Embryo element preparation method based on soybean germs

Abstract

The invention discloses a preparation method of an embryo extract based on soybean germs. The preparation method comprises the following steps: screening the soybean germs, carrying out antioxidant cleaning, hatching and activating in a dark place under specific temperature and humidity, and carrying out low-temperature ultrasonic pretreatment; mixing the activated wet germs with a low-temperature buffer solution, and performing wet grinding under a temperature control condition to obtain primary slurry; step-by-step enzymolysis is carried out by adopting a compound enzyme containing alkaline protease, cellulase, beta-glucanase and glutamine transaminase, and macromolecular substances are directionally hydrolyzed under the protection of nitrogen; carrying out inactivation and solid-liquid separation on the enzymatic hydrolysate, and then carrying out fine separation, desalination and concentration on the enzymatic hydrolysate through a microfiltration membrane system, an ultrafiltration membrane system and a nanofiltration membrane system in sequence; by means of systematic activation, low-temperature treatment, directional enzymolysis and membrane separation technologies, micromolecular active substances in soybean germs can be efficiently extracted and enriched, and the obtained product is high in active ingredient retention rate, high in purity and good in stability.

Description

Technical Field

[0001] This invention relates to the field of soybean germ processing technology, specifically to a method for preparing embryonic stem cells based on soybean germ. Background Technology

[0002] Soybeans, as an important food and economic crop, have attracted much attention in terms of processing and utilization. Although soybean germ accounts for only about 2% of the weight of soybean grains, it is the most active part of the whole soybean, with a high concentration of nutrients and bioactive components. Studies have shown that soybean germ is not only rich in high-quality protein, lipids, vitamins, and minerals, but also contains a variety of functional components, such as soy isoflavones, saponins, nucleic acids, and active peptides and oligosaccharides with specific physiological regulatory functions. These active substances show potential value in anti-oxidation, immune regulation, and promotion of cell metabolism, making soybean germ a high-quality raw material for developing bioactive products, especially in the fields of functional foods, health products, and cosmetic raw materials that pursue natural and healthy ingredients.

[0003] "Embryonic extract" usually refers to a complex of small molecule active substances, such as small molecule peptides, oligosaccharides, and nucleotides, extracted from plant germ and other tissues with vigorous vitality. These substances are valued for their easy absorption, good biocompatibility, and potential to regulate physiological functions. The preparation of embryonic extract from soybean germ is essentially a process of efficient and targeted extraction and purification of its essential active components.

[0004] However, maximizing the extraction and preservation of these heat-sensitive and easily degradable active ingredients from soybean germ remains a key technological challenge. Traditional extraction methods often involve high temperatures, strong acids and alkalis, or organic solvents, which can easily lead to the inactivation and structural damage of active substances. Furthermore, the extracts are often complex in composition, contain many impurities, and have low purity of the target components, affecting the efficacy and stability of the final product. In addition, simple pulverization, single enzymatic hydrolysis, or conventional drying methods are also difficult to balance extraction efficiency, activity preservation, and product quality. Therefore, developing a mild and efficient preparation method that can systematically protect activity, achieve fine separation, and ultimately obtain highly stable embryonic extract products is of great significance for enhancing the resource value of soybean germ and meeting the market demand for high-quality natural active ingredients. Summary of the Invention

[0005] In view of the problems existing in the prior art, the present invention discloses a method for preparing embryonic fibroblasts based on soybean germ, the technical solution of which includes the following steps: Step A: Select intact, mold-free soybean germs and clean and activate them; Step B: The activated wet soybean germ is wet-milled under low temperature conditions to obtain primary germ slurry; Step C: Add a compound enzyme preparation to the primary germ slurry and carry out a stepwise enzymatic hydrolysis reaction at pH 5.0-8.0 and temperature 45-52℃ to directionally hydrolyze macromolecules into small molecule active peptides and oligosaccharides. Step D: The enzymatically hydrolyzed solution is sequentially passed through membrane systems with different pore sizes for separation, desalting, and concentration, and the permeate or retentate rich in embryonic active components is collected. Step E: Dry the purified liquid embryonic active component to obtain a solid embryonic product.

[0006] As a preferred embodiment of the present invention, step A includes the following steps: Step A1: Screen soybean germ raw materials with uniform particle size and integrity of more than 95%, and separate the germ shells. Use a pure aqueous solution containing 0.1%-0.3% (w / v) food-grade citric acid and 0.05% (w / v) vitamin C to perform vortex washing twice at 25-30℃, 3-5 minutes each time, to remove surface impurities and initially establish an antioxidant environment. Step A2: Spread the washed and drained soybean germ evenly in a constant temperature and humidity incubator. Incubate for 12-18 hours in the dark under the conditions of constant temperature 28℃±1℃ and relative humidity 75%±5%. During this process, spray with an atomized aqueous solution containing 0.01mol / L potassium chloride and 5ppm gibberellin every 4 hours until the germ length germinates and grows to 1.2-1.5 times the original length. Step A3: Quickly place the activated soybean germ in an ultrasonic treatment tank with a frequency of 40-45KHz and a power of 300W, and treat it in a low-temperature ice-water bath environment of 4-10℃ for 5-8 minutes to obtain wet soybean germ.

