Preparation method of sturgeon protein peptide for enhancing immunity

Abstract

The invention belongs to the technical field of biological extraction, and particularly relates to a preparation method of sturgeon protein peptide for enhancing immunity. Soluble salts of Cu < 2 + >, Zn < 2 + > and Ti < 4 + > are added into an ethanol-water mixed solution, the pH is adjusted by ammonia water to form a precipitate, and a hydrothermal reaction is carried out. A microbial carrier is soaked or coated in sol, a uniform gel layer is formed on the surface of the carrier through a sol-gel method, and the carrier is converted into a Cu-Zn-Ti three-way catalyst layer after being dried and roasted. And then, adding a microbial carrier loaded with a catalyst and a compound enzyme into the minced sturgeon, carrying out enzymolysis reaction under the conditions that the temperature is 25-50 DEG C, the pH is 6.8-7.2 and the speed is 100-200 rpm, and carrying out membrane separation and drying on a product to obtain the sturgeon protein peptide. According to the method, the Cu-Zn-Ti three-way catalyst is combined with the microbial carrier, so that the enzymolysis efficiency and the uniformity of the molecular weight of the product are improved, the active ingredients are effectively protected, and the production cost is reduced.

Description

Technical Field

[0001] The invention belongs to the technical field of biological extraction, and in particular relates to a method for preparing a sturgeon protein peptide for enhancing immunity. Background Art

[0002] As people pay more attention to health and nutrition, the market for products that enhance immunity continues to expand. As a natural bioactive substance, sturgeon protein peptide has attracted widespread attention due to its low molecular weight, easy absorption and high biological activity. It is believed to have multiple functions such as enhancing immunity, anti-oxidation and promoting growth. However, the existing sturgeon protein peptide preparation methods still have some problems, such as complex preparation process, high cost, low extraction efficiency, low product purity and poor stability, which limit the wide application of sturgeon protein peptide.

[0003] For example, the existing patent publication number is CN105734102B, which discloses a method for preparing collagen peptides. The patent provides a method for preparing collagen peptides, including mixing fish skin with water, enzymolysis, deodorization, decolorization, ultrafiltration and freeze-drying. The obtained collagen peptides have the advantages of high yield, good antibacterial properties, and good skin care effects. However, this method mainly uses fish skin as raw material, and does not fully utilize other parts of sturgeon, such as fish meat and viscera, which are rich in protein, resulting in low resource utilization. In addition, this method has high requirements for the selection and processing of fish skin, has many process steps, and has high costs, making it difficult to achieve large-scale industrial production.

[0004] Another existing patent publication number is CN100475972C, which is a method for preparing shrimp peptide protein powder. The patent provides a method for preparing shrimp peptide protein powder using fresh shrimp heads as raw materials through crushing, sterilization, enzymolysis, drying, crushing and other steps. The prepared protein powder has a small molecular weight and has the advantages of improving animal immunity and disease resistance and strengthening mineral absorption. However, this method is only applicable to shrimp heads and is not suitable for the preparation of protein peptides from other aquatic products such as sturgeon. In addition, this method does not involve the utilization of microbial resources, it is difficult to improve the activity and yield of protein peptides, and it does not take environmental friendliness into consideration enough.

[0005] In summary, the existing sturgeon protein peptide preparation methods still have deficiencies in terms of raw material utilization, process complexity, cost control, and environmental friendliness. Therefore, the present invention provides a method for preparing a sturgeon protein peptide for enhancing immunity, aiming to make full use of the protein resources of various parts of sturgeon, optimize the preparation process, improve the activity and yield of protein peptides, reduce production costs, and meet the needs of modern functional foods for efficient and green preparation processes. Summary of the invention

[0006] The purpose of the present invention is to provide a method for preparing a sturgeon protein peptide for enhancing immunity, which can effectively improve the extraction rate, purity and biological activity of the sturgeon protein peptide, while reducing production costs and meeting market demand; the present invention also provides specific steps of the preparation method to ensure that the preparation process is simple, efficient, economical and environmentally friendly.

