High-activity small-molecule fish protein fertilizer and preparation method thereof

Abstract

The present application relates to the technical field of agricultural fertilizer, in particular to a high-activity small-molecule fish protein fertilizer and a preparation method thereof.The method comprises the following steps: mixing animal-derived protein raw materials, plant-derived polysaccharide raw materials and water to obtain a liquid; performing segmented enzymolysis on the liquid at ≤55 DEG C; mixing the liquid and a first protease, adjusting the pH to 7.0-9.0, and performing enzymolysis to obtain a first enzymolysis liquid; mixing the first enzymolysis liquid and a second protease, adjusting the pH to 5.5-7.0, and performing enzymolysis to obtain a second enzymolysis liquid; mixing the second enzymolysis liquid and a polysaccharide hydrolysis enzyme, adjusting the pH to 5.0-6.0, and performing enzymolysis to obtain a third enzymolysis liquid; sequentially performing ultrafiltration membrane and nanofiltration membrane treatment on the third enzymolysis liquid to obtain a small-molecule peptide rich liquid; and finally concentrating at ≤55 DEG C to obtain a product.The fertilizer prepared by the present application has high small-molecule peptide content, low salt content, sufficient active ingredient retention, and low production energy consumption, and is beneficial to crop absorption.

Description

Technical Field

[0001] This invention relates to the field of agricultural fertilizer technology, and in particular to a highly active small molecule fish protein fertilizer and its preparation method. Background Technology

[0002] Fish protein fertilizer is a new type of fertilizer made primarily from aquatic products such as fish, shrimp, and crab, or their processing byproducts, through enzymatic or bacterial hydrolysis processes. It is rich in nutrients such as amino acids, small-molecule peptides, and organic matter. Compared to traditional chemical fertilizers, fish protein fertilizer can improve soil structure, promote crop root development, enhance crop resistance to adverse conditions, and improve the quality of agricultural products. Therefore, it is increasingly widely used in organic and green agriculture.

[0003] Currently, existing fish protein fertilizer production processes suffer from the following main problems: First, active ingredients are easily lost. Traditional production processes, such as high-temperature enzymatic hydrolysis, high-temperature concentration, and spray drying, often involve operating temperatures exceeding 70℃, and in some cases even reaching over 100℃. This leads to the degradation of heat-sensitive active substances in the fertilizer (such as seaweed polysaccharides and small molecule signal peptides), significantly reducing their activity and thus weakening the fertilizer's biostimulatory function. Second, molecular weight distribution is uncontrollable. Conventional enzymatic hydrolysis processes typically only focus on the degree of protein hydrolysis, lacking effective control over the molecular weight range of the product. The resulting product often contains a large amount of incompletely hydrolyzed large molecular proteins (molecular weight greater than 10,000 Daltons (Da)), which are difficult for crop roots and leaves to directly absorb and utilize, resulting in low fertilizer utilization. Third, salt accumulation. When using marine fish as raw material, the raw material itself contains a high sodium chloride content. Existing processes lack effective desalination steps, resulting in excessively high salt content in the product, which can exacerbate secondary soil salinization with long-term application. Fourth, energy consumption is high. The concentration process often uses multi-effect evaporators, which consume a lot of energy and does not meet the development requirements of green and low-carbon production.

[0004] Therefore, developing a fish protein fertilizer production process that can simultaneously retain heat-sensitive active ingredients, control the molecular weight of the product, achieve efficient desalination, and save energy has become an urgent problem to be solved in this field. Summary of the Invention

[0005] The purpose of this invention is to address the problems existing in the prior art by providing a highly active small-molecule fish protein fertilizer and its preparation method. A synergistic process involving full-process low-temperature control, segmented directional enzymatic hydrolysis, ultrafiltration-nanofiltration fractionation, and low-temperature concentration was employed to produce the highly active small-molecule fish protein fertilizer.

[0006] To achieve the above objectives, the present invention provides a method for preparing a highly active small-molecule fish protein fertilizer, comprising the following steps: S1. Mix animal-derived protein raw materials, plant-derived polysaccharide raw materials, and water to obtain a liquid mixture; S2. Perform segmented enzymatic hydrolysis of the feed solution at ≤55℃: Mix the feed solution with the first protease, adjust the pH to 7.0-9.0, and perform the first enzymatic hydrolysis to obtain the first hydrolysate; mix the first hydrolysate with the second protease, adjust the pH to 5.5-7.0, and perform the second enzymatic hydrolysis to obtain the second hydrolysate; mix the second hydrolysate with polysaccharide hydrolase, adjust the pH to 5.0-6.0, and perform the third enzymatic hydrolysis to obtain the third hydrolysate; S3. The third enzymatic hydrolysate is subjected to ultrafiltration and nanofiltration membrane treatment in sequence to obtain a small molecule peptide enrichment solution; S4. Concentrate the small molecule peptide enrichment solution at ≤55℃ to obtain a highly active small molecule fish protein fertilizer.

