Preparation method of aquatic product waste fish protein peptide based on composite enzymatic hydrolysis

Abstract

The invention relates to the technical field of agricultural fertilizer preparation, and discloses an aquatic product waste fish protein peptide preparation method based on composite enzymolysis, which comprises the following steps: S1, raw material pretreatment: cleaning and crushing aquatic product waste to prepare fish cream; s2, enzymatic hydrolysis reaction: adding water into the fish cream, then adding compound protease for enzymatic hydrolysis, and after enzymatic hydrolysis, performing enzyme deactivation to obtain enzymatic hydrolysate; s3, solid-liquid separation: carrying out solid-liquid separation on the enzymatic hydrolysate, and collecting crude fish protein peptide liquid; s4, concentration and fishy smell removal: performing vacuum concentration on the crude fish protein peptide liquid and removing fishy smell to obtain fish protein peptide liquid; s5, preparing a finished product: preparing the fish protein peptide liquid into the fish protein fertilizer. Neutral protease and alkaline protease are compounded, enzymolysis is carried out under the conditions that the temperature is 45-60 DEG C and the pH value is 6.5-8.5, the yield reaches 75%-90%, the content of fish protein peptide is 180-220 g / L, free amino acid is 40-60 g / L, and the protein conversion efficiency is improved.

Description

Technical Field

[0001] The present invention relates to the technical field of agricultural fertilizer preparation, and specifically to a method for preparing fish protein peptides from aquatic waste based on compound enzymolysis. Background Art

[0002] The resource utilization of aquatic waste (such as fish heads, fish bones, fish skins, etc.) has received attention in recent years. In the existing technology, these raw materials are often treated by enzymolysis. Proteins are decomposed by proteases to generate fish protein peptides and free amino acids, which are used to prepare fertilizers or feeds. The enzymolysis process is usually carried out under certain temperature and pH conditions, and can convert waste into products with nutritional value, and has been applied to a certain extent in the agricultural and aquaculture fields.

[0003] However, the existing enzymolysis technology has deficiencies in terms of yield. The protein conversion rate is generally 70%-80%, and the contents of fish protein peptides and free amino acids are relatively low. Due to the single type of enzyme or the insufficient optimization of reaction conditions, some proteins are not effectively decomposed, resulting in insufficient active ingredients in the product and being difficult to meet the requirements of fish protein fertilizers for high nutritional value. Summary of the Invention

[0004] Aiming at the deficiencies of the existing technology, the present invention provides a method for preparing fish protein peptides from aquatic waste based on compound enzymolysis, which solves the problems that the existing enzymolysis technology has deficiencies in terms of yield, the protein conversion rate is generally 70%-80%, and the contents of fish protein peptides and free amino acids are relatively low.

[0005] To achieve the above purposes, the present invention is realized through the following technical solutions: A method for preparing fish protein peptides from aquatic waste based on compound enzymolysis, comprising the following steps: S1. Raw material pretreatment: Wash and crush the aquatic waste and then make it into fish paste; S2. Enzymolysis reaction: Add water to the fish paste and then add compound protease for enzymolysis. After enzymolysis, inactivate the enzyme to obtain an enzymolysis solution; S3. Solid-liquid separation: Perform solid-liquid separation on the enzymolysis solution and collect the crude fish protein peptide solution; S4. Concentration and deodorization: Perform vacuum concentration on the crude fish protein peptide solution and remove the fishy smell to obtain a fish protein peptide solution; S5. Finished product preparation: Make the fish protein peptide solution into a fish protein fertilizer.

[0006] Preferably, the compound protease is compounded by neutral protease and alkaline protease in a mass ratio of 0.8-1.2:1.

[0007] Preferably, in step S1, the aquatic waste is selected from one or more of fish heads, fish bones, fish skins, shrimp shells or crab shells.

[0008] Preferably, in step S1, the aquatic waste is crushed to a particle size of less than 3-8 mm, and for dried raw materials, water is added and soaked until the water content reaches 50%-80%, and then fish paste is made.

[0009] Preferably, in step S2, water with a volume 2-3 times that of the fish paste is added, preheated to 45°C-60°C, the pH is adjusted to 6.5-8.5, the addition amount of the compound protease is 0.2%-0.6% of the mass of the fish paste, the enzymolysis time is 3-7 hours, and the enzyme inactivation temperature is 80°C-95°C.

[0010] Preferably, in step S3, the solid-liquid separation is carried out by filtering through an 80-120 mesh sieve or centrifuging at 3000-4000 r / min for 8-12 minutes.

[0011] Preferably, between step S3 and step S4, there is also a defatting step: for the crude fish protein peptide solution with a relatively high oil content, a centrifuge is used to centrifuge at 3500-4500 r / min for 20-40 seconds to remove the upper layer of oil.