[0007] To achieve the above purpose, the technical solution adopted by the present invention is: (1) Preparation of Cu-Zn-Ti ternary catalyst carrier: A) will contain Cu 2+ 、Zn 2+ 、Ti 4+ The soluble salt is dissolved in an ethanol-water mixed solution containing a surfactant, and stirred to form a uniform mixed solution; B) Add ammonia water dropwise under stirring to generate a precipitate and form a suspension; C) transferring the suspension to a reactor for hydrothermal reaction to obtain a metal oxide precursor sol; D) soaking or coating a carrier without any active ingredient in the sol, and causing the metal oxide precursor to undergo hydrolysis and polycondensation on the surface of the carrier by a sol-gel method to form a uniform gel layer; E) After drying and calcination, a carrier loaded with a Cu-Zn-Ti ternary catalyst is obtained; (2) Preparation of microbial carriers loaded with complex enzymes: a) immersing the microbial carrier in a compound enzyme solution in which flavor protease and neutral protease are compounded in a mass ratio of 1:1; b) coating the surface of the carrier to form a protective layer and fix the complex enzyme; (3) Enzymatic reaction and product separation: Sturgeon minced fish is added to the raw material input layer of the microbial decomposition tank, and enters the enzymatic reaction layer through the input port, and then the carrier loaded with the Cu-Zn-Ti catalyst and the microbial carrier loaded with the composite enzyme are loaded in the enzymatic reaction layer, the temperature is 25-50°C, the pH is 6.8-7.2, the stirring speed is 100-200rpm, the reaction product is filtered through the product collection layer to obtain a clear liquid, and the sturgeon protein peptide is obtained after drying. Preferably, the composite enzyme is a flavor protease and a neutral protease compounded in a mass ratio of 1:1.

[0008] Specifically, the enzymatic reaction layer is loaded with a Cu-Zn-Ti ternary catalyst carrier and a microbial carrier, the Cu-Zn-Ti ternary catalyst carrier is a porous ceramic carrier, the loading weight is 1-10 parts, the microbial carrier loaded with the complex enzyme is a porous ceramic carrier or a polymer material carrier, the loading weight is 50-100 parts. Preferably, the polymer material carrier is any one of polyethylene, polymethyl methacrylate and polyacrylamide.

[0009] Specifically, the process of fixing the complex enzyme on the microbial carrier is: immersing the carrier fixed with the ternary catalyst in the complex enzyme solution, fixing the complex enzyme on the inner side of the catalyst layer through a cross-linking reaction, using glutaraldehyde as a cross-linking agent, and stirring at room temperature for 2-4 hours.

[0010] Specifically, Bacillus natto and Bifidobacterium are fixed on the microbial carrier, and Bacillus natto and Bifidobacterium are compounded in a mass ratio of 2:1, and the total addition amount is 10-30 parts. The synergistic effect of the two microorganisms significantly improves the enzymolysis efficiency and the quality of the product.

[0011] Specifically, the microbial decomposition tank includes a raw material input layer, an enzymatic hydrolysis reaction layer and a product collection layer. The raw material input layer is located at the top of the tank body and is provided with a raw material input port. The enzymatic hydrolysis reaction layer is located below the raw material input layer and is filled with microbial carriers. It is provided with a stirring device and a ventilation system. The product collection layer is located below the enzymatic hydrolysis reaction layer.

[0012] Specifically, the microbial carrier is coated with a sodium alginate coating layer or a chitosan coating layer, the coating thickness is 1-10 microns, and the amount of sodium alginate or chitosan added is 2 parts by weight. The sodium alginate coating layer has good biocompatibility and degradability, and can form a protective film to maintain the activity and stability of the microorganisms; the chitosan coating layer has excellent antibacterial properties and air permeability, and can further enhance the immobilization effect of the microorganisms. The coating material is used to fix the microorganisms on the carrier to prevent them from being lost and improve the reaction efficiency.