[0007] As a preferred technical solution of the present invention, in step B, the wet grinding conditions are as follows: the wet soybean germ treated in step A3 is pre-cooled to 2-5℃ and mixed with a phosphate buffer solution with pH 6.0-6.5 at a mass ratio of 1:3-1:5, and fed into a colloid mill pre-cooled to below 5℃. The mill is circulated and ground 3-5 times with the grinding disc gap adjusted to 10-20 micrometers. The temperature of the entire grinding system is always maintained below 10℃ by an external cooling circulation device, and finally a homogenized primary germ slurry with a particle size D90≤30μm is obtained.

[0008] As a preferred embodiment of the present invention, step C, the stepwise enzymatic hydrolysis reaction includes the following steps: Step C1: Proteolytic enzyme hydrolysis. Adjust the temperature of the primary germ pulp to 48℃-52℃ and the pH to 7.5-8.0. First, add alkaline protease at 0.8%-1.2% of the total protein content of the pulp and stir for 80-100 minutes. Step C2: After completing step C1, do not terminate the reaction. Adjust the system temperature to 45℃-48℃ and the pH to 5.0-5.5. Then, add cellulase and β-glucanase at 0.5%-0.8% of the total carbohydrate mass of the slurry, and synergistically add transglutaminase at 0.02%-0.05% of the total slurry mass. Continue the reaction for 50-70 minutes. The entire enzymatic hydrolysis process is carried out under nitrogen protection. The enzymatic hydrolysis reaction is terminated when the degree of hydrolysis reaches 30%-35%.

[0009] As a preferred embodiment of the present invention, after the enzymatic hydrolysis reaction is completed in step C, the process further includes enzyme inactivation and inactivation and preliminary solid-liquid separation. The hydrolyzed liquid is rapidly heated to 85℃-90℃ and maintained at this temperature for 5-8 minutes. Then, solid-liquid separation is performed by centrifuging at a speed of 3000-5000 rpm. The supernatant is collected, and the filter residue is washed twice with pure water at 50℃-60℃ using countercurrent washing. The washing liquid and the supernatant are combined to obtain crude embryo extract solution.

[0010] As a preferred embodiment of the present invention, the membrane system in step D includes the following steps: Step D1: First-stage microfiltration: The crude embryo extract solution is pumped into a ceramic microfiltration membrane module with a pore size of 0.1-0.2μm, and cross-flow filtration is performed at an operating pressure of 0.1-0.3MPa and a temperature of 25-30℃. The permeate is collected. Step D2: Second-stage ultrafiltration. The first-stage permeate is pumped into a polyethersulfone hollow fiber ultrafiltration membrane system with a molecular weight cutoff of 5kDa-10kDa. Separation is carried out at an operating pressure of 0.2-0.4MPa and a temperature of 20-25℃. The permeate of the active component with a molecular weight of less than 10kDa is collected. Step D3: Third-stage nanofiltration desalination and concentration. The permeate from the second-stage ultrafiltration is pumped into a nanofiltration membrane system with a molecular weight cutoff of 200-300 Da. The system is operated at an operating pressure of 1.0-1.5 MPa and a temperature of 15-20℃ to remove inorganic salts, monosaccharides, and water. At the same time, the target active component is concentrated 5-8 times to obtain a concentrated solution of embryonic active component.

[0011] As a preferred embodiment of the present invention, the drying and shaping in step E employs a two-step method combining spray drying and vacuum freeze drying: Step 1: Spray drying preforming: The concentrated solution of embryonic active components is dried through a high-speed centrifugal spray drying tower. The inlet air temperature is controlled at 155℃-165℃, the outlet air temperature is maintained at 75℃-85℃, and the atomizing disc speed is 18000-22000 rpm to obtain microencapsulated pre-finished powder with good flowability. The second step is vacuum freeze-drying for quality improvement: The pre-finished powder obtained by spray drying is uniformly dispersed in an aqueous solution containing 5% trehalose and 2% hydroxypropyl-β-cyclodextrin, which is 2-3 times its weight in the solution. The solution is then emulsified by high-speed shearing to form a homogeneous suspension, which is then injected into a freeze-drying tray and pre-frozen at -40°C for 4-6 hours. After that, it is placed in a vacuum freeze dryer and subjected to main drying and desorption drying for 20-28 hours under conditions where the cold trap temperature is below -50°C and the vacuum degree is below 10Pa, finally obtaining the solid embryonic product.

