A low-temperature enzymolysis production method of all-water-soluble oil cake organic fertilizer

Abstract

The application discloses a low-temperature enzymolysis production method of full-water-soluble oil cake organic fertilizer, and particularly relates to the technical field of organic fertilizer production, and comprises the following steps: vacuum ultrasonic and low-temperature puffing pretreatment is carried out on oil cake; under the condition of 25-35 DEG C, a limited immobilized composite enzyme preparation is added into the pretreated liquid to carry out a multi-phase circulation gradient enzymolysis reaction, the enzymolysis reaction comprises ultrasonic assistance, circulation backflow of the enzymolysis liquid and the introduction of micro-pressure gas to form gas-liquid-solid three-phase disturbance, the composite enzyme preparation comprises three layers of enzyme systems separated by a separation film layer and forms a time sequence release mechanism; and the enzyme preparation is recovered and reused after the enzymolysis is completed. The application can realize the full-water-soluble conversion of the oil cake at low temperature, the product has high full-water-soluble rate, rich free amino acid content, short production cycle and low energy consumption, the enzyme can be reused, the anti-nutritional factors are completely removed, and the application is suitable for water-fertilizer integrated planting and high-value utilization of the oil cake.

Description

Technical Field

[0001] This invention relates to the field of organic fertilizer production technology, and more specifically, to a low-temperature enzymatic hydrolysis method for producing fully water-soluble oilseed cake organic fertilizer. Background Technology

[0002] Oilseed cake is a byproduct of oilseed pressing and oil extraction. Rich in nutrients such as protein and organic matter, it is a high-quality raw material for producing organic fertilizer. Current technologies for producing organic fertilizer from oilseed cake mainly include high-temperature composting fermentation, enzymatic hydrolysis, and enzyme-microbial co-fermentation.

[0003] High-temperature composting fermentation requires 15-30 days at 55-75℃, resulting in a long production cycle, high energy consumption, and the high temperature environment destroys heat-sensitive active ingredients in the oilseed cake, leading to significant losses of nutrients such as amino acids. Existing enzymatic hydrolysis technologies are mostly geared towards the feed industry, with hydrolysis temperatures still reaching 40-50℃. The products contain large amounts of insoluble residues and have poor water solubility, making them unsuitable for integrated water and fertilizer management systems such as drip irrigation and sprinkler irrigation. While enzyme-microbial co-fermentation offers some improvement, it is essentially a simple superposition of enzymes and bacteria, lacking spatiotemporal matching between enzyme systems, resulting in poor product uniformity. Furthermore, oilseed cake commonly contains various anti-nutritional factors such as glucosinolates, gossypol, and saponins. Current detoxification methods primarily rely on high-temperature heating, which has limited detoxification efficiency and further exacerbates the destruction of heat-sensitive components.

[0004] Therefore, there is an urgent need to develop a production method that can simultaneously achieve efficient degradation, full water-soluble conversion, and deep detoxification of oilseed cake under low-temperature conditions. Summary of the Invention

[0005] To achieve the above objectives, the present invention provides the following technical solution: a low-temperature enzymatic hydrolysis method for producing fully water-soluble oilseed cake organic fertilizer, comprising the following steps: S1. The oilseed cake raw material is subjected to vacuum ultrasonic combined low temperature puffing pretreatment to obtain a pretreated mixture; S2. Under conditions of 25–35°C, a confined immobilized complex enzyme preparation is sequentially added to the pretreated mixture to carry out a multiphase cyclic gradient enzymatic hydrolysis reaction. The multiphase cyclic gradient enzymatic hydrolysis reaction includes: under the assistance of ultrasound, the enzymatic hydrolysate is circulated back through the solid phase enzymatic hydrolysis bed, and micro-pressure gas is introduced to form a gas-liquid-solid three-phase disturbance. The confined immobilized complex enzyme preparation contains three layers of enzyme systems, which are added in the following order: the first layer is a cellulase system, the second layer is a protease system, and the third layer is an anti-nutritional factor targeting enzyme system. Each enzyme system is separated by an isolation membrane layer in a mesoporous carrier to form a time-sequential release mechanism. S3. After the enzymatic hydrolysis reaction is completed, the enzymatic hydrolysate and residual confined immobilized complex enzyme preparation are recovered. The recovered confined immobilized complex enzyme preparation is reused in the next batch of reaction.

