A bio-extraction process for enzymatic hydrolysis of distiller's grains

Abstract

This invention belongs to the field of distillers' grains recycling technology, specifically a process technology for enzymatic hydrolysis and bio-extraction of distillers' grains. The process technology includes the following steps: pretreatment of distillers' grains; preparation of a magnetic biosorbent; detoxification and stepwise enzymatic hydrolysis of the distillers' grains; and multi-stage membrane separation of the enzymatically hydrolyzed distillers' grains slurry. This invention, through combined treatment of wheat distillers' grains with plasma, pulsed electric field, and cryogenic-hot pressing, exposes more enzymatic hydrolysis sites between components during subsequent slurry preparation. This significantly promotes the enzymatic hydrolysis efficiency of amylase, cellulase, and protease, ensuring peptide yield while effectively reducing the viscosity of the enzymatically hydrolyzed distillers' grains slurry. This allows for better solid-liquid separation in subsequent plate and frame filtration processes, reducing nutrient waste.

Description

Technical Field

[0001] This invention belongs to the field of distillery lees recycling technology, specifically a process technology for enzymatic hydrolysis separation and bio-extraction of distillery lees. Background Technology

[0002] Distillers' grains are a major byproduct of the brewing process. They are produced in large quantities and have high levels of moisture, protein, and organic matter, making them extremely susceptible to spoilage. Currently, only a small amount of distillers' grains are dried to produce dried products, while most are sold at low prices or even discarded directly, resulting in a waste of protein resources. Therefore, realizing the resource utilization of distillers' grains can not only significantly improve their utilization rate and reduce environmental pollution, but also create economic benefits.

[0003] Existing technologies generally employ enzymatic extraction to utilize proteins in distiller's grains. This involves using cellulase and amylase to enzymatically hydrolyze starch and cellulose to release protein components from the distiller's grains, then extracting the protein for further enzymatic hydrolysis. While this can improve the protein dissolution rate to some extent, it overlooks the fact that cellulose and starch are also in an aggregated state, with few exposed sites for enzymatic hydrolysis. This results in cellulase and amylase being unable to degrade the proteins efficiently, leading to a low protein dissolution rate and low resource utilization rate.

[0004] Furthermore, the invisible Fusarium graminearum and Aspergillus flavus hidden in the storage of distiller's grains can produce heat-resistant deoxynivalenol (DON) toxin and AFB1 / AFB2 toxin. Existing processes often overlook these hidden biological toxins. Although the activated carbon used in conventional adsorption processes can partially adsorb toxins, it also binds to 20-30% of the target products, such as small molecule peptides, resulting in a decrease in the yield of nutrients. In addition, activated carbon is difficult to recover when mixed with other insoluble substances, which can easily increase the cost of resource utilization. Summary of the Invention

[0005] To address the aforementioned technical deficiencies, this invention presents a bio-extraction process for enzymatic separation of distiller's grains. This process not only effectively removes biotoxins from the distiller's grains but also fully exposes the enzymatic hydrolysis sites for cellulose, starch, and protein, thereby improving hydrolysis efficiency and ensuring peptide yield. Furthermore, its multi-stage membrane filtration can effectively separate and recover various nutrients.

[0006] A bio-extraction process for enzymatic hydrolysis of distiller's grains includes the following steps:

[0007] S1: Pretreatment of distiller's grains

[0008] Wheat lees were subjected to plasma treatment at 4-5kV for 20-25s, then spread flat on the lower electrode plate connected to a high-voltage pulse power supply. The distance between the upper and lower electrode plates was controlled to be 2-3cm. The wheat lees were subjected to pulse electric field treatment at 15-20kV and 6-8μs pulse width for 10-15min. After being frozen to -15 to -10℃ in a freezer, the wheat lees were transferred to a double-roll crusher with a roller temperature of 80-85℃ for rolling to obtain pretreated wheat lees.

[0009] S2: Preparation of magnetic biosorbent

[0010] A glucose, ferric chloride hexahydrate, and ferrous chloride dihydrate were dissolved in ultrapure water to prepare an iron salt solution. The solution was reacted under alkaline conditions, followed by centrifugation, removal of agglomerates, and dialysis with ultrapure water to obtain a glucose-ferric oxide nanoparticle suspension. Lactobacillus delbrueckii subsp. bulgaricus and Rhizobium were cultured to an OD600 of 6-8, then mixed and centrifuged to obtain a mixed bacterial suspension. The glucose-ferric oxide nanoparticle suspension and the mixed bacterial suspension were shaken evenly, added to a mixed culture medium, and cultured. The solid matter was then collected by magnetic separation, washed, filtered, and freeze-dried to obtain a magnetic biosorbent.

[0011] S3: Detoxification and stepwise enzymatic hydrolysis of distiller's grains

[0012] Pretreated wheat lees were mixed with distilled water and the pH was adjusted to 6-6.5. Magnetic biosorbent was added at 30-32℃ and stirred continuously. Then, magnetic separation was performed to obtain detoxified wheat lees slurry. After adjusting the pH, pullulanase, saccharifying enzyme and cellulase were added for enzymatic hydrolysis. After inactivating the enzymes and adjusting the pH, alkaline protease and trypsin were added in sequence for enzymatic hydrolysis to obtain secondary enzymatic hydrolyzed lees slurry.

[0013] S4: Multi-stage membrane separation of enzymatically hydrolyzed distiller's grains slurry

[0014] The secondary enzymatic hydrolysis of distiller's grains slurry was subjected to plate and frame filter press filtration to obtain plate and frame filter cake and plate and frame filter clear liquid. The plate and frame filter cake was flash dried to obtain DDG dried material. The plate and frame filter clear liquid was filtered through an ultrafiltration membrane to obtain membrane concentrate I and membrane permeate I. Membrane concentrate I was concentrated and dried to obtain large molecular weight DDS soluble material. Membrane permeate I was filtered through a nanofiltration membrane to obtain membrane concentrate II and membrane permeate II. Membrane concentrate II was concentrated and dried to obtain small molecular weight DDS soluble material. Membrane permeate II was filtered through a reverse osmosis membrane to obtain membrane concentrate III and membrane permeate III. Membrane concentrate III was evaporated and concentrated to obtain DWS soluble material. Membrane permeate III was directly discharged to a wastewater treatment plant.

