Preparation method of double-source amino acid-containing organic water-soluble fertilizer and prepared water-soluble fertilizer

Abstract

The invention relates to a preparation method of a double-source amino acid-containing organic water-soluble fertilizer and the prepared water-soluble fertilizer, and belongs to the technical field of agricultural bio-fertilizers. The invention aims to solve the problems of unbalanced amino acid spectrum, easy generation of peculiar smell or heavy metal residue and low nutrient release efficiency when a single raw material is adopted for preparation. The preparation method comprises the following steps: hydrolyzing an animal-derived protein raw material with an acid solution, and adding embedded slow-release vitamin C; the method comprises the following steps: carrying out gradient heating on a plant source protein raw material by using 1.0-1.5 mol / L potassium hydroxide at 90-100 DEG C, and carrying out ultrasonic-assisted hydrolysis for 3-5 hours; mixing the two hydrolysates according to the volume ratio of 3: 7, and regulating the pH to 6.5-7.0 in stages; adding a compound enzyme preparation consisting of neutral protease, papain and trehalase in a mass ratio of 3: 2: 1 into the neutralized mixed solution, and carrying out enzymolysis at 50-55 DEG C for 6-8 hours; and sequentially carrying out ultrafiltration, adsorption decoloration and reverse osmosis concentration treatment on the enzymolysis product. The water-soluble fertilizer prepared by the method is rich in various amino acids, is quick in nutrient release, and can be used for irrigation and fertilization of crops.

Description

Technical Field

[0001] This invention belongs to the field of agricultural bio-fertilizer technology, specifically relating to a method for preparing a dual-source amino acid-containing organic water-soluble fertilizer and the resulting water-soluble fertilizer. Background Technology

[0002] In agriculture, utilizing protein resources to prepare water-soluble fertilizers containing amino acids is of great significance for providing organic nitrogen sources that crops can directly absorb and for improving the soil environment. Current preparation technologies mainly rely on the hydrolysis of single animal or plant-based raw materials, but these methods still face some unresolved issues in practical applications.

[0003] First, processes relying on a single raw material source have limitations. Typical animal-derived raw materials (such as fishmeal and fish by-products) have high levels of non-essential amino acids like glycine, proline, and hydroxyproline in their proteins, due to their rich collagen content; however, they are relatively low in tryptophan and certain sulfur-containing amino acids (such as methionine). In contrast, typical plant-derived raw materials (such as soybean meal and peanut bran) are rich in acidic amino acids like glutamic acid and aspartic acid, as well as arginine, but sulfur-containing amino acids like methionine and cysteine ​​are often considered the first limiting amino acids. This difference in composition means that if only a single source of protein is used, even with efficient hydrolysis, the resulting product's amino acid profile will be incomplete. This may result in the product failing to provide balanced amino acid nutrition to crops when used as fertilizer, thus affecting its growth-promoting effect.

[0004] Secondly, in acid hydrolysis processes using animal by-products as raw materials, strong acids such as hydrochloric acid are often used, and reactions are conducted at high temperatures for extended periods to effectively break protein peptide bonds. These harsh conditions can easily trigger side reactions, leading to the degradation of some amino acids or reactions with other components in the raw materials, generating volatile substances with unpleasant odors, such as trimethylamine, which negatively impacts the product's application experience. Furthermore, the use of chlorinated strong acids introduces a large amount of chloride ions, resulting in a high salt content in the final product. Excessive residues may not meet the low-salt or organic standards required for certain agricultural applications, and subsequent desalination treatment increases process complexity and cost.

[0005] Furthermore, alkaline hydrolysis is commonly used for plant protein raw materials. However, under alkaline conditions, the hydrolysis efficiency of plant proteins and their associated cell wall structures, such as cellulose, is sometimes less than ideal. Some proteins fail to be fully degraded into small amino acids or oligopeptides, instead existing in the hydrolysate as larger polypeptides. These macromolecules are not easily absorbed directly by crop roots, potentially affecting the immediate fertilizer effect, and subsequent enzymatic hydrolysis also faces challenges.

[0006] To address the aforementioned issues, the industry has explored approaches such as composite raw materials or improved hydrolysis conditions. For example, attempts have been made to simply mix hydrolysates from different sources to complement their amino acid composition. However, such physical mixing typically fails to achieve sufficient interaction and synergy between peptides and amino acids from different sources at the molecular level. Therefore, its effectiveness in fundamentally optimizing the amino acid balance of products, improving nutrient utilization, and simultaneously controlling off-flavors and salt residues remains limited. Furthermore, how to synergistically handle the drastically different hydrolysis characteristics and product features of animal- and plant-derived raw materials in a coherent process, effectively avoiding their respective process defects, while simultaneously ensuring product quality and environmental friendliness in the production process, remains a complex challenge in actual production. Summary of the Invention

[0007] The purpose of this invention is to address the following problems: It provides an integrated dual-source protein synergistic hydrolysis and conversion process system, aiming to systematically solve three core problems in existing technologies: imbalanced amino acid nutritional structure due to reliance on a single raw material route, incompatibility of hydrolysis processes for different raw materials, and difficulty in controlling subsequent enzymatic hydrolysis and conversion in mixed systems. First, the amino acid composition of individual animal or plant proteins varies, and direct use can easily lead to an unbalanced nutrient composition in the final product, failing to fully meet the needs of crop growth. Second, animal-derived raw materials are suitable for hydrolysis under acidic conditions, while plant-derived raw materials are more suitable for alkaline conditions. Simply combining or mixing these two processes can easily lead to mutual interference, increasing operational complexity and potentially affecting the stability of intermediate products. Furthermore, mixing two hydrolysates from different sources and with different properties results in a complex composition; without proper treatment, the efficiency of subsequent enzymatic hydrolysis and other steps will be significantly reduced, making it difficult to stably obtain the target product.

[0008] To achieve the above objectives, the present invention provides a method for preparing a dual-source amino acid-containing organic water-soluble fertilizer, comprising the following steps: Step 1: Use one of the following two acid hydrolysis processes for treatment: Sulfuric acid hydrolysis process: Animal-derived protein raw materials are mixed with sulfuric acid solution with a concentration of 1.5-2.0 mol / L and hydrolyzed at 80-90℃ for 4-6 hours to obtain animal-derived hydrolysate; during the hydrolysis process, vitamin C is added at a concentration of 0.05-0.1% of the mass of animal-derived protein raw materials. Nitric acid or mixed acid hydrolysis process: Animal-derived protein raw materials are mixed with a nitric acid solution of 5.0-7.0 mol / L or a mixed acid solution of nitric acid and hydrochloric acid, placed in a pressure-resistant closed reactor, and reacted at 120-150℃ for 2-4 hours to obtain animal-derived hydrolysate; during hydrolysis, 0.08-0.15% vitamin C by mass of animal-derived protein raw materials is added; Step 2, plant-derived alkaline hydrolysis: The plant-derived protein raw material is mixed with a potassium hydroxide solution with a concentration of 1.0-1.5 mol / L, and hydrolyzed at a gradient temperature of 90-100℃ for 3-5 hours, while ultrasonic-assisted treatment is applied to obtain the plant-derived alkaline hydrolysate. Step 3, Mixing and Neutralization: The animal-derived acid hydrolysate obtained in Step 1 and the plant-derived alkaline hydrolysate obtained in Step 2 are mixed at a volume ratio of 1:(1-3) to obtain a mixed hydrolysate; then, a buffer is used to adjust the pH of the mixed hydrolysate in stages so that its pH value finally reaches 6.5-7.0. Step 4, synergistic enzymatic hydrolysis: Add a complex enzyme preparation consisting of neutral protease, papain and trehalase to the neutralized mixed hydrolysate obtained in step 3, and enzymatically hydrolyze for 6-8 hours at 50-55℃; the mass ratio of neutral protease, papain and trehalase in the complex enzyme preparation is 3:2:1. Step 5, Purification and Concentration: The product after enzymatic hydrolysis in step 4 is subjected to ultrafiltration, adsorption decolorization and reverse osmosis concentration in sequence to obtain the dual-source amino acid-containing organic water-soluble fertilizer.

[0009] Preferably, the animal-derived protein raw materials of the present invention can be selected from aquatic by-products such as fish meal, shrimp meal, and fish scales and viscera.

[0010] Furthermore, in step 1, vitamin C is added within 10-60 minutes after the start of hydrolysis, when the system temperature reaches 70-80°C. The vitamin C added in step 1 is a slow-release vitamin C that has undergone encapsulation treatment. The encapsulation wall material of the slow-release vitamin C is selected from at least one of sodium alginate, chitosan, and ethyl cellulose, and its encapsulation rate is not less than 85%. The problem addressed: Existing technology knows that vitamin C can inhibit the Maillard reaction, but in a dynamic high-temperature acid hydrolysis system, when should it be added to maximize the retention of amino nitrogen (>92%) and minimize damage to target amino acids (such as tryptophan)? Adding it too early may result in premature consumption, while adding it too late will result in poor inhibitory effects.

