchased from Leshengyuan Biotechnology (Nanjing) Co., Ltd.; superoxide dismutase was purchased from Guangzhou Huayu Biotechnology Co., Ltd.; peroxidase and chitinase were purchased from Wuhan Lanabai Pharmaceutical Chemical Co., Ltd.; and compound amino acids were purchased from Shaanxi Chenming Biotechnology Co., Ltd., item number 122401.

[0025] Unless otherwise specified, all chemical reagents used in this invention are commercially available analytical grade reagents.

[0026] Zinc-ethylenediaminetetraacetic acid chelate: 10g of zinc sulfate was dissolved in 100mL of deionized water, and 14.6g of disodium ethylenediaminetetraacetate was dissolved in 100mL of deionized water. The mixture was slowly mixed and the pH was adjusted to 6. The mixture was heated to 65℃ and stirred for 2 hours. After filtration and drying, zinc-ethylenediaminetetraacetic acid chelate was obtained.

[0027] Boron-plant polysaccharide chelates: Add 5g of seaweed polysaccharide to 100mL of deionized water and stir at 55℃ for 30 minutes until completely dissolved to prepare a polysaccharide solution; dissolve 5g of boric acid in 50mL of deionized water and slowly add it to the polysaccharide solution, adjust the pH to 6.5, and stir at 60℃ for 3 hours; add 2 times the volume of anhydrous ethanol to precipitate, filter and collect the precipitate, and vacuum dry at 60℃ for 8 hours to obtain boron-plant polysaccharide chelate.

[0028] Calcium-polysaccharide chelates: Add 11.1g of calcium chloride to 200mL of deionized water and stir to dissolve. Then add 18.2g of mannitol and 18.2g of sorbitol and stir at 45℃ until completely dissolved. Adjust the pH to 6.2, raise the temperature to 75℃, and keep the reaction at this temperature for 3.5 hours. Concentrate under reduced pressure to a solid content of 50% and freeze-dry (-40℃, vacuum degree ≤10Pa) to obtain calcium-polysaccharide chelate.

[0029] Iron-citrate chelate: The process was carried out in the dark. 27.8g of ferrous sulfate and 0.3g of ascorbic acid were added to 300mL of deionized water and stirred to dissolve. Then 58.8g of sodium citrate was added. The pH was adjusted to 5.5 and the reaction was stirred at 55℃ for 2 hours. The mixture was purified by ion exchange resin, vacuum concentrated to a solid content of 40%, and spray dried (inlet air 160℃, outlet air 80℃) to obtain iron-citrate chelate.

[0030] Manganese-ethylenediaminetetraacetic acid chelate: Dissolve 16.9 g of manganese sulfate and 37.2 g of disodium ethylenediaminetetraacetate in 100 mL of deionized water. Slowly add the disodium ethylenediaminetetraacetate solution to the manganese sulfate solution while stirring. Adjust the pH to 6.5 and keep the reaction at 60 °C for 2.5 hours. Cool to room temperature and crystallize at 4 °C for 12 hours. Filter, wash, and dry in a forced-air environment at 55 °C for 6 hours to obtain manganese-ethylenediaminetetraacetic acid chelate.