[0015] Furthermore, the pretreatment of the distiller's grains in step S1 includes the following steps:

[0016] S1.1: Place wheat lees with a moisture content of 10-15% in a plasma surface processor, adjust the voltage to 4-5kV, the spray distance to 4.5-6cm, and treat with helium plasma for 20-25s. Then spread it flat on the lower electrode plate connected to the high-voltage pulse power supply with a thickness of 5-6mm. Set the distance between the upper and lower electrode plates to 2-3cm, the voltage of the high-voltage pulse power supply to 15-20kV, the pulse width to 6-8μs, and perform pulse electric field treatment for 10-15min to obtain plasma pulse treated lees.

[0017] S1.2: Plasma pulse treated lees are placed in a freezing chamber and frozen at -15 to -10℃ for 1-1.5 hours. Then, they are put into a double-roll crusher with preheated rollers for rolling. The roller preheating temperature is 80-85℃, the roller gap is 0.1-0.15mm, and the roller speed is 35-40rpm to obtain pretreated wheat lees.

[0018] Further, the preparation of the magnetic biosorbent in step S2 includes the following steps:

[0019] S2.1: Under a nitrogen atmosphere, 18-20 parts by weight of glucose, 2.4-2.5 parts by weight of ferric chloride hexahydrate, and 0.85-0.9 parts by weight of ferrous chloride dihydrate are dissolved in 900-1000 parts by weight of ultrapure water to obtain an iron salt solution. Under magnetic stirring at 70-75℃ and 1200-1500 rpm, 1-1.2 mol / L NaOH solution is added dropwise at a rate of 3-4 mL / min until the pH reaches 10-11. The reaction is carried out at a constant temperature for 1-1.5 h. Then, the mixture is centrifuged at 3000-4000 rpm to remove the larger aggregates at the bottom layer. The upper suspension is collected and placed in a dialysis bag with a molecular weight cutoff of 7000-8000 Da. The suspension is dialyzed with ultrapure water for 3-4 days, with the ultrapure water being replaced every 6-8 h, to obtain a glucose-ferric oxide nanoparticle suspension.

[0020] S2.2: Lactobacillus delbrueckii subsp. bulgaricus and Rhizobium were cultured routinely to an OD600 of 6-8 to obtain Lactobacillus delbrueckii subsp. bulgaricus and Rhizobium cultures were then mixed and placed in a sterile high-speed centrifuge at 4-5℃ and 8000-8500 rpm for 12-15 min. The supernatant was discarded, and the cells were resuspended in 0.1 mol / L phosphate buffer (pH 7.4) until the cell concentration reached 2 × 10⁻⁶ cells / mL. 9 -5×10 9 CFU / mL was used to obtain a mixed bacterial suspension;

[0021] S2.3: Glucose-Fe3O4 nanoparticle suspension and mixed bacterial suspension were shaken and mixed evenly at a volume ratio of 1:(8-10), and then added to the mixed culture medium. The pH was maintained at 6.5-7. After culturing at 30-32℃ for 25-30h, magnetic separation was performed, and the magnetically adsorbed solid material was collected. The solid material was washed 2-3 times with ultrapure water, filtered and the filter cake was collected. The filter cake was freeze-dried at -40 to -45℃ to obtain the magnetic biosorbent.

[0022] Furthermore, step S3, the detoxification and stepwise enzymatic hydrolysis of the distiller's grains, includes the following steps:

[0023] S3.1: Mix pretreated wheat lees and distilled water at a mass ratio of 1:(6-10) and stir evenly. Add acetic acid solution with a concentration of 10-15% to adjust the pH to 6-6.5. Add magnetic biosorbent at a solid-liquid ratio of 1:(400-450) g / mL. Stir at 120-150 rpm for 10-12 h at 30-32℃. Then, perform magnetic separation on the magnetic biosorbent to obtain detoxified wheat lees slurry.

[0024] S3.2: Continue to add 10-15% acetic acid solution to the detoxified wheat lees slurry to adjust the pH to 5.5-6, then add pullulanase, saccharifying enzyme and cellulase, and enzymatically hydrolyze for 2-3 hours at 55-57℃. Then place it in an autoclave and inactivate the enzymes at 135℃ for 5 minutes to obtain the first enzymatically hydrolyzed lees slurry.

[0025] S3.3: Add distilled water to the first enzymatic hydrolysate of distiller's grains to adjust the concentration to 15-17%, then add food-grade sodium hydroxide to adjust the pH to 8-8.5, add alkaline protease at 55-60℃ and hydrolyze for 4-6 hours, then add trypsin and continue hydrolysis for 3-5 hours, and finally inactivate the enzyme at 85℃ for 15 minutes to obtain the second enzymatic hydrolysate of distiller's grains.

[0026] Furthermore, step S4, the multi-stage membrane separation of the enzymatic hydrolysis of the distiller's grains slurry, includes the following steps:

[0027] S4.1: The secondary enzymatically hydrolyzed distiller's grains slurry is placed in a plate and frame filter press for filtration to obtain a plate and frame filter cake and a clear liquid. The plate and frame filter cake is then transferred to a flash dryer, with the blower airflow set to 6000-6500 m³ / h. 3 The plate and frame filter cake is flash-dried at a speed of 6000-8000Pa, an air pressure of 6000-8000Pa, an inlet air temperature of 100-120℃, and an outlet air temperature of 55-65℃ until the moisture content is reduced to 6-8%, thus obtaining DDG dried product.

[0028] S4.2: The plate and frame clarified liquid obtained in step S4.1 is filtered through an ultrafiltration membrane to obtain membrane concentrate I and membrane permeate I. Membrane concentrate I is concentrated and dried at 60-85℃ to obtain macromolecular DDS solubles. Then, membrane permeate I is filtered through a nanofiltration membrane to obtain membrane concentrate II and membrane permeate II. Membrane concentrate II is concentrated and dried at 60-85℃ to obtain small molecule DDS solubles.

[0029] S4.3: The membrane permeate II obtained in step S4.2 is filtered through a reverse osmosis membrane to obtain membrane concentrate III and membrane permeate III. The membrane concentrate III is evaporated and concentrated to 30-35% of its original volume to obtain DWS solubles. The membrane permeate III is directly discharged to the wastewater treatment plant.

[0030] Furthermore, in step S2, the culture medium used for the routine culture of Lactobacillus delbrueckii subsp. bulgaricus is MRS medium, and the culture medium used for the routine culture of Rhizobium is yeast mannitol agar medium. The mixed culture medium is prepared by mixing PDA medium and MRS medium in a volume ratio of 1:(1-2) according to the art.