[0006] Preferably, in step S1, the specific process parameters for the vacuum ultrasonic synergistic low-temperature puffing pretreatment are as follows: solid-liquid ratio 1:1.5~2.5, immersion temperature 15~25℃, immersion time 30~60min, vacuum degree -0.085~-0.095MPa, ultrasonic power density 0.12~0.18kW / kg, ultrasonic frequency in the range of 25~35kHz with alternating frequency sweeps of 30s, temperature 25~30℃, and time 30~45min.

[0007] Preferably, in step S2, the mesoporous carrier is mesoporous silica with a pore size of 12-18 nm, and the enzyme systems in each layer are separated by an isolation membrane layer, which is composed of polyvinyl alcohol-polylysine and has a thickness of 2-3 nm.

[0008] Preferably, in step S2, the first layer of cellulase system includes endoglucanase, exoglucanase and β-glucosidase, the second layer of protease system includes alkaline protease, neutral protease and flavor protease, and the third layer of anti-nutritional factor targeting enzyme system includes myrosinase, β-glucosidase and LPMO.

[0009] Preferably, the mass ratio of endoglucanase, exoglucanase, and β-glucosidase in the first cellulase system is 2:1:1; the mass ratio of alkaline protease, neutral protease, and flavor protease in the second protease system is 2:2:1; and the mass ratio of myrosinase, β-glucosidase, and LPMO in the third antinutritional factor targeting enzyme system is 2:1:1.

[0010] Preferably, the timing and amount of enzyme addition in step S2 are as follows: At the start of the reaction, add the first layer of cellulase system at 0h, with an enzyme dosage of 50-80 U / g dry material, and react for 30min. After 0.5 hours of reaction, a second layer of protease system is added at an enzyme dosage of 200–400 U / g of dry material, and the reaction continues for 2 hours. After 2.5 hours of reaction, add the third layer of anti-nutritional factor targeted enzyme system at an enzyme dosage of 30-60 U / g dry material, and continue the reaction until completion.

[0011] Preferably, in step S2, the multiphase cyclic gradient enzymatic hydrolysis reaction is carried out in a reactor. The reactor is equipped with a circulating liquid collection tank at the bottom and a spray device at the top. During the reaction, the enzymatic hydrolysate in the collection tank is pumped to the top spray device by a circulating pump, so that it passes through the solid phase enzymatic hydrolysis bed again to form a circulating reflux. The circulation flow rate is 3 to 5 times the material volume / hour. Micro-pressure air is introduced into the bottom of the reactor at a pressure of 0.02–0.05 MPa. Ultrasonic assistance is activated during the reaction, with the ultrasonic frequency alternating between 20 and 40 kHz with a period of 60 seconds. The ultrasonic power density is 0.10–0.15 kW / kg. The pH value is maintained at 6.5–7.8 during the reaction using an online pH controller.

[0012] Preferably, in step S3, the total duration of the enzymatic hydrolysis reaction is 16-24 hours. The endpoint is determined by the degree of protein hydrolysis ≥45% and the residual rate of anti-nutritional factors ≤8%. After the enzymatic hydrolysis is completed, the pH of the reaction system is adjusted to 5.0-5.5, the confined immobilized complex enzyme preparation is precipitated and recovered, and the enzyme preparation is washed and regenerated with a buffer solution of pH 6.5-7.0, and the enzyme activity retention rate is ≥85%.