[0012] As a preferred technical solution of the present invention, after drying and molding in step E, a sustained-release encapsulation treatment is further included. The solid embryonic extract powder is dry-mixed with pre-gelled octenyl succinate starch ester and dietary fiber extracted from the germ shell separated in step A1 at a mass ratio of 10:2:1. Then, fluidized bed coating technology is used, with an 8%-12% lecithin ethanol solution as the coating liquid. Bottom spray coating is performed under the conditions of an inlet air temperature of 50-55℃, a material temperature of 35-40℃, and an atomization pressure of 0.15-0.25MPa. The coating weight gain rate is 15%-25%, and finally, a microencapsulated embryonic extract product is prepared.

[0013] The beneficial effects of this invention are: 1. This invention effectively activates and enhances the primary metabolic level inside soybean germ by performing light-proof incubation and physiological activation treatment under specific temperature and humidity conditions, promoting the accumulation of small molecule active substances; combined with low-temperature ultrasonic pretreatment and low-temperature wet grinding throughout the process, the biological activity of heat-sensitive and easily oxidized active substances is protected to the greatest extent, and the activity loss in the early stage of preparation is reduced. 2. A stepwise compound enzymatic hydrolysis strategy is adopted, targeting proteins and carbohydrates sequentially with the synergistic use of transglutaminase. This not only efficiently hydrolyzes macromolecules into target small-molecule active peptides and oligosaccharides, but also helps improve the functional properties of the product, increasing the yield and purity of the target active components. Furthermore, a membrane separation system composed of microfiltration, ultrafiltration, and nanofiltration is used to achieve precise separation, desalting, and concentration of components with different molecular weights in the enzymatic hydrolysis products. The process is mild and does not require high temperature or violent chemical treatment, effectively removing impurities, inorganic salts, and macromolecular fragments, thereby enriching the concentrate with core embryonic components such as small-molecule active peptides, oligosaccharides, and nucleotides, resulting in a high-purity product. 3. A two-step drying method combining spray drying and vacuum freeze drying is adopted, with optional slow-release encapsulation treatment. This method firstly uses spray drying to quickly form a microcapsule prestructure that is conducive to subsequent processing, and then uses vacuum freeze drying to completely remove moisture and protect the natural structure of active substances to the maximum extent. The final solid product has high activity retention, good solubility, and strong stability. Further encapsulation treatment can endow the product with slow-release characteristics, enhance its tolerance to processing and storage environments, and broaden its application range. Attached Figure Description Detailed Implementation