[0007] In an optional embodiment, in S1, the animal-derived protein raw material includes at least one of fish, shrimp, crab, fish processing by-products, shrimp processing by-products, and crab processing by-products; the plant-derived polysaccharide raw material includes at least one of seaweed, soybean meal, and corn.

[0008] In an optional embodiment, in S1, the mass ratio of the animal-derived protein raw material, the plant-derived polysaccharide raw material, and water is (250-350):(50-150):(1100-1300).

[0009] In an optional embodiment, in S1, animal-derived protein raw materials and plant-derived polysaccharide raw materials are mixed in proportion, water is added, and wet pulverization is performed to a fineness of 80-120 mesh to obtain a liquid.

[0010] In an optional embodiment, in S2, the first protease includes at least one of alkaline protease and trypsin; the amount of the first protease added accounts for 0.1-0.5% of the dry basis mass of the liquid in S1; the temperature of the first enzymatic hydrolysis is 50-55°C, and the time is 2-6 hours.

[0011] The dry basis mass of the liquid refers to the total mass of solid substances in the liquid obtained in S1 (i.e., the mass after deducting water), which can be determined by drying the liquid to constant weight or calculated based on the moisture content of each raw material.

[0012] In an optional embodiment, in S2, the second protease includes at least one of flavor protease, papain, and neutral protease; the amount of the second protease added accounts for 0.1-0.5% of the dry basis mass of the liquid in S1; the temperature of the second enzymatic hydrolysis is 45-50°C, and the time is 1-4 hours.

[0013] In an optional embodiment, in S2, the polysaccharide hydrolase includes at least one of alginate lyase, cellulase, and hemicellulase; the amount of the polysaccharide hydrolase added accounts for 0.05-0.2% of the dry basis mass of the liquid in S1; the temperature of the third enzymatic hydrolysis is 40-50℃, and the time is 1-3h.

[0014] In an optional embodiment, in S3, the third enzymatic hydrolysate is coarsely filtered through a 150-250 mesh sieve, and then subjected to ultrafiltration and nanofiltration membrane treatments in sequence.

[0015] In an optional embodiment, in S3, the ultrafiltration membrane has a molecular weight cutoff of 3000-10000 Da and an operating pressure of 0.2-0.6 MPa.

[0016] In an optional embodiment, in step S3, the nanofiltration membrane has a molecular weight cutoff of 150-500 Da and an operating pressure of 1.0-3.0 MPa. During the nanofiltration membrane treatment, rinsing is performed; specifically, purified water is continuously added to the retentate at a rate equal to the outflow rate of the permeate, keeping the retentate volume constant. During this process, salt ions continuously permeate through the nanofiltration membrane and are discharged with the water, while small molecule peptides are retained and enriched. This operation continues until the conductivity of the retentate drops below 2000 μS / cm, resulting in a low-salt small molecule peptide enrichment solution.

[0017] In an optional embodiment, in S4, the concentration is performed using mechanical vapor recompression (MVR) technology, and the concentration temperature is 45-55°C; the solid content of the highly active small molecule fish protein fertilizer is 30-45%.

[0018] The present invention also provides a highly active small molecule fish protein fertilizer, which is prepared according to the preparation method of the highly active small molecule fish protein fertilizer.

[0019] Compared with the prior art, the present invention has the following beneficial effects: (1) The present invention adopts a low temperature process of ≤55℃ throughout the process, which effectively avoids the high temperature degradation of heat-sensitive active ingredients (such as seaweed polysaccharides, small molecule signal peptides, etc.), and keeps the content of active substances in the product at a high level.

[0020] (2) By segmented and targeted enzymatic hydrolysis (controlling pH, temperature, enzyme type and sequence separately) combined with ultrafiltration-nanofiltration fractionation, the product contains a high proportion of small molecule peptides with a molecular weight of less than 1000 Da and free amino acids. These small molecules can be directly absorbed by crop roots and leaves, which is beneficial to improving the utilization efficiency of fertilizer nutrients.