[0013] Preferably, the process further includes solid-liquid separation of the enzymatic hydrolysate obtained in step S3, collection of the supernatant, and vacuum concentration of the supernatant at 55-65°C to a total solids content of 30-35% to obtain a fully water-soluble organic liquid fertilizer; or further spray drying to produce a fully water-soluble organic fertilizer powder, wherein the inlet air temperature of the spray dryer is 120-140°C and the outlet air temperature is 60-70°C.

[0014] This invention also provides a confined immobilized complex enzyme preparation for the low-temperature enzymatic hydrolysis of oilseed cake, comprising a mesoporous carrier and a three-layer enzyme system immobilized in the mesoporous carrier. The mesoporous carrier has a first region, a second region, and a third region arranged sequentially in space. The three-layer enzyme system, in order of release, is a first layer of cellulase system, a second layer of protease system, and a third layer of anti-nutritional factor targeting enzyme system. Each enzyme system is separated by an isolation membrane layer. The first layer of cellulase system is confined in the first region of the mesoporous carrier, with a release half-life ≤30 min. The second layer of protease system is confined in the second region of the mesoporous carrier, with a release half-life of 1–2 h. The third layer of anti-nutritional factor targeting enzyme system is confined in the third region of the mesoporous carrier, with a release half-life of 2–4 h.

[0015] The technical effects and advantages of this invention are as follows: 1. This invention achieves efficient conversion of oilseed cake into fully water-soluble small molecule active substances through the time-sequential release of a confined immobilized three-layer enzyme system and low-temperature multiphase cyclic gradient enzymatic hydrolysis. The resulting product has high water solubility, rich free amino acid content, and low average molecular weight, which can fully meet the needs of drip irrigation, sprinkler irrigation and other integrated water and fertilizer planting. Moreover, the low temperature conditions effectively preserve the heat-sensitive active ingredients, and the bioavailability of the product is significantly better than that of traditional fermentation processes. 2. The invention operates under mild and low temperature conditions throughout the process, without the need for high-temperature cooking or long-term fermentation. The total enzymatic hydrolysis time is shortened by more than 90% compared with the traditional composting process, and the energy consumption per unit product is reduced by more than half. At the same time, the multi-phase enhanced mass transfer methods of ultrasonic assistance, circulation reflux and micro-pressure ventilation greatly improve the contact efficiency between enzyme and substrate, and realize efficient and low-carbon production. 3. This invention uses confined immobilized compound enzyme preparations, which can be recycled and reused, significantly reducing enzyme costs. It simultaneously and efficiently removes multiple anti-nutritional factors from oilseed cake, resulting in high product safety, no fermentation waste discharge, and high solids conversion rate. This achieves high-value and full utilization of oilseed cake resources, and has good economic and social benefits. Attached Figure Description

[0016] Figure 1 This is a flowchart of the overall method of the present invention. Detailed Implementation

[0017] The technical solutions of the present invention will be clearly and completely described below with reference to the accompanying drawings in the embodiments of the present invention.