[0014] Example 1

[0015] This invention discloses a method for preparing embryogenic protein based on soybean germ, the technical solution of which includes the following steps: Step A: Select intact, mold-free soybean germs and clean and activate them; Step A1: Screen soybean germ raw materials with uniform particle size and integrity of more than 95%, and separate the germ shells. Use a pure aqueous solution containing 0.1% (w / v) food-grade citric acid and 0.05% (w / v) vitamin C to perform vortex washing twice at 25°C for 3 minutes each time to remove surface impurities and initially establish an antioxidant environment. Step A2: Spread the washed and drained soybean germ evenly in a constant temperature and humidity incubator. Incubate for 12 hours in the dark under the conditions of constant temperature of 28℃±1℃ and relative humidity of 75%±5%. During this process, spray an atomized aqueous solution containing 0.01mol / L potassium chloride and 5ppm gibberellin every 4 hours until the germ length grows to 1.2 times the original length, so as to fully activate the activity of endogenous enzymes in the germ and accumulate small molecule active substances. Step A3: Quickly place the activated soybean germ in an ultrasonic treatment tank with a frequency of 40KHz and a power of 300W, and treat it in a low-temperature ice-water bath environment of 4℃ for 5 minutes to obtain wet soybean germ. Step B: The activated wet soybean germ is wet-milled under low temperature conditions to obtain primary germ slurry. The wet milling conditions are as follows: the wet soybean germ treated in step A3 is pre-cooled to 2°C and mixed with phosphate buffer solution with pH 6.0 at a mass ratio of 1:3. The mixture is fed into a colloid mill pre-cooled to below 5°C and circulated for 3 times with the grinding disc gap adjusted to 10 micrometers. The temperature of the entire grinding system is maintained below 10°C by an external cooling circulation device. Finally, a homogenized primary germ slurry with a particle size D90≤30μm is obtained. Step C: Add a compound enzyme preparation to the primary germ slurry and carry out a stepwise enzymatic hydrolysis reaction at pH 5.0-8.0 and temperature 45-52℃ to directionally hydrolyze macromolecules into small molecule active peptides and oligosaccharides. Step C1: Proteolytic enzyme hydrolysis. Adjust the temperature of the primary germ pulp to 48°C and the pH to 7.5. First, add alkaline protease at 0.8% of the total protein content of the pulp and stir for 80 minutes. Step C2: After completing step C1, without terminating the reaction, adjust the system temperature to 45℃ and the pH to 5.0. Then, add cellulase and β-glucanase at 0.5% of the total carbohydrate mass of the slurry, and glutamine transaminase at 0.02% of the total slurry mass, and continue the reaction for 50 minutes. The entire enzymatic hydrolysis process is carried out under nitrogen protection. The enzymatic hydrolysis reaction is terminated when the degree of hydrolysis reaches 30%. After the enzymatic hydrolysis reaction is terminated, enzyme inactivation and preliminary solid-liquid separation are also performed. The hydrolyzed liquid is rapidly heated to 85℃ and maintained at this temperature for 5 minutes to completely inactivate various enzyme preparations. Then, solid-liquid separation is performed by centrifuging at 3000 rpm. The supernatant is collected, and the filter residue is washed twice countercurrently with pure water at 50℃. The washing liquid and the supernatant are combined to obtain crude embryo extract solution. Step D: The enzymatically hydrolyzed solution is sequentially passed through membrane systems with different pore sizes for separation, desalting, and concentration, and the permeate or retentate rich in embryonic active components is collected. Step D1: First-stage microfiltration: The crude embryo extract solution is pumped into a ceramic microfiltration membrane module with a pore size of 0.1μm. Cross-flow filtration is performed at an operating pressure of 0.1MPa and a temperature of 25-30℃ to remove residual fine particles, insoluble fibers and some macromolecular colloids. The permeate is collected. Step D2: Second-stage ultrafiltration. The first-stage permeate is pumped into a polyethersulfone hollow fiber ultrafiltration membrane system with a molecular weight cutoff of 5 kDa. Separation is carried out at an operating pressure of 0.2 MPa and a temperature of 20°C. The permeate containing active components with a molecular weight of less than 10 kDa, which are rich in small molecule peptides, oligosaccharides and nucleotides, is collected. The retentate mainly consists of protein fragments with a larger molecular weight. Step D3: Third-stage nanofiltration desalination and concentration. The permeate from the second-stage ultrafiltration is pumped into a nanofiltration membrane system with a molecular weight cutoff of 200 Da. The system is operated at an operating pressure of 1.0 MPa and a temperature of 15°C to remove inorganic salts, monosaccharides and water. At the same time, the target active component is concentrated 5 times to obtain a concentrated solution of embryonic active component. Step E: The purified embryonic active component liquid is dried. This drying process employs a two-step method combining spray drying and vacuum freeze drying. Step 1 Spray Drying Pre-forming: The concentrated solution of embryonic active components is dried through a high-speed centrifugal spray drying tower. The inlet air temperature is controlled at 155℃, the outlet air temperature is maintained at 75℃, and the atomizing disc speed is 18000rpm, to obtain microencapsulated pre-finished powder with good flowability. This step can quickly form a porous surface structure, which is beneficial for subsequent processing. The second step, vacuum freeze-drying for quality improvement, involves uniformly dispersing the pre-finished powder obtained from spray drying in twice its weight of an aqueous protective agent solution containing 5% trehalose and 2% hydroxypropyl-β-cyclodextrin. This solution is then emulsified by high-speed shearing to form a homogeneous suspension, which is then injected into a freeze-drying tray and pre-frozen at -40°C for 4 hours. Afterward, it is placed in a vacuum freeze dryer and subjected to primary drying and desorption drying for 20 hours under conditions of a cold trap temperature below -50°C and a vacuum degree below 10 Pa. The final product is then dried and shaped. The process also includes a sustained-release encapsulation treatment, in which solid embryonic extract powder is dry-mixed with pre-gelled octenyl succinate starch ester and dietary fiber extracted from the germ shell separated in step A1 at a mass ratio of 10:2:1. Then, fluidized bed coating technology is used, with an 8% lecithin ethanol solution as the coating liquid. Bottom spray coating is carried out under the conditions of an inlet air temperature of 50℃, a material temperature of 35℃, and an atomization pressure of 0.15MPa. The coating weight gain rate is 15%, and finally, microencapsulated embryonic extract products are prepared.