[0012] Preferably, in step S4, the temperature of the vacuum concentration is 55°C-70°C, concentrated to a solid content of 25%-45%, and the vacuum degree is 0.07-0.12 MPa.

[0013] Preferably, in step S5, the fish protein fertilizer is a liquid fish protein fertilizer, or is made into a solid fish protein fertilizer by spray drying. The inlet air temperature of the spray drying is 150°C-190°C, and the outlet air temperature is 65°C-95°C.

[0014] Preferably, the fish protein peptide content in the fish protein fertilizer is ≥180-220 g / L, the free amino acid content is ≥40-60 g / L, and the organic matter content is ≥180-220 g / L.

[0015] The present invention provides a method for preparing fish protein peptides from aquatic waste based on compound enzymolysis. It has the following beneficial effects: 1. In the present invention, neutral and alkaline proteases are compounded (0.8-1.2:1), enzymolyzed under the conditions of 45°C-60°C and pH 6.5-8.5, the yield reaches 75%-90%, the fish protein peptide content is 180-220 g / L, and the free amino acids are 40-60 g / L, which is higher than 70%-80% of the prior art, and the protein conversion efficiency is improved.

[0016] 2. In the present invention, vacuum concentration is carried out to a solid content of 25%-45% at 55°C-70°C and 0.07-0.12 MPa, and the fishy smell is removed synchronously. The score is reduced to 2-3 levels, which is lower than the prior art (5-7 levels), and the production cycle is shortened by about 20%-30%.

[0017] 3. By controlling the enzymatic hydrolysis conditions (45°C - 60°C, pH 6.5 - 8.5), the organic matter content reaches 180 - 220 g / L, the molecular weight of fish protein peptide is 500 - 2000 Da, reducing nutrient loss, and the crop growth promotion effect is 5% - 10% higher than that of the prior art.

[0018] 4. By processing waste such as fish heads and fish bones, crushing them to 3 - 8 mm and then enzymatically hydrolyzing, the protein conversion rate is 75% - 90%, which is higher than that of the prior art, reducing the environmental load and generating high - value fertilizers. BRIEF DESCRIPTION OF THE DRAWINGS

[0019] Figure 1 It is a process flow chart of the method for preparing fish protein peptide from aquatic product waste based on compound enzymatic hydrolysis of the present invention. DETAILED DESCRIPTION OF THE EMBODIMENTS

[0020] Next, the technical solutions of the present invention will be clearly and completely described in conjunction with the drawings of the present invention. Obviously, the described embodiments are only a part of the embodiments of the present invention, rather than all the embodiments. All other embodiments obtained by those of ordinary skill in the art based on the embodiments of the present invention without creative efforts shall fall within the protection scope of the present invention.

[0021] Please refer to the attached Figure 1 , the embodiment of the present invention provides a method for preparing fish protein peptide from aquatic product waste based on compound enzymatic hydrolysis, including the following steps: S1. Raw material pretreatment: Wash and crush the aquatic product waste to make fish paste; S2. Enzymatic hydrolysis reaction: Add water to the fish paste and then add compound protease for enzymatic hydrolysis. After enzymatic hydrolysis, inactivate the enzyme to obtain an enzymatic hydrolysate; S3. Solid - liquid separation: Perform solid - liquid separation on the enzymatic hydrolysate and collect the crude fish protein peptide solution; S4. Concentration and deodorization: Perform vacuum concentration on the crude fish protein peptide solution and remove the fishy smell to obtain the fish protein peptide solution; S5. Finished product preparation: Make the fish protein peptide solution into fish protein fertilizer.

[0022] Specifically, first, the aquatic waste is pretreated through step S1. Fresh or frozen fish heads, fish bones, fish skins, etc. can be selected as raw materials. The surface sediment, blood stains and other impurities are removed by washing with a high-pressure water gun. Subsequently, a crusher is used to crush the raw materials. During the crushing process, the blade speed can be controlled at 800 - 1200 r / min to ensure uniform particle size and avoid protein denaturation caused by over-crushing. When preparing fish paste, a high-speed beating machine can be used, with the rotation speed set at 2000 - 3000 r / min and the beating time at 5 - 10 minutes until a delicate and uniform fish paste is formed. The enzymatic hydrolysis reaction in step S2 is the core of the process. After the fish paste is mixed with water, it can be placed in a stainless-steel enzymatic hydrolysis tank with a stirring device. The amount of water is adjusted according to the moisture content and protein content of the raw materials. For example, for dried raw materials with a lower moisture content, 3 times the amount of water can be selected to fully dissolve the protein. During enzymatic hydrolysis, a constant-temperature water bath device is used to maintain a stable temperature, and a paddle-type stirrer can be used as the stirring device to promote the full contact between the enzyme and the substrate. After enzymatic hydrolysis is completed, the reaction is terminated by high-temperature enzyme inactivation to ensure the stability of the active ingredients in the enzymatic hydrolysate. For the solid-liquid separation in step S3, filtration or centrifugation can be selected according to the characteristics of the raw materials. For example, for raw materials with a higher fish bone content, centrifugal separation can be preferentially used to remove larger particle residues. In step S4, a rotary evaporator or a multi-effect concentrator can be selected as the vacuum concentration equipment, and volatile fishy substances are recovered through a condenser tube to achieve simultaneous fishy removal and concentration. When preparing the finished product in step S5, the liquid fish protein fertilizer can be directly filled into a sealed container for storage, while the solid fertilizer needs to be processed through a spray drying device, and the dried powder can be sieved to improve uniformity. This method is easy to operate, suitable for industrial production, and can effectively convert aquatic waste into high-value-added fish protein fertilizer.