[0013] The metal ions form hydroxide or oxide precipitation, thereby obtaining a uniformly dispersed metal oxide precursor sol. Next, the microbial carrier that has been loaded with the complex enzyme and microorganisms is immersed or coated in this sol, and the metal oxide precursor is hydrolyzed and polycondensed on the surface of the carrier by the sol-gel method to form a uniform gel layer. After drying and calcination, this layer of gel will be converted into a stable Cu-Zn-Ti ternary catalyst layer, thereby fixing the catalyst on the outermost layer of the microbial carrier. The outermost layer is the area 0-5μm from the carrier surface. This process not only realizes the close combination of the ternary catalyst and the microbial carrier, but also provides the catalyst with a large specific surface area and good active centers, which is conducive to its efficient catalytic performance in the subsequent protein peptide preparation process.

[0014] Specifically, the temperature of the enzymatic reaction layer is controlled at 25-50°C, the pH value is controlled at 6.8-7.2, and the stirring speed is controlled at 100-200rpm / h. pH control: The optimal pH value is 6.8-7.2, and the pH value is within the range to avoid fluctuations caused by the accumulation of metabolites. Stirring speed: The stirring speed is controlled at 100-200rpm / h to ensure that the raw materials are fully in contact with the microorganisms without destroying the microbial structure.

[0015] The preparation process of the sturgeon protein peptide of the present invention is as follows A) pre-treating 100 portions of fresh sturgeon meat to obtain sturgeon surimi; B) adding sturgeon surimi into a microbial decomposition tank, the microbial decomposition tank comprising a raw material input layer, an enzymatic reaction layer and a product collection layer, the raw material input layer being located at the top of the tank body and provided with a raw material input port, the enzymatic reaction layer being located below the raw material input layer and filled with microbial carriers, and being provided with a stirring device and a ventilation system, and the product collection layer being located below the enzymatic reaction layer; C) The collected product is separated from the membrane and then dried to obtain sturgeon protein peptide powder.

[0016] Wherein: the pretreatment includes cleaning, chopping and defatting to remove impurities and fat in the fish meat and ensure the smooth progress of the subsequent enzymatic hydrolysis process.

[0017] Specifically, the enzymatic reaction layer is filled with a microbial carrier, which includes a porous ceramic carrier or a polymer material carrier, and the filling amount is 50-100 parts by weight. Preferably, the polymer material carrier is any one of polyethylene, polymethyl methacrylate and polyacrylamide.

[0018] Specifically, Bacillus natto and Bifidobacterium are fixed on the microbial carrier, and Bacillus natto and Bifidobacterium are compounded in a mass ratio of 2:1, and the total addition amount is 10-30 parts. The synergistic effect of the two microorganisms significantly improves the enzymolysis efficiency and the quality of the product.

[0019] The total addition amount of the Cu-Zn-Ti ternary catalyst is 1-10 parts, and the composite enzyme catalyst includes flavor protease and neutral protease, with 10 parts of flavor protease and neutral protease respectively. The selection and compounding of the catalysts are helpful to further improve the efficiency of the enzymatic reaction and the uniformity of the product; metal ion catalyst: Cu 2+ 、Zn 2+ and Ti 4+ , as an enzyme cofactor, can enhance the activity and stability of proteases; composite enzyme catalysts: such as the combination of flavor proteases and neutral proteases, can synergistically act on protein substrates of different molecular weights and optimize the molecular weight distribution of the product.

[0020] Specifically, the microbial carrier is coated with a sodium alginate coating layer or a chitosan coating layer, the coating thickness is 1-10 microns, and the amount of sodium alginate or chitosan added is 2 parts. The sodium alginate coating layer has good biocompatibility and degradability, and can form a protective film to maintain the activity and stability of the microorganisms; the chitosan coating layer has excellent antibacterial properties and air permeability, and can further enhance the immobilization effect of the microorganisms. The coating material is used to immobilize the microorganisms on the carrier to prevent their loss and improve the reaction efficiency.

[0021] Specifically, the temperature of the enzymatic hydrolysis reaction layer is controlled at 25-50°C, the pH value is controlled at 6.8-7.2, and the stirring speed is controlled at 100-200rpm. Temperature control: The optimal enzymatic hydrolysis temperature is 25-50°C. Within this range, the enzyme activity is the highest and the product molecular weight distribution is the most uniform. pH regulation: The optimal pH value is 6.8-7.2, and the pH value is dynamically adjusted within the range to avoid fluctuations caused by the accumulation of metabolites. Stirring speed: The stirring speed is controlled at 100-200rpm to ensure that the raw materials are fully in contact with the microorganisms without destroying the microbial structure.