[0016] Example 2

[0017] This invention discloses a method for preparing embryogenic protein based on soybean germ, the technical solution of which includes the following steps: Step A: Select intact, mold-free soybean germs and clean and activate them; Step A1: Screen soybean germ raw materials with uniform particle size and integrity of more than 95%, and separate the germ shell. Use a pure aqueous solution containing 0.2% (w / v) food-grade citric acid and 0.05% (w / v) vitamin C to perform vortex washing twice at 28°C for 4 minutes each time to remove surface impurities and initially establish an antioxidant environment. Step A2: Spread the washed and drained soybean germ evenly in a constant temperature and humidity incubator. Incubate for 16 hours in the dark under the conditions of constant temperature of 28℃±1℃ and relative humidity of 75%±5%. During this process, spray an atomized aqueous solution containing 0.01mol / L potassium chloride and 5ppm gibberellin every 4 hours until the germ length grows to 1.3 times the original length, so as to fully activate the activity of endogenous enzymes in the germ and accumulate small molecule active substances. Step A3: Quickly place the activated soybean germ in an ultrasonic treatment tank with a frequency of 42KHz and a power of 300W, and treat it in a low-temperature ice-water bath environment of 6℃ for 6 minutes to obtain wet soybean germ. Step B: The activated wet soybean germ is wet-milled under low temperature conditions to obtain primary germ slurry. The wet milling conditions are as follows: the wet soybean germ treated in step A3 is pre-cooled to 4°C and mixed with phosphate buffer solution with pH 6.3 at a mass ratio of 1:4. The mixture is fed into a colloid mill pre-cooled to below 5°C and circulated for 4 times with the grinding disc gap adjusted to 15 micrometers. The temperature of the entire grinding system is maintained below 10°C by an external cooling circulation device. Finally, a homogenized primary germ slurry with a particle size D90≤30μm is obtained. Step C: Add a compound enzyme preparation to the primary germ slurry and carry out a stepwise enzymatic hydrolysis reaction at pH 5.0-8.0 and temperature 45-52℃ to directionally hydrolyze macromolecules into small molecule active peptides and oligosaccharides. Step C1: Proteolytic enzyme hydrolysis. Adjust the temperature of the primary germ pulp to 50°C and the pH to 7.8. First, add alkaline protease at 1% of the total protein content of the pulp and stir for 90 minutes. Step C2: After completing step C1, without terminating the reaction, adjust the system temperature to 47℃ and the pH to 5.3. Then, add cellulase (0.6% of the total carbohydrate mass of the slurry) and β-glucanase (0.4% of the total carbohydrate mass of the slurry) sequentially, and add transglutaminase (0.03% of the total carbohydrate mass of the slurry) in combination. Continue the reaction for 65 minutes. The entire enzymatic hydrolysis process is carried out under nitrogen protection. The enzymatic hydrolysis reaction is terminated when the degree of hydrolysis reaches 33%. After the enzymatic hydrolysis reaction is terminated, enzyme inactivation and preliminary solid-liquid separation are also performed. The hydrolyzed liquid is rapidly heated to 88℃ and maintained at this temperature for 7 minutes to completely inactivate various enzyme preparations. Then, solid-liquid separation is performed by centrifuging at 4000 rpm. The supernatant is collected, and the filter residue is washed twice countercurrently with pure water at 55℃. The washing liquid and the supernatant are combined to obtain crude embryo extract solution. Step D: The enzymatically hydrolyzed solution is sequentially passed through membrane systems with different pore sizes for separation, desalting, and concentration, and the permeate or retentate rich in embryonic active components is collected. Step D1: First-stage microfiltration: The crude embryo extract solution is pumped into a ceramic microfiltration membrane module with a pore size of 0.15μm. Cross-flow filtration is performed at an operating pressure of 0.2MPa and a temperature of 28℃ to remove residual fine particles, insoluble fibers and some macromolecular colloids. The permeate is collected. Step D2: Second-stage ultrafiltration. The first-stage permeate is pumped into a polyethersulfone hollow fiber ultrafiltration membrane system with a molecular weight cutoff of 8 kDa. Separation is carried out at an operating pressure of 0.3 MPa and a temperature of 23°C. The permeate containing active components with a molecular weight of less than 10 kDa, which are rich in small molecule peptides, oligosaccharides and nucleotides, is collected. The retentate mainly consists of protein fragments with a larger molecular weight. Step D3: Third-stage nanofiltration desalination and concentration. The permeate from the second-stage ultrafiltration is pumped into a nanofiltration membrane system with a molecular weight cutoff of 250 Da. The system is operated at an operating pressure of 1.3 MPa and a temperature of 18 °C to remove inorganic salts, monosaccharides and water. At the same time, the target active component is concentrated 7 times to obtain a concentrated solution of embryonic active component. Step E: The purified embryonic active component liquid is dried. This drying process employs a two-step method combining spray drying and vacuum freeze drying. Step 1 Spray Drying Pre-forming: The concentrated solution of embryonic active components is dried through a high-speed centrifugal spray drying tower. The inlet air temperature is controlled at 160℃, the outlet air temperature is maintained at 80℃, and the atomizing disc speed is 20000rpm, to obtain microencapsulated pre-finished powder with good flowability. This step can quickly form a porous surface structure, which is beneficial for subsequent processing. The second step, vacuum freeze-drying for quality improvement, involves uniformly dispersing the pre-finished powder obtained from spray drying in an aqueous solution containing 5% trehalose and 2% hydroxypropyl-β-cyclodextrin, at 2.5 times its weight. This solution is then emulsified by high-speed shearing to form a homogeneous suspension, which is then injected into a freeze-drying tray and pre-frozen at -40°C for 5 hours. Afterward, it is placed in a vacuum freeze dryer and subjected to primary drying and desorption drying for 24 hours under conditions of a cold trap temperature below -50°C and a vacuum degree below 10 Pa. The dried product is then formed... Following this, a sustained-release encapsulation process is included. The solid embryonic extract powder is dry-mixed with pre-gelled octenyl succinate starch ester and dietary fiber extracted from the germ shell separated in step A1 at a mass ratio of 10:2:1. Then, fluidized bed coating technology is used, with a 10% lecithin ethanol solution as the coating liquid. Bottom-spray coating is performed under conditions of an inlet air temperature of 53℃, a material temperature of 38℃, and an atomization pressure of 0.2MPa. The coating weight gain rate is 20%, ultimately preparing a microencapsulated embryonic extract product.