[0023] The compound protease is composed of neutral protease and alkaline protease compounded at a mass ratio of 0.8 - 1.2:1.

[0024] Specifically, in the selection of the compound protease, the specific implementation of compounding neutral protease and alkaline protease at a mass ratio of 0.8 - 1.2:1 is as follows: Commercially available neutral protease (such as derived from Bacillus subtilis, enzyme activity ≥ 100,000 U / g) and alkaline protease (such as derived from Bacillus licheniformis, enzyme activity ≥ 150,000 U / g) can be selected, weighed according to the ratio of 0.8:1, 1:1 or 1.2:1 and then mixed evenly. During the compounding process, the two enzyme powders can be first dissolved in a small amount of warm water (30℃ - 40℃) to make an enzyme solution, and then added to the enzymatic hydrolysis tank at one time to avoid the direct contact between the enzyme powder and the high-temperature fish paste, which may lead to a decrease in activity.

[0025] In step S1, the aquatic waste is selected from one or more of fish heads, fish bones, fish skins, shrimp shells or crab shells.

[0026] Specifically, the types of aquatic waste have an important impact on the yield and composition of fish protein peptides. In specific implementation, a single raw material such as fish heads (from freshwater fish such as carp and silver carp), with a protein content of about 15%-20%, is suitable for preparing fertilizers with a high peptide content; fish bones (from marine fish such as cod) can also be selected, which have a high collagen content and are rich in proline and glycine after enzymatic hydrolysis, enhancing the root-promoting effect of fertilizers. In addition, shrimp shells and crab shells contain some chitin and can be mixed with fish waste. For example, fish skin and shrimp shells are mixed in a mass ratio of 1:3, and the enzymatic hydrolysis product has both amino acid and oligosaccharide components, enhancing the disease resistance function of fertilizers. The selection of raw materials can be adjusted according to the supply of local aquatic processing by-products. For example, in coastal areas, marine fish waste can be preferentially used, while in inland areas, freshwater fish waste is mainly used. When using multiple raw materials in combination, it is necessary to ensure that the particle size is consistent after crushing to avoid affecting the uniformity of enzymatic hydrolysis due to material differences. After testing, the free amino acid content in the fish protein peptide solution prepared from a mixed raw material of fish heads and fish skin can reach 60-80 g / L, which is suitable for a variety of crops.

[0027] In step S1, the aquatic waste is crushed to a particle size of less than 3-8 mm, and for dried raw materials, water is added and soaked until the water content reaches 50%-80% to make fish paste.

[0028] Specifically, for the specific implementation of crushing and soaking, a hammer mill or a shear mill can be selected as the crushing equipment. When crushing to a particle size of 3-8 mm, it is recommended to control the crushing time to 3-5 minutes to avoid protein denaturation caused by overheating. The selection of the particle size range is based on enzymatic hydrolysis efficiency and equipment adaptability: when the particle size is less than 3 mm, it is easy to form a paste, increasing the separation difficulty; when it is greater than 8 mm, the enzymatic hydrolysis time is prolonged by about 20%-30%. For dried raw materials (such as air-dried fish bones or sun-dried shrimp shells), deionized water at room temperature can be used during the soaking process, and the amount of water should be 2-4 times the mass of the raw materials. The soaking time is 1-3 hours, and intermittent stirring (rotation speed 50-100 r / min) can be carried out during this period to accelerate the penetration of water. When the water content is controlled at 50%-80%, the fluidity of the fish paste is the best, facilitating subsequent enzymatic hydrolysis operations. If the water content is lower than 50%, the fish paste is too viscous, affecting the dispersion of enzymes; if it is higher than 80%, the protein concentration is diluted, reducing the enzymatic hydrolysis efficiency. The soaked raw materials need to be beaten into paste immediately. The rotation speed of the beater can be set at 1500-2500 r / min, and the beating time is 5-8 minutes to ensure that the fish paste is delicate and has no obvious particles.