[0018] Example 1: Preparation of fully water-soluble organic liquid fertilizer using rapeseed cake as raw material This embodiment provides a low-temperature enzymatic hydrolysis method for producing fully water-soluble oilseed cake organic fertilizer, including the following steps: S1, Vacuum ultrasonic synergistic low-temperature puffing pretreatment Commercially available low-glucosinolate rapeseed cake with a crude protein content of 38.5% and a moisture content of 8.2% was used. The rapeseed cake was pulverized to a fineness of 60 mesh, and pure water was added at a solid-liquid ratio of 1:2.0, i.e., 200 kg of pure water was added for every 100 kg of dry material. The mixture was placed in a sealed conditioning tank and soaked at a temperature of 15–25°C (specifically, 25°C in this embodiment) for 45 minutes. Subsequently, ultrasonic-assisted puffing was performed for 40 minutes under a vacuum of -0.09 MPa and a temperature of 28°C. The ultrasonic power density was 0.16 kW / kg, and the ultrasonic frequency was in the range of 25–35 kHz (specifically, 28–32 kHz with alternating frequency sweeps every 30 seconds in this embodiment). Scanning electron microscopy revealed that the cell wall destruction rate of the treated material reached 87.3%, yielding a pretreated mixture. S2, Multiphase cyclic gradient enzymatic hydrolysis reaction First, a confined immobilized complex enzyme preparation was prepared. Mesoporous silica microspheres with a pore size of 12–18 nm were prepared as a carrier using a template method; in this embodiment, a pore size of 15 nm was specifically used. The mesoporous silica microspheres were then subjected to a three-layer confined enzyme immobilization process: the first layer was a cellulase system containing endoglucanase, exoglucanase, and β-glucosidase in a mass ratio of 2:1:1, with an enzyme dosage of 60 U / g (dry weight). This was immobilized in the first region of the mesoporous silica via electrostatic adsorption and glutaraldehyde cross-linking. A 2–3 nm thick isolation membrane was formed on the surface of the first enzyme layer using a layer-by-layer self-assembly method; in this embodiment, the thickness was 2.5 nm. The isolation membrane was composed of polyvinyl alcohol and polylysine. The second layer is a protease system, comprising alkaline protease, neutral protease, and flavor protease in a mass ratio of 2:2:1, with an enzyme dosage of 300 U / g dry material. It is immobilized in the second region of mesoporous silica, and a 2.5 nm thick isolation membrane of the same material is formed again on the surface of the second layer. The third layer is an anti-nutritional factor targeting enzyme system, comprising myrosinase, β-glucosidase, and LPMO in a mass ratio of 2:1:1, with an enzyme dosage of 40 U / g dry material. It is immobilized in the third region of mesoporous silica. In the resulting confined immobilized composite enzyme preparation, the release half-life of the first layer enzyme system is ≤30 min, the half-life of the second layer is 1–2 h (1.5 h in this example), and the half-life of the third layer is 2–4 h (3 h in this example). The pretreated mixture obtained in step S1 is transferred to a 300L enzymatic hydrolysis reactor. The reactor has a circulating liquid collection tank at the bottom and a spray device at the top. During the reaction, a circulating pump draws the hydrolysate from the collection tank to the top spray device, allowing it to pass through the solid-phase enzymatic hydrolysis bed again to form a circulating reflux. The circulation flow rate is 3–5 times the material volume / hour; in this embodiment, it is 4 times. Micro-pressure air is introduced into the bottom of the reactor at a pressure of 0.02–0.05 MPa; in this embodiment, it is 0.03 MPa. Ultrasonic assistance is activated during the reaction, with the ultrasonic frequency alternating between 20–40 kHz with a period of 60 seconds; in this embodiment, it is 28→32→28 kHz alternating frequency sweep. The ultrasonic power density is 0.10–0.15 kW / kg; in this embodiment, it is 0.14 kW / kg. During the reaction, the pH value is maintained at 6.5–7.8 using an online pH controller; in this embodiment, it is specifically controlled at 7.0–7.2. The reaction temperature was controlled between 25 and 35°C throughout the process, and in this embodiment it was specifically 30 ± 2°C. The confined immobilized complex enzyme preparation was added to the pretreatment mixture in the following sequence: the first layer of cellulase system was added at 0h of the reaction start and reacted for 30min; the second layer of protease system was added at 0.5h of the reaction and reacted for 2h; the third layer of anti-nutritional factor targeting enzyme system was added at 2.5h of the reaction and reacted until the end. The total enzymatic hydrolysis reaction time was 18h, specifically 18h in this example. The endpoint was determined by a protein hydrolysis degree ≥45% and an anti-nutritional factor residual rate ≤8%. Samples were taken at 16h of the reaction, and the protein hydrolysis degree was 47.2%, the glucosinolate residue was 0.12mg / g (initial value 1.52mg / g), the removal rate was 92.1%, and the free amino acid content was 21.6g / 100mL. The reaction continued for 18h, the protein hydrolysis degree increased to 49.8%, the glucosinolate removal rate was 93.5%, and the free amino acid content was 23.4%, reaching the endpoint. S3, recovery of enzymatic hydrolysate and residual confined immobilized complex enzyme preparation After the enzymatic hydrolysis reaction is completed, the pH of the reaction system is adjusted to 5.0–5.5, specifically 5.2 in this embodiment, to precipitate and recover the confined immobilized complex enzyme preparation. The recovered enzyme preparation is regenerated by washing with a buffer solution of pH 6.5–7.0, specifically pH 6.8 in this embodiment, with an enzyme activity retention rate of ≥85%, specifically 87.2% in this embodiment, and is directly reused in the next batch of reaction.