[0018] Example 3

[0019] This invention discloses a method for preparing embryogenic protein based on soybean germ, the technical solution of which includes the following steps: Step A: Select intact, mold-free soybean germs and clean and activate them; Step A1: Screen soybean germ raw materials with uniform particle size and integrity of more than 95%, and separate the germ shells. Use a pure aqueous solution containing 0.3% (w / v) food-grade citric acid and 0.05% (w / v) vitamin C to perform vortex washing twice at 30°C for 5 minutes each time to remove surface impurities and initially establish an antioxidant environment. Step A2: Spread the washed and drained soybean germ evenly in a constant temperature and humidity incubator. Incubate for 18 hours in the dark under the conditions of constant temperature of 28℃±1℃ and relative humidity of 75%±5%. During this process, spray an atomized aqueous solution containing 0.01mol / L potassium chloride and 5ppm gibberellin every 4 hours until the germ length grows to 1.5 times the original length, so as to fully activate the activity of endogenous enzymes in the germ and accumulate small molecule active substances. Step A3: Quickly place the activated soybean germ in an ultrasonic treatment tank with a frequency of 45KHz and a power of 300W, and treat it in a low-temperature ice-water bath environment of 10℃ for 8 minutes to obtain wet soybean germ. Step B: The activated wet soybean germ is wet-milled under low temperature conditions to obtain primary germ slurry. The wet milling conditions are as follows: the wet soybean germ treated in step A3 is pre-cooled to 5°C and mixed with a phosphate buffer solution with pH 6.5 at a mass ratio of 1:5. The mixture is fed into a colloid mill that has been pre-cooled to below 5°C. The milling is circulated and milled 5 times with the grinding disc gap adjusted to 20 micrometers. The temperature of the entire milling system is always maintained below 10°C by an external cooling circulation device. Finally, a homogenized primary germ slurry with a particle size D90≤30μm is obtained. Step C: Add a compound enzyme preparation to the primary germ slurry and carry out a stepwise enzymatic hydrolysis reaction at pH 5.0-8.0 and temperature 45-52℃ to directionally hydrolyze macromolecules into small molecule active peptides and oligosaccharides. Step C1: Proteolytic enzyme hydrolysis. Adjust the temperature of the primary germ pulp to 52°C and the pH to 8.0. First, add alkaline protease at 1.2% of the total protein content of the pulp and stir for 100 minutes. Step C2: After completing step C1, without terminating the reaction, adjust the system temperature to 48℃ and the pH to 5.5. Then, add cellulase (0.8% of the total carbohydrate mass of the slurry) and β-glucanase (0.5% of the total carbohydrate mass of the slurry) sequentially, and glutamine transaminase (0.05% of the total carbohydrate mass of the slurry) in combination. Continue the reaction for 70 minutes. The entire enzymatic hydrolysis process is carried out under nitrogen protection. The enzymatic hydrolysis reaction is terminated when the degree of hydrolysis reaches 35%. After the enzymatic hydrolysis reaction is completed, enzyme inactivation and preliminary solid-liquid separation are also performed. The hydrolyzed liquid is rapidly heated to 90℃ and maintained at this temperature for 5-8 minutes to completely inactivate various enzyme preparations. Then, solid-liquid separation is performed by centrifuging at 5000 rpm. The supernatant is collected, and the filter residue is washed twice countercurrently with pure water at 60℃. The washing liquid and the supernatant are combined to obtain crude embryo extract solution. Step D: The enzymatically hydrolyzed solution is sequentially passed through membrane systems with different pore sizes for separation, desalting, and concentration, and the permeate or retentate rich in embryonic active components is collected. Step D1: First-stage microfiltration: The crude embryo extract solution is pumped into a ceramic microfiltration membrane module with a pore size of 0.2 μm. Cross-flow filtration is performed at an operating pressure of 0.3 MPa and a temperature of 30 °C to remove residual fine particles, insoluble fibers and some macromolecular colloids. The permeate is collected. Step D2: Second-stage ultrafiltration. The first-stage permeate is pumped into a polyethersulfone hollow fiber ultrafiltration membrane system with a molecular weight cutoff of 10 kDa. Separation is carried out at an operating pressure of 0.4 MPa and a temperature of 25°C. The permeate containing active components with molecular weights less than 10 kDa, rich in small peptides, oligosaccharides and nucleotides, is collected. The retentate mainly consists of protein fragments with larger molecular weights. Step D3: Third-stage nanofiltration desalination and concentration. The permeate from the second-stage ultrafiltration is pumped into a nanofiltration membrane system with a molecular weight cutoff of 300 Da. The system is operated at an operating pressure of 1.5 MPa and a temperature of 20 °C to remove inorganic salts, monosaccharides and water. At the same time, the target active component is concentrated 8 times to obtain a concentrated solution of embryonic active component. Step E: The purified embryonic active component liquid is dried. This drying process employs a two-step method combining spray drying and vacuum freeze drying. Step 1 Spray Drying Pre-forming: The concentrated solution of embryonic active components is dried through a high-speed centrifugal spray drying tower. The inlet air temperature is controlled at 165℃, the outlet air temperature is maintained at 85℃, and the atomizing disc speed is 22000rpm, to obtain microencapsulated pre-finished powder with good flowability. This step can quickly form a porous surface structure, which is beneficial for subsequent processing. The second step, vacuum freeze-drying for quality improvement, involves uniformly dispersing the pre-finished powder obtained from spray drying in an aqueous solution containing 5% trehalose and 2% hydroxypropyl-β-cyclodextrin, three times its weight in volume. This solution is then emulsified by high-speed shearing to form a homogeneous suspension, which is then injected into a freeze-drying tray and pre-frozen at -40°C for 6 hours. Following this, the powder is placed in a vacuum freeze dryer and subjected to primary drying and desorption drying for 28 hours under conditions of a cold trap temperature below -50°C and a vacuum degree below 10 Pa. After drying and shaping... The process also includes sustained-release encapsulation, where solid embryonic extract powder is dry-mixed with pre-gelled octenyl succinate starch ester and dietary fiber extracted from the germ shell separated in step A1 at a mass ratio of 10:2:1. Then, fluidized bed coating technology is used, with a 12% lecithin ethanol solution as the coating liquid. Bottom-spray coating is performed under conditions of 55℃ inlet air temperature, 40℃ material temperature, and 0.25MPa atomization pressure. The coating weight gain rate is 25%, ultimately producing a microencapsulated embryonic extract product.