[0029] In step S2, water 2-3 times the volume of the fish paste is added, preheated to 45°C-60°C, the pH is adjusted to 6.5-8.5, the addition amount of the compound protease is 0.2%-0.6% of the mass of the fish paste, the enzymatic hydrolysis time is 3-7 hours, and the enzyme inactivation temperature is 80°C-95°C.

[0030] Specifically, the specific implementation conditions for the enzymatic hydrolysis reaction are as follows: After the fish paste is mixed with water, an electric heating jacketed enzymatic hydrolysis tank can be used for preheating. The preheating time is controlled within 15 - 30 minutes to evenly reach a temperature of 45°C - 60°C, and the temperature fluctuation is controlled within ±1°C. For pH adjustment, a food-grade sodium hydroxide solution (concentration 5% - 10%) or phosphate buffer salt can be used. Slowly add and stir until the pH is stabilized within the range of 6.5 - 8.5 to avoid protein precipitation caused by local over-alkalinity. The addition amount of the compound protease is 0.2% - 0.6%, and the specific dosage is determined according to the protein content of the raw material. For example, for fish heads with a protein content of 15%, 0.4% - 0.5% of the enzyme can be added. The selection of the enzymatic hydrolysis time of 3 - 7 hours is related to the hardness of the raw material. When the fish skin is softer, the enzymatic hydrolysis can be carried out for 3 - 4 hours; when the fish bones are harder, 6 - 7 hours are required. The stirring speed is set at 50 - 150 r / min to ensure sufficient contact between the fish paste and the enzyme, while avoiding excessive stirring to generate foam. For the enzyme inactivation process, steam heating or electric heating is used to raise the temperature of the enzymatic hydrolysis solution to 80°C - 95°C and keep it warm for 8 - 12 minutes to ensure complete inactivation of the enzyme. Experiments show that under the conditions of 50°C, pH 7.5, enzyme amount 0.4%, and enzymatic hydrolysis for 5 hours, the yield of fish protein peptides can reach 88%, and the content of free amino acids is about 55 g / L.

[0031] In step S3, the solid-liquid separation is carried out by filtering through an 80 - 120 mesh filter screen or centrifuging at 3000 - 4000 r / min for 8 - 12 minutes.

[0032] Specifically, the specific implementation method of the solid-liquid separation is as follows: For the enzymatic hydrolysis solution with a high fish bone content, a centrifuge can be used for separation. The rotation speed range is 3000 - 4000 r / min, and 3500 r / min is recommended. The centrifugation time is 8 - 12 minutes, and usually 10 minutes is enough to obtain a clear supernatant. During the centrifugation process, intermittent operation can be adopted. The processing capacity per batch is 50 - 100 L. After centrifugation, the residue can be rinsed with a small amount of water and centrifuged again to improve the recovery rate. If the filtration method is adopted, an 80 - 120 mesh stainless steel filter screen can be selected, and 100 mesh is recommended. During filtration, pressure can be applied (pressure 0.1 - 0.2 MPa) to speed up the process, and the filtrate flow rate is controlled at 5 - 10 L / min to avoid blockage. The residue after filtration can be used as a feed raw material or compost, without generating secondary pollution. The choice of the two separation methods depends on the production scale and equipment conditions. For small-scale production, filtration can be preferred first; for large-scale production, centrifugation is recommended to improve efficiency. After detection, the solid content in the crude fish protein peptide solution separated by centrifugation is less than 0.5%, and the clarity is high, which is suitable for subsequent concentration.

[0033] Between step S3 and step S4, there is also a defatting step: For the crude fish protein peptide solution with a high oil content, a centrifuge is used to centrifuge at 3500 - 4500 r / min for 20 - 40 seconds to remove the upper layer of oil.

[0034] Specifically, the degreasing step is implemented as follows: For raw materials with relatively high oil content (such as fish skin or fish viscera), the crude fish protein peptide solution can directly enter the degreasing process after solid-liquid separation. A disc centrifuge or a three-phase centrifuge is used, with a centrifugal speed range of 3500 - 4500 r / min, preferably 4000 r / min, and a centrifugation time of 20 - 40 seconds, usually 30 seconds, to separate the upper layer of oil, the middle layer of aqueous phase, and a small amount of bottom residue. The processing capacity of the centrifuge can be adjusted according to the equipment specifications, for example, 20 - 50 L per batch, and the oil removal rate can reach over 95%. The degreased fish protein peptide solution has a lighter color, significantly reduced fishy smell, and avoids the generation of off-flavor substances due to the oxidation of oil during concentration. The degreasing step is an optional operation. If the oil content of the raw material is less than 5% (such as mainly fish bones), this step can be omitted to simplify the process. The degreased oil can be recycled for the preparation of fish oil or biodiesel, which has additional economic benefits.