[0019] The supernatant of the enzyme hydrolysate after enzyme recovery is subjected to solid-liquid separation, and the supernatant is collected. In this example, centrifugation at 5000 rpm for 15 min is used to collect approximately 285 L of clear enzyme hydrolysate with a solid content of approximately 8.5%. The supernatant is then concentrated under vacuum at 55–65 °C to a total solid content of 30–35%. In this example, it is specifically concentrated at 60 °C to a total solid content of 32%, yielding approximately 75 L of fully water-soluble organic liquid fertilizer.

[0020] Alternatively, it can be further processed into a fully water-soluble organic fertilizer powder by spray drying. The inlet air temperature for spray drying is 120–140°C, and the outlet air temperature is 60–70°C. Spray drying was not used in this embodiment.

[0021] The product in this embodiment was tested using national and industry standard methods, and the results are as follows: The total water solubility was determined to be 97.6% according to GB / T 8576-2020; The total amount of free amino acids, on a dry basis, was 23.8%. The average molecular weight, as determined by gel filtration chromatography-mass spectrometry, was 742 Da, with Mw / Mn = 1.28. Organic matter content: 46.2%; The total nutrients, namely N+P2O5+K2O content, are 9.4%; The residual glucosinolate was 0.11 mg / g, with a removal rate of 92.8%. Anti-nutritional factors such as gossypol and saponins were not detected, with a detection limit of 0.01 mg / g; After 180 days of storage at room temperature, the retention rate of free amino acids was 92.3%.

[0022] Example 2: Preparation of fully water-soluble organic fertilizer powder using cottonseed cake as raw material This embodiment provides a low-temperature enzymatic hydrolysis method for producing fully water-soluble oilseed cake organic fertilizer, including the following steps: S1, Vacuum ultrasonic synergistic low-temperature puffing pretreatment Commercially available cottonseed cake was used, with a crude protein content of 36.2%, a free gossypol content of 0.12%, and a moisture content of 7.5%. The cake was pulverized to 80 mesh and pure water was added at a solid-liquid ratio of 1:1.5 (150 kg of pure water per 100 kg of dry material). The mixture was soaked at 20°C for 60 min. It was then ultrasonically puffed at a vacuum of -0.095 MPa and a temperature of 25°C for 30 min. The ultrasonic power density was 0.12 kW / kg, and the ultrasonic frequency was in the range of 25–35 kHz. In this embodiment, a frequency sweep of 25–28 kHz with a period of 30 seconds was used. The cell wall destruction rate of the treated material reached 85.1%, resulting in a pretreated mixture. S2, Multiphase cyclic gradient enzymatic hydrolysis reaction The preparation method of the confined immobilized complex enzyme preparation is the same as in Example 1, but the LPMO in the third layer of the anti-nutritional factor targeting enzyme system is replaced with gossypol degrading enzyme. The enzyme dosage of each layer is adjusted as follows: 50 U / g dry material for the first layer cellulase system, 200 U / g dry material for the second layer protease system, and 60 U / g dry material for the third layer anti-nutritional factor targeting enzyme system; The enzymatic hydrolysis reactor and operating parameters were adjusted as follows: reaction temperature 25±2℃, pH value 6.8~7.0, circulation flow rate 3 times material volume / hour, micro-pressure air pressure 0.02MPa, ultrasonic frequency alternating sweep with a period of 60s in the range of 20~40kHz (specifically, 20→40kHz alternating sweep in this embodiment), ultrasonic power density 0.10kW / kg. The addition sequence was the same as in Example 1. The total enzymatic hydrolysis time was 24h. Endpoint detection: protein hydrolysis degree 46.5%, free gossypol residue 0.008mg / g, initial free gossypol content 0.12mg / g, removal rate 93.3%, free amino acid content 19.2%; S3, recovery of enzymatic hydrolysate and residual confined immobilized complex enzyme preparation The enzyme recovery process was the same as in Example 1. After washing and regeneration with pH 6.8 buffer, the enzyme activity retention rate was 85.6%. After centrifugation, the supernatant was concentrated under vacuum at 55°C to a total solids content of 30%, and then spray-dried at an inlet air temperature of 120°C and an outlet air temperature of 60°C to obtain approximately 32 kg of fully water-soluble organic fertilizer powder.