[0020] Components not described in detail in this article are existing technologies.

[0021] While the specific embodiments of the present invention have been described in detail above, the present invention is not limited to the above embodiments. Within the scope of knowledge possessed by those skilled in the art, various changes can be made without departing from the spirit of the present invention, and modifications or variations without creative effort are still within the protection scope of the present invention.

Claims

1. A method for preparing embryonic stem cells based on soybean germ, characterized in that, Includes the following steps: Step A: Select intact, mold-free soybean germs and clean and activate them; Step B: The activated wet soybean germ is wet-milled under low temperature conditions to obtain primary germ slurry; Step C: Add a compound enzyme preparation to the primary germ slurry and carry out a stepwise enzymatic hydrolysis reaction at pH 5.0-8.0 and temperature 45-52℃ to directionally hydrolyze macromolecules into small molecule active peptides and oligosaccharides. Step D: The enzymatically hydrolyzed solution is sequentially passed through membrane systems with different pore sizes for separation, desalting, and concentration, and the permeate or retentate rich in embryonic active components is collected. Step E: Dry the purified liquid embryonic active component to obtain a solid embryonic product.

2. The method for preparing embryogenesis based on soybean germ according to claim 1, characterized in that: Step A includes the following steps: Step A1: Screen soybean germ raw materials with uniform particle size and integrity of more than 95%, and separate the germ shells. Use a pure aqueous solution containing 0.1%-0.3% (w / v) food-grade citric acid and 0.05% (w / v) vitamin C to perform vortex washing twice at 25-30℃, 3-5 minutes each time, to remove surface impurities and initially establish an antioxidant environment. Step A2: Spread the washed and drained soybean germ evenly in a constant temperature and humidity incubator. Incubate for 12-18 hours in the dark under the conditions of constant temperature 28℃±1℃ and relative humidity 75%±5%. During this process, spray with an atomized aqueous solution containing 0.01mol / L potassium chloride and 5ppm gibberellin every 4 hours until the germ length germinates and grows to 1.2-1.5 times the original length. Step A3: Quickly place the activated soybean germ in an ultrasonic treatment tank with a frequency of 40-45KHz and a power of 300W, and treat it in a low-temperature ice-water bath environment of 4-10℃ for 5-8 minutes to obtain wet soybean germ.

3. The method for preparing embryogenesis based on soybean germ according to claim 2, characterized in that: In step B, the wet grinding conditions are as follows: the wet soybean germ treated in step A3 is pre-cooled to 2-5℃ and mixed with a phosphate buffer solution with pH 6.0-6.5 at a mass ratio of 1:3-1:

5. The mixture is then fed into a colloid mill that has been pre-cooled to below 5℃. The mill is then circulated and ground 3-5 times with the grinding disc gap adjusted to 10-20 micrometers. The temperature of the entire grinding system is maintained below 10℃ by an external cooling circulation device. Finally, a homogenized primary germ slurry with a particle size D90≤30μm is obtained.