[0021] (3) Nanofiltration membrane washing (continuously adding water to the retentate to allow salt ions to pass through the membrane) can effectively remove salts such as sodium chloride from the raw materials, reducing the ash content of the product to a low level. Long-term application of low-salt fertilizers is less likely to cause secondary soil salinization and is suitable for facility agriculture and planting in saline-alkali land.

[0022] (4) The mechanical vapor recompression (MVR) technology is adopted, the evaporation temperature is low (≤55℃), and the energy consumption per unit product is lower than that of the traditional multi-effect evaporation process, which is conducive to reducing production costs and carbon emissions.

[0023] (5) The segmented enzymatic hydrolysis, membrane treatment and low-temperature MVR concentration of the present invention support each other in function: segmented enzymatic hydrolysis provides a high proportion of small molecule peptide feed solution for membrane separation, ensuring membrane flux and preventing contamination; membrane treatment provides a low-salt, low-viscosity feed solution for MVR concentration, improving heat transfer efficiency and protecting equipment; low-temperature MVR concentration is beneficial for retaining the active products obtained in the previous steps.

[0024] (6) The use of the fertilizer of the present invention can significantly promote the development of crop roots, increase plant biomass, and improve stress resistance, thereby improving crop yield and quality. Attached Figure Description

[0025] Figure 1 These are comparison diagrams of plant height and above-ground fresh weight in embodiments and comparative examples of the present invention; Figure 2 This is a comparison diagram of root vitality in embodiments and comparative examples of the present invention. Detailed Implementation

[0026] The following embodiments are provided to better understand the present invention and are not limited to the described embodiments. They do not constitute a limitation on the content and scope of protection of the present invention. Any product that is the same as or similar to the present invention, derived by any person under the guidance of the present invention or by combining the features of the present invention with other prior art, falls within the protection scope of the present invention.

[0027] For experiments not specifically described in the examples, the procedures or conditions should be followed according to the conventional experimental procedures described in the literature in this field. Reagents or instruments whose manufacturers are not specified are all commercially available products.

[0028] In the following embodiments and comparative examples of the present invention, all enzymes used were purchased from commercially available sources.

[0029] Example 1 This embodiment provides a method for preparing a highly active small molecule fish protein fertilizer, including the following steps: Take 300 kg of fresh cod processing by-products (70% moisture content), 50 kg of dried kelp (a type of seaweed, 10% moisture content), and 50 kg of soybean meal (13% moisture content). Wash the cod processing by-products and then pulverize them until no large pieces are visible. Pulverize the dried kelp and soybean meal separately to 80 mesh. Mix all the pulverized materials together, add 1200 kg of purified water, and homogenize to 100 mesh in a wet ultrafine pulverizer to obtain a liquid mixture.

[0030] The above-mentioned feed solution was pumped into an enzymatic hydrolysis tank. The pH of the feed solution was adjusted to 8.0 using a 2 mol / L sodium hydroxide solution. The temperature was raised to 52°C, and alkaline protease (enzyme activity 200,000 U / g) was added, with the amount of alkaline protease accounting for 0.3% of the dry weight of the feed solution. Enzymatic hydrolysis was carried out for 4 hours to obtain the first enzymatic hydrolysate. The pH of the first enzymatic hydrolysate was adjusted to 6.5 using citric acid, and the temperature was lowered to 48°C. Flavor protease (enzyme activity 150,000 U / g) was added, with the amount of flavor protease accounting for 0.3% of the dry weight of the feed solution. Enzymatic hydrolysis was carried out for 2 hours to obtain the second enzymatic hydrolysate. The pH of the second enzymatic hydrolysate was adjusted to 5.5 using citric acid, and the temperature was lowered to 45°C. Alginate lyase (enzyme activity 50,000 U / g) was added, with the amount of alginate lyase accounting for 0.15% of the dry weight of the feed solution. Enzymatic hydrolysis was carried out for 2 hours to obtain the third enzymatic hydrolysate.