[0031] Furthermore, in step S3.2, the amounts of pullulanase, saccharifying enzyme, and cellulase added account for 0.6-0.8 wt%, 0.4-0.5 wt%, and 0.5-0.7 wt% of the dry matter in the detoxified wheat distillers' grains slurry, respectively.

[0032] Furthermore, based on the first enzymatic hydrolysis of distiller's grains slurry, the amount of alkaline protease added in step S3.3 is 6000-7000 U / g, and the amount of trypsin added is 3000-5000 U / g.

[0033] Furthermore, in step S4.2, the molecular weight cutoff of the ultrafiltration membrane is 1000 Da, the molecular weight cutoff of the nanofiltration membrane is 150-200 Da, and the molecular weight cutoff of the reverse osmosis membrane is 100 Da.

[0034] Furthermore, in step S4, the main component of the DDG dried product is poorly soluble protein, the main components of the macromolecular DDS soluble product and the small molecule DDS soluble product are glucose, xylooligosaccharides, polypeptides and small peptides, and the main components of the DWS soluble product are organic acids, amino acids and growth factors.

[0035] The beneficial effects are as follows: 1. This invention uses a combined synergistic treatment of wheat distillers' grains with plasma, pulsed electric field, and freeze-heat rolling. The high-energy particles of helium plasma bombardment causes the cellulose and hemicellulose molecular chains to break bonds, reducing their degree of polymerization and structural strength, creating channels for subsequent enzymatic hydrolysis. When the pulsed electric field acts on the distillers' grains, its strong pulses penetrate the material, reducing the proportion of β-sheets and random coils in the secondary structure of proteins, increasing the proportion of α-helices and β-turns, and decreasing crystallinity. This forms a loose structure between the protein and starch. After freeze-heat rolling, the different components generate interfacial stress due to the difference in thermal expansion coefficients. Micro-gaps are formed between the broken cellulose skeleton and the loosened protein-starch matrix. During subsequent slurry preparation, more enzymatic hydrolysis sites are exposed between the components, thereby significantly promoting the enzymatic hydrolysis efficiency of amylase, cellulase, and protease. This not only improves the yield of peptides but also effectively reduces the viscosity of the enzymatically hydrolyzed distillers' grains slurry, enabling better solid-liquid separation in the subsequent plate and frame filtration process and reducing the waste of nutrients.

[0036] 2. This invention prepares a glucose-ferric oxide nanoparticle suspension by reacting glucose, ferric chloride hexahydrate, and ferrous chloride dihydrate under alkaline conditions. This suspension is then mixed with a mixed culture of *Lactobacillus delbrueckii* subsp. bulgaricus and *Rhizobium* in a mixed culture medium and cultured. After magnetic separation, a magnetic biosorbent is obtained. This biosorbent is added to a wheat distillers' grains slurry and stirred. The peptidoglycan layer on the surface of *Lactobacillus delbrueckii* subsp. bulgaricus forms a multi-scale adsorption interface with the polysaccharide-protein complex of *Rhizobium* mycelium. Its abundant carboxyl, amino, and phosphate groups can capture DON toxins through ionic and hydrogen bonds, while the hydrophobic regions of the bacterial cells can bind mycotoxins AFB1 / AFB2 to cell wall peptidoglycans, polysaccharides, and tannic acid. Combined with magnetic separation, this effectively adsorbs and removes biotoxins, thus preventing contamination of subsequent nutrients by toxins produced by a small number of *Fusarium graminearum* and mycotoxins that may be present in the wheat distillers' grains.

[0037] 3. This invention utilizes pullulanase, saccharifying enzyme, cellulase, and alkaline protease sequentially to enzymatically hydrolyze pretreated distillers' grains, effectively degrading starch, polysaccharides, and cellulose in wheat distillers' grains while avoiding protein loss during protein separation. This allows proteins to be effectively dissolved and hydrolyzed by alkaline protease, and then filtered sequentially through plate and frame filter press, ultrafiltration membrane, nanofiltration membrane, and reverse osmosis membrane, effectively separating and recovering various nutrients. This makes it convenient for application in different scenarios and results in a high resource utilization rate for wheat distillers' grains. Attached Figure Description

[0038] Figure 1 The flowchart illustrates the enzymatic hydrolysis and bio-extraction process of distiller's grains used in embodiments of the present invention. Detailed Implementation

[0039] The technical solutions of the embodiments of the present invention will be clearly and completely described below with reference to the accompanying drawings. Obviously, the described embodiments are only some embodiments of the present invention, and not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention.

[0040] Example 1

[0041] A process technology for enzymatic hydrolysis and bio-extraction of distiller's grains, such as Figure 1 As shown, it includes the following steps:

[0042] S1: Pretreatment of distiller's grains

[0043] S1.1: Wheat lees with a moisture content of 10% were placed in a plasma surface processor. The voltage was adjusted to 4kV and the spray distance was 4.5cm. Helium plasma was used for 20s treatment. Then, the lees were spread flat on the lower electrode plate connected to the high-voltage pulse power supply with a thickness of 5mm. The distance between the upper and lower electrode plates was set to 2cm. The voltage of the high-voltage pulse power supply was 15kV and the pulse width was 6μs. The pulse electric field treatment was carried out for 10min to obtain plasma pulse-treated lees.

[0044] S1.2: Plasma pulse treated lees are placed in a freezing chamber and frozen at -15℃ for 1 hour. Then, they are put into a double-roll crusher with preheated rollers for rolling. The roller preheating temperature is 80℃, the roller gap is 0.1mm, and the roller speed is 35rpm to obtain pretreated wheat lees.

[0045] S2: Preparation of magnetic biosorbent

[0046] S2.1: Under a nitrogen atmosphere, 18 parts by weight of glucose, 2.4 parts by weight of ferric chloride hexahydrate and 0.85 parts by weight of ferrous chloride dihydrate were dissolved in 900 parts by weight of ultrapure water to obtain an iron salt solution. Under magnetic stirring at 70℃ and 1200 rpm, 1 mol / L NaOH solution was added dropwise at a rate of 3 mL / min until the pH reached 10. The reaction was carried out at a constant temperature for 1 h. Then, the mixture was centrifuged at 3000 rpm to remove the largest aggregates at the bottom layer. The upper suspension was collected and placed in a dialysis bag with a molecular weight cutoff of 7000 Da. The suspension was dialyzed with ultrapure water for 3 days, with the ultrapure water being replaced every 6 h to obtain a glucose-ferric oxide nanoparticle suspension.