[0023] Product test results: total water solubility 96.8%, total free amino acids 18.9% on a dry basis, average molecular weight 798 Da, organic matter 44.5%, total nutrients 8.7%, free gossypol residue 0.007 mg / g, removal rate 94.2%, other anti-nutritional factors such as cyclopropene fatty acid removal rate ≥90%.

[0024] Comparative Example 1: Traditional High-Temperature Composting Fermentation Method The rapeseed cake raw material was processed using the same conventional high-temperature composting process as in Example 1. 100 kg of rapeseed cake powder was mixed with 50 kg of crushed straw as a conditioner to adjust the carbon-to-nitrogen ratio to 25:1 and the moisture content to 60%. Windrow composting was employed, with the pile turned over every 3 days, and the composting temperature maintained at 55–65°C for 25 days of continuous fermentation. After fermentation, the compost was dried, crushed, and sieved to obtain the traditional organic fertilizer product.

[0025] Product test results: total water solubility is about 38%, free amino acid content is 4.2% on a dry basis, average molecular weight is not applicable, glucosinolate residue is 0.45 mg / g, removal rate is about 70%, total nutrients are 5.1%, production cycle is 25 days, and energy consumption per unit product is about 2.8 times that of Example 1.

[0026] The performance of the products in the above embodiments and comparative examples is tested in the following manner: Determination of protein hydrolysis degree: The formaldehyde titration method was used to determine the free amino nitrogen content in the enzymatic hydrolysate according to GB / T 5009.124-2016. The ratio of the free amino nitrogen content to the total nitrogen content is the degree of protein hydrolysis.

[0027] Determination of free amino acid content: The content was determined using an automatic amino acid analyzer according to the method in GB / T 18246-2019.

[0028] Determination of average molecular weight distribution: A calibration curve was plotted using a gel filtration chromatography-mass spectrometry system with standards of known molecular weights, and the weight-average molecular weight Mw and number-average molecular weight Mn were calculated.

[0029] Total water solubility determination: Dissolve 5g of sample in 100mL of deionized water at 25℃ for 30min, filter through a 0.45μm filter membrane, dry and weigh the insoluble matter, and calculate the water solubility.

[0030] Determination of anti-nutritional factor residues: glucosinolates were determined by high performance liquid chromatography, referring to NY / T 1582-2007; free gossypol was determined by the national standard GB / T 13086-2020; tea saponins were determined by spectrophotometry at a wavelength of 550 nm.

[0031] Enzyme activity retention rate determination: The catalytic activity of the recovered enzyme preparation and the fresh enzyme preparation on a specific substrate were measured separately, and the retention rate was calculated by the ratio of their activities.

[0032] Organic matter and total nutrients: in accordance with NY / T 525-2021 standard.