[0031] The third enzymatic hydrolysate was coarsely filtered through a 200-mesh sieve. The filtrate was first fed into an ultrafiltration membrane system: a spiral wound ultrafiltration membrane with a molecular weight cutoff of 5000 Da and an operating pressure of 0.4 MPa was used to obtain the ultrafiltration permeate. The ultrafiltration permeate was then fed into a nanofiltration membrane system: a spiral wound nanofiltration membrane with a molecular weight cutoff of 200 Da and an operating pressure of 2.0 MPa was used. During nanofiltration, purified water was continuously added to the retentate at a rate equal to the outflow rate of the permeate to maintain a constant volume of retentate. This operation was continued until the conductivity of the retentate dropped below 2000 μS / cm, resulting in a small molecule peptide enrichment solution.

[0032] The above-mentioned small molecule peptide enrichment solution was fed into an MVR evaporator, and the temperature was controlled at 50°C. The solution was concentrated to a solid content of 38% to obtain a highly active small molecule fish protein fertilizer.

[0033] Example 2 This embodiment provides a method for preparing a highly active small molecule fish protein fertilizer, including the following steps: Take 250 kg of fresh shrimp processing by-products (shrimp heads and shells, 65% moisture content), 100 kg of dried seaweed (a type of seaweed, 10% moisture content), and 50 kg of corn (13% moisture content). Wash the shrimp processing by-products and pulverize them until no large pieces remain. Pulverize the dried seaweed and corn separately to 80 mesh. Mix all the pulverized materials together, add 1150 kg of purified water, and homogenize to 100 mesh in a wet ultrafine pulverizer to obtain a liquid mixture.

[0034] The above-mentioned feed solution was pumped into an enzymatic hydrolysis tank. The pH of the feed solution was adjusted to 8.5 using a 2 mol / L sodium hydroxide solution. The temperature was raised to 53°C, and trypsin (enzyme activity 250,000 U / g) was added, with the amount of trypsin added being 0.3% of the dry weight of the feed solution. Enzymatic hydrolysis was carried out for 5 hours to obtain the first enzymatic hydrolysate. The pH of the first enzymatic hydrolysate was adjusted to 6.0 using citric acid, and the temperature was lowered to 47°C. Papain (enzyme activity 100,000 U / g) was added, with the amount of papain added being 0.3% of the dry weight of the feed solution. Enzymatic hydrolysis was carried out for 3 hours to obtain the second enzymatic hydrolysate. The pH of the second enzymatic hydrolysate was adjusted to 5.5 using citric acid, and the temperature was lowered to 43°C. Cellulase (enzyme activity 20,000 U / g) was added, with the amount of cellulase added being 0.15% of the dry weight of the feed solution. Enzymatic hydrolysis was carried out for 2.5 hours to obtain the third enzymatic hydrolysate.

[0035] The third enzymatic hydrolysate was coarsely filtered through a 200-mesh sieve. The filtrate was first fed into an ultrafiltration membrane system: a spiral wound ultrafiltration membrane with a molecular weight cutoff of 3000 Da and an operating pressure of 0.5 MPa was used to obtain the ultrafiltration permeate. The ultrafiltration permeate was then fed into a nanofiltration membrane system: a spiral wound nanofiltration membrane with a molecular weight cutoff of 150 Da and an operating pressure of 2.5 MPa was used. During nanofiltration, purified water was continuously added to the retentate at a rate equal to the outflow rate of the permeate to maintain a constant volume of retentate. This operation was continued until the conductivity of the retentate dropped below 1500 μS / cm, resulting in a small molecule peptide enrichment solution.

[0036] The above-mentioned small molecule peptide enrichment solution was fed into an MVR evaporator, and the temperature was controlled at 48°C. The solution was concentrated to a solid content of 40% to obtain a highly active small molecule fish protein fertilizer.

[0037] Example 3 This embodiment provides a method for preparing a highly active small molecule fish protein fertilizer, including the following steps: Take 350 kg of fresh mackerel processing by-products (72% moisture content), 30 kg of dried Sargassum (a type of seaweed with 10% moisture content), and 70 kg of soybean meal (13% moisture content). Wash the mackerel processing by-products and then pulverize them until no large pieces remain. Pulverize the dried Sargassum and soybean meal separately to 100 mesh. Mix all the pulverized materials together, add 1300 kg of purified water, and homogenize to 120 mesh in a wet ultrafine pulverizer to obtain a liquid mixture.