[0022] Specifically, the top of the microbial decomposition tank is a raw material input port, which is connected to the raw material input layer.

[0023] Specifically, the bottom of the microbial decomposition tank is a product output port, which is connected to the product collection layer.

[0024] Specifically, the body material of the microbial decomposition pool is food-grade stainless steel.

[0025] Specifically, the surfactant in step 1) is hexadecyltrimethylammonium bromide.

[0026] Specifically, the concentration of hexadecyltrimethylammonium bromide in step 1) is 0.5-3.0 wt %.

[0027] Specifically, the hydrothermal reaction conditions in step 1) include: hydrothermal reaction at 135° C. for 18 hours.

[0028] Specifically, the calcination temperature in step 1) is 500° C. and the calcination time is 2-5 hours.

[0029] Specifically, the body material of the microbial decomposition pool is food-grade stainless steel. The beneficial effects of the present invention are as follows: 1. Design of microbial decomposition pool: The multi-layer three-dimensional structure of the microbial decomposition pool achieves uniform dispersion of raw materials, efficient enzymatic reaction and rapid collection of products through the layered design of raw material input layer, enzymatic reaction layer and product collection layer. The pool body is made of food-grade stainless steel, which is corrosion-resistant and easy to clean. The enzymatic reaction layer maintains a suitable temperature and pH environment through a stirring device and a ventilation system 7 to ensure efficient enzymatic hydrolysis.

[0030] 2. Microbial carrier: Porous ceramic carriers and polymer material carriers enhance the adhesion ability of microorganisms through chemical modification. The high specific surface area and air permeability of porous ceramic carriers are suitable for the growth of aerobic microorganisms, the flexibility and biocompatibility of polymer material carriers are suitable for the immobilization of a variety of microorganisms, and the pore structure ensures the free diffusion of substrates and products, providing a stable adhesion environment for microorganisms.

[0031] 3. Construction of the compound system: The microbial combination includes Bacillus natto and Bifidobacterium, which respectively secrete alkaline protease and assist in decomposing intermediates, generate small molecular weight peptides and produce a small amount of metabolites, such as glutathione. The metal ion catalyst adopts the Cu-Zn-Ti ternary catalytic system to enhance the activity and stability of the protease. The combination of complex enzyme catalysts such as flavor protease and neutral protease synergistically acts on protein substrates of different molecular weights to optimize the molecular weight distribution of the product.

[0032] 4. Optimization of process parameters: Through temperature control, pH regulation and optimization of stirring speed, ensure the highest enzyme activity, the most uniform product molecular weight distribution, and sufficient contact between raw materials and microorganisms without destroying the microbial structure. Dynamically adjust the pH value to avoid fluctuations caused by the accumulation of metabolites. The microbial decomposition tank adopts a multi-layer three-dimensional structure design, which is divided into a raw material input layer, an enzymatic reaction layer and a product collection layer. This design can achieve uniform dispersion of raw materials, efficient enzymatic reaction and rapid collection of products, thereby improving the overall reaction efficiency. DETAILED DESCRIPTION

[0033] The present invention is further described below in conjunction with specific embodiments, but the present invention is not limited to these embodiments. Those skilled in the art should recognize that the present invention covers all possible alternatives, improvements and equivalents within the scope of the claims.

[0034] The following examples were tested according to the following experimental scheme. All parts by weight are used in the examples. The following are the experimental conditions for all examples.