[0033] The test results are shown in the table below: Summarize: As can be seen from the above embodiments and comparative examples, the low-temperature enzymatic hydrolysis production method of fully water-soluble oilseed cake organic fertilizer provided by the present invention has the following significant advantages compared with the prior art: 1. Fully water-soluble and small-molecule. The product has a full water solubility of ≥96%, a free amino acid content of 18% to 24%, and an average molecular weight of ≤800 Da, which is far superior to traditional compost products and fully meets the needs of drip irrigation, sprinkler irrigation and other integrated water and fertilizer planting. 2. Extremely short production cycle. The total enzymatic hydrolysis time is only 16-24 hours, which is more than 90% shorter than the 15-30 days of traditional high-temperature composting, greatly improving production efficiency; 3. Low temperature, low carbon, and energy saving. The entire process operates at 25-35℃, eliminating the need for high-temperature cooking or heating and insulation, and the unit energy consumption is only less than 40% of that of traditional processes; 4. High-activity ingredient retention. Low-temperature conditions prevent the destruction of heat-sensitive amino acids, vitamins, and plant polyphenols, with a retention rate of ≥95%; 5. Simultaneous and efficient detoxification. The removal rate of various anti-nutritional factors such as glucosinolates, gossypol, and saponins is ≥92%, ensuring high product safety; 6. Enzyme preparations are reusable. After simple recycling and regeneration, the enzyme activity retention rate of confined immobilized complex enzyme preparations is ≥85%, significantly reducing production costs.

[0034] In summary, this invention has made groundbreaking progress in the field of oilseed cake resource utilization, with outstanding substantive features and significant advancements, and is suitable for industrial-scale promotion and application.

[0035] The above description is merely a preferred embodiment of the present invention and is not intended to limit the present invention. Any modifications, equivalent substitutions, improvements, etc., made within the spirit and principles of the present invention should be included within the protection scope of the present invention.

Claims

1. A method for low-temperature enzymatic hydrolysis production of fully water-soluble oilseed cake organic fertilizer, characterized in that: Includes the following steps: S1. The oilseed cake raw material is subjected to vacuum ultrasonic combined low temperature puffing pretreatment to obtain a pretreated mixture; S2. Under conditions of 25–35°C, a confined immobilized complex enzyme preparation is sequentially added to the pretreated mixture to carry out a multiphase cyclic gradient enzymatic hydrolysis reaction. The multiphase cyclic gradient enzymatic hydrolysis reaction includes: under the assistance of ultrasound, the enzymatic hydrolysate is circulated back through the solid phase enzymatic hydrolysis bed, and micro-pressure gas is introduced to form a gas-liquid-solid three-phase disturbance. The confined immobilized complex enzyme preparation contains three layers of enzyme systems, which are added in the following order: the first layer is a cellulase system, the second layer is a protease system, and the third layer is an anti-nutritional factor targeting enzyme system. Each enzyme system is separated by an isolation membrane layer in a mesoporous carrier to form a time-sequential release mechanism. S3. After the enzymatic hydrolysis reaction is completed, the enzymatic hydrolysate and residual confined immobilized complex enzyme preparation are recovered. The recovered confined immobilized complex enzyme preparation is reused in the next batch of reaction.

2. The method according to claim 1, characterized in that: In step S1, the specific process parameters for vacuum ultrasonic synergistic low-temperature puffing pretreatment are as follows: solid-liquid ratio 1:1.5~2.5, immersion temperature 15~25℃, immersion time 30~60min, vacuum degree -0.085~-0.095MPa, ultrasonic power density 0.12~0.18kW / kg, ultrasonic frequency in the range of 25~35kHz with alternating frequency sweeps of 30s, temperature 25~30℃, and time 30~45min.

3. The method according to claim 1, characterized in that: In step S2, the mesoporous carrier is mesoporous silica with a pore size of 12-18 nm. Each enzyme system is separated by an isolation membrane layer, which is composed of polyvinyl alcohol-polylysine and has a thickness of 2-3 nm.