[0031] Example 1 The preparation of the composite organic polypeptide solution includes the following steps: Raw material pretreatment: Soybean meal and cottonseed meal are crushed to 60 mesh, and fish meal and bone meal are defatted at 65°C for 2.5 hours. Then, 3 parts by weight of pretreated soybean meal, 3 parts by weight of pretreated cottonseed meal, 2 parts by weight of pretreated fish meal and 2 parts by weight of pretreated bone meal are mixed to obtain mixed protein raw materials. Compound enzymatic hydrolysis: Add 10 times the weight of deionized water to the mixed protein raw materials, set the pH to 7, and the temperature to 50℃. Then add 2% of the weight of the mixed protein raw materials as compound protease (alkaline protease: neutral protease weight ratio 3:2). Hydrolyze for 6 hours, adjusting the pH to 7 every 2 hours. Enzyme inactivation and purification: The temperature was raised to 85℃ and maintained for 15 minutes to inactivate the enzyme. The solution was then purified by filtration through an ultrafiltration membrane with a molecular weight cutoff of 3000 Da. The retentate containing plant-derived enzymatically hydrolyzed peptides and animal-derived enzymatically hydrolyzed peptides and the permeate containing microbial fermentation peptides were collected. The solutions were then vacuum concentrated to a solid content of 35% and mixed with plant-derived enzymatically hydrolyzed peptides, animal-derived enzymatically hydrolyzed peptides, and microbial fermentation peptides in a weight ratio of 1:1:1 to obtain a composite organic peptide solution. A method for preparing complex enzyme active substances includes the following steps: Enzyme activity protection: Protease, cellulase, superoxide dismutase, peroxidase, and chitinase were dissolved in 0.1 mol / L phosphate buffer containing 0.2 wt% trehalose and 0.08 wt% ascorbic acid at a weight ratio of 3:2:1:1.5:0.5 to prepare protease buffer (3 mg / mL), cellulase buffer (2 mg / mL), superoxide dismutase buffer (1 mg / mL), peroxidase buffer (1.5 mg / mL), and chitinase buffer (0.5 mg / mL). The pH of the protease buffer was then adjusted to 7, the cellulase buffer to 5, the superoxide dismutase buffer to 8, the peroxidase buffer to 6.5, and the chitinase buffer to 5.2 using pH adjusters. Microcapsule encapsulation: Chitosan was dissolved in 1.5% dilute acetic acid solution to prepare a 1.5 wt% chitosan solution; sodium alginate was dissolved in deionized water and heated and stirred at 55°C to prepare a 2.0 wt% sodium alginate solution; the two solutions were mixed at a weight ratio of chitosan to sodium alginate of 1:2.5 and stirred evenly to obtain a wall material solution; The above-mentioned protease buffer, cellulase buffer, superoxide dismutase buffer, peroxidase buffer, and chitinase buffer were mixed to obtain an enzyme mixture. The enzyme mixture was then mixed with the wall material solution at a mass ratio of 1:2 (enzyme mixture: wall material solution), and emulsified by high-speed stirring at 1000 rpm for 20 minutes to form a homogeneous oil-water emulsion. Enzyme microcapsules were prepared by spray drying at an inlet air temperature of 170℃ and an outlet air temperature of 85℃. The encapsulation rate was 90%, the enzyme activity retention rate was 93%, and the microcapsule particle size was 5 μm, thus obtaining a composite enzyme active product. Raw material ratio (100% by weight): 30% compound organic polypeptide solution; 10% complex enzyme active ingredients; Amino acid composition 20% (proline:glutamic acid:complex amino acid weight ratio 2.5:3.5:8); Trace element chelates 8% (zinc-ethylenediaminetetraacetic acid chelate: boron-plant polysaccharide chelate: calcium-polysaccharide chelate: iron-citrate chelate: manganese-ethylenediaminetetraacetic acid chelate weight ratio 3:2:6:2:1.5); 12% plant-derived bioactive substances (seaweed extract: humic acid: chitosan oligosaccharide weight ratio 1.8:1.6:1); 0.3% natural plant growth regulators (brassinolide: methyl jasmonate: salicylic acid weight ratio 1.2:2.3:3.2); Additives 7.7% (6% glycerin, 1.5% sodium citrate, 0.2% potassium sorbate); 12% deionized water; A method for preparing a complex biostimulant based on organic polypeptide enzymes includes the following steps: (1) Segmented mixing: Add the compound organic polypeptide liquid, amino acid components, and plant-derived bioactive substances to the mixing tank, stir at 250 rpm, and stir at 40°C for 35 minutes to obtain phase A; dissolve the trace element chelate and natural plant growth regulator in one-third of the deionized water and stir until completely dissolved to obtain phase B; disperse the compound enzyme active substances in glycerol and one-third of the deionized water, and sonicate for 10 minutes at 250W to obtain phase C; (2) Sequential mixing: Add phase B slowly to phase A at a rate of 10 ml / min, while increasing the stirring speed to 400 rpm and mixing for 20 minutes; then add phase C at a rate of 10 ml / min, reduce the stirring speed to 150 rpm and mix for 30 minutes; finally add glycerol, sodium citrate, potassium sorbate and one-third deionized water, and continue stirring for 20 minutes to obtain the mixture; (3) Homogenization and aging: The mixture was homogenized twice under a pressure of 35 MPa by a high-pressure homogenizer, with each homogenization time of 8 minutes, so that the particle size of the product was concentrated at 2.5 μm; then it was aged for 48 hours at 20°C and 55% humidity to obtain the compound biostimulant based on organic polypeptide enzymes.