[0035] Embodiment 1;

[0036] Preparation of Cu-Zn-Ti ternary catalyst: First, take 60mL of ethanol and 40mL of deionized water and mix them evenly, add 1.3g of hexadecyltrimethylammonium bromide, and stir until completely dissolved; then add 0.01mol of copper nitrate 2.42g, 0.01mol of zinc nitrate 3.24g and 0.01mol of tetraisopropoxytitanium 2.56g in sequence, and stir evenly to obtain a mixed solution. First, add nitric acid to adjust the pH to 2.8 to dissolve the titanium precursor, and then add 1mol / L ammonia water dropwise under stirring conditions, adjust the solution pH to 9.5 until precipitation produces a suspension, transfer the suspension to the reactor, transfer the suspension to the reactor, and ultrasonically clean the porous ceramic carrier before use, dry it at 60°C, and soak the pretreated porous ceramic carrier (particle size 1-5mm) in the sol for 3 hours, and repeat the coating twice. The metal oxide precursor is hydrolyzed and polycondensed on the surface of the carrier by the sol-gel method to form a uniform gel layer. The suspension with adjusted pH is transferred to a hydrothermal reactor and reacted at 135°C for 18 hours. After the reaction, it is washed once with deionized water and ethanol, each washing amount is 50mL, filtered and placed in a 60°C drying oven to constant weight. Finally, the dried product is placed in a muffle furnace and calcined at 500°C in a nitrogen atmosphere for 2-5 hours, and the heating rate is controlled at about 5°C / min to obtain a Cu-Zn-Ti ternary catalyst supported on a porous ceramic carrier.

[0037] Preparation of sturgeon protein peptide: In this embodiment, 100 portions of fresh sturgeon meat are pretreated to obtain sturgeon surimi. Pretreatment includes washing, chopping and defatting to remove impurities and fat in the fish meat. Then, the pretreated sturgeon surimi is added to a microbial decomposition tank. The microbial decomposition tank includes a raw material input layer, an enzymatic reaction layer and a product collection layer. The raw material input layer is located at the top of the tank body and is provided with a raw material input port. The enzymatic reaction layer is located below the raw material input layer and is filled with a microbial carrier. The microbial carrier is coated with a sodium alginate coating layer with a coating thickness of 5 microns and a sodium alginate addition amount of 2 parts. A stirring device and a ventilation system are provided, and the product collection layer is located below the enzymatic reaction layer. The enzymatic reaction layer is filled with a porous ceramic carrier with a filling amount of 75 parts by weight. Natto Bacillus and Bifidobacterium are fixed on the microbial carrier, compounded in a mass ratio of 2:1, and the total addition amount is 20 parts by weight. The Cu-Zn-Ti ternary catalyst and the composite enzyme catalyst loaded on the microbial carrier were added to the enzymatic reaction layer, the total addition amount of the Cu-Zn-Ti ternary catalyst was 5 parts by weight, and the composite enzyme catalyst included flavor protease and neutral protease, and the flavor protease and neutral protease were 10 parts by weight respectively. The temperature of the enzymatic reaction layer was controlled at 33°C, the pH value was adjusted to 7.0, and the stirring speed was controlled at 150rpm. After 2 hours of reaction, the collected products were subjected to membrane separation, and a membrane with a pore size of 0.05μm was used for separation to remove impurities and obtain a sturgeon protein peptide solution. Finally, the solution was spray dried to obtain a sturgeon protein peptide powder. After testing, the protein peptide content was 95.37%, the purity was 97.64%, and the immunoactivity was 93.78%.

[0038] Embodiment 2;

[0039] In this embodiment, 100 parts of fresh sturgeon meat are pretreated to obtain sturgeon surimi. The pretreatment includes washing, chopping and defatting. Then, the pretreated sturgeon surimi is added to the microbial decomposition tank. The structure of the microbial decomposition tank is the same as that of Example 1. The enzymatic reaction layer is filled with a polymer material carrier, and the carrier is coated with a sodium alginate coating layer with a coating thickness of 3 microns. The amount of sodium alginate added is 2 parts. The filling amount is 50 parts. Bacillus natto and bifidobacterium are fixed on the microbial carrier, compounded at a mass ratio of 2:1, and the total addition amount is 10 parts. The Cu-Zn-Ti ternary catalyst and the composite enzyme catalyst loaded on the microbial carrier are added to the enzymatic reaction layer, and the total addition amount of the Cu-Zn-Ti ternary catalyst is 1 part, and the composite enzyme catalyst includes flavor protease and neutral protease, and the flavor protease and neutral protease are 10 parts each. The temperature of the enzymatic reaction layer is controlled at 32°C, the pH value is controlled at 6.8, and the stirring speed is controlled at 100rpm. After 2 hours of reaction, the collected products were separated by membrane separation, and impurities were removed using a membrane with a pore size of 0.05 μm to obtain a sturgeon protein peptide solution. Finally, the solution was spray-dried to obtain a sturgeon protein peptide powder. After testing, the protein peptide content was 94.96%, the purity was 95.45%, and the immunoactivity was 93.16%.