4. The method according to claim 1, characterized in that: In step S2, the first layer of cellulase system includes endoglucanase, exoglucanase and β-glucosidase, the second layer of protease system includes alkaline protease, neutral protease and flavor protease, and the third layer of anti-nutritional factor targeting enzyme system includes myrosinase, β-glucosidase and LPMO.

5. The method according to claim 4, characterized in that: The mass ratio of endoglucanase, exoglucanase, and β-glucosidase in the first cellulase system is 2:1:1; the mass ratio of alkaline protease, neutral protease, and flavor protease in the second protease system is 2:2:1; and the mass ratio of myrosinase, β-glucosidase, and LPMO in the third antinutritional factor targeting enzyme system is 2:1:

1.

6. The method according to claim 1, characterized in that: The timing and amount of enzyme additions for each layer in step S2 are as follows: At the start of the reaction, add the first layer of cellulase system at 0h, with an enzyme dosage of 50-80 U / g dry material, and react for 30min. After 0.5 hours of reaction, a second layer of protease system is added at an enzyme dosage of 200–400 U / g of dry material, and the reaction continues for 2 hours. After 2.5 hours of reaction, add the third layer of anti-nutritional factor targeted enzyme system at an enzyme dosage of 30-60 U / g dry material, and continue the reaction until completion.

7. The method according to claim 1, characterized in that: In step S2, the multiphase cyclic gradient enzymatic hydrolysis reaction is carried out in a reactor. The reactor is equipped with a circulating liquid collection tank at the bottom and a spray device at the top. During the reaction, the enzymatic hydrolysate in the collection tank is pumped to the top spray device by a circulating pump, so that it passes through the solid phase enzymatic hydrolysis bed again to form a circulating reflux. The circulation flow rate is 3 to 5 times the material volume / hour. Micro-pressure air is introduced into the bottom of the reactor at a pressure of 0.02–0.05 MPa. Ultrasonic assistance is activated during the reaction, with the ultrasonic frequency alternating between 20 and 40 kHz with a period of 60 seconds. The ultrasonic power density is 0.10–0.15 kW / kg. The pH value is maintained at 6.5–7.8 during the reaction using an online pH controller.

8. The method according to claim 1, characterized in that: In step S3, the total duration of the enzymatic hydrolysis reaction is 16-24 hours. The endpoint is determined by the degree of protein hydrolysis ≥45% and the residual rate of anti-nutritional factors ≤8%. After the enzymatic hydrolysis is completed, the pH of the reaction system is adjusted to 5.0-5.5, and the confined immobilized complex enzyme preparation is precipitated and recovered. After the recovered enzyme preparation is washed and regenerated with a buffer solution of pH 6.5-7.0, the enzyme activity retention rate is ≥85%.

9. The method according to claim 1, characterized in that: It also includes solid-liquid separation of the enzymatic hydrolysate obtained in step S3, collection of the supernatant, and vacuum concentration of the supernatant at 55-65°C to a total solids content of 30-35% to obtain a fully water-soluble organic liquid fertilizer; or further spray drying to produce a fully water-soluble organic fertilizer powder, wherein the inlet air temperature of the spray drying is 120-140°C and the outlet air temperature is 60-70°C.

10. A confined immobilized complex enzyme preparation for the low-temperature enzymatic hydrolysis production of oilseed cake, characterized in that: The device comprises a mesoporous carrier and a three-layer enzyme system immobilized within the mesoporous carrier. The mesoporous carrier has a first region, a second region, and a third region arranged sequentially in space. The three enzyme systems, in order of release, are a first layer of cellulase, a second layer of protease, and a third layer of anti-nutritional factor targeting enzyme. Each enzyme system is separated by an isolation membrane. The first layer of cellulase is confined to the first region of the mesoporous carrier, with a release half-life ≤30 min. The second layer of protease is confined to the second region of the mesoporous carrier, with a release half-life of 1–2 h. The third layer of anti-nutritional factor targeting enzyme is confined to the third region of the mesoporous carrier, with a release half-life of 2–4 h.

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