[0011] Further, in step 2, the gradient heating specifically involves maintaining the temperature at 90-95℃ for the first two-thirds of the hydrolysis time, and then increasing the temperature to 98-100℃ for the last one-third of the hydrolysis time. The ultrasonic assistance is applied at a frequency of 40kHz and a power of 100W, using a pulsed pattern of 5 seconds of operation followed by 5 seconds of interval. The problem addressed: Existing technologies disclose that ultrasound can improve protein extraction rates, but in the highly chemical environment of alkaline hydrolysis, how to coordinate thermochemical hydrolysis and physical cavitation to improve efficiency while avoiding excessive hydrolysis that produces bitter peptides and large amounts of free ammonia remains an unsolved problem.

[0012] Further, in step 3, the pH adjustment is performed in stages: first, adjust the pH of the mixed hydrolysate to 5.5±0.1, maintaining stirring for 10-15 minutes; then adjust the pH to 6.0±0.1, maintaining stirring for 10-15 minutes; finally, adjust the pH to 6.8±0.1; and add 0.05% trehalose by mass of the mixed hydrolysate at the beginning of neutralization. The problem addressed is how to simultaneously achieve efficient passivation of heavy metals and stabilization of functional peptides (avoiding isoelectric point precipitation and inactivation) in a single buffer system without adding strong chelating agents (such as EDTA, which does not meet organic standards), solely through precise step control of pH. This is a goal that has not been achieved by combining these two approaches in existing technologies.

[0013] Furthermore, in step 4, the total amount of the compound enzyme preparation added is in a mass ratio of 1:60 to 1:70 to the total solids in the mixed hydrolysate. During the enzymatic hydrolysis process, 20% of the initial amount of neutral protease and papain is added at the 4th hour of hydrolysis. The problem addressed: While some existing technologies disclose the synergistic effect of two proteases, existing technologies have not solved the problem of how to optimize the synergistic release rate of hydrophobic amino acids (such as leucine) and functional peptides (GSH precursors) through enzyme ratio and dynamic enzyme supplementation strategies for highly complex mixed substrates composed of animal and plant pre-hydrolysates.

[0014] Furthermore, in step 5, after reverse osmosis concentration, 0.1% chitosan oligosaccharide and 0.05% humic acid by mass of the concentrate are added to the concentrate, and the conductivity (EC value) of the final product is adjusted to 1.8-2.2 mS / cm. The problem addressed: Chitosan oligosaccharide and humic acid are known additives in existing technologies. However, how to combine them with the concentrate prepared by the aforementioned specific process, which contains a specific amino acid profile and functional peptides, and precisely control the ionic strength (EC value) of the final product so that it can synergistically exert stress-resistant and growth-promoting functions while providing nutrition, without causing root osmotic stress, is an application-level adaptability problem that has not been solved by existing technologies.

[0015] Further, the acid used in step 2 to adjust the pH of the potassium hydroxide solution to the initial reaction value is the waste acid solution generated after animal-derived acid hydrolysis in step 1. Before using the waste acid solution generated in step 1 to adjust the pH of the potassium hydroxide solution in step 2, the waste acid solution undergoes targeted purification treatment: 0.5%-1.0% by volume of activated clay is added to the waste acid solution, and the mixture is stirred and adsorbed at 60-70℃ for 30-45 minutes, followed by filtration; the filtrate is then dynamically adsorbed through a column packed with hydrophobic macroporous adsorption resin, and the effluent is collected for later use. The problem addressed is: how to safely and effectively integrate the high chemical oxygen demand (COD) waste acid solution generated from animal-derived acid hydrolysis into a plant-derived alkaline hydrolysis process, while simultaneously achieving waste resource utilization and avoiding cross-contamination. Meanwhile, a two-stage directional adsorption purification method is used to specifically address the fat-soluble odor precursors, pigments, and small-molecule hydrophobic organic compounds in animal-derived waste acid. If these substances are directly introduced into the plant alkaline hydrolysis system without treatment, unpredictable secondary reactions (such as alkaline catalytic condensation) will occur under strong alkaline and high-temperature conditions, generating new chromophores and stable odor substances that are difficult to remove in subsequent decolorization steps, ultimately endangering the stability of the product's odor and color.

[0016] Further, in step 1, when the crude fat content of the animal-derived protein raw material is higher than 10% on a dry basis, the following operations are performed sequentially: a) Preparation of the composite treatment agent: Take rice husk ash or sugarcane bagasse ash, by-products generated during the processing of plant-derived protein raw materials in step 2, mix them with concentrated sulfuric acid at a mass ratio of 1:1.5-2.0, react at 180-220℃ for 2-3 hours, cool, wash, dry, and pulverize to 200-300 mesh to obtain a solid acid component; uniformly mix this solid acid component with an equal mass of diatomaceous earth to obtain the composite treatment agent for later use; b) Establishment of the reaction system: Add animal-derived protein raw material to the reactor, accounting for 70% of the total sulfuric acid solution, with a concentration of 1.5-2.0. a) A mol / L sulfuric acid solution and the composite treatment agent obtained in step a) accounting for 3%-5% of the dry weight of the animal-derived protein raw material; start stirring, heat the system to 60-65℃ and maintain it for 10-15 minutes; c) Start and maintain the reaction: add 2%-3% (v / v) glycerol to the system of step b), and add vitamin C; continue to heat to the acid hydrolysis reaction temperature of 80-90℃, and immediately add the remaining 30% sulfuric acid solution; maintain the reaction temperature, and when the acid hydrolysis is carried out to the 3rd hour, add 30%-50% of the initial glycerol to the system; d) Post-treatment and separation: after the acid hydrolysis reaction is completed, cool the reaction system to 60-65℃ at a rate of 1-2℃ / min, and continue stirring at this temperature for 20-30 minutes, and then filter while hot; collect the filtrate as animal-derived acid hydrolysate, and proceed to step 3; after drying the filter residue, its solid acid component can be regenerated and reused according to the method of step a). Technical problem to be solved: When the crude fat content of animal-derived raw materials (such as poultry processing by-products) is high, conventional acid hydrolysis processes are prone to generating a large amount of free fatty acids and producing off-flavor substances such as short-chain fatty acids, which seriously affect the odor, color and storage stability of intermediate acid hydrolysate and final products.

[0017] Further, in step 2, when the phytic acid content (on a dry basis) in the plant-derived protein raw material is higher than 1%, the following operation shall be performed: Step 21, the magnesium aluminum hydrotalcite raw powder calcined at 500-600℃ for 2-3 hours is mixed with a potassium dihydrogen phosphate solution with a concentration of 0.5-1.0 mol / L at a solid-liquid ratio of 1:5-1:8, stirred at 50-60℃ for 1-2 hours, filtered, washed, dried, and then pulverized to 300-400 mesh to obtain the hydrotalcite adsorbent; the hydrotalcite adsorbent is uniformly mixed with an equal mass of attapulgite clay activated at 200-250℃ to obtain a composite adsorbent for later use; Step 22, the plant-derived protein raw material with a concentration of 1.0-1.5 mol / L is mixed with phytic acid powder calcined at 500-600℃ for 2-3 hours at a solid-liquid ratio of 1:5-1:8, stirred at 50-60℃ for 1-2 hours, filtered, washed, dried, and ... A mol / L potassium hydroxide solution and the composite adsorbent obtained in step 21, accounting for 4%-6% of the dry weight of the plant-derived protein raw material, are added to the reactor. In step 23, stirring is started, and a gradient temperature increase and pulsed ultrasonic assisted program are initiated. During the last 1 / 3 of the alkaline hydrolysis reaction, the power of the pulsed ultrasonic waves is temporarily increased from 100W to 150-180W for 10-15 minutes, and then returned to the original parameters until the reaction ends. In step 24, after the alkaline hydrolysis reaction is completed, the mixture is filtered. The resulting filtrate is the plant-derived alkaline hydrolysate, which proceeds to step 3. After the filter residue is collected and acid-washed, the hydrotalcite adsorbent can be regenerated and reused after treatment with the same phosphate solution. When the phytic acid content of the plant-derived raw material (such as soybean meal) is high, the conventional alkaline hydrolysis process has limited effect on the degradation or removal of phytic acid, resulting in high phytic acid residue in the product. Phytic acid strongly chelates trace elements such as calcium and zinc, reducing their bioavailability, and may form precipitates in irrigation systems, causing a risk of blockage.

[0018] The beneficial effects of this invention are as follows: 1. This invention achieves comprehensive, balanced, and efficient release of amino acid nutrition. By employing a dual-source parallel process of animal-derived sulfuric acid hydrolysis and plant-derived potassium hydroxide alkaline hydrolysis, mixed in a specific volume ratio (3:7), the glycine and proline abundant in animal protein complement the glutamic acid and aspartic acid abundant in plant protein, constructing a more comprehensive amino acid profile. Based on this, a composite enzyme preparation composed of neutral protease, papain, and trehalase in a specific ratio (3:2:1) is used for synergistic hydrolysis, with dynamic supplementation of protease during the mid-stage of enzymatic hydrolysis. This combined strategy specifically enhances the release rate of hydrophobic amino acids (such as leucine) and the generation of functional peptides (such as glutathione precursors). The total amount of free amino acids in the final product is significantly increased, with a superior proportion of essential amino acids, and the 24-hour nitrogen release rate is improved, meaning that nutrients are more easily absorbed and utilized by crops.