[0040] Embodiment 3;

[0041] In this embodiment, 100 parts of fresh sturgeon meat are pretreated to obtain sturgeon surimi. The pretreatment includes washing, chopping and defatting. Then, the pretreated sturgeon surimi is added to the microbial decomposition tank. The structure of the microbial decomposition tank is the same as that of Example 1. The enzymatic reaction layer is filled with a porous ceramic carrier, and the carrier is coated with a chitosan coating layer with a coating thickness of 1-10 microns. The amount of chitosan added is 2 parts. The filling amount is 100 parts by weight. Bacillus natto and bifidobacterium are fixed on the microbial carrier, compounded at a mass ratio of 2:1, and the total addition amount is 30 parts by weight. The Cu-Zn-Ti ternary catalyst and the composite enzyme catalyst loaded on the microbial carrier are added to the enzymatic reaction layer, and the total addition amount of the Cu-Zn-Ti ternary catalyst is 10 parts by weight. The composite enzyme catalyst includes flavor protease and neutral protease, and the flavor protease and neutral protease are 10 parts by weight each. The temperature of the enzymatic reaction layer is controlled at 35°C, the pH value is controlled at 7.2, and the stirring speed is controlled at 200rpm. After 2 hours of reaction, the collected products were subjected to membrane separation, and impurities were removed using a membrane with a pore size of 0.05 μm to obtain a sturgeon protein peptide solution. Finally, the solution was spray-dried to obtain a sturgeon protein peptide powder. After testing, the protein peptide content was 93.78%, the purity was 94.47%, and the immunoactivity was 92.34%.

[0042] Embodiment 4;

[0043] In this embodiment, 100 parts of fresh sturgeon meat are pretreated to obtain sturgeon surimi. The pretreatment includes washing, chopping and defatting. Then, the pretreated sturgeon surimi is added to the microbial decomposition tank. The structure of the microbial decomposition tank is the same as that of Example 1. The enzymatic reaction layer is filled with a polymer material carrier, and the carrier is coated with a sodium alginate coating layer with a coating thickness of 1 micron. The amount of sodium alginate added is 2 parts. The filling amount is 75 parts by weight. Bacillus natto and bifidobacterium are fixed on the microbial carrier, compounded at a mass ratio of 2:1, and the total addition amount is 20 parts by weight. The Cu-Zn-Ti ternary catalyst and the composite enzyme catalyst loaded on the microbial carrier are added to the enzymatic reaction layer. The total addition amount of the Cu-Zn-Ti ternary catalyst is 5 parts by weight. The composite enzyme catalyst includes flavor protease and neutral protease, and the flavor protease and neutral protease are 10 parts by weight each. The temperature of the enzymatic reaction layer is controlled at 25°C, the pH value is controlled at 7.0, and the stirring speed is controlled at 180rpm. After 2 hours of reaction, the collected products were subjected to membrane separation, and impurities were removed using a membrane with a pore size of 0.05 μm to obtain a sturgeon protein peptide solution. Finally, the solution was spray-dried to obtain a sturgeon protein peptide powder. After testing, the protein peptide content was 91.78%, the purity was 93.27%, and the immunoactivity was 90.93%.