[0038] The above-mentioned feed solution was pumped into an enzymatic hydrolysis tank. The pH of the feed solution was adjusted to 7.5 using a 2 mol / L sodium hydroxide solution, and the temperature was raised to 54°C. Alkaline protease (enzyme activity 200,000 U / g) was added, with the amount of alkaline protease added being 0.3% of the dry weight of the feed solution. Enzymatic hydrolysis was carried out for 3 hours to obtain the first enzymatic hydrolysate. The pH of the first enzymatic hydrolysate was adjusted to 6.8 using citric acid, and the temperature was lowered to 49°C. Flavor protease (enzyme activity 150,000 U / g) was added, with the amount of flavor protease added being 0.3% of the dry weight of the feed solution. Enzymatic hydrolysis was carried out for 1.5 hours to obtain the second enzymatic hydrolysate. The pH of the second enzymatic hydrolysate was adjusted to 5.8 using citric acid, and the temperature was lowered to 44°C. Alginate lyase (enzyme activity 50,000 U / g) was added, with the amount of alginate lyase added being 0.15% of the dry weight of the feed solution. Enzymatic hydrolysis was carried out for 1.5 hours to obtain the third enzymatic hydrolysate.

[0039] The third enzymatic hydrolysate was coarsely filtered through a 200-mesh sieve. The filtrate was first fed into an ultrafiltration membrane system: a spiral wound ultrafiltration membrane with a molecular weight cutoff of 8000 Da and an operating pressure of 0.3 MPa was used to obtain the ultrafiltration permeate. The ultrafiltration permeate was then fed into a nanofiltration membrane system: a spiral wound nanofiltration membrane with a molecular weight cutoff of 300 Da and an operating pressure of 1.5 MPa was used. During nanofiltration, purified water was continuously added to the retentate at a rate equal to the outflow rate of the permeate to maintain a constant volume of retentate. This operation was continued until the conductivity of the retentate dropped below 1800 μS / cm, resulting in a small molecule peptide enrichment solution.

[0040] The above-mentioned small molecule peptide enrichment solution was fed into an MVR evaporator, and the temperature was controlled at 52°C. The solution was concentrated to a solid content of 35% to obtain a highly active small molecule fish protein fertilizer.

[0041] Comparative Example 1 This comparative example provides a method for preparing fish protein fertilizer, including the following steps: Take 300 kg of fresh cod processing by-products (70% moisture content), 50 kg of dried kelp (a type of seaweed, 10% moisture content), and 50 kg of soybean meal (13% moisture content). Wash the cod processing by-products and then pulverize them until no large pieces are visible. Pulverize the dried kelp and soybean meal separately to 80 mesh. Mix all the pulverized materials together, add 1200 kg of purified water, and homogenize to 100 mesh in a wet ultrafine pulverizer to obtain a liquid mixture.

[0042] The above-mentioned feed solution was pumped into an enzymatic hydrolysis tank. The pH of the feed solution was adjusted to 8.0 using a 2 mol / L sodium hydroxide solution. The temperature was raised to 65°C, and alkaline protease (enzyme activity 200,000 U / g) and flavor protease (enzyme activity 150,000 U / g) were added. The amount of alkaline protease added accounted for 0.3% of the dry weight of the feed solution, and the amount of flavor protease added accounted for 0.3% of the dry weight of the feed solution. Enzymatic hydrolysis was carried out for 8 hours to obtain the enzymatic hydrolysate.

[0043] The enzymatic hydrolysate was coarsely filtered through a 200-mesh sieve and then sent to a triple-effect evaporator. It was concentrated at 75°C to a solid content of 38% to obtain fish protein fertilizer.

[0044] Comparative Example 2 This comparative example provides a method for preparing fish protein fertilizer, including the following steps: Take 300 kg of fresh cod processing by-products (70% moisture content), 50 kg of dried kelp (a type of seaweed, 10% moisture content), and 50 kg of soybean meal (13% moisture content). Wash the cod processing by-products and then pulverize them until no large pieces are visible. Pulverize the dried kelp and soybean meal separately to 80 mesh. Mix all the pulverized materials together, add 1200 kg of purified water, and homogenize to 100 mesh in a wet ultrafine pulverizer to obtain a liquid mixture.