[0019] 2. This invention significantly improves the safety and environmental friendliness of the product. First, by using sulfuric acid instead of hydrochloric acid for animal-derived acid hydrolysis, the large-scale introduction of chloride ions is avoided at the source, making it easier to control the product's salt content (EC value) to a safe range. Second, precise staged pH adjustment of the mixed hydrolysate (e.g., 5.5→6.0→6.8) achieves efficient passivation of harmful heavy metals such as cadmium and lead by controlling the complexation and precipitation windows of different heavy metal ions without the addition of exogenous strong chelating agents, significantly reducing heavy metal residues in the final product. Finally, the animal-derived acid hydrolysis waste liquid is recycled after two-stage targeted purification using activated clay and hydrophobic resin to adjust the initial pH of plant-derived alkaline hydrolysis. This not only achieves resource utilization of high-COD waste acid and reduces wastewater discharge, but also effectively removes impurities such as grease and odor precursors through the purification step, avoiding the negative impact of recycling on product quality.

[0020] 3. This invention optimizes process efficiency and improves the quality of intermediate and final products. In plant-derived alkaline hydrolysis, a gradient temperature increase (90-95℃→98-100℃) combined with pulsed ultrasound assistance at specific parameters (40kHz, 100W) is employed. This synergistic effect of heat, chemistry, and sound gently and effectively disrupts plant cell walls and protein aggregates, increasing the protein degradation rate to a high level while avoiding the negative problems caused by excessive hydrolysis. In animal-derived acid hydrolysis, vitamin C is added when the system temperature reaches 70-80℃. This timing effectively inhibits the critical stage of the Maillard reaction, reducing product browning and loss of amino nitrogen. Furthermore, a specific ratio of chitosan oligosaccharides and humic acid is added to the final concentrate, and the product's EC value is precisely controlled to 1.8-2.2 mS / cm. This not only endows the product with additional functions of stress resistance and growth promotion but also ensures that high-concentration products can be directly and safely applied, avoiding the risk of salt damage.

[0021] 4. This invention enhances adaptability to complex raw materials and process robustness. For animal raw materials with high crude fat content, a strategy of adding a composite treatment agent of solid acid prepared from biochar and diatomaceous earth, and introducing glycerol stepwise, promotes in-situ esterification of free fatty acids, significantly reducing the acid value and free fatty acid content of the acid hydrolysate from the source, effectively eliminating the off-odor caused by oil oxidation and rancidity, and improving product odor and storage stability. For plant raw materials with high phytic acid content, a synergistic adsorption-degradation system is constructed by adding a phosphate-intercalated hydrotalcite / attapulgite composite adsorbent and temporarily enhancing ultrasound in the later stage of alkaline hydrolysis. This efficiently removes phytic acid, reduces its chelation loss of trace elements, improves phosphorus availability, and avoids potential clogging problems in drip irrigation systems.

[0022] Other advantages, objectives and features of the present invention will become apparent in part from the following description, and in part from those skilled in the art through study and practice of the invention. Detailed Implementation

[0023] The present invention will be further described in detail below with reference to examples, so that those skilled in the art can implement it based on the description.

[0024] It should be noted that, unless otherwise specified, the experimental methods described in the following implementation plan are all conventional methods, and the reagents and materials described are all commercially available unless otherwise specified.

[0025] It should be understood that terms such as “having,” “comprising,” and “including” as used herein do not exclude the presence or addition of one or more other elements or combinations thereof.

[0026] The detection method of the present invention is as follows: 1. Analysis of total free amino acids and amino acid composition The amino acid assay was performed using an automated amino acid analyzer. Procedure: Accurately measure 1.0 mL of sample, add 10 mL of 6 mol / L hydrochloric acid, seal the tube under vacuum, and hydrolyze at 110℃ for 24 hours. Transfer the hydrolysate to an evaporating dish and evaporate to dryness in a water bath to remove excess hydrochloric acid. Make up to 50 mL with sodium citrate buffer (pH 2.2) and filter through a 0.22 μm filter membrane. Analyze using an automated amino acid analyzer (equipped with a cation exchange column and ninhydrin post-column derivatization system). Quantification was performed using the external standard method (mixed amino acid standards). Calculations: The total free amino acid content is the sum of the contents of each monomeric amino acid. The percentage of essential amino acids is calculated as (sum of lysine, tryptophan, phenylalanine, methionine, threonine, isoleucine, leucine, and valine contents) / total free amino acids × 100%. The percentage of glycosylated + proline is the ratio of the sum of their contents to the total free amino acid content. The release rate of hydrophobic amino acids needs to be calculated separately. It refers to the percentage of the total amount of typical hydrophobic amino acids such as leucine, isoleucine, valine, phenylalanine, alanine, and proline to the theoretical maximum potential release amount (the total amount of the corresponding amino acids measured after complete acid hydrolysis of the raw material).

[0027] 2. Product color (OD) 420 ) Method: Spectrophotometry. Procedure: Dilute the final product appropriately with deionized water until the transmittance is within the visible light range (usually diluted 10-50 times). Using deionized water as a reference, measure its absorbance at a wavelength of 420 nm using a UV-Vis spectrophotometer. This value directly reflects the degree of browning caused by Maillard reactions, etc.

[0028] 3. Heavy metal detection The method for detecting lead (Pb) content is graphite furnace atomic absorption spectrometry (refer to GB 5009.12). Procedure: Weigh 5.0 g of sample into a digestion vessel, add 5 mL of nitric acid and 2 mL of hydrogen peroxide, and digest using a microwave digester. After complete digestion, transfer the digest to a volumetric flask and dilute to volume with 1% nitric acid solution. Measure the absorbance at a wavelength of 283.3 nm using a graphite furnace atomic absorption spectrometer, and quantify using the standard curve method.

[0029] The method for detecting cadmium (Cd) content is graphite furnace atomic absorption spectrometry (refer to GB 5009.15 "Determination of Cadmium in Food"). Procedure: Weigh 5.0 g of sample into a digestion vessel, add 5 mL of nitric acid and 2 mL of hydrogen peroxide, and digest using a microwave digester according to the programmed procedure. After complete digestion and cooling, transfer the digest to a 25 mL volumetric flask, dilute to volume with 1% nitric acid solution, and mix well. Measure the absorbance at a wavelength of 228.8 nm using a graphite furnace atomic absorption spectrometer, and quantify using the standard curve method. If the cadmium content in the sample is below the method detection limit, it is recorded as "Not detected (<0.02 mg / kg)".

[0030] Method for detecting arsenic (As) content: Hydride generation-atomic fluorescence spectrometry (refer to GB 5009.11 "Determination of Total Arsenic and Inorganic Arsenic in Food"). Procedure: Weigh 5.0 g of sample into a digestion vessel, add 5 mL of nitric acid and 2 mL of hydrogen peroxide, and microwave digest. Transfer the digest to a 25 mL volumetric flask and dilute to volume with 1% nitric acid. Take 5.0 mL of the diluted solution into a 25 mL colorimetric tube, add 5 mL of a 5% thiourea-5% ascorbic acid mixed solution, dilute to volume with 1% nitric acid, mix well, and let stand at room temperature for 30 minutes. Using an atomic fluorescence spectrometer, with potassium borohydride (or sodium) as a reducing agent, reduce arsenic to arsine. Measure the fluorescence intensity under a specially designed arsenic hollow cathode lamp, and quantify using a standard curve method. If the arsenic content in the sample is below the method detection limit, record it as "Not detected (<0.05 mg / kg)".

[0031] The method for determining the content of heavy metal chromium (Cr) is graphite furnace atomic absorption spectrometry (refer to GB 5009.123 "Determination of Chromium in Food"). Procedure: Weigh 5.0 g of sample into a digestion vessel, add 5 mL of nitric acid and 2 mL of hydrogen peroxide, and digest using microwave. Transfer the digest to a 25 mL volumetric flask and dilute to volume with 1% nitric acid. Measure the absorbance at 357.9 nm using a graphite furnace atomic absorption spectrometer, and quantify using the standard curve method. If the chromium content in the sample is below the method detection limit, record it as "Not detected (<0.05 mg / kg)".

[0032] 4. 24-hour nitrogen release rate Method: Soil simulation culture method. Procedure: Weigh 100g of clean quartz sand (inert medium) that has passed through a 2mm sieve into a petri dish, add a sample solution equivalent to 50mg of nitrogen, and mix well. Place in a constant temperature incubator at 25±1℃ and maintain a certain humidity for 24 hours. After culture, extract with 2 mol / L KCl solution, and determine the total nitrogen content in the extract using the Kjeldahl method or an automated nitrogen analyzer. Nitrogen release rate (%) = (amount of nitrogen extracted / total amount of nitrogen added) × 100%.

[0033] 5. Electrical conductivity (EC value) Method: Direct measurement using a conductivity meter. Procedure: Shake the sample well, take an appropriate amount into a clean beaker, and insert the calibrated conductivity meter electrode directly into the sample under constant temperature conditions of 25±1℃. Record the reading after it stabilizes. The unit is mS / cm.

[0034] 6. Stability observation (30 days) Method: Room temperature static observation method. Procedure: Dispense the final product into transparent glass bottles, seal them, and place them in a dark place indoors (temperature 20-25℃). Observe and record the appearance changes on days 1, 7, 15, and 30, including whether sediment, turbidity, layering, oil rings, and color changes occur.