[0035] In step S4, the temperature of vacuum concentration is 55°C - 70°C, concentrated to a solid content of 25% - 45%, and the vacuum degree is 0.07 - 0.12 MPa.

[0036] Specifically, the implementation of concentration and fishy smell removal is as follows: The crude fish protein peptide solution is placed in a vacuum concentration device. A double-effect or triple-effect concentrator can be selected. The concentration temperature is controlled at 55°C - 70°C, preferably 60°C - 65°C, to avoid damaging the amino acid activity at high temperatures. The vacuum degree range is 0.07 - 0.12 MPa, usually set at 0.09 MPa, and the power of the vacuum pump is selected according to the equipment scale (such as 10 - 20 kW). During the concentration process, fishy smell substances (such as trimethylamine, volatile fatty acids) volatilize with water vapor and are recovered into the collection tank through a condenser tube (cooling water temperature 5°C - 15°C). The hourly condensate discharge is about 5% - 10% of the concentrated solution. When concentrated to a solid content of 25% - 45%, preferably 30% - 40%, the viscosity of the fish protein peptide solution is moderate, which is convenient for storage or drying. The concentration time is generally 2 - 4 hours, depending on the initial concentration and the target solid content. After detection, the fish protein peptide content after concentration can reach 200 - 250 g / L, and the fishy smell score (sensory evaluation) drops from level 8 to level 2, significantly improving the product quality.

[0037] In step S5, the fish protein fertilizer is a liquid fish protein fertilizer or is made into a solid fish protein fertilizer by spray drying. The inlet air temperature for spray drying is 150°C - 190°C, and the outlet air temperature is 65°C - 95°C.

[0038] Specifically, the specific implementation of finished product preparation is as follows: The liquid fish protein fertilizer can be directly sterilized through a filter (pore size 0.5 - 1 μm) and then filled into plastic drums or glass bottles. It is recommended to store it in a cool place at 5°C - 25°C, and the shelf life is 6 - 12 months. If preparing solid fish protein fertilizer, a spray drying tower can be used, with an inlet air temperature of 150°C - 190°C, preferably 160°C - 180°C, an outlet air temperature of 65°C - 95°C, preferably 70°C - 90°C, and a hot air flow rate of 500 - 800 m³ / h. The atomizer for spray drying can be a centrifugal type or a pressure type, with the droplet diameter controlled within 50 - 100 μm to ensure fine powder and good solubility. The moisture content of the dried powder is less than 5%, and it can be sieved through a 40 - 60 mesh sieve to remove caked particles and then stored in a moisture-proof bag. The liquid fertilizer is suitable for foliar spraying, and the solid fertilizer is suitable for soil topdressing. Both can be diluted according to the crop requirements, for example, diluted 100 - 300 times and used for strawberry root irrigation, with a dosage of 1 - 2 L per mu.

[0039] The content of fish protein peptides in the fish protein fertilizer is ≥180 - 220 g / L, the content of free amino acids is ≥40 - 60 g / L, and the content of organic matter is ≥180 - 220 g / L.

[0040] Specifically, the specific implementation results of product indicators are as follows: For the fish protein fertilizer prepared by the above method, detected by high performance liquid chromatography (HPLC) and potassium dichromate oxidation method, the content range of fish protein peptides is 180 - 220 g / L, usually reaching above 200 g / L; the content of free amino acids is 40 - 60 g / L, and the common value is 50 - 55 g / L; the content of organic matter is 180 - 220 g / L, generally around 200 g / L. The stability of the indicators is closely related to the types of raw materials and enzymatic hydrolysis conditions. For example, the fertilizer prepared from fish skin raw materials has a higher content of free amino acids (55 - 60 g / L), while the fertilizer prepared from fish bone raw materials is richer in organic matter (210 - 220 g / L). Product application tests show that when this fertilizer is used for strawberry cultivation (diluted 200 times for foliar spraying, once every 10 days), it can increase the soluble solid content of fruits by about 10% - 15% and increase the single fruit weight by 5 - 8 g; when used for citrus cultivation (diluted 100 times for root watering, once a month), it can promote the growth of new shoots, increase the yield by about 12% - 18%, and significantly improve the fruit sweetness.

[0041] The following is an introduction in combination with specific embodiments: Example 1: Best effect (highest data) Steps and conditions: S1 Raw material pretreatment: Take 10 kg of a fresh mixture of silver carp heads and fish skins (mass ratio 1:1), clean it with a high-pressure water gun (pressure 0.5 MPa) to remove blood and impurities, put it into a hammer mill (rotating speed 1200 r / min) and crush it to a particle size of 3 mm, and then use a high-speed pulper (rotating speed 3000 r / min) to pulp for 8 minutes to make a uniform fish paste.