[0032] Example 2 The difference between this embodiment and Embodiment 1 is that: Preparation of compound organic polypeptide liquid: 2 parts by weight of pretreated soybean meal, 4 parts by weight of pretreated cottonseed meal, 1 part by weight of pretreated fish meal, and 3 parts by weight of pretreated bone meal were mixed; the weight ratio of plant-derived enzymatically hydrolyzed polypeptide, animal-derived enzymatically hydrolyzed polypeptide, and microbial fermented polypeptide was 2:1:1. Preparation of compound enzyme active ingredients: When encapsulating microcapsules, the mass ratio of enzyme mixture to wall material solution is 1:1.8.

[0033] Example 3 The difference between this embodiment and Embodiment 1 is that: Preparation of compound organic polypeptide liquid: The weight ratio of plant-derived enzymatically hydrolyzed polypeptides, animal-derived enzymatically hydrolyzed polypeptides, and microbial fermented polypeptides is 2:2:2; Raw material ratio: 30% compound organic polypeptide solution; 10% complex enzyme active ingredients; Amino acid composition 20% (proline:glutamic acid:complex amino acid weight ratio 2.5:3.5:8); Trace element chelates 9% (zinc-ethylenediaminetetraacetic acid chelate: boron-plant polysaccharide chelate: calcium-polysaccharide chelate: iron-citrate chelate: manganese-ethylenediaminetetraacetic acid chelate weight ratio 4:2:5:2:1); 11% plant-derived bioactive substances (seaweed extract: humic acid: chitosan oligosaccharide weight ratio 1.5:1.5:1). 0.2% natural plant growth regulators (brassinolide: methyl jasmonate: salicylic acid weight ratio 1:2:3); Additives 7.8% (6% glycerin, 1.5% sodium citrate, 0.3% potassium sorbate); 12% deionized water.

[0034] Example 4 The difference between this embodiment and Embodiment 1 is that: Preparation of compound organic polypeptide liquid: The weight ratio of plant-derived enzymatically hydrolyzed polypeptide, animal-derived enzymatically hydrolyzed polypeptide, and microbial fermentation polypeptide is 1:3:0.5.

[0035] Example 5 The difference between this embodiment and Embodiment 1 is that: The preparation of complex enzyme active substances involves a weight ratio of protease, cellulase, superoxide dismutase, peroxidase, and chitinase of 5:2:1.5:1:0.5.

[0036] Comparative Example 1 The difference between this embodiment and Embodiment 1 is that: In the preparation of complex enzyme active substances, the "microcapsule encapsulation" step is omitted, and only enzyme activity protection treatment is performed.

[0037] Comparative Example 2 The difference between this embodiment and Embodiment 1 is that: The trace element chelate 8% (zinc-ethylenediaminetetraacetic acid / amino acid chelate: boron-plant polysaccharide chelate: calcium-polysaccharide chelate: iron-citrate chelate: manganese-ethylenediaminetetraacetic acid chelate weight ratio 3:2:6:2:1.5) was replaced with inorganic 8% (zinc sulfate, boric acid, calcium chloride, ferrous sulfate, manganese sulfate weight ratio 3:2:6:2:1.5), while the remaining raw materials and proportions remained unchanged; during compound preparation, the inorganic trace elements were directly dissolved in deionized water to prepare phase B.

[0038] The following performance tests were performed on the compound biostimulants obtained in Examples 1-5 and Comparative Examples 1-2: 1. Basic product performance testing (1) Enzyme activity retention rate test Test subjects: Protease, cellulase, superoxide dismutase, peroxidase, and chitinase in complex enzyme active substances; Test methods: The corresponding international standard methods for each enzyme were used (protease: Folin-phenol method; cellulase: DNS method; superoxide dismutase: nitroblue tetrazolium (NBT) photoreduction method; peroxidase: guaiacol method; chitinase: DNS method) to detect the enzyme activity at the time of product preparation and after 18 months of storage at room temperature, and the activity retention rate was calculated as (activity after storage / initial activity × 100%).