[0035] 7. Acid value and free fatty acid content of the acid hydrolysate Acid value: Performed according to the cold solvent indicator titration method in GB 5009.229 (National Food Safety Standard: Determination of Acid Value in Food). The acid hydrolysate is used instead of the oil sample, and titrated to the phenolphthalein endpoint with potassium hydroxide standard solution. Free fatty acid content: Calculated by acid value conversion, or directly determined by gas chromatography (GC). GC method description: The acid hydrolysate is extracted with n-hexane, esterified (e.g., boron trifluoride-methanol method), and then analyzed for fatty acid methyl esters using a gas chromatograph (equipped with an FID detector). Quantification is performed using the external standard method.

[0036] 8. Odor substances were detected by GC-MS. Method: Headspace solid-phase microextraction-gas chromatography-mass spectrometry (HS-SPME-GC-MS). Procedure: Take 5 mL of acid hydrolysate or final product into a 20 mL headspace vial and add internal standard (2-octanol). After equilibration at a certain temperature (e.g., 60 °C), adsorb volatile components in the headspace gas using a specially coated SPME fiber tip. After adsorption, insert the fiber tip into the GC inlet for thermal desorption, and separate and identify volatile components using GC-MS. Focus on comparing the characteristic peaks of off-odor substances such as short-chain fatty acids (hexanoic acid, octanoic acid).

[0037] 9. Odor Evaluation Method: Sensory evaluation panel method. Procedure: A panel of 5 sensory evaluators was formed. Samples were prepared and presented under identical conditions. Evaluators independently described the overall odor characteristics of the samples and graded the intensity of negative odors such as "rancid odor" or "fishy odor" (e.g., 0-none, 1-slight, 2-moderate, 3-strong). The average grade was taken as the evaluation result.

[0038] 10. Phytic acid content Methods: Ion chromatography (refer to GB 5009.153). Procedure: Accurately weigh an appropriate amount of alkaline hydrolysate or final product (subject to appropriate pretreatment such as dilution and centrifugation), and dilute to volume with ultrapure water. Filter through a 0.22 μm aqueous membrane and an OnGuard RP column (for organic matter removal). Analyze using an ion chromatograph (equipped with an anion exchange column and conductivity detector). Plot a standard curve using sodium phytate standard solution, and quantify using the external standard method.

[0039] 11. Total phosphorus content and percentage of available phosphorus Total phosphorus: The ammonium molybdate spectrophotometric method was used (refer to GB / T 6437). After nitric acid-perchloric acid digestion, the sample reacted with ammonium molybdate, potassium antimony tartrate, and ascorbic acid under acidic conditions to form a blue complex, which was then determined colorimetrically at 700 nm. Water-soluble available phosphorus: After centrifugation or filtration, the supernatant was collected and, without digestion, the phosphorus content was directly determined using the ammonium molybdate spectrophotometric method described above. Calculation: Available phosphorus percentage (%) = (Water-soluble available phosphorus content / Total phosphorus content) × 100%. Here, available phosphorus mainly refers to phosphorus in the form of inorganic orthophosphate that can be directly absorbed and utilized by plants.

[0040] 12. Micronutrients (Ca) 2+ Zn 2+ ) chelation rate Method: Dialysis combined with ICP-OES determination. Procedure: Add a certain amount (of known concentration) of CaCl2 or ZnSO4 standard solution to a known volume of the final product. Place the mixture into a dialysis bag with a molecular weight cutoff of 100-500 Da and dialyze in deionized water for 24 hours (changing the water several times during this period). Measure the Ca or Zn concentration in the dialysis bag solution (total metal content, M_total) and the dialysis fluid (content of unchelated, free metal ions, M_free) using ICP-OES. Calculate the chelation rate (%) = [(M_total - M_free) / M_total] × 100%.

[0041] 13. Drip irrigation clogging simulation test Method: Membrane filtration pressure rise simulation method. Procedure: Prepare a simulated irrigation solution containing 0.1% of the sample. Using a peristaltic pump, pass the solution through a standard dropper or a filter / membrane with a known pore size (e.g., 0.5 mm or 50 mesh) at a constant flow rate (e.g., 1 L / h). Install a pressure sensor before the filter membrane and continuously monitor the inlet pressure change over 4 hours. Record the pressure change curve over time and observe the filter membrane surface for any deposits after the test. A significant pressure rise or visible deposits on the filter membrane are considered a risk of clogging.

[0042] All the above detection methods were performed in triplicate, and the data are expressed as mean ± standard deviation.

[0043] Example 1 (based on fish scales and viscera) 1. Raw materials and equipment Animal-derived protein raw materials: Fishery processing by-products—fresh fish scales (mainly tilapia scales) and fish viscera (mainly a mixture of fish liver, stomach, etc.), in a weight ratio of approximately 4:1. The fish scales are rinsed with deionized water and dried, then pulverized into a fine powder of at least 80 mesh using a high-speed grinder. The fish viscera are chopped and set aside. Take a total of 500g of the mixed raw materials (400g of fish scale powder and 100g of chopped fish viscera) for later use. Plant-derived protein raw materials: Commercially available peanut bran, pulverized through a 40-mesh sieve. Take 300g for later use.

[0044] Reagents: Concentrated sulfuric acid (98%), potassium hydroxide (granules), citric acid monohydrate, tripotassium citrate (buffer), trehalose (food grade), neutral protease (enzyme activity ≥100,000 U / g, EC 3.4.21.4), papain (enzyme activity ≥800,000 U / g, EC 3.4.22.2), trehalase (enzyme activity ≥50,000 U / g, EC 3.2.1.28), activated clay (food grade), D301 weakly basic anion exchange resin (hydrophobic), chitosan oligosaccharide (molecular weight approximately 1500 Da, degree of deacetylation ≥90%), humic acid (fulvic acid content ≥50%). All reagents are industrial or food grade. Sustained-release vitamin C microcapsules, food grade. The microcapsules use sodium alginate and chitosan as composite wall materials, achieving an encapsulation rate of 95%. They are commercially available and can also be prepared using the following method: the encapsulation wall material is at least one of sodium alginate, chitosan, or ethyl cellulose. The preparation method for sustained-release vitamin C includes: slowly adding 2.0 g of sodium alginate to 200 mL of deionized water and continuously stirring in a 50°C water bath until completely dissolved, obtaining a 1.0% (w / v) sodium alginate solution. Separately, dissolving 1.0 g of chitosan in 100 mL of 1% (v / v) acetic acid solution and stirring until clear, obtaining a 1.0% (w / v) chitosan solution. Adding 10.0 g of vitamin C powder (theoretical core loading) to the sodium alginate solution and homogenizing using a high-speed shear emulsifier at 8000 rpm for 3 minutes yields a homogeneous suspension. While continuously stirring, slowly adding the chitosan-acetic acid solution dropwise to the above suspension. After the addition was complete, stirring continued for 30 minutes to allow the electrostatic interaction between sodium alginate and chitosan to cause complex aggregation and encapsulate vitamin C. The system was then slowly poured into 400 mL of 2% (w / v) calcium chloride solution and allowed to stand for 1 hour to solidify. The solidified microcapsule particles were collected by filtration and washed three times with deionized water. The wet capsules were redispersed in an appropriate amount of deionized water to form a slurry, which was then spray-dried (inlet air temperature 160℃, outlet air temperature 80℃, atomizer frequency 50Hz). The dried powder was collected and passed through a 100-mesh sieve to obtain sustained-release vitamin C microcapsule powder. The vitamin C encapsulation rate was no less than 85%.

[0045] Equipment: Glass reactor with stirring and reflux condenser, constant temperature water bath, ultrasonic processor (40kHz frequency, adjustable power, with pulse function), pH meter, high-speed centrifuge, ultrafiltration device (10kDa molecular weight cutoff membrane), chromatography column (for resin adsorption), reverse osmosis concentration equipment, conductivity meter, amino acid analyzer, atomic absorption spectrometer.

[0046] 2. Preparation method (1) Animal-derived acid hydrolysis 500g of mixed raw materials (300g fish scale powder + 200g crushed fish viscera) were added to a 2L reactor. Due to the dense structure of fish scales, the sulfuric acid concentration was increased to 1.9 mol / L, with a total addition volume of 4L. Stirring (250 rpm) and heating were started. When the system temperature reached 78℃, slow-release vitamin C microcapsules containing 0.5g of pure vitamin C (i.e., 0.1% of the raw material mass) were added (these microcapsules use sodium alginate and chitosan as composite wall materials, with an encapsulation rate of 95%, and are commercially available). The temperature was further increased to 88℃, and hydrolysis was maintained at this temperature for 5.5 hours. After the reaction was completed, the mixture was cooled to approximately 60℃ and filtered through a 300-mesh filter cloth to separate approximately 3.7L of dark, slightly viscous animal-derived acid hydrolysate (filtrate, pH approximately 0.9) and filter residue. Collect the filtrate and rinsing waste acid (approximately 0.6 L in total, pH < 1.0, containing more fat-soluble substances and pigments dissolved from the viscera) for later use.

[0047] (2) Alkaline hydrolysis of plant-derived products As before, take 300g of peanut bran and add 2.7L of potassium hydroxide solution with a concentration of 1.2 mol / L.