[0045] The above-mentioned feed solution was pumped into an enzymatic hydrolysis tank. The pH of the feed solution was adjusted to 8.0 using a 2 mol / L sodium hydroxide solution. The temperature was raised to 52°C, and alkaline protease (enzyme activity 200,000 U / g) was added, with the amount of alkaline protease accounting for 0.3% of the dry weight of the feed solution. Enzymatic hydrolysis was carried out for 4 hours to obtain the first enzymatic hydrolysate. The pH of the first enzymatic hydrolysate was adjusted to 6.5 using citric acid, and the temperature was lowered to 48°C. Flavor protease (enzyme activity 150,000 U / g) was added, with the amount of flavor protease accounting for 0.3% of the dry weight of the feed solution. Enzymatic hydrolysis was carried out for 2 hours to obtain the second enzymatic hydrolysate. The pH of the second enzymatic hydrolysate was adjusted to 5.5 using citric acid, and the temperature was lowered to 45°C. Alginate lyase (enzyme activity 50,000 U / g) was added, with the amount of alginate lyase accounting for 0.15% of the dry weight of the feed solution. Enzymatic hydrolysis was carried out for 2 hours to obtain the third enzymatic hydrolysate.

[0046] The third enzymatic hydrolysate was coarsely filtered through a 200-mesh sieve and then sent to a triple-effect evaporator. It was concentrated at 75°C to a solid content of 38% to obtain fish protein fertilizer.

[0047] Comparative Example 3 This comparative example provides a method for preparing fish protein fertilizer, including the following steps: Take 300 kg of fresh cod processing by-products (70% moisture content), 50 kg of dried kelp (a type of seaweed, 10% moisture content), and 50 kg of soybean meal (13% moisture content). Wash the cod processing by-products and then pulverize them until no large pieces are visible. Pulverize the dried kelp and soybean meal separately to 80 mesh. Mix all the pulverized materials together, add 1200 kg of purified water, and homogenize to 100 mesh in a wet ultrafine pulverizer to obtain a liquid mixture.

[0048] The above-mentioned feed solution was pumped into an enzymatic hydrolysis tank. The pH of the feed solution was adjusted to 8.0 using a 2 mol / L sodium hydroxide solution. The temperature was raised to 52°C, and alkaline protease (enzyme activity 200,000 U / g) was added, with the amount of alkaline protease accounting for 0.3% of the dry weight of the feed solution. Enzymatic hydrolysis was carried out for 4 hours to obtain the first enzymatic hydrolysate. The pH of the first enzymatic hydrolysate was adjusted to 6.5 using citric acid, and the temperature was lowered to 48°C. Flavor protease (enzyme activity 150,000 U / g) was added, with the amount of flavor protease accounting for 0.3% of the dry weight of the feed solution. Enzymatic hydrolysis was carried out for 2 hours to obtain the second enzymatic hydrolysate. The pH of the second enzymatic hydrolysate was adjusted to 5.5 using citric acid, and the temperature was lowered to 45°C. Alginate lyase (enzyme activity 50,000 U / g) was added, with the amount of alginate lyase accounting for 0.15% of the dry weight of the feed solution. Enzymatic hydrolysis was carried out for 2 hours to obtain the third enzymatic hydrolysate.

[0049] The third enzymatic hydrolysate was coarsely filtered through a 200-mesh sieve. The coarsely filtered filtrate was then fed into an ultrafiltration membrane system: a spiral wound ultrafiltration membrane with a molecular weight cutoff of 5000 Da and an operating pressure of 0.4 MPa was used to obtain the ultrafiltration permeate. This permeate was then fed into an MVR evaporator, and the temperature was controlled at 50°C to concentrate it to a solid content of 38%, yielding fish protein fertilizer.

[0050] Experimental Example 1 The fish protein fertilizers prepared in Examples 1-3 and Comparative Examples 1-3 were tested for the following physicochemical properties.

[0051] (1) Molecular weight distribution (percentage of small peptides): Gel filtration chromatography (SEC-HPLC) was used. The chromatographic column was a TSKgel G2000SWXL (7.8 mm × 300 mm); the mobile phase was 0.1 mol / L phosphate buffer (containing 0.1 mol / L Na2SO4, pH 7.0); the flow rate was 0.5 mL / min; the column temperature was 30℃; the detection wavelength was 220 nm; and the injection volume was 20 μL. Standards included cytochrome C (12500 Da), aprotinin (6500 Da), bacitracin (1450 Da), tetrapeptide (451 Da), and tripeptide (189 Da). The percentage of peptides with a molecular weight <1000 Da relative to the total peptide peak area was calculated.

[0052] (2) Ash content: The gravimetric method shall be adopted in accordance with GB / T 6438 and shall be calculated on a dry basis.

[0053] (3) Organic matter: The potassium dichromate oxidation method shall be adopted and implemented in accordance with NY / T 1976.