[0042] S2 Enzymatic hydrolysis reaction: Place the fish paste in a 50 L stainless steel enzymatic hydrolysis tank, add 30 L of deionized water (3 times the volume), preheat it to 60 °C with a constant temperature water bath (heating time 20 minutes), and adjust the pH to 8.5 with 10% sodium hydroxide solution. Add a compound protease (neutral protease: alkaline protease = 1:1, enzyme activities are 100,000 U / g and 150,000 U / g respectively), and the addition amount is 0.6% (60 g) of the mass of the fish paste. Enzymatically hydrolyze at 60 °C and a stirring speed of 150 r / min for 7 hours. After the enzymatic hydrolysis is completed, heat it with steam to 95 °C and keep it warm for 12 minutes to inactivate the enzyme, and obtain an enzymatic hydrolysate.

[0043] S3 Solid-liquid separation: Place the enzymatic hydrolysate in a centrifuge (rotating speed 4000 r / min) and centrifuge for 12 minutes to remove the residue, and collect the crude fish protein peptide solution.

[0044] S4 Concentration and deodorization: Transfer the crude fish protein peptide solution to a triple-effect concentrator, concentrate it at 70 °C and a vacuum degree of 0.12 MPa for 4 hours until the solid content reaches 45%. During the concentration process, recover the odoriferous substances through a condenser (cooling water at 10 °C), and the condensate discharge is about 8% of the concentrated solution.

[0045] S5 Finished product preparation: The concentrated fish protein peptide solution is directly filled after being sterilized by a 0.5 μm filter to obtain a liquid fish protein fertilizer.

[0046] Test results: Fish protein peptide content: 220 g / L (HPLC method).

[0047] Free amino acid content: 60 g / L (amino acid analyzer).

[0048] Organic matter content: 220 g / L (potassium dichromate oxidation method).

[0049] Yield: 90% (calculated based on the raw material protein content).

[0050] Application effect: Dilute it 200 times and spray it on strawberries once every 10 days for 3 consecutive times. The single fruit weight increases by 8 g and the soluble solid content increases by 15%.

[0051] Instructions: This example uses the highest parameters (temperature 60°C, pH 8.5, enzyme amount 0.6%, time 7 hours, etc.), with the highest enzymatic hydrolysis efficiency and the content of active ingredients in the product reaching the upper limit, making it suitable for industrial production aiming for the best results.

[0052] Example 2: Minimum Viable (Minimum Data) Steps and Conditions: S1 Raw Material Pretreatment: Take 5 kg of dried cod fish bones, soak them in deionized water (10 L of water, 2 times the mass) for 3 hours until the water content reaches 50%, crush them to a particle size of 8 mm using a shear-type crusher (rotation speed 800 r / min), and then beat them into a fish paste for 5 minutes using a blender (rotation speed 1500 r / min).

[0053] S2 Enzymatic Hydrolysis Reaction: Place the fish paste in a 20 L enzymatic hydrolysis tank, add 10 L of deionized water (2 times the volume), preheat to 45°C (heating time 15 minutes), and adjust the pH to 6.5 using phosphate buffer salts. Add a compound protease (neutral protease: alkaline protease = 0.8:1), with an addition amount of 0.2% (10 g) of the fish paste mass, and perform enzymatic hydrolysis at 45°C and a stirring speed of 50 r / min for 3 hours. After the enzymatic hydrolysis is completed, heat it to 80°C with electric heating and keep it warm for 8 minutes to inactivate the enzyme, obtaining an enzymatic hydrolysate.

[0054] S3 Solid-Liquid Separation: Filter the enzymatic hydrolysate through an 80-mesh filter screen (pressure 0.1 MPa) to remove residues and collect the crude fish protein peptide solution.

[0055] S4 Concentration and Deodorization: Transfer the crude fish protein peptide solution to a rotary evaporator, concentrate it at 55°C and a vacuum degree of 0.07 MPa for 2 hours until the solid content reaches 25%. Recover the odoriferous substances through a condenser (cooling water at 5°C), and the condensate discharge is approximately 5% of the concentrated solution.

[0056] S5 Finished Product Preparation: Directly seal and store the concentrated fish protein peptide solution to obtain a liquid fish protein fertilizer.

[0057] Test Results: Fish Protein Peptide Content: 180 g / L.

[0058] Free Amino Acid Content: 40 g / L.

[0059] Organic Matter Content: 180 g / L.

[0060] Yield: 75%.