[0039] (2) Test of polypeptide content and molecular weight distribution Polypeptide content: The total polypeptide content was calculated by measuring the absorbance at a wavelength of 540 nm using the biuret method with bovine serum albumin as a standard.

[0040] Molecular weight distribution: Gel permeation chromatography (GPC) was used with a TSK gel G2000SWxl column, a mobile phase of 0.1 mol / L phosphate buffer (pH 7.0), a flow rate of 1.0 mL / min, a column temperature of 30 ℃, and a detection wavelength of 220 nm. The molecular weight distribution and proportion of the three peptides were analyzed by calibration with standard molecular weight peptides.

[0041] (3) Product stability test Accelerated stability testing: Store the product in a 54℃ constant temperature chamber for 14 days and test the enzyme activity retention rate and peptide content change rate (≤5% is acceptable).

[0042] Stability at room temperature: The product was stored in an environment of 25°C and 55% humidity for 18 months, and the above indicators were tested.

[0043] (4) Particle size distribution test A laser particle size analyzer (Malvern Mastersizer 3000) was used with deionized water as the dispersion medium. The mixture was stirred at 2000 rpm and ultrasonically dispersed for 5 minutes. The particle size distribution of the product was measured and the D50 value (particle size distribution) was recorded.

[0044] 2. Field application effect test (1) Test crops and scenarios Stress resistance test: Wheat (variety: Jimai 22), low temperature stress (-5℃, lasting for 3 days) scenario.

[0045] Flowering and fruit setting test: Apple (variety: Red Fuji, tree age 5 years), tracked throughout the entire growth period.

[0046] Quality and yield improvement test: Tomato (variety: Zhongza 105), greenhouse planting scenario.

[0047] (2) Test group Each group had 3 replicate plots, each plot with an area of ​​20m², arranged in a randomized block design. The control group received "conventional fertilization + water spraying", while the example group and the comparative group received "conventional fertilization + targeted application of the corresponding product". All other field management practices were the same.

[0048] (3) Test indicators and methods Stress resistance indicators: Seven days after low temperature stress, wheat survival rate (number of surviving plants / total number of plants × 100%), leaf chlorophyll content (SPAD method), and root activity (TTC method) were measured.

[0049] Indicators for promoting flowering and fruit setting: Apple fruit setting rate (number of fruits set / number of flowers × 100%), marketable fruit rate (number of fruits with a diameter ≥ 80mm / total number of fruits × 100%), and yield per mu (total weight at harvest / plot area × 667).

[0050] Quality and yield improvement indicators: tomato yield per mu, soluble solids in fruit (handheld refractometer method), and vitamin C content (2,6-dichlorophenol titration method).

[0051] Table 1: Results of Basic Product Performance Tests

[0052] Table 2: Results of wheat stress resistance test (low temperature stress)

[0053] Table 3: Test Results of Apple Flower Promotion and Fruit Retention Effects

[0054] Table 4: Test Results of Tomato Quality Improvement and Yield Increase Effects