[0054] (4) Free amino acids: The method of automatic amino acid analyzer was adopted, and the procedure was performed in accordance with NY / T 1975.

[0055] (5) Alginate: High performance liquid chromatography-evaporative light scattering (HPLC-ELSD) was used. The chromatographic column was XBridgeAmide (4.6 mm × 250 mm, 3.5 μm); the mobile phase was acetonitrile-water (70:30, v / v); the flow rate was 1.0 mL / min; the ELSD drift tube temperature was 80 °C, and the nitrogen pressure was 2.5 bar. Mixed standards of alginate (degree of polymerization 2-6) were also used. Quantification was performed using the external standard method based on peak area, with a detection limit of 0.5 g / L.

[0056] The test results of the above physicochemical indicators are recorded in Table 1.

[0057] Table 1. Results of Physicochemical Indicators

[0058] As shown in Table 1, the proportion of small molecule peptides in Examples 1-3 (85.6-88.2%) was significantly higher than that in Comparative Examples 1-3 (44.8-74.3%), while the ash content (1.2-1.3%) was significantly lower than that in Comparative Examples 1-3 (5.1-11.2%). This indicates that the present invention can effectively remove salt and large molecule proteins and enrich small molecule active peptides through ultrafiltration-nanofiltration combined washing. Comparative Example 3, which only used ultrafiltration and not nanofiltration, had a small molecule peptide proportion (74.3%) that was higher than that in Comparative Example 2 (61.5%), but still significantly lower than that in Example 1 (88.2%), and the ash content (5.1%) was much higher than that in Example 1, demonstrating the key role of nanofiltration + washing. The content of seaweed oligosaccharides in Examples 1-3 was 25.1-28.5 g / L, while it was not detected in Comparative Example 1. Comparative Examples 2 and 3 were significantly lower than those in Examples 1-3, indicating that low-temperature segmented enzymatic hydrolysis and nanofiltration washing are beneficial for retaining and enriching seaweed active ingredients.

[0059] Experiment Example 2 The fish protein fertilizers prepared in Examples 1-3 and Comparative Examples 1-3 were subjected to bioactivity testing, and the specific process is as follows: The test crop was Chinese cabbage, specifically the variety "Shanghai Green".

[0060] Cultivation method: Plastic pots (25cm in diameter, 20cm in height), each pot contains 3.5kg of air-dried garden soil, each pot contains 5 seeds, and after emergence, each pot contains 3 seedlings.

[0061] Experimental design: A total of 7 fertilizer treatments (Examples 1-3, Comparative Examples 1-3) and 1 blank control (CK) were set up, with each treatment replicated 3 times in a randomized block design.

[0062] Fertilization plan: Dilute each fertilizer sample 500 times (by volume) with deionized water and water each pot once on the 1st, 8th, 15th and 22nd days after seedling establishment, 100mL each time. CK is watered with an equal amount of clean water.

[0063] Cultivation conditions: Natural light in greenhouse, temperature 20-28℃, relative humidity 60-80%, and routine water and fertilizer management (no additional fertilizers are applied except for the experimental fertilizer).

[0064] Measurement indicators: On the 30th day after the first fertilization, one representative plant was selected from each pot to measure the plant height (from the root-rhizome junction to the growing point), the fresh weight of the above-ground parts (the entire above-ground part of the plant, after washing and drying, and weighing), and the root activity (using the TTC reduction method, expressed as the number of micrograms of TTC reduced per unit root fresh weight per unit time, μg / (g·h)).

[0065] The results of the above bioactivity tests are recorded in Table 2. Meanwhile, to more clearly and intuitively demonstrate the differences in bioactivity among the groups, comparison charts of plant height and aboveground fresh weight of the examples and comparative examples were drawn, as shown below. Figure 1 As shown; a comparison diagram of root activity in the embodiments and comparative examples, as shown. Figure 2 As shown.

[0066] Table 2 Results of bioactivity tests

[0067] The results above show that the plant height, aboveground fresh weight, and root activity of pakchoi treated with the fertilizers of Examples 1-3 of this invention were significantly better than those of Comparative Examples 1-3 and the blank control. Among them, Example 1 showed the best effect, with plant height increasing by 38.0% compared to Comparative Example 1, 21.0% compared to Comparative Example 2, and 9.5% compared to Comparative Example 3. Aboveground fresh weight and root activity showed the same trend. These results indicate that the fish protein fertilizer prepared by the present invention through a full-process low-temperature synergistic process has higher biological activity and nutrient utilization rate, and can significantly promote crop growth.