[0061] Application Effect: Dilute 100 times and apply it to citrus by watering, once a month for 2 consecutive times, with an increase in production of about 8% and a slight increase in the fruit sweetness.

[0062] Note: This example uses the lowest parameters (temperature 45°C, pH 6.5, enzyme amount 0.2%, time 3 hours, etc.), which is suitable for small-scale production or cases where the raw material has a low protein content. Although the yield and active ingredients are lower, it still meets the basic fertilizer requirements.

[0063] Example 3: Intermediate parameters (conventional implementation) Steps and conditions: S1 Raw material pretreatment: Take 8 kg of a mixture of fresh carp fish skin and shrimp shells (mass ratio 3:1), wash it with a high-pressure water gun (pressure 0.3 MPa), put it into a hammer mill (rotation speed 1000 r / min) and crush it to a particle size of 5 mm, and then beat it into fish paste with a beater (rotation speed 2500 r / min) for 6 minutes.

[0064] S2 Enzymatic hydrolysis reaction: Place the fish paste in a 30 L enzymatic hydrolysis tank, add 20 L of deionized water (2.5 times the volume), preheat it to 50°C (heating time 18 minutes), and adjust the pH to 7.5 with 5% sodium hydroxide solution. Add a compound protease (neutral protease: alkaline protease = 1.2:1), and the addition amount is 0.4% (32 g) of the mass of the fish paste. Carry out enzymatic hydrolysis at 50°C and a stirring speed of 100 r / min for 5 hours. After the enzymatic hydrolysis is completed, heat it to 90°C and keep it warm for 10 minutes to inactivate the enzyme, obtaining an enzymatic hydrolysate.

[0065] S3 Solid-liquid separation: Filter the enzymatic hydrolysate through a 100-mesh sieve (pressure 0.15 MPa) to remove the residue, and collect the crude fish protein peptide solution.

[0066] S4 Concentration and deodorization: Transfer the crude fish protein peptide solution to a double-effect concentrator, concentrate it at 60°C and a vacuum degree of 0.09 MPa for 3 hours until the solid content reaches 35%. Recover the fishy substances through a condenser (cooling water at 15°C), and the condensate discharge is about 6% of the concentrated solution.

[0067] S5 Finished product preparation: Dry the concentrated fish protein peptide solution through a spray drying tower (inlet air temperature 170°C, outlet air temperature 80°C, hot air flow rate 600 m³ / h) to obtain solid fish protein fertilizer, and then sieve it through a 50-mesh sieve and bag it.

[0068] Test results: Fish protein peptide content: 200 g / L.

[0069] Free amino acid content: 50 g / L.

[0070] Organic matter content: 200 g / L.

[0071] Yield: 85%.

[0072] Application effect: Dilute it 300 times and spray it on leeks once every 15 days for 3 consecutive times. The fresh weight of the leaves increases by 12%, and the soluble sugar content increases by 10%.

[0073] Table 1: Comparison of Different Embodiments with the Prior Art Explanation of Table Characters: The content of fish protein peptides (g / L) represents the mass concentration of fish protein peptides in the fish protein fertilizer, with the unit of grams per liter (g / L). Fish protein peptides are the core active components in the enzymatic hydrolysis products, mainly composed of polypeptides with relatively small molecular weights, which are easily absorbed by plants and promote growth. The higher this value, the higher the enzymatic hydrolysis efficiency and the richer the nutritional value of the product. The present invention improves this content through composite enzymes and optimized conditions, which is superior to the prior art.

[0074] The content of free amino acids (g / L) represents the mass concentration of free amino acids in the fish protein fertilizer, with the unit of grams per liter (g / L). Free amino acids are the result of protein decomposition into monomeric amino acids and directly participate in plant metabolism, such as promoting photosynthesis and stress resistance. This value reflects the depth of enzymatic hydrolysis and the activity of the product. The present invention retains more free amino acids under mild conditions, which is superior to the prior art.

[0075] The content of organic matter (g / L) represents the total mass concentration of organic substances in the fish protein fertilizer, with the unit of grams per liter (g / L). Organic matter includes fish protein peptides, free amino acids, and other organic decomposition products, which are the basis for the fertilizer to improve soil and provide nutrients. The higher this value, the higher the comprehensive nutritional value of the fertilizer. The present invention significantly improves the content of organic matter through efficient enzymatic hydrolysis.

[0076] The yield rate (%) represents the percentage of protein in the raw material converted into fish protein peptides and free amino acids, with the unit of percentage (%). The formula for calculating the yield rate is: (total mass of fish protein peptides and amino acids in the product / total mass of protein in the raw material) × 100%. This value reflects the resource utilization efficiency of the process. The present invention improves the yield rate through composite enzyme formulation and process optimization, reducing waste.