[0055] As shown in Tables 1-4, the compound biostimulant based on organic polypeptide enzymes of the present invention exhibits excellent performance in terms of activity stability, synergistic effect, crop stress resistance, and quality and yield improvement. Specifically, comparing Example 2 with Example 1, it can be seen that after adjusting the ratio of plant protein raw materials and the weight ratio of plant-derived, animal-derived, and microbial fermented polypeptides to 2:1:1, the product still maintains a good activity retention rate and application effect, proving that the raw material ratio and polypeptide ratio of the compound organic polypeptide liquid have flexible adaptability within a reasonable range and do not affect the core efficacy; comparing Example 3 with Example 1, it can be seen that... After optimizing the ratio of trace element chelates and the proportions of plant-derived bioactive substances and natural plant growth regulators, the crop's flowering and fruit-setting effects were stabilized, verifying the scientific adjustability of the multi-component ratios and allowing for precise adaptation to crop needs. A comparison of Example 4 and Example 1 shows that when the weight ratio of plant-derived, animal-derived, and microbial fermented peptides was adjusted to 1:3:0.5 (close to the lower limit of the ratio), although the performance was still better than the comparative example, it was slightly lower than that of Example 1. This demonstrates that the three peptides need to be within a reasonable ratio range to maximize the synergistic effect of the molecular weight gradient. Excessive animal-derived peptides or insufficient microbial fermented peptides can lead to adverse reactions. Fermented peptides weaken the synergistic effect of rapid stress resistance and long-term nutrient supply. A comparison of Example 5 and Example 1 shows that the weight ratio of the five enzymes, including protease and cellulase, exceeded the reasonable limit (adjusted to 5:2:1.5:1:0.5), leading to a decrease in the synergistic effect of enzyme activity, a slight reduction in product storage stability and application effect, proving that precise control of the enzyme component ratio is key to ensuring synergistic effects. A comparison of Comparative Example 1 and Example 1 shows that because the compound enzyme actives were not microencapsulated, the enzyme activity retention rate was only 62.3% after 18 months of storage at room temperature, resulting in a decrease in wheat survival rate. The fruit setting rate of apples and the yield of tomatoes were significantly lower than those in Example 1, proving that microencapsulation technology is the core technology to ensure the long-term stability of enzyme activity and avoid inactivation during application. Without this technology, the efficacy requirements of crops throughout their entire growth period cannot be met. Comparing Comparative Example 2 with Example 1, it can be seen that after replacing chelated trace elements with inorganic forms, the absorption rate of trace elements by crops decreased significantly, and the stress resistance, yield, and quality indicators were all weaker than those in Example 1. This proves that chelated trace elements can effectively avoid antagonism with other components and improve absorption efficiency, which is an important foundation for achieving synergistic effects of multiple components and ensuring the application effect of products.

[0056] This invention can also be implemented in various other ways. Without departing from the spirit and essence of this invention, those skilled in the art can make various corresponding changes and modifications according to this invention, but these corresponding changes and modifications should all fall within the protection scope of the appended claims.

Claims

1. A complex biostimulant based on organic polypeptide enzymes, characterized in that, Based on 100% by weight, this compound biostimulant based on organic polypeptide enzymes contains the following components: 20-40% compound organic polypeptide liquid, 8-15% compound enzyme active ingredients, 15-25% amino acid components, 5-12% trace element chelates, 8-15% plant-derived bioactive substances, 0.1-0.5% natural plant growth regulators, and the balance being adjuvants and deionized water.

2. The compound biostimulant based on organic polypeptide enzymes according to claim 1, characterized in that, The preparation of the composite organic polypeptide solution includes the following steps: Raw material pretreatment: Crush the plant protein raw material to 60-80 mesh, defatt the animal protein raw material at 60-70℃ for 2-3 hours, and mix them in proportion to obtain mixed protein raw material; Compound enzymatic hydrolysis: Add deionized water to the mixed protein raw materials, adjust the pH to 6.5-7.5, and the temperature to 45-55℃. Then add 1.5-2.5% of the weight of the mixed protein raw materials with compound protease. Hydrolyze for 6-8 hours, adjusting the pH to 6.5-7.5 every 1-2 hours. Control the molecular weight distribution of plant-derived hydrolyzed peptides, animal-derived hydrolyzed peptides, and microbial fermentation peptides during the hydrolysis process. Enzyme inactivation and purification: Heat to 85-90℃ and maintain for 15 minutes to inactivate enzymes. Purify by ultrafiltration membrane filtration. Collect the retentate containing plant-derived enzymatically hydrolyzed peptides and animal-derived enzymatically hydrolyzed peptides, and the permeate containing microbial fermentation peptides. Concentrate them separately under vacuum to a solid content of 35-40%, and then mix them in proportion to obtain a composite organic peptide solution.

3. The compound biostimulant based on organic polypeptide enzymes according to claim 2, characterized in that, The weight ratio of the plant-derived enzymatically hydrolyzed polypeptide, the animal-derived enzymatically hydrolyzed polypeptide, and the microbial fermentation polypeptide is (2-4):(1-3):(1-2).