[0048] pH adjustment: Take all the waste acid liquid (0.6L) collected in step (1) and perform enhanced purification treatment: Add 6mL (1%, v / v) activated clay to it, stir at 300 rpm for 45 minutes at 68℃, and then filter while hot. Pass the filtrate through a chromatography column packed with 250mL hydrophobic D301 macroporous adsorption resin at a flow rate of 1.5 BV / h to more fully remove lipids and small molecule organic matter, and collect the effluent. Use this purified acidic effluent to adjust the initial pH of the peanut bran-alkali solution mixture to about 12.5.

[0049] The ultrasonic-assisted and gradient-temperature hydrolysis process was the same as before (92℃ for 160 minutes, then 99℃ for 80 minutes, for a total of 4 hours). After the reaction, the solution was filtered to obtain approximately 2.5 L of plant-derived alkaline hydrolysate.

[0050] (3) Mixing and neutralization Given that the animal-derived acid hydrolysate comes from fish scales and viscera and may contain more metal ions and colloids, the mixing volume ratio was fine-tuned to 3.5:6.5 (animal source: plant source) to balance the amino acid profile and reduce risk. That is, approximately 1.3 L of animal-derived acid hydrolysate was mixed with approximately 2.4 L of plant-derived alkaline hydrolysate to obtain approximately 3.7 L of mixed hydrolysate (pH approximately 10.3).

[0051] The neutralization process is the same as before, with the pH adjusted to 5.5, 6.0, and 6.8 in three stages, and 1.85g of trehalose added at the beginning. This process helps to further chelate any additional heavy metals (such as arsenic and lead) that may dissolve from the viscera.

[0052] (4) Synergistic enzymatic hydrolysis The neutralized and mixed hydrolysate was heated to 52°C. The total solids content was determined to be approximately 7.0% (due to the contribution of partial hydrolysis products of fish scale collagen), and the total solids mass was approximately 259g. Approximately 4.0g of total enzyme (neutral protease:papain:trehalase = 3:2:1) was weighed out at an enzyme-to-protein ratio of 1:65. Enzymatic hydrolysis was performed for 7 hours, with 20% neutral protease and papain added at the 4th hour. Enzyme inactivation conditions were the same as before.

[0053] (5) Purification and concentration The purification steps are the same as before (ultrafiltration, resin adsorption, reverse osmosis concentration). Concentrate to approximately 1.0L.

[0054] Add 1.0 g chitosan oligosaccharide (0.1%) and 0.5 g humic acid (0.05%) to the concentrate. Adjust the pH of the final product to 6.0 and the EC value to 2.0 mS / cm.

[0055] Approximately 1.05 L of dual-source amino acid-containing organic water-soluble fertilizer (product of Example 1) was obtained. It was a dark amber transparent liquid with a characteristic amino acid odor and no obvious fishy or offal odor.

[0056] Comparative Example 1 The difference from Example 1 is that the plant-derived alkaline hydrolysis and all subsequent synergistic steps are omitted, and a purely animal-derived process is used instead.

[0057] Specific procedures: Only step (1) of Example 1 was performed: acid hydrolysis of animal-derived food (fish scales and viscera, sulfuric acid method, with the addition of sustained-release vitamin C microcapsules). The hydrolysate was directly neutralized to pH 6.8 with potassium hydroxide solution (without staged adjustment, and without the addition of trehalose). Then, enzymatic hydrolysis was performed under the same conditions (using the same complex enzyme, the same enzyme-to-base ratio, and the same time and temperature). Subsequent purification and concentration were performed without the addition of chitosan oligosaccharide and humic acid, and only diluted or concentrated to EC=2.0 mS / cm.

[0058] Comparative Example 2 The difference from Example 1 is that the timing of adding the sustained-release vitamin C microcapsules has been changed.

[0059] Specific operation: exactly the same as in Example 1, except that all sustained-release vitamin C microcapsules are added at the beginning of animal-derived acid hydrolysis (at room temperature) in step (1). All other steps (including dual-source, pH adjustment, enzymatic hydrolysis, purification, functional addition, etc.) are strictly consistent with Example 1.

[0060] Comparative Example 3 Cancel gradient heating and pulsed ultrasound.

[0061] Specific procedures: Step (2) of plant-derived alkaline hydrolysis is changed to hydrolysis at a constant temperature of 98°C for 4 hours without the use of ultrasonic assistance. All other steps are strictly consistent with those in Example 1.

[0062] Comparative Example 4 The pH adjustment process was simplified, and the addition of trehalose was eliminated.

[0063] Specific operation: Step (3) mixing and neutralization is changed to: after mixing the acid hydrolysate and the alkaline hydrolysate, the pH is quickly adjusted to 6.8 in one go with the buffer solution, without going through the intermediate stage of 5.5 and 6.0, and without adding trehalose. The stirring time is the same as the total neutralization time in Example 1. All other steps are strictly consistent with Example 1.

[0064] Comparative Example 5 Use a single enzyme and do not supplement with additional enzymes.

[0065] Specific procedures: Step (4) co-enzymatic hydrolysis is changed to: only neutral protease is added, and the total amount added is such that the mass ratio of the enzyme to the substrate is the same as the ratio of total enzyme to substrate in Example 1 (i.e., equivalent total enzyme activity). Enzyme supplementation is not performed in the 4th hour. The hydrolysis temperature and time are the same as in Example 1. All other steps are strictly consistent with Example 1.

[0066] Comparative Example 6 It does not contain chitosan oligosaccharide or humic acid, and does not precisely regulate the EC value.

[0067] Specific procedures: After purification and concentration in step (5), no chitosan oligosaccharide or humic acid is added. The product is only pH adjusted to 6.0, and the EC value is not specifically controlled (referred to as the natural EC value of the final product). All other steps are strictly consistent with those in Example 1.

[0068] Comparative Example 7 Waste acid is reused directly without purification and without using a buffer system.

[0069] Specific procedures: In step (2), the waste acid collected in step (1) is used directly to adjust the initial pH of alkaline hydrolysis without any treatment (without activated clay and resin adsorption). In step (3), a strong acid / strong base (such as dilute sulfuric acid / potassium hydroxide) is used instead of a citric acid buffer system for pH adjustment (still attempting to adjust to 6.8 in stages). All other steps are strictly consistent with those in Example 1.

[0070] Comparison of experimental results data Parallel testing was conducted on the products of Example 1 and Comparative Examples 1-7, and the performance data are shown in the table below: Table 1. Product performance of Example 1 and Comparative Examples 1-7 As shown in the table above, Comparative Example 1, using a single animal-derived process, produced significantly lower levels of free amino acids (28.3 g / L) and essential amino acid content (41.2%) compared to Example 1 (44.5 g / L, 58.9%). This indicates that optimizing the hydrolysis of a single raw material alone cannot yield a balanced and high-content amino acid profile. The present invention employs a dual-pathway approach, combining animal-derived acid hydrolysis and plant-derived alkaline hydrolysis in a specific ratio (3:7), providing a complementary substrate basis for subsequent enzymatic hydrolysis. Comparative Example 5 shows that even with a dual-source substrate, using only a single neutral protease without dynamic supplementation significantly reduces the hydrophobic amino acid release rate (62%) and total amino acid content (37.2 g / L). This demonstrates that the complex enzyme system ratio (3:2:1) and the enzyme supplementation during the mid-hydrolysis phase are essential for achieving efficient and comprehensive hydrolysis of this specific mixed substrate, and cannot be replaced by a single protease or a fixed enzyme amount.

[0071] Comparative Example 2, where vitamin C was added at the start of the reaction, resulted in a significantly darker product color (OD420 = 0.25) compared to Example 1 (0.12), indicating that the browning caused by the Maillard reaction was more severe. This demonstrates that the timely addition of vitamin C before the system reaches the Maillard reaction acceleration stage (70-80°C) can more effectively inhibit harmful browning and protect the product's appearance and amino nitrogen.

[0072] Comparative Example 3 eliminated gradient heating and ultrasound assistance, employing isothermal alkaline hydrolysis, and its total amino acid content (38.6 g / L) was lower than that of Example 1. This indicates that the gradient heating (90-95℃→98-100℃) combined with pulsed ultrasound of the present invention can more gently and effectively destroy the plant protein structure, improve hydrolysis efficiency, and avoid the over-reaction or efficiency bottleneck that may be caused by constant high temperature.

[0073] Comparative Example 4 changed the phased pH adjustment to a one-step adjustment to the endpoint, with other conditions remaining the same. The lead content in its product (0.18 mg / kg) was more than twice that of Example 1 (0.08 mg / kg). This result directly verifies the key role of the phased (5.5→6.0→6.8) pH adjustment in claim 4: by controlling the pH window for complexation precipitation of different metal ions, it achieves selective and efficient passivation of heavy metals (such as Pb), a selective removal that cannot be achieved by a one-step method. Comparative Example 4 not only had a significantly higher lead (Pb) content, but also significantly higher contents of heavy metals such as cadmium (Cd), arsenic (As), and chromium (Cr), reaching 0.15 mg / kg, 0.12 mg / kg, and 0.25 mg / kg, respectively, all significantly higher than the product of Example 1 (Cd and As were not detected, and Cr was 0.05 mg / kg). This further proves that the present invention systematically controls the pH window for complexation precipitation of different heavy metal ions, achieving simultaneous and efficient passivation of multiple harmful heavy metals. In Example 1, the content of multiple key heavy metals in the product was lower than or far below the relevant agricultural input safety standard limits, demonstrating the effectiveness and reliability of the process in ensuring product safety.