[0047] S2.2: Lactobacillus delbrueckii subsp. bulgaricus and Rhizobium were cultured to an OD600 of 6 to obtain Lactobacillus delbrueckii subsp. bulgaricus and Rhizobium cultures were mixed and placed in a sterile high-speed centrifuge at 4°C and 8000 rpm for 12 min. The supernatant was discarded, and the culture was resuspended in 0.1 mol / L phosphate buffer (pH 7.4) until the cell concentration reached 2 × 10⁻⁶ cells / mL. 9 CFU / mL was used to obtain a mixed bacterial suspension;

[0048] S2.3: Glucose-Fe3O4 nanoparticle suspension and mixed bacterial suspension were shaken and mixed evenly at a volume ratio of 1:8, and then added to a mixed culture medium. The mixed culture medium was prepared by mixing PDA medium and MRS medium in a volume ratio of 1:1, which is conventional in this field. The pH was maintained at 6.5. After culturing at 30°C for 25 h, magnetic separation was performed, and the magnetically adsorbed solid material was collected. The material was washed twice with ultrapure water, filtered, and the filter cake was collected. The cake was then freeze-dried at -40°C to obtain the magnetic biosorbent.

[0049] S3: Detoxification and stepwise enzymatic hydrolysis of distiller's grains

[0050] S3.1: Mix pretreated wheat lees and distilled water at a mass ratio of 1:6 and stir evenly. Add 10% acetic acid solution to adjust the pH to 6. Add magnetic biosorbent at a solid-liquid ratio of 1:400 g / mL. Stir at 120 rpm for 8 hours at 30℃ and then perform magnetic separation on the magnetic biosorbent to obtain detoxified wheat lees slurry.

[0051] S3.2: Continue to add a 10% acetic acid solution to the detoxified wheat distillers' grains slurry to adjust the pH to 5.5. Then add pullulanase, saccharifying enzyme and cellulase. The amount of pullulanase, saccharifying enzyme and cellulase added accounts for 0.6wt%, 0.4wt% and 0.5wt% of the dry matter in the detoxified wheat distillers' grains slurry, respectively. Enzymatic hydrolysis is carried out at 55℃ for 2 hours. Then it is placed in an autoclave and the enzymes are inactivated at 135℃ for 5 minutes to obtain the first enzymatic hydrolyzed distillers' grains slurry.

[0052] S3.3: Distilled water was added to the first enzymatically hydrolyzed distiller's grains slurry to adjust the concentration to 15%, then food-grade sodium hydroxide was added to adjust the pH to 8, alkaline protease was added at 55℃ at a dosage of 6000 U / g, and enzymatic hydrolysis was carried out for 4 hours. Then trypsin was added at a dosage of 3000 U / g, and enzymatic hydrolysis was continued for 3 hours. Finally, the enzyme was inactivated at 85℃ for 15 minutes to obtain the second enzymatically hydrolyzed distiller's grains slurry.

[0053] S4: Multi-stage membrane separation of enzymatically hydrolyzed distiller's grains slurry

[0054] S4.1: The secondary enzymatically hydrolyzed distiller's grains slurry is placed in a plate and frame filter press for filtration to obtain a plate and frame filter cake and a clear liquid. The plate and frame filter cake is then transferred to a flash dryer, with the blower airflow set to 6000 m³ / h. 3 The plate and frame filter cake was flash-dried at a speed of 6000 Pa, an air pressure of 6000 Pa, an inlet air temperature of 100 ℃, and an outlet air temperature of 55 ℃ until the moisture content was reduced to 6%, thus obtaining DDG dried product.

[0055] S4.2: The plate and frame clarified solution obtained in step S4.1 is filtered through an ultrafiltration membrane with a molecular weight cutoff of 1000 Da to obtain membrane concentrate I and membrane permeate I. Membrane concentrate I is concentrated and dried at 60°C to obtain soluble macromolecular DDS. Then, membrane permeate I is filtered through a nanofiltration membrane with a molecular weight cutoff of 150 Da to obtain membrane concentrate II and membrane permeate II. Membrane concentrate II is concentrated and dried at 60°C to obtain soluble small molecule DDS.

[0056] S4.3: The membrane permeate II obtained in step S4.2 is filtered through a reverse osmosis membrane with a molecular weight cutoff of 100 Da to obtain membrane concentrate III and membrane permeate III. The membrane concentrate III is evaporated and concentrated to 30% of its original volume to obtain DWS solubles. The membrane permeate III is directly discharged to the wastewater treatment plant.

[0057] Example 2

[0058] A process technology for enzymatic hydrolysis and bio-extraction of distiller's grains, such as Figure 1 As shown, it includes the following steps:

[0059] S1: Pretreatment of distiller's grains

[0060] S1.1: Wheat lees with a moisture content of 12% were placed in a plasma surface processor, the voltage was adjusted to 4.5kV, the spray distance was 5cm, and helium plasma was used for 23s treatment. Then, the lees were spread flat on the lower electrode plate connected to the high-voltage pulse power supply with a thickness of 5.5mm. The distance between the upper and lower electrode plates was set to 2.5cm, the voltage of the high-voltage pulse power supply was 18kV, the pulse width was 7μs, and the pulse electric field treatment was performed for 12min to obtain plasma pulse treated lees.

[0061] S1.2: Plasma pulse treated lees are placed in a freezing chamber and frozen at -12℃ for 1.2 hours. Then, they are put into a double-roll crusher with preheated rollers for rolling. The roller preheating temperature is 83℃, the roller gap is 0.12mm, and the roller speed is 38rpm to obtain pretreated wheat lees.

[0062] S2: Preparation of magnetic biosorbent

[0063] S2.1: Under a nitrogen atmosphere, 19 parts by weight of glucose, 2.5 parts by weight of ferric chloride hexahydrate and 0.9 parts by weight of ferrous chloride dihydrate were dissolved in 950 parts by weight of ultrapure water to obtain an iron salt solution. Under magnetic stirring at 72℃ and 1350 rpm, 1.1 mol / L NaOH solution was added dropwise at a rate of 4 mL / min until the pH reached 10.5. The reaction was carried out at a constant temperature for 1.2 h. Then, the mixture was centrifuged at 3500 rpm to remove the largest aggregates at the bottom. The upper suspension was collected and placed in a dialysis bag with a molecular weight cutoff of 7000 Da. The suspension was dialyzed with ultrapure water for 4 days, with the ultrapure water being replaced every 7 h, to obtain a glucose-ferric oxide nanoparticle suspension.