[0044] Embodiment 5;

[0045] In this embodiment, 100 portions of fresh sturgeon meat are pretreated to obtain sturgeon surimi. The pretreatment includes washing, chopping and defatting. Then, the pretreated sturgeon surimi is added to the microbial decomposition tank. The structure of the microbial decomposition tank is the same as that of Example 1. The enzymatic reaction layer is filled with a porous ceramic carrier, and the carrier is coated with a chitosan coating layer with a coating thickness of 10 microns. The amount of chitosan added is 2 parts, and the filling amount is 75 parts by weight. Bacillus natto and bifidobacterium are fixed on the microbial carrier, compounded at a mass ratio of 2:1, and the total addition amount is 20 parts by weight. The Cu-Zn-Ti ternary catalyst and the composite enzyme catalyst loaded on the microbial carrier are added to the enzymatic reaction layer, and the total addition amount of the Cu-Zn-Ti ternary catalyst is 5 parts by weight. The composite enzyme catalyst includes flavor protease and neutral protease, and the flavor protease and neutral protease are 10 parts by weight each. The temperature of the enzymatic reaction layer is controlled at 50°C, the pH value is controlled at 7.2, and the stirring speed is controlled at 120rpm. After 2 hours of reaction, the collected products were separated by membrane, and impurities were removed by membrane separation with a pore size of 0.05 μm to obtain a sturgeon protein peptide solution. Finally, the solution was spray-dried to obtain sturgeon protein peptide powder. After testing, the protein peptide content was 90.86%, the purity was 92.71%, and the immunoactivity was 90.24%.

[0046] Comparative Example Comparative Example 1 (without microbial carrier and metal catalyst) In this comparative example, 100 portions of fresh sturgeon meat were pretreated to obtain sturgeon surimi. The pretreatment included cleaning, chopping and defatting. Then, the pretreated sturgeon surimi was allowed to stand at room temperature for 2 hours. The collected product was subjected to membrane separation using a membrane with a pore size of 0.05 μm to remove impurities and obtain a sturgeon protein peptide solution. Finally, the solution was spray dried to obtain sturgeon protein peptide powder. After testing, the protein peptide content was 58.76%, the purity was 78.45%, and the immunoactivity was 33.34%.

[0047] Comparative Example 2 (without Bacillus natto and Bifidobacterium) In this comparative example, 100 portions of fresh sturgeon meat were pretreated to obtain sturgeon surimi. Then, the pretreated sturgeon surimi was added to a microbial decomposition tank. The structure of the microbial decomposition tank is the same as that in Example 1. The enzymatic reaction layer is filled with a porous ceramic carrier, and the carrier is coated with a sodium alginate coating layer with a coating thickness of 5 microns. The amount of sodium alginate added is 2 parts, and the filling amount is 75 parts by weight. Bacillus natto and Bifidobacterium were not added to the enzymatic reaction layer, and other conditions were the same as in Example 1. After 2 hours of reaction, the collected product was subjected to membrane separation, and a membrane with a pore size of 0.05 μm was used for separation to remove impurities and obtain a sturgeon protein peptide solution. Finally, the solution was spray dried to obtain sturgeon protein peptide powder. After testing, the protein peptide content was 67.89%, the purity was 82.34%, and the immunoactivity was 35.47%.

[0048] Comparative Example 3 (without metal ion catalyst) In this comparative example, 100 parts of fresh sturgeon meat were pretreated to obtain sturgeon surimi. Then, the pretreated sturgeon surimi was added to the microbial decomposition tank. The structure of the microbial decomposition tank is the same as that in Example 1. The enzymatic reaction layer is filled with a porous ceramic carrier, and the microbial carrier is coated with a sodium alginate coating layer with a coating thickness of 5 microns. The amount of sodium alginate added is 2 parts, and the filling amount is 75 parts by weight. Bacillus natto and Bifidobacterium are fixed on the microbial carrier, compounded in a mass ratio of 2:1, and the total addition amount is 20 parts by weight. No metal ion catalyst is added to the enzymatic reaction layer, and the composite enzyme catalyst includes flavor protease and neutral protease, with 10 parts by weight of flavor protease and neutral protease respectively. Other conditions are the same as in Example 1. After 2 hours of reaction, the collected product is subjected to membrane separation, and a membrane with a pore size of 0.05 μm is used for separation to remove impurities and obtain a sturgeon protein peptide solution. Finally, the solution is spray-dried to obtain a sturgeon protein peptide powder. After testing, the protein peptide content was 73.49%, the purity was 86.78%, and the immune activity was 30.12%.