[0074] Comparative Example 6 did not add chitosan oligosaccharides or humic acid, nor did it adjust the EC value; its product had an EC value as high as 3.5 mS / cm. Excessively high EC values ​​pose a risk of salt damage during irrigation and fertilization, directly affecting application safety. This invention, by adding specific functional substances, precisely controls the EC value to 1.8-2.2 mS / cm. This ensures that the high-concentration nutrient product can be directly and safely applied, representing a crucial step in transitioning the product from laboratory formulation to field application.

[0075] Comparative Example 7 reused the acid hydrolysis waste liquid directly without purification, resulting in a product with a severe odor and oily sheen, and the darkest color (0.31). This indicates that the grease, pigments, and odor precursors carried in the waste acid liquid can seriously damage the quality of the final product. The two-step purification method of activated clay adsorption + hydrophobic resin refining employed in this invention effectively removes these impurities, allowing the waste acid liquid to be safely reused as a pH adjuster in the upstream alkaline hydrolysis process, achieving internal resource recycling while avoiding cross-contamination. This solution resolves the conflict between environmental protection requirements and product quality.

[0076] The following are the amino acid composition data of the present invention (in grams per 100g crude protein, g / 100g CP).

[0077] Table 2 Comparison of amino acid composition between animal-derived and plant-derived raw materials of the present invention Using single animal or plant-based raw materials, it is difficult to prepare fertilizer products with a comprehensive and balanced amino acid profile due to their inherent amino acid composition patterns. This invention designs a dual-pathway system of animal-derived acid hydrolysis and plant-derived alkaline hydrolysis, mixed at a specific volume ratio. One of its fundamental purposes is to achieve targeted complementarity in amino acid composition as shown in the table above. Animal-derived materials contribute abundant glycine and proline, as well as relatively high levels of lysine and methionine; plant-derived materials contribute abundant glutamic acid, aspartic acid, phenylalanine, and relatively high levels of isoleucine and leucine.

[0078] In summary, the specific mixing ratio of dual-source substrates, the specific timing of vitamin C addition, the gradient temperature combined with ultrasonic alkaline hydrolysis, the staged pH adjustment, the dynamic hydrolysis by the compound enzyme, the precise control of the product's EC value, and the targeted purification and reuse of waste acid in the technical solution of this invention have all been proven by experimental data to be effective and necessary means to solve specific technical problems (amino acid imbalance, browning, low hydrolysis efficiency, heavy metal residues, poor product safety, and conflicts between waste liquid pollution and quality). These features are interconnected and work synergistically to achieve a comprehensive and verifiable improvement in the final product's nutritional composition, safety, stability, and environmental friendliness.

[0079] Example 2 This method is specifically designed for animal-derived protein raw materials with a crude fat content (on a dry basis) exceeding 10%. This example uses poultry processing byproducts—chicken fat residue—as raw material, with a crude fat content of 63% as tested. The crude fat content of the fish scales and viscera mixture used in Example 1 was tested to be 7.2%. The specific preparation process is as follows: First, prepare the raw materials and equipment. For animal-derived protein, take 500g of chicken fat residue (crushed to a particle size of approximately 3-5 mm using a meat grinder before use). For plant-derived protein, take 300g of commercially available peanut bran, crushed and passed through a 40-mesh sieve. The composite treatment agent consists of rice husk ash (obtained from a local rice processing plant, passed through a 100-mesh sieve), concentrated sulfuric acid (98%), and food-grade diatomaceous earth. Other reagents and equipment are the same as in Example 1, plus a rotary evaporator, acid value analyzer, and gas chromatography-mass spectrometry (GC-MS).

[0080] Preparation begins with the acid hydrolysis of animal-derived materials. First, a composite treatment agent is prepared: 100g of rice husk ash and 150g of concentrated sulfuric acid (mass ratio 1:1.5) are weighed and mixed evenly, then transferred to a reaction vessel lined with polytetrafluoroethylene (PTFE) and reacted at 200℃ for 2.5 hours. After cooling, the mixture is washed with deionized water until the pH of the effluent reaches 6.5-7.0, and then dried at 105℃. The resulting dark brown solid is pulverized and passed through a 250-mesh sieve to obtain the solid acid component. This component is then mixed with an equal mass (100g) of food-grade diatomaceous earth in a mixer for 20 minutes to obtain the composite treatment agent, which is then sealed for later use.

[0081] The reaction system was then established as follows: 500g of crushed chicken fat residue, 2.8L of 1.8 mol / L sulfuric acid solution (70% of the total sulfuric acid solution volume of 4.0L), and 20g of the above-mentioned composite treatment agent (4% of the dry weight of the chicken fat residue) were added to a 2L stirred reactor. The stirring was started (250 rpm), and the system was heated to 63°C in a water bath and maintained for 12 minutes.

[0082] Next, the reaction was initiated and maintained: 80 mL of glycerol (approximately 2.5% of the estimated total volume, v / v) was added to the system. When the system temperature reached 78°C, sustained-release vitamin C microcapsules containing 0.5 g of pure vitamin C (0.1% of the raw material mass) were added. The temperature was continued to rise, and when it reached 82°C, the remaining 1.2 L of sulfuric acid solution (30% of the total volume) was slowly added. The temperature was maintained at 85°C for acid hydrolysis. At the end of the 3rd hour of the reaction, 30 mL of glycerol (37.5% of the initial volume) was added. The total acid hydrolysis time was 5 hours.

[0083] Finally, post-processing and separation were performed: After the reaction, the water bath was cooled to 62°C at a rate of approximately 1.5°C / min, and this temperature was maintained while stirring at 250 rpm for 25 minutes. Then, while still hot, the solution was vacuum filtered through a Buchner funnel lined with 300-mesh filter cloth and filter aid, yielding approximately 3.6 L of dark yellowish-brown animal-derived acid hydrolysate, which was then used in subsequent steps. The filter residue, after drying, weighed approximately 118 g, and the solid acid component could be regenerated using the method described above.

[0084] The subsequent alkaline hydrolysis steps were the same as in Example 1, using 300g of peanut bran and 2.7L of 1.2 mol / L potassium hydroxide solution, and adjusting the initial pH with the purified waste acid solution generated from the conventional acid hydrolysis in step (1) of Example 1. The mixing and neutralization, synergistic enzymatic hydrolysis, purification and concentration steps were all consistent with those in Example 1, and finally about 1.05L of dual-source amino acid-containing organic water-soluble fertilizer was obtained, which was recorded as the product of Example 2.

[0085] Comparative Example 8 To evaluate the effectiveness of Example 2 on high-fat raw materials, Comparative Example 8 was established, with Example 1 as a reference. Comparative Example 8 used the exact same raw material (500g chicken fat residue), sulfuric acid conditions, temperature, and time as Example 2 for the acid hydrolysis step, but the key difference was the absence of any compound treatment agent and glycerol; that is, it employed an acid hydrolysis process similar to Example 1. All subsequent treatment steps for the resulting filtrate were strictly maintained identically to those in Example 2, thus isolating the differences between the two acid hydrolysis processes while controlling for other variables.

[0086] Table 3 Comparison of properties of animal-derived acid hydrolysate and performance of final product Note: The acid hydrolysate data in Example 1 is derived from its fish scale viscera raw material. The crude fat content was tested to be 7.2%, which is lower than that of chicken fat residue. It is used for reference and comparison.

[0087] The results in the table above show that for high-fat raw materials, the general acid hydrolysis process (Comparative Example 8) results in a free fatty acid content as high as 6.1% in the hydrolysate, producing a large amount of short-chain off-flavor fatty acids, leading to a noticeable rancid taste and oily sheen in the final product. However, Example 2, by adding a specially formulated composite treatment agent (solid acid and diatomaceous earth) and glycerol, and implementing a stepwise and dynamic replenishment process, promotes in-situ catalytic esterification of free fatty acids, significantly reducing their content to 1.8% and lowering the acid value by more than 60%, thus eliminating off-flavor precursors and physical instability at the source. This is directly reflected in a significant improvement in the sensory quality and storage stability of the final product. Simultaneously, this specialized process did not adversely affect the main protein hydrolysis efficiency (total amino acid content did not decrease) nor introduce new contamination risks. This proves that the entire active intervention and conversion scheme designed in this invention is necessary and not obvious for the specific problems of high-fat raw materials, and its technical effects exceed the scope achievable by conventional process parameter optimization or simple post-treatment.

[0088] Example 3 This study specifically targets plant-derived protein raw materials with a phytic acid content (on a dry basis) exceeding 1%. Example 3 uses soybean meal as raw material, with a phytic acid content of 1.8%, while the peanut bran used in Example 1 has a phytic acid content of 0.4%. The specific preparation process is as follows: First, prepare the raw materials and equipment. Take 500g of soybean meal as the plant-based protein raw material, grind it through a 40-mesh sieve, and set aside. The animal-based protein raw material is the same as in Example 1, consisting of 500g of a mixture of fish scales and viscera. The composite treatment agent raw materials are magnesium aluminum hydrotalcite powder, potassium dihydrogen phosphate, and attapulgite ore. Other reagents and equipment are the same as in Example 1, plus a muffle furnace, ion chromatograph, and inductively coupled plasma optical emission spectrometer (ICP-OES).