[0064] S2.2: Lactobacillus delbrueckii subsp. bulgaricus and Rhizobium were cultured routinely to an OD600 of 7 to obtain Lactobacillus delbrueckii subsp. bulgaricus and Rhizobium cultures were then mixed and placed in a sterile high-speed centrifuge at 4°C and 8250 rpm for 13 min. The supernatant was discarded, and the culture was resuspended in 0.1 mol / L phosphate buffer (pH 7.4) until the cell concentration reached 3.5 × 10⁻⁶ cells / mL. 9 CFU / mL was used to obtain a mixed bacterial suspension;

[0065] S2.3: Glucose-Fe3O4 nanoparticle suspension and mixed bacterial suspension were shaken and mixed evenly at a volume ratio of 1:9, and then added to a mixed culture medium. The mixed culture medium was prepared by mixing PDA medium and MRS medium in a volume ratio of 1:1.5, which are conventional in this field. The pH was maintained at 7. After culturing at 31°C for 28 hours, magnetic separation was performed, and the magnetically adsorbed solid material was collected. The material was washed three times with ultrapure water, filtered, and the filter cake was collected. The filter cake was then freeze-dried at -42°C to obtain the magnetic biosorbent.

[0066] S3: Detoxification and stepwise enzymatic hydrolysis of distiller's grains

[0067] S3.1: Mix pretreated wheat lees and distilled water at a mass ratio of 1:8 and stir evenly. Add 12% acetic acid solution to adjust the pH to 6.5. Add magnetic biosorbent at a solid-liquid ratio of 1:425 g / mL. Stir at 135 rpm for 9 hours at 31℃ and then perform magnetic separation on the magnetic biosorbent to obtain detoxified wheat lees slurry.

[0068] S3.2: Continue to add 12% acetic acid solution to the detoxified wheat distillers' grains slurry to adjust the pH to 6, then add pullulanase, saccharifying enzyme and cellulase. The amount of pullulanase, saccharifying enzyme and cellulase added accounts for 0.7wt%, 0.45wt% and 0.6wt% of the dry matter in the detoxified wheat distillers' grains slurry, respectively. Enzymatic hydrolysis is carried out at 56℃ for 2.5h, and then placed in an autoclave and inactivated at 135℃ for 5min to obtain the first enzymatic hydrolyzed distillers' grains slurry.

[0069] S3.3: Distilled water was added to the first enzymatically hydrolyzed distiller's grains slurry to adjust the concentration to 16%, then food-grade sodium hydroxide was added to adjust the pH to 8.2. Alkaline protease was added at 57℃ at a dosage of 6500 U / g, and enzymatic hydrolysis was carried out for 5 hours. Then trypsin was added at a dosage of 4000 U / g, and enzymatic hydrolysis was continued for 4 hours. Finally, the enzyme was inactivated at 85℃ for 15 minutes to obtain the second enzymatically hydrolyzed distiller's grains slurry.

[0070] S4: Multi-stage membrane separation of enzymatically hydrolyzed distiller's grains slurry

[0071] S4.1: The secondary enzymatically hydrolyzed distiller's grains slurry is placed in a plate and frame filter press for filtration to obtain a plate and frame filter cake and a clear liquid. The plate and frame filter cake is then transferred to a flash dryer, with the blower airflow set to 6250 m³ / h. 3 The plate and frame filter cake was flash-dried at a speed of 7000 Pa, an air pressure of 7000 Pa, an inlet air temperature of 110 ℃, and an outlet air temperature of 60 ℃ until the moisture content was reduced to 7%, thus obtaining DDG dried product.

[0072] S4.2: The plate and frame clarified solution obtained in step S4.1 is filtered through an ultrafiltration membrane with a molecular weight cutoff of 1000 Da to obtain membrane concentrate I and membrane permeate I. Membrane concentrate I is concentrated and dried at 75°C to obtain soluble macromolecular DDS. Then, membrane permeate I is filtered through a nanofiltration membrane with a molecular weight cutoff of 180 Da to obtain membrane concentrate II and membrane permeate II. Membrane concentrate II is concentrated and dried at 75°C to obtain soluble small molecule DDS.

[0073] S4.3: The membrane permeate II obtained in step S4.2 is filtered through a reverse osmosis membrane with a molecular weight cutoff of 100 Da to obtain membrane concentrate III and membrane permeate III. The membrane concentrate III is evaporated and concentrated to 32% of its original volume to obtain DWS solubles. The membrane permeate III is directly discharged to the wastewater treatment plant.

[0074] Example 3

[0075] A process technology for enzymatic hydrolysis and bio-extraction of distiller's grains, such as Figure 1 As shown, it includes the following steps:

[0076] S1: Pretreatment of distiller's grains

[0077] S1.1: Wheat lees with a moisture content of 15% were placed in a plasma surface processor. The voltage was adjusted to 5kV and the spray distance was 6cm. Helium plasma was used for treatment for 25s. Then, the lees were spread flat on the lower electrode plate connected to the high-voltage pulse power supply with a thickness of 6mm. The distance between the upper and lower electrode plates was set to 3cm. The voltage of the high-voltage pulse power supply was 20kV and the pulse width was 8μs. The pulse electric field treatment was carried out for 15min to obtain plasma pulse-treated lees.

[0078] S1.2: Plasma pulse treated lees are placed in a freezing chamber and frozen at -10℃ for 1.5h. Then, they are put into a double-roll crusher with preheated rollers for rolling. The roller preheating temperature is 85℃, the roller gap is 0.15mm, and the roller speed is 40rpm to obtain pretreated wheat lees.

[0079] S2: Preparation of magnetic biosorbent

[0080] S2.1: Under a nitrogen atmosphere, 20 parts by weight of glucose, 2.5 parts by weight of ferric chloride hexahydrate and 0.9 parts by weight of ferrous chloride dihydrate were dissolved in 1000 parts by weight of ultrapure water to obtain an iron salt solution. Under magnetic stirring at 75℃ and 1500 rpm, 1.2 mol / L NaOH solution was added dropwise at a rate of 4 mL / min until the pH reached 11. The reaction was carried out at a constant temperature for 1.5 h. Then, the mixture was centrifuged at 4000 rpm to remove the largest aggregates at the bottom. The upper suspension was collected and placed in a dialysis bag with a molecular weight cutoff of 8000 Da. The suspension was dialyzed with ultrapure water for 4 days, with the ultrapure water being replaced every 8 h, to obtain a glucose-ferric oxide nanoparticle suspension.