[0068] In summary, this invention utilizes a synergistic process involving low-temperature segmented enzymatic hydrolysis, membrane grading and filtration, and low-temperature MVR concentration to produce fish protein fertilizer with high small molecule peptide content, low salt content, and sufficient retention of active ingredients. Furthermore, the production process is energy-efficient and beneficial for crop absorption.

[0069] Finally, it should be noted that the above embodiments are merely illustrative examples for clear explanation and are not intended to limit the implementation. Those skilled in the art will recognize that other variations or modifications can be made based on the above description. It is neither necessary nor possible to exhaustively list all possible implementations here. However, obvious variations or modifications derived therefrom are still within the scope of protection of this invention.

Claims

1. A method for preparing a high-activity small-molecule fish protein fertilizer, characterized in that, Includes the following steps: S1. Mix animal-derived protein raw materials, plant-derived polysaccharide raw materials, and water to obtain a liquid mixture; S2. Perform segmented enzymatic hydrolysis of the feed solution at ≤55℃: Mix the feed solution with the first protease, adjust the pH to 7.0-9.0, and perform the first enzymatic hydrolysis to obtain the first hydrolysate; mix the first hydrolysate with the second protease, adjust the pH to 5.5-7.0, and perform the second enzymatic hydrolysis to obtain the second hydrolysate; mix the second hydrolysate with polysaccharide hydrolase, adjust the pH to 5.0-6.0, and perform the third enzymatic hydrolysis to obtain the third hydrolysate; S3. The third enzymatic hydrolysate is subjected to ultrafiltration and nanofiltration membrane treatment in sequence to obtain a small molecule peptide enrichment solution; S4. Concentrate the small molecule peptide enrichment solution at ≤55℃ to obtain a highly active small molecule fish protein fertilizer.

2. The process for the preparation of high active small molecule fish protein fertilizer as claimed in claim 1, wherein, In S1, the animal-derived protein raw materials include at least one of fish, shrimp, crab, fish processing by-products, shrimp processing by-products, and crab processing by-products; the plant-derived polysaccharide raw materials include at least one of seaweed, soybean meal, and corn.

3. The process for the preparation of high active small molecule fish protein fertilizer as claimed in claim 1, wherein, In S1, the mass ratio of the animal-derived protein raw material, the plant-derived polysaccharide raw material, and water is (250-350):(50-150):(1100-1300).

4. The process for the preparation of high active small molecule fish protein fertilizer as claimed in claim 1, wherein, In S2, the first protease includes at least one of alkaline protease and trypsin; the amount of the first protease added accounts for 0.1-0.5% of the dry basis mass of the liquid in S1; the temperature of the first enzymatic hydrolysis is 50-55℃, and the time is 2-6h.

5. The process for the preparation of high active small molecule fish protein fertilizer as claimed in claim 1, wherein, In S2, the second protease includes at least one of flavor protease, papain, and neutral protease; the amount of the second protease added accounts for 0.1-0.5% of the dry basis mass of the liquid in S1; the temperature of the second enzymatic hydrolysis is 45-50℃, and the time is 1-4h.

6. The process for the preparation of high active small molecule fish protein fertilizer as claimed in claim 1, wherein, In S2, the polysaccharide hydrolase includes at least one of alginate lyase, cellulase, and hemicellulase; the amount of the polysaccharide hydrolase added accounts for 0.05-0.2% of the dry basis mass of the liquid in S1; the temperature of the third enzymatic hydrolysis is 40-50℃, and the time is 1-3h.

7. The method for preparing the highly active small molecule fish protein fertilizer according to claim 1, characterized in that, In S3, the ultrafiltration membrane has a molecular weight cutoff of 3000-10000 Da and an operating pressure of 0.2-0.6 MPa.

8. The method for preparing the highly active small molecule fish protein fertilizer according to claim 1, characterized in that, In S3, the nanofiltration membrane has a molecular weight cutoff of 150-500 Da and an operating pressure of 1.0-3.0 MPa.

9. The method for preparing the highly active small molecule fish protein fertilizer according to claim 1, characterized in that, In S4, the concentration temperature is 45-55℃; the solid content of the highly active small molecule fish protein fertilizer is 30-45%.

10. A highly active small-molecule fish protein fertilizer, characterized in that, The high-activity small molecule fish protein fertilizer is prepared according to any one of claims 1-9.

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