[0089] The entire preparation process begins with animal-derived acid hydrolysis, the same step as in Example 1, which involves processing a mixture of fish scales and viscera to obtain an animal-derived acid hydrolysate.

[0090] Subsequently, alkaline hydrolysis using plant-derived materials was performed. First, a composite treatment agent was prepared: 100g of magnesium aluminum hydrotalcite powder was calcined at 550℃ for 2.5 hours, cooled, and mixed with 500mL of a 0.8 mol / L potassium dihydrogen phosphate solution. The mixture was stirred at 55℃ for 1.5 hours. After treatment, the mixture was filtered, washed until no phosphorus was detected, dried at 105℃, and ground through a 350-mesh sieve to obtain a phosphate-intercalated hydrotalcite adsorbent. Separately, attapulgite was activated at 220℃ for 3 hours and then passed through a 350-mesh sieve. This adsorbent was mixed with an equal mass of activated attapulgite for 30 minutes to obtain the composite treatment agent for later use.

[0091] Next, an enhanced alkaline hydrolysis system was constructed: 500g of soybean meal powder, 4.5L of potassium hydroxide solution with a concentration of 1.2 mol / L, and 25g of the above-mentioned composite treatment agent were added to the reactor.

[0092] Then, the adsorption and hydrolysis synergistic reaction was carried out: stirring and gradient temperature ultrasonic program were started. The temperature was maintained at 93°C for the first 160 minutes, and then increased to 99°C for the next 80 minutes; the ultrasonic parameters were 40kHz, 100W, 5s operation / 5s interval. At the 20th minute of the last 80 minutes of the alkaline hydrolysis reaction, the ultrasonic power was temporarily increased to 165W and maintained for 12 minutes, then returned to 100W until the reaction was completed. The total alkaline hydrolysis time was 4 hours.

[0093] Finally, separation and processing were carried out: After the reaction was completed, the mixture was filtered while hot to obtain approximately 4.2 L of plant-derived alkaline hydrolysate, which was then used in subsequent steps. The filter residue was dried and weighed to approximately 68 g. The hydrotalcite component in the residue could be regenerated through acid washing and treatment with phosphate solution.

[0094] The subsequent mixing and neutralization, synergistic enzymatic hydrolysis, purification and concentration steps were consistent with those in Example 1, and approximately 1.1 L of dual-source amino acid-containing organic water-soluble fertilizer was finally obtained, which was designated as the product of Example 3.

[0095] Comparative Example 9 To evaluate the effectiveness of Example 3, Comparative Example 9 was set up, with Example 1 serving as a reference. The plant-derived alkaline hydrolysis step in Comparative Example 9 used the exact same soybean meal raw material, alkaline conditions, and basic ultrasonic procedure as Example 3. The only difference was the absence of any compound treatment agent and the lack of a temporary increase in ultrasonic power at the final stage. All subsequent treatment steps for the filtrate obtained after alkaline hydrolysis were strictly maintained identically to those in Example 3, thus isolating the differences between the two alkaline hydrolysis processes while controlling for other variables.

[0096] Table 4 Comparison of properties of plant-derived alkaline hydrolysate and performance of final product The results in the table above show that for high-phytic acid raw materials, the conventional alkaline hydrolysis process (Comparative Example 9) cannot effectively degrade phytic acid, resulting in a phytic acid residue of up to 1.85 g / L in the hydrolysate, and an effective phosphorus content of only 45.2%, leading to a phytic acid residue of 320 mg / kg in the final product. Furthermore, it severely chelates trace elements such as calcium and zinc (chelation rate of only 65-78%), even causing blockages in drip irrigation tests. However, the solution of this invention (Example 3), by adding a specially formulated phosphate-intercalated hydrotalcite / attapulgite composite treatment agent and implementing temporary ultrasonic enhancement in the final stage of alkaline hydrolysis, constructs an adsorption-hydrolysis-physical enhancement synergistic system. This allows phytic acid to be efficiently adsorbed and removed, reducing the phytic acid content in the hydrolysate to 0.21 g / L and increasing the effective phosphorus content to 92.5%. This fundamentally eliminates the harm of phytic acid to product quality: the final product has no detectable phytic acid, the trace element chelation rate recovers to approximately 95%, and the risk of drip irrigation blockage is completely avoided. Meanwhile, the total amount of amino acids in this specialized process is considerable, and it does not adversely affect the protein hydrolysis efficiency. This proves that, for the unique problems of high phytic acid raw materials, the comprehensive solution designed in this invention, which combines specific adsorption, process coupling, and dynamic enhancement, is necessary and not obvious. Its technical effect significantly surpasses the conventional approach of simply optimizing hydrolysis conditions.

[0097] Example 4 (using nitric acid acidolysis process) This embodiment uses the same animal-derived protein raw materials as in Example 1 (500g of fish scales and viscera mixture, ratio 4:1) and plant-derived protein raw materials (300g of peanut bran) to prepare water-soluble fertilizer using nitric acid hydrolysis process.

[0098] The preparation process is as follows: First, animal-derived acid hydrolysis is performed. 500g of the mixed raw material is added to a 2.0L pressure-resistant stainless steel reactor equipped with a stirrer, condenser, and tail gas alkaline absorption device. 1.5L of pre-prepared 6.5 mol / L nitric acid solution is added. The reactor is sealed, stirring is started (200 rpm), and heating begins. The heating rate is controlled, and when the system temperature reaches 100℃, slow-release vitamin C microcapsules (containing 0.6g of pure vitamin C, or 0.12% of the raw material mass) are added via the feeding system. These microcapsules use sodium alginate and chitosan as composite wall materials, with an encapsulation rate of 95%, and are commercially available. The temperature is further increased to 130℃ (at which point the pressure inside the reactor is approximately 0.45MPa), and the hydrolysis reaction is maintained at this temperature and pressure for 3 hours. The gas generated during the reaction is condensed and then treated in a 5% sodium hydroxide solution absorption tower. After the reaction is complete, heating is stopped, and the reactor is opened only after the reaction system has naturally cooled to below 80℃ and the pressure has been completely released. The reaction solution was transferred and filtered through a 300-mesh filter cloth to obtain approximately 1.6 L of animal-derived acid hydrolysate (pH approximately 0.5). The filtrate was collected for later use. Subsequent steps for plant-derived alkaline hydrolysis, mixing and neutralization, synergistic enzymatic hydrolysis, purification, and concentration were identical to those in Example 1. Specifically, the plant-derived alkaline hydrolysis used a 1.2 L / L filter... A mol / L potassium hydroxide solution was used, and the initial pH was adjusted using purified waste acid from the conventional acid hydrolysis in Example 1. The hydrolysis was carried out under gradient heating (92℃ for 160 minutes, then 99℃ for 80 minutes) and pulsed ultrasound assistance. The volume ratio of animal-derived to plant-derived hydrolysates was approximately 3.5:6.5. After staged pH adjustment (5.5→6.0→6.8, with the initial addition of 0.05% trehalose), the mixture was enzymatically hydrolyzed at 52℃ for 7 hours using a compound enzyme preparation (neutral protease, papain, and trehalase in a mass ratio of 3:2:1, total enzyme to substrate solids in a mass ratio of 1:65, with 20% protease added at the 4th hour). The hydrolysate was then subjected to ultrafiltration, adsorption decolorization, and reverse osmosis concentration. 0.1% chitosan oligosaccharide and 0.05% humic acid were added to the concentrate, and the pH of the final product was adjusted to 6.0, and the conductivity (EC value) was controlled to 2.0. The concentration of mS / cm was measured to obtain approximately 1.05L of a deep amber-colored, transparent, dual-source amino acid-containing organic water-soluble fertilizer, which is referred to as the product of Example 4.

[0099] To verify the advantages of the nitric acid hydrolysis process in compatibility with other fertilizer products, a stability test was designed for mixing with conventional fertilizers. Products from Example 1 (sulfuric acid system) and Example 4 (nitric acid system) were diluted 100 times with deionized water to prepare working solutions. The following two sets of experiments were conducted: (1) Mixing with calcium fertilizer: 0.5g of hydrated calcium chloride (CaCl2·2H2O) was added to 50mL of the diluted solution and stirred until completely dissolved; (2) Mixing with potassium fertilizer: 0.5g of potassium chloride (KCl) was added to another 50mL of the diluted solution and stirred until completely dissolved. Each mixed solution was allowed to stand for 24 hours, and the phenomena were observed and recorded. After 24 hours, the solution was shaken well, and its transmittance was measured at a wavelength of 660 nm using a UV-Vis spectrophotometer (with deionized water as a reference, recorded as 100%). The mass of the precipitate produced was also determined by quantification.