[0081] S2.2: Lactobacillus delbrueckii subsp. bulgaricus and Rhizobium were cultured to an OD600 of 8 to obtain Lactobacillus delbrueckii subsp. bulgaricus and Rhizobium cultures were mixed and placed in a sterile high-speed centrifuge at 5°C and 8500 rpm for 15 min. The supernatant was discarded, and the culture was resuspended in 0.1 mol / L phosphate buffer (pH 7.4) until the cell concentration reached 5 × 10⁻⁶ cells / mL. 9 CFU / mL was used to obtain a mixed bacterial suspension;

[0082] S2.3: Glucose-Fe3O4 nanoparticle suspension and mixed bacterial suspension were shaken and mixed evenly at a volume ratio of 1:10, and then added to a mixed culture medium. The mixed culture medium was prepared by mixing PDA medium and MRS medium in a volume ratio of 1:2, which is conventional in this field. The pH was maintained at 7. After culturing at 32°C for 30 h, magnetic separation was performed, and the magnetically adsorbed solid material was collected. The material was washed three times with ultrapure water, filtered, and the filter cake was collected. The filter cake was then freeze-dried at -45°C to obtain the magnetic biosorbent.

[0083] S3: Detoxification and stepwise enzymatic hydrolysis of distiller's grains

[0084] S3.1: Mix pretreated wheat lees and distilled water at a mass ratio of 1:10 and stir evenly. Add 15% acetic acid solution to adjust the pH to 6.5. Add magnetic biosorbent at a solid-liquid ratio of 1:450 g / mL. Stir at 150 rpm for 10 h at 32℃ and then perform magnetic separation on the magnetic biosorbent to obtain detoxified wheat lees slurry.

[0085] S3.2: Continue to add 15% acetic acid solution to the detoxified wheat distillers' grains slurry to adjust the pH to 6, and then add pullulanase, saccharifying enzyme and cellulase. The amount of pullulanase, saccharifying enzyme and cellulase added accounts for 0.8wt%, 0.5wt% and 0.7wt% of the dry matter in the detoxified wheat distillers' grains slurry, respectively. Enzymatic hydrolysis is carried out at 57℃ for 3 hours, and then placed in an autoclave and inactivated at 135℃ for 5 minutes to obtain the first enzymatic hydrolyzed distillers' grains slurry.

[0086] S3.3: Distilled water was added to the first enzymatically hydrolyzed distiller's grains slurry to adjust the concentration to 17%, then food-grade sodium hydroxide was added to adjust the pH to 8.5. Alkaline protease was added at 60℃ at a dosage of 7000 U / g, and enzymatic hydrolysis was carried out for 6 hours. Then trypsin was added at a dosage of 5000 U / g, and enzymatic hydrolysis was continued for 5 hours. Finally, the enzyme was inactivated at 85℃ for 15 minutes to obtain the second enzymatically hydrolyzed distiller's grains slurry.

[0087] S4: Multi-stage membrane separation of enzymatically hydrolyzed distiller's grains slurry

[0088] S4.1: The secondary enzymatically hydrolyzed distiller's grains slurry is placed in a plate and frame filter press for filtration to obtain a plate and frame filter cake and a clear liquid. The plate and frame filter cake is then transferred to a flash dryer, with the blower airflow set to 6500 m³ / h. 3 The plate and frame filter cake was flash-dried at a speed of 8000 Pa, an air pressure of 8000 Pa, an inlet air temperature of 120 ℃, and an outlet air temperature of 65 ℃ until the moisture content was reduced to 8%, thus obtaining DDG dried product.

[0089] S4.2: The plate and frame clarified solution obtained in step S4.1 is filtered through an ultrafiltration membrane with a molecular weight cutoff of 1000 Da to obtain membrane concentrate I and membrane permeate I. Membrane concentrate I is concentrated and dried at 85°C to obtain macromolecular DDS solubles. Then, membrane permeate I is filtered through a nanofiltration membrane with a molecular weight cutoff of 200 Da to obtain membrane concentrate II and membrane permeate II. Membrane concentrate II is concentrated and dried at 85°C to obtain small molecule DDS solubles.

[0090] S4.3: The membrane permeate II obtained in step S4.2 is filtered through a reverse osmosis membrane with a molecular weight cutoff of 100 Da to obtain membrane concentrate III and membrane permeate III. The membrane concentrate III is evaporated and concentrated to 35% of its original volume to obtain DWS solubles. The membrane permeate III is directly discharged to the wastewater treatment plant.

[0091] Comparative Example 1

[0092] Compared with Example 1, Comparative Example 1 removed step S1.1, did not perform plasma and pulsed electric field treatment on the wheat distillers' grains, and replaced the plasma pulse-treated distillers' grains in step S1.2 with wheat distillers' grains with a moisture content of 10%. The remaining steps were the same as in Example 1.

[0093] Comparative Example 2

[0094] Compared with Example 1, Comparative Example 2 removed step S1.2, did not perform freeze-heat rolling treatment on the wheat distillers' grains, and replaced the pretreated wheat distillers' grains in step S3.1 with plasma pulse treated distillers' grains. All other steps were the same as in Example 1.

[0095] Comparative Example 3

[0096] Compared with Example 1, Comparative Example 3 removed step S3.3, and adjusted step S4 to a conventional enzymatic hydrolysis method: the secondary enzymatic hydrolysis of distiller's grains in S4.1 was replaced with an equal mass of primary enzymatic hydrolysis of distiller's grains to obtain DDG dried product. Then, distilled water was added to the DDG dried product to adjust the concentration to 15%, food-grade sodium hydroxide was added to adjust the pH to 8, alkaline protease was added at 55°C at a dosage of 6000 U / g, and enzymatic hydrolysis was carried out for 4 hours. Then, trypsin was added at a dosage of 3000 U / g, and enzymatic hydrolysis was continued for 3 hours. The enzyme was inactivated at 85°C for 15 minutes, and the supernatant was collected by centrifugation and spray-dried to obtain soluble polypeptide powder.

[0097] Comparative Example 4

[0098] Compared to Example 1, in Comparative Example 4, the bacterial cell concentration in step S2, the preparation of the magnetic biosorbent, was 2 × 10⁻⁶. 9 Replace the mixed bacterial culture with a bacterial cell concentration of 2×10⁻⁶ CFU / mL. 9 The magnetic biosorbent was prepared by using a CFU / mL Lactobacillus delbrueckii subsp. bulgaricus culture followed by the same steps.