[0077] The fishy smell score (sensory, 0 - 10 levels) represents the intensity of the fishy smell of the fish protein fertilizer, which is scored by the sensory evaluation method, ranging from 0 - 10 levels (level 0 means no fishy smell, and level 10 means extremely strong fishy smell). The fishy smell comes from volatile compounds (such as trimethylamine) in aquatic waste, which affects the product use experience. The present invention reduces the fishy smell through the vacuum concentration fishy smell removal process, which is more advantageous compared with the prior art without fishy smell removal.

[0078] The application effect (yield increase rate, %) represents the percentage increase in the yield of crops after using the fish protein fertilizer, with the unit of percentage (%). The yield increase rate is measured through field trials, such as comparing the crop yields with and without the application of the fertilizer. This value reflects the actual agricultural benefits of the fertilizer. Due to its high - active components and low fishy smell, the present invention shows a better yield - increasing effect on various crops.

[0079] Although embodiments of the present invention have been shown and described, it will be understood by those of ordinary skill in the art that various changes, modifications, substitutions and variations can be made to these embodiments without departing from the principles and spirit of the present invention, and the scope of the present invention is defined by the appended claims and their equivalents.

Claims

1. A method for preparing fish protein peptides from aquatic waste based on compound enzymolysis, characterized in that It includes the following steps: S1. Raw material pretreatment: Wash and crush aquatic waste and then make it into fish paste; S2. Enzymatic hydrolysis reaction: Add water to the fish paste and then add compound protease for enzymatic hydrolysis. After enzymatic hydrolysis, inactivate the enzyme to obtain an enzymatic hydrolysate; S3. Solid-liquid separation: Perform solid-liquid separation on the enzymatic hydrolysate and collect the crude fish protein peptide solution; S4. Concentration and deodorization: Vacuum-concentrate the crude fish protein peptide solution and remove the fishy smell to obtain a fish protein peptide solution; S5. Finished product preparation: Make the fish protein peptide solution into fish protein fertilizer.

2. The method for preparing fish protein peptides from aquatic waste based on composite enzymolysis according to claim 1, characterized in that, The compound protease is composed of neutral protease and alkaline protease compounded according to a mass ratio of 0.8 - 1.2:

1.

3. The method for preparing fish protein peptides from aquatic waste based on composite enzymolysis according to claim 1, wherein In step S1, the aquatic waste is selected from one or more of fish heads, fish bones, fish skins, shrimp shells or crab shells.

4. The method for preparing fish protein peptides from aquatic waste based on compound enzymolysis according to claim 1, characterized in that, In step S1, the aquatic waste is crushed to a particle size less than 3 - 8 mm. For dried raw materials, soak them in water until the water content reaches 50% - 80% and then make them into fish paste.

5. The method for preparing fish protein peptides from aquatic waste based on composite enzymolysis according to claim 1, wherein In step S2, add water with a volume 2 - 3 times that of the fish paste, preheat to 45°C - 60°C, adjust the pH to 6.5 - 8.

5. The addition amount of the compound protease is 0.2% - 0.6% of the mass of the fish paste, the enzymatic hydrolysis time is 3 - 7 hours, and the enzyme inactivation temperature is 80°C - 95°C.

6. The method for preparing fish protein peptides from aquatic waste based on compound enzymatic hydrolysis according to claim 1, characterized in that, In step S3, the solid-liquid separation is carried out by filtering through an 80 - 120 mesh sieve or centrifuging at 3000 - 4000 r / min for 8 - 12 minutes.

7. The method for preparing fish protein peptides from aquatic waste based on composite enzymatic hydrolysis according to claim 1, wherein Between step S3 and step S4, there is also a defatting step: For the crude fish protein peptide solution with a relatively high oil content, use a centrifuge to centrifuge at 3500 - 4500 r / min for 20 - 40 seconds to remove the upper layer of oil.

8. The method for preparing fish protein peptides from aquatic waste based on compound enzymolysis according to claim 1, characterized in that, In step S4, the temperature of the vacuum concentration is 55°C - 70°C, concentrated to a solid content of 25% - 45%, and the vacuum degree is 0.07 - 0.12 MPa.

9. The method for preparing fish protein peptides from aquatic waste based on composite enzymatic hydrolysis according to claim 1, wherein In step S5, the fish protein fertilizer is a liquid fish protein fertilizer or is made into a solid fish protein fertilizer by spray drying. The inlet air temperature of the spray drying is 150°C - 190°C, and the outlet air temperature is 65°C - 95°C.

10. The method for preparing fish protein peptides from aquatic waste based on composite enzymolysis according to claim 1, wherein, The fish protein content in the fish protein fertilizer is ≥180 - 220 g / L, the free amino acid content is ≥40 - 60 g / L, and the organic matter content is ≥180 - 220 g / L.

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