4. The method for preparing embryogenesis based on soybean germ according to claim 1, characterized in that: In step C, the stepwise enzymatic hydrolysis reaction includes the following steps: Step C1: Proteolytic enzyme hydrolysis. Adjust the temperature of the primary germ pulp to 48℃-52℃ and the pH to 7.5-8.

0. First, add alkaline protease at 0.8%-1.2% of the total protein content of the pulp and stir for 80-100 minutes. Step C2: After completing step C1, do not terminate the reaction. Adjust the system temperature to 45℃-48℃ and the pH to 5.0-5.

5. Then, add cellulase and β-glucanase at 0.5%-0.8% of the total carbohydrate mass of the slurry, and synergistically add transglutaminase at 0.02%-0.05% of the total slurry mass. Continue the reaction for 50-70 minutes. The entire enzymatic hydrolysis process is carried out under nitrogen protection. The enzymatic hydrolysis reaction is terminated when the degree of hydrolysis reaches 30%-35%.

5. The method for preparing embryogenesis based on soybean germ according to claim 4, characterized in that: After the enzymatic hydrolysis reaction is completed in step C, the process also includes enzyme inactivation and inactivation and preliminary solid-liquid separation. The hydrolyzed liquid is rapidly heated to 85℃-90℃ and maintained at this temperature for 5-8 minutes. Then, solid-liquid separation is performed by centrifuging at a speed of 3000-5000 rpm. The supernatant is collected, and the filter residue is washed twice countercurrently with pure water at 50℃-60℃. The washing liquid and the supernatant are combined to obtain crude embryo extract solution.

6. The method for preparing embryogenic protein based on soybean germ according to claim 5, characterized in that: The membrane system in step D includes the following steps: Step D1: First-stage microfiltration: The crude embryo extract solution is pumped into a ceramic microfiltration membrane module with a pore size of 0.1-0.2μm, and cross-flow filtration is performed at an operating pressure of 0.1-0.3MPa and a temperature of 25-30℃. The permeate is collected. Step D2: Second-stage ultrafiltration. The first-stage permeate is pumped into a polyethersulfone hollow fiber ultrafiltration membrane system with a molecular weight cutoff of 5kDa-10kDa. Separation is carried out at an operating pressure of 0.2-0.4MPa and a temperature of 20-25℃. The permeate of the active component with a molecular weight of less than 10kDa is collected. Step D3: Third-stage nanofiltration desalination and concentration. The permeate from the second-stage ultrafiltration is pumped into a nanofiltration membrane system with a molecular weight cutoff of 200-300 Da. The system is operated at an operating pressure of 1.0-1.5 MPa and a temperature of 15-20℃ to remove inorganic salts, monosaccharides, and water. At the same time, the target active component is concentrated 5-8 times to obtain a concentrated solution of embryonic active component.

7. The method for preparing embryogenesis based on soybean germ according to claim 1, characterized in that: The drying and shaping process in step E employs a two-step method combining spray drying and vacuum freeze drying. Step 1: Spray drying preforming: The concentrated solution of embryonic active components is dried through a high-speed centrifugal spray drying tower. The inlet air temperature is controlled at 155℃-165℃, the outlet air temperature is maintained at 75℃-85℃, and the atomizing disc speed is 18000-22000 rpm to obtain microencapsulated pre-finished powder with good flowability. The second step is vacuum freeze-drying for quality improvement: The pre-finished powder obtained by spray drying is uniformly dispersed in an aqueous solution containing 5% trehalose and 2% hydroxypropyl-β-cyclodextrin, which is 2-3 times its weight in the solution. The solution is then emulsified by high-speed shearing to form a homogeneous suspension, which is then injected into a freeze-drying tray and pre-frozen at -40°C for 4-6 hours. After that, it is placed in a vacuum freeze dryer and subjected to main drying and desorption drying for 20-28 hours under conditions where the cold trap temperature is below -50°C and the vacuum degree is below 10Pa, finally obtaining the solid embryonic product.

8. The method for preparing embryogenesis based on soybean germ according to claim 1, characterized in that: After drying and molding in step E, a sustained-release encapsulation process is also included. The solid embryonic extract powder is dry-mixed with pre-gelled octenyl succinate starch ester and dietary fiber extracted from the germ shell separated in step A1 at a mass ratio of 10:2:

1. Then, fluidized bed coating technology is used, with an 8%-12% lecithin ethanol solution as the coating liquid. Bottom spray coating is carried out under the conditions of an inlet air temperature of 50-55℃, a material temperature of 35-40℃, and an atomization pressure of 0.15-0.25MPa. The coating weight gain rate is 15%-25%, and finally, a microencapsulated embryonic extract product is prepared.