[0099] Comparative Example 5

[0100] Compared to Example 1, in Comparative Example 4, the bacterial cell concentration in step S2, the preparation of the magnetic biosorbent, was 2 × 10⁻⁶. 9 Replace the mixed bacterial culture with a bacterial cell concentration of 2×10⁻⁶ CFU / mL. 9 The magnetic biosorbent was prepared by using a CFU / mL Rhizobium culture followed by the same steps.

[0101] The macromolecular DDS solubles and small molecule DDS solubles prepared in Examples 1-3 and Comparative Examples 1-2 were purified and collected by gel column chromatography, and then spray-dried to obtain soluble polypeptide powder. The soluble polypeptide powder prepared in Comparative Example 3 was then taken, and the polypeptide yield was calculated. The polypeptide yield = mass of soluble polypeptide powder / crude protein content in wheat lees × 100%. The crude protein content in wheat lees was determined by the Kjeldahl method. Three parallel experiments were conducted for each group and the yield was calculated, as shown in Table 1.

[0102]

[0103] As can be seen from the data of Examples 1-3 and Comparative Examples 1-3 in Table 1, the peptide yield of Examples 1-3 of this application is all above 11.8%, which proves that this application has a high peptide yield. As can be seen from the data of Comparative Examples 1 and 2, the peptide yield decreases when the wheat lees are not treated with plasma + pulsed electric field or cryogenic rolling, which proves that the combined synergistic treatment of plasma, pulsed electric field and cryogenic rolling can expose more enzymatic sites among the components, thereby greatly promoting the enzymatic hydrolysis efficiency of amylase, cellulase and protease and improving the peptide yield.

[0104] The data from Comparative Example 3 show that enzymatic hydrolysis of pretreated lees using pullulanase, saccharifying enzyme, cellulase, and alkaline protease sequentially can improve peptide yield compared to separating proteins for enzymatic hydrolysis.

[0105] 100g of wheat lees and 600g of distilled water were mixed and stirred evenly. Acetic acid solution was added to adjust the pH to 6. DON toxin and AFB1 / AFB2 toxin were added to make the concentrations of DON, AFB1 and AFB2 2μg / g to obtain a moldy lees solution. The magnetic biosorbent prepared in Examples 1-3 and Comparative Examples 4-5 was added to the moldy lees solution at a solid-liquid ratio of 1:400g / mL. After stirring at 120rpm for 8 hours at 30℃, magnetic separation was performed. The toxin content in the moldy lees solution was detected. Three parallel experiments were performed. The toxin removal rate was calculated as (toxin content in the moldy lees solution - toxin content in the moldy lees solution after magnetic separation) / toxin content in the moldy lees solution × 100%. The average value was taken and the data were recorded as shown in Table 2.

[0106]

[0107] As can be seen from the data in Tables 2 (Examples 1-3), the magnetic biosorbent prepared in this application can effectively adsorb and remove DON toxin and AFB1 / AFB2 toxin, thereby avoiding the contamination of subsequent nutrients by toxins produced by a small amount of Fusarium graminearum and mold that may be hidden in wheat distillers' grains.

[0108] The above embodiments are merely illustrative of the principles and effects of the present invention and are not intended to limit the invention. Any person skilled in the art can modify or alter the above embodiments without departing from the spirit and scope of the present invention. Therefore, all equivalent modifications or alterations made by those skilled in the art without departing from the spirit and technical concept disclosed in the present invention should still be covered by the claims of the present invention.

Claims

1. A process technology for enzymatic hydrolysis and bio-extraction of distiller's grains, characterized in that, Includes the following steps: S1: Pretreatment of distiller's grains S1.1: Place wheat lees with a moisture content of 10-15% in a plasma surface processor, adjust the voltage to 4-5kV, the spray distance to 4.5-6cm, and treat with helium plasma for 20-25s. Then spread it flat on the lower electrode plate connected to the high-voltage pulse power supply with a thickness of 5-6mm. Set the distance between the upper and lower electrode plates to 2-3cm, the voltage of the high-voltage pulse power supply to 15-20kV, the pulse width to 6-8μs, and perform pulse electric field treatment for 10-15min to obtain plasma pulse treated lees. S1.2: Plasma pulse treated lees are placed in a freezing chamber and frozen at -15 to -10℃ for 1-1.5 hours. Then, they are put into a double-roll crusher with preheated rollers for rolling. The roller preheating temperature is 80-85℃, the roller gap is 0.1-0.15mm, and the roller speed is 35-40rpm to obtain pretreated wheat lees. S2: Preparation of magnetic biosorbent S2.1: Under a nitrogen atmosphere, 18-20 parts by weight of glucose, 2.4-2.5 parts by weight of ferric chloride hexahydrate, and 0.85-0.9 parts by weight of ferrous chloride dihydrate are dissolved in 900-1000 parts by weight of ultrapure water to obtain an iron salt solution. Under magnetic stirring at 70-75℃ and 1200-1500 rpm, 1-1.2 mol / L NaOH solution is added dropwise at a rate of 3-4 mL / min until the pH reaches 10-11. The reaction is carried out at a constant temperature for 1-1.5 h. Then, the mixture is centrifuged at 3000-4000 rpm to remove the bottom aggregates. The upper suspension is collected and placed in a dialysis bag with a molecular weight cutoff of 7000-8000 Da. The suspension is dialyzed with ultrapure water for 3-4 days, with the ultrapure water being replaced every 6-8 h, to obtain a glucose-ferric oxide nanoparticle suspension. S2.2: Lactobacillus delbrueckii subsp. bulgaricus and Rhizobium were cultured routinely to an OD600 of 6-8 to obtain Lactobacillus delbrueckii subsp. bulgaricus and Rhizobium cultures were then mixed and placed in a sterile high-speed centrifuge at 4-5℃ and 8000-8500 rpm for 12-15 min. The supernatant was discarded, and the cells were resuspended in 0.1 mol / L phosphate buffer (pH 7.4) until the cell concentration reached 2 × 10⁻⁶ cells / mL. 9 -5×10 9 CFU / mL was used to obtain a mixed bacterial suspension; S2.3: Glucose-Fe3O4 nanoparticle suspension and mixed bacterial suspension were shaken and mixed evenly at a volume ratio of 1:(8-10), and then added to mixed culture medium. The pH was maintained at 6.5-7. After culturing at 30-32℃ for 25-30h, magnetic separation was performed, and the magnetically adsorbed solid material was collected. The solid material was washed 2-3 times with ultrapure water, filtered and the filter cake was collected. The filter cake was freeze-dried at -40 to -45℃ to obtain the magnetic biosorbent. S3: Detoxification and stepwise enzymatic hydrolysis of distiller's grains Pretreated wheat lees were mixed with distilled water and the pH was adjusted to 6-6.