[0100] The results are shown in Table 5: Table 5 Comparison of product stability when mixed with conventional fertilizers In Example 4 (nitric acid system), the product, when mixed with calcium chloride or potassium chloride, maintained a clear and transparent solution for 24 hours, with no visible turbidity or precipitation. The transmittance was consistently above 98%, and almost no insoluble matter was weighed out. In contrast, the product in Example 1 (sulfuric acid system), when mixed with calcium chloride, developed a white flocculent turbidity within one hour. After standing, a distinct white precipitate formed at the bottom of the bottle, and the transmittance decreased to approximately 65% ​​after 24 hours. The filtered insoluble matter showed a significant weight reduction after drying. While no visible precipitate was observed when mixed with potassium chloride, the transmittance decreased slightly (approximately 92%), indicating the possible presence of fine suspensions. These results demonstrate that the nitric acid hydrolysis process fundamentally avoids the introduction of sulfate ions, thereby completely eliminating the risk of forming insoluble sulfate precipitates with cations such as calcium and potassium. This significantly improves the compatibility and stability of water-soluble fertilizers when mixed with common inorganic fertilizers in practical application, effectively solving the potential problem of reduced efficiency when mixed with sulfuric acid systems.

[0101] Although embodiments of the present invention have been disclosed above, they are not limited to the applications listed in the specification and embodiments. It can be applied to various fields suitable for the present invention. Further modifications can be readily implemented by those skilled in the art.

Claims

1. A method for preparing a dual-source amino acid-containing organic water-soluble fertilizer, characterized in that, Includes the following steps: Step 1: Use one of the following two acid hydrolysis processes for treatment: Sulfuric acid hydrolysis process: Animal-derived protein raw materials are mixed with sulfuric acid solution with a concentration of 1.5-2.0 mol / L and hydrolyzed at 80-90℃ for 4-6 hours to obtain animal-derived hydrolysate; during the hydrolysis process, vitamin C is added at a concentration of 0.05-0.1% of the mass of animal-derived protein raw materials. Nitric acid or mixed acid hydrolysis process: Animal-derived protein raw materials are mixed with a nitric acid solution of 5.0-7.0 mol / L or a mixed acid solution of nitric acid and hydrochloric acid, placed in a pressure-resistant closed reactor, and reacted at 120-150℃ for 2-4 hours to obtain animal-derived hydrolysate; during hydrolysis, 0.08-0.15% vitamin C by mass of animal-derived protein raw materials is added; Step 2, plant-derived alkaline hydrolysis: The plant-derived protein raw material is mixed with a potassium hydroxide solution with a concentration of 1.0-1.5 mol / L, and hydrolyzed at a gradient temperature of 90-100℃ for 3-5 hours, while ultrasonic-assisted treatment is applied to obtain the plant-derived alkaline hydrolysate. Step 3, Mixing and Neutralization: The animal-derived acid hydrolysate obtained in Step 1 and the plant-derived alkaline hydrolysate obtained in Step 2 are mixed at a volume ratio of 1:(1-3) to obtain a mixed hydrolysate; then, a buffer is used to adjust the pH of the mixed hydrolysate in stages so that its pH value finally reaches 6.5-7.

0. Step 4, synergistic enzymatic hydrolysis: Add a complex enzyme preparation consisting of neutral protease, papain and trehalase to the neutralized mixed hydrolysate obtained in step 3, and enzymatically hydrolyze for 6-8 hours at 50-55℃; the mass ratio of neutral protease, papain and trehalase in the complex enzyme preparation is 3:2:

1. Step 5, Purification and Concentration: The product after enzymatic hydrolysis in step 4 is subjected to ultrafiltration, adsorption decolorization and reverse osmosis concentration treatment in sequence to obtain dual-source amino acid-containing organic water-soluble fertilizer.

2. The method according to claim 1, characterized in that, In step 1, vitamin C should be added within 10-60 minutes after the start of hydrolysis and when the system temperature reaches 70-80℃.

3. The method according to claim 2, characterized in that, In step 2, the gradient temperature is as follows: the first 2 / 3 of the hydrolysis time is maintained at 90-95℃, and the temperature is increased to 98-100℃ in the last 1 / 3 of the hydrolysis time; the ultrasonic assistance frequency is 40kHz, the power is 100W, and it is applied in a pulse mode with a working time of 5s and an interval of 5s.

4. The method according to claim 3, characterized in that, In step 3, the pH adjustment is carried out in stages as follows: first, adjust the pH of the mixed hydrolysate to 5.5±0.1 and maintain stirring for 10-15 minutes; then adjust the pH to 6.0±0.1 and maintain stirring for 10-15 minutes; finally, adjust the pH to 6.8±0.1; and add 0.05% of the mass of the mixed hydrolysate at the beginning of neutralization.

5. The method according to claim 4, characterized in that, In step 4, the total amount of the compound enzyme preparation added is in a mass ratio of 1:60 to 1:70 to the total solids in the mixed hydrolysate. During the enzymatic hydrolysis, 20% of the initial amount of neutral protease and papain is added when the enzymatic hydrolysis is carried out for 4 hours.

6. The method according to claim 5, characterized in that, In step 5, after reverse osmosis concentration, 0.1% chitosan oligosaccharide and 0.05% humic acid by mass of the concentrate are added to the concentrate, and the conductivity of the final product is adjusted to 1.8-2.2 mS / cm.

7. The method according to claim 6, characterized in that, The acid used in step 2 to adjust the pH of the potassium hydroxide solution to the initial value of the reaction is the waste acid solution generated after the animal-derived acid hydrolysis in step 1; Before using the waste acid solution generated in step 1 to adjust the pH of the potassium hydroxide solution in step 2, the waste acid solution is subjected to targeted purification treatment: 0.5%-1.0% of its volume of activated clay is added to the waste acid solution, and the mixture is stirred and adsorbed at 60-70℃ for 30-45 minutes, followed by filtration; the filtrate is passed through a column packed with hydrophobic macroporous adsorption resin for dynamic adsorption, and the effluent is collected for later use.

8. The method according to claim 1 or 2, characterized in that, In step 1, when the crude fat content of the animal-derived protein raw material is higher than 10% on a dry basis, the following operations are performed sequentially: a) Preparation of composite treatment agent: Take rice husk ash or sugarcane bagasse ash, by-products generated during the processing of plant-derived protein raw materials in step 2, mix them with concentrated sulfuric acid at a mass ratio of 1:1.5-2.0, react at 180-220℃ for 2-3 hours, cool, wash, dry and pulverize to 200-300 mesh to obtain a solid acid component; mix the solid acid component with an equal mass of diatomaceous earth to obtain a composite treatment agent for later use; b) Establishing the reaction system: Add animal-derived protein raw materials, sulfuric acid solution with a concentration of 1.5-2.0 mol / L accounting for 70% of the total sulfuric acid solution, and the composite treatment agent obtained in step a) accounting for 3%-5% of the dry weight of the animal-derived protein raw materials to the reactor; start stirring, heat the system to 60-65℃ and maintain it for 10-15 minutes; c) Start and maintain the reaction: Add 2%-3% (v / v) of glycerol to the system from step b), and add vitamin C; continue to heat to the acidolysis reaction temperature of 80-90℃, and immediately add the remaining 30% sulfuric acid solution; maintain the reaction temperature, and when the acidolysis has proceeded to the 3rd hour, add 30%-50% of the initial glycerol to the system; d) Post-treatment and separation: After the acid hydrolysis reaction is completed, the reaction system is cooled to 60-65℃ at a rate of 1-2℃ / min, and stirred for 20-30 minutes at this temperature. Then, the mixture is filtered while hot. The filtrate is collected as animal-derived acid hydrolysate and proceeds to step 3. After the filter residue is dried, its solid acid components can be regenerated and reused according to the method in step a).

9. The method according to claim 3, characterized in that, In step 2, when the phytic acid content in the plant-derived protein raw material is higher than 1% on a dry basis, the following procedure shall be followed: Step 21: The magnesium aluminum hydrotalcite raw powder, calcined at 500-600℃ for 2-3 hours, is mixed with a potassium dihydrogen phosphate solution with a concentration of 0.5-1.0 mol / L at a solid-liquid ratio of 1:5-1:

8. The mixture is stirred at 50-60℃ for 1-2 hours, filtered, washed, dried, and then pulverized to 300-400 mesh to obtain the hydrotalcite adsorbent. The hydrotalcite adsorbent is then uniformly mixed with an equal mass of attapulgite clay activated at 200-250℃ to obtain a composite adsorbent for later use. Step 22: Plant-derived protein raw material, potassium hydroxide solution with a concentration of 1.0-1.5 mol / L, and composite adsorbent obtained in step 21, accounting for 4%-6% of the dry weight of plant-derived protein raw material, are added to the reactor together. Step 23: Start stirring and start the gradient heating and pulsed ultrasound assisted program; when the alkaline hydrolysis reaction is in the last 1 / 3 period, temporarily increase the power of the pulsed ultrasound from 100W to 150-180W for 10-15 minutes, and then restore it to the original parameters until the reaction is over. Step 24: After the alkaline hydrolysis reaction is completed, filter the solution. The resulting filtrate is a plant-derived alkaline hydrolysate, which proceeds to step 3. After the filter residue is collected and acid-washed, the hydrotalcite adsorbent can be regenerated by the same phosphate solution and reused.

10. The method according to claim 1 or 2, characterized in that, The vitamin C added in step 1 is a sustained-release vitamin C that has been encapsulated. The encapsulation wall material of the sustained-release vitamin C is selected from at least one of sodium alginate, chitosan, and ethyl cellulose, and its encapsulation rate is not less than 85%.

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