4. The compound biostimulant based on organic polypeptide enzymes according to claim 1, characterized in that, The preparation method of the complex enzyme active substance includes the following steps: Enzyme activity protection: Protease, cellulase, superoxide dismutase, peroxidase, and chitinase were dissolved separately in buffer solutions containing 0.1-0.3 wt% trehalose and 0.05-0.1 wt% ascorbic acid to prepare protease buffer, cellulase buffer, superoxide dismutase buffer, peroxidase buffer, and chitinase buffer, respectively. The pH of the protease buffer was adjusted to 7.0-7.5, the cellulase buffer to 4.8-5.5, the superoxide dismutase buffer to 7.8-8.2, the peroxidase buffer to 6.0-6.5, and the chitinase buffer to 5.0-5.5 for later use. Microcapsule encapsulation: Using a chitosan-sodium alginate mixed solution as the wall material solution, the above-mentioned protease buffer, cellulase buffer, superoxide dismutase buffer, peroxidase buffer, and chitinase buffer were mixed with the wall material solution, and enzyme microcapsules were prepared by spray drying. The spray drying inlet temperature was 160-180℃ and the outlet temperature was 80-90℃. The encapsulation rate was ≥85%, the enzyme activity retention rate was ≥90%, and the microcapsule particle size was 1-10μm, thus obtaining a composite enzyme active product.

5. The compound biostimulant based on organic polypeptide enzymes according to claim 4, characterized in that, The weight ratio of the protease, cellulase, superoxide dismutase, peroxidase, and chitinase is (2-4):(1-3):(0.5-1.5):(1-2):(0.5-1), and the protease activity is ≥50000 U / g, cellulase ≥30000 U / g, superoxide dismutase ≥20000 U / g, peroxidase ≥25000 U / g, and chitinase ≥15000 U / g.

6. The compound biostimulant based on organic polypeptide enzymes according to claim 1, characterized in that, The amino acid composition is a mixture of proline, glutamic acid, and complex amino acids; the weight ratio of proline, glutamic acid, and complex amino acids is (2-3):(3-4.5):(5-10).

7. The composite biostimulant based on organic polypeptide enzymes according to claim 1, characterized in that, The plant-derived bioactive substance is a mixture of seaweed extract, humic acid / fulvic acid, and chitosan / chitooligosaccharide, with a weight ratio of (1.5-2):(1.5-1.8):1; the natural plant growth regulator is a mixture of brassinolide, methyl jasmonic acid, and salicylic acid, with a weight ratio of (1-1.5):(2-2.5):(3-3.5).

8. The compound biostimulant based on organic polypeptide enzymes according to claim 1, characterized in that, The trace element chelate is one or more of the following additives: zinc-ethylenediaminetetraacetic acid / amino acid chelate, boron-plant polysaccharide chelate, calcium-polysaccharide alcohol chelate, iron-citrate chelate, and manganese-ethylenediaminetetraacetic acid chelate.

9. The method for preparing the complex biostimulant based on organic polypeptide enzymes according to any one of claims 1-8, characterized in that, Includes the following steps: (1) Segmented mixing: Add the compound organic polypeptide liquid, amino acid components, and plant-derived bioactive substances to the mixing tank, stir at 200-300 rpm, and stir at 40-45℃ for 30-45 minutes to obtain phase A; dissolve the trace element chelate and natural plant growth regulator in part of the deionized water, and stir until completely dissolved to obtain phase B; disperse the compound enzyme active substances in the adjuvant and the remaining deionized water, and sonicate for 10-15 minutes at 200-300W to obtain phase C; (2) Sequential mixing: Add phase B slowly to phase A at a rate of 5-10 ml / min, while increasing the stirring speed to 300-500 rpm, and mix for 15-25 minutes; then add phase C at a rate of 10-15 ml / min, reduce the stirring speed to 150-200 rpm, and mix for 25-30 minutes; finally add the additives and continue stirring for 10-20 minutes to obtain the mixture. (3) Homogenization and aging: The mixture is homogenized 2-3 times under a pressure of 30-40MPa using a high-pressure homogenizer, with each homogenization time being 5-10 minutes, so that the product particle size is concentrated at 1-5μm; then it is aged for 24-48 hours at 20-25℃ and 50-60% humidity to obtain the compound biostimulant based on organic polypeptide enzymes.

10. The application of the organic polypeptide enzyme-based complex biostimulant in crops according to any one of claims 1-8.

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