5. Magnetic biosorbent was added at 30-32℃ and stirred continuously. Then, magnetic separation was performed to obtain detoxified wheat lees slurry. After adjusting the pH, pullulanase, saccharifying enzyme and cellulase were added for enzymatic hydrolysis. After inactivating the enzymes and adjusting the pH, alkaline protease and trypsin were added in sequence for enzymatic hydrolysis to obtain secondary enzymatic hydrolyzed lees slurry. S4: Multi-stage membrane separation of enzymatically hydrolyzed distiller's grains slurry The secondary enzymatic hydrolysis of distiller's grains slurry was subjected to plate and frame filter press filtration to obtain plate and frame filter cake and plate and frame filter clear liquid. The plate and frame filter cake was flash dried to obtain DDG dried material. The plate and frame filter clear liquid was filtered through an ultrafiltration membrane to obtain membrane concentrate I and membrane permeate I. Membrane concentrate I was concentrated and dried to obtain large molecular weight DDS soluble material. Membrane permeate I was filtered through a nanofiltration membrane to obtain membrane concentrate II and membrane permeate II. Membrane concentrate II was concentrated and dried to obtain small molecular weight DDS soluble material. Membrane permeate II was filtered through a reverse osmosis membrane to obtain membrane concentrate III and membrane permeate III. Membrane concentrate III was evaporated and concentrated to obtain DWS soluble material. Membrane permeate III was directly discharged to a wastewater treatment plant.

2. The enzymatic hydrolysis and bio-extraction process for distiller's grains according to claim 1, characterized in that, Step S3, detoxification and stepwise enzymatic hydrolysis of distiller's grains, includes the following steps: S3.1: Mix pretreated wheat lees and distilled water at a mass ratio of 1:(6-10) and stir evenly. Add acetic acid solution with a concentration of 10-15% to adjust the pH to 6-6.

5. Add magnetic biosorbent at a solid-liquid ratio of 1:(400-450) g / mL. Stir at 120-150 rpm for 8-10 hours at 30-32℃. Then, perform magnetic separation on the magnetic biosorbent to obtain detoxified wheat lees slurry. S3.2: Continue to add 10-15% acetic acid solution to the detoxified wheat lees slurry to adjust the pH to 5.5-6, then add pullulanase, saccharifying enzyme and cellulase, and enzymatically hydrolyze for 2-3 hours at 55-57℃. Then place it in an autoclave and inactivate the enzymes at 135℃ for 5 minutes to obtain the first enzymatically hydrolyzed lees slurry. S3.3: Add distilled water to the first enzymatic hydrolysate of distiller's grains to adjust the concentration to 15-17%, then add food-grade sodium hydroxide to adjust the pH to 8-8.5, add alkaline protease at 55-60℃ and hydrolyze for 4-6 hours, then add trypsin and continue hydrolysis for 3-5 hours, and finally inactivate the enzyme at 85℃ for 15 minutes to obtain the second enzymatic hydrolysate of distiller's grains.

3. The enzymatic hydrolysis and bio-extraction process for distiller's grains according to claim 2, characterized in that, Step S4, enzymatic hydrolysis of distiller's grains slurry, involves multi-stage membrane separation, including the following steps: S4.1: The secondary enzymatically hydrolyzed distiller's grains slurry is placed in a plate and frame filter press for filtration to obtain a plate and frame filter cake and a clear liquid. The plate and frame filter cake is then transferred to a flash dryer, with the blower airflow set to 6000-6500 m³ / h. 3 The plate and frame filter cake is flash-dried at a speed of 6000-8000Pa, an air pressure of 6000-8000Pa, an inlet air temperature of 100-120℃, and an outlet air temperature of 55-65℃ until the moisture content is reduced to 6-8%, thus obtaining DDG dried product. S4.2: The plate and frame clarified liquid obtained in step S4.1 is filtered through an ultrafiltration membrane to obtain membrane concentrate I and membrane permeate I. Membrane concentrate I is concentrated and dried at 60-85℃ to obtain macromolecular DDS solubles. Then, membrane permeate I is filtered through a nanofiltration membrane to obtain membrane concentrate II and membrane permeate II. Membrane concentrate II is concentrated and dried at 60-85℃ to obtain small molecule DDS solubles. S4.3: The membrane permeate II obtained in step S4.2 is filtered through a reverse osmosis membrane to obtain membrane concentrate III and membrane permeate III. The membrane concentrate III is evaporated and concentrated to 30-35% of its original volume to obtain DWS solubles. The membrane permeate III is directly discharged to the wastewater treatment plant.

4. The enzymatic hydrolysis and bio-extraction process for distiller's grains according to claim 1, characterized in that, In step S2, the culture medium used for the routine culture of Lactobacillus delbrueckii subsp. bulgaricus is MRS medium, and the culture medium used for the routine culture of Rhizobium is yeast mannitol agar medium. The mixed culture medium is prepared by mixing PDA medium and MRS medium in a volume ratio of 1:(1-2).

5. The enzymatic hydrolysis and bio-extraction process for distiller's grains according to claim 2, characterized in that, In step S3.2, the amounts of pullulanase, saccharifying enzyme, and cellulase added account for 0.6-0.8 wt%, 0.4-0.5 wt%, and 0.5-0.7 wt% of the dry matter in the detoxified wheat distillers' grains slurry, respectively.

6. The enzymatic hydrolysis and bio-extraction process for distiller's grains according to claim 2, characterized in that, Based on the enzymatic hydrolysis of distiller's grains slurry, the amount of alkaline protease added in step S3.3 is 6000-7000 U / g, and the amount of trypsin added is 3000-5000 U / g.

7. The enzymatic hydrolysis and bio-extraction process for distiller's grains according to claim 3, characterized in that, In step S4.2, the molecular weight cutoff of the ultrafiltration membrane is 1000 Da, the molecular weight cutoff of the nanofiltration membrane is 150-200 Da, and the molecular weight cutoff of the reverse osmosis membrane is 100 Da.

8. The enzymatic hydrolysis and bio-extraction process for distiller's grains according to claim 3, characterized in that, In step S4, the main component of DDG dried matter is poorly soluble protein, the main components of macromolecular DDS soluble matter and small molecule DDS soluble matter are glucose, xylooligosaccharides, polypeptides and small peptides, and the main components of DWS soluble matter are organic acids, amino acids and growth factors.

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