[0049] Based on Example 1, the preparation process of the catalyst was changed, and the remaining conditions and steps were the same as in Example 1, and the catalytic effects of different metal combinations were measured.

[0050] Catalyst composition Enzyme activity retention rate (%) Product yield (%) Thermal stability (80℃, 1h) Cu-Zn-Ti 98.7 93.15 95.2 Cu-Zn 76.4 81.23 68.7 Cu-Ti 83.2 85.47 77.9 Zn-Ti 71.5 78.16 63.4 The above protein peptide content test method Test principle: High performance liquid chromatography (HPLC) is used to separate the protein peptides in the sample through a chromatographic column, and the content of the protein peptides is calculated based on the peak area.

[0051] The test steps include the following: Sample preparation: Accurately weigh a certain amount of sturgeon protein peptide powder, dissolve it in an appropriate amount of phosphate buffered saline (PBS) to prepare a sample solution of a certain concentration.

[0052] Chromatographic conditions: C18 column was used, the mobile phase was a mixture of 0.1% trifluoroacetic acid (TFA) aqueous solution and acetonitrile, gradient elution, detection wavelength was 220 nm, flow rate was 1.0 mL / min, column temperature was 30 °C.

[0053] Content calculation: The sample solution was measured according to the above chromatographic conditions, and the content of protein peptide in the sample was calculated based on the peak area and the standard curve.

[0054] The test methods for protein peptide purity include the following: Test principle: High performance liquid chromatography (HPLC) is also used to separate protein peptides and other impurities in the sample through a chromatographic column, and the purity of the protein peptide is calculated based on the peak area.

[0055] Test steps: Sample preparation: Same as the protein peptide content test, accurately weigh a certain amount of sturgeon protein peptide powder, dissolve it in an appropriate amount of phosphate buffered saline (PBS) to prepare a sample solution of a certain concentration.

[0056] Chromatographic conditions: Same as the protein peptide content test, using a C18 column, the mobile phase is a mixture of 0.1% trifluoroacetic acid (TFA) aqueous solution and acetonitrile, gradient elution. The detection wavelength is 220nm, the flow rate is 1.0mL / min, and the column temperature is 30℃.

[0057] Peak area integration: In the chromatogram, the protein peptide peaks and other impurity peaks are integrated to calculate their respective peak areas.

[0058] Purity calculation: protein peptide purity (%) = (protein peptide peak area / total peak area) × 100%.

[0059] Immunocompetence test method Test principle: In vitro cell experiments are used to evaluate the immune activity of samples by detecting their effects on the proliferation of immune cells.

[0060] Test steps: Cell culture: Human peripheral blood mononuclear cells (PBMCs) were selected and cultured in RPMI-1640 medium containing 10% fetal bovine serum (FBS) at 37°C and 5% CO2.

[0061] Sample processing: Sturgeon protein peptide powder was prepared into sample solutions of different concentrations and added into the cell culture system respectively to set up blank control group and positive control group.

[0062] Cell proliferation detection: After 72 hours of culture, CCK-8 reagent was added, and the culture was continued for 2 hours, and the absorbance value (OD value) of each group was measured.

[0063] Calculation of immunoactivity: immunoactivity (%) = [(OD value of sample group - OD value of blank control group) / (OD value of positive control group - OD value of blank control group)] × 100%.

[0064] In addition to the above embodiments, the present invention may also have other implementation modes. Any technical solution formed by equivalent replacement or equivalent transformation falls within the protection scope required by the present invention.

Claims

1. A method for preparing a sturgeon protein peptide for enhancing immunity, characterized in that: The following steps are involved: (1) Preparation of Cu-Zn-Ti ternary catalyst carrier: A) will contain Cu 2+ 、Zn 2+ 、Ti 4+ The soluble salt is dissolved in an ethanol-water mixed solution containing a surfactant, and stirred to form a uniform mixed solution; B) Add ammonia water dropwise under stirring to generate a precipitate and form a suspension; C) transferring the suspension to a reactor for hydrothermal reaction to obtain a metal oxide precursor sol; D) soaking or coating a carrier without any active ingredient in the sol, and causing the metal oxide precursor to undergo hydrolysis and polycondensation on the surface of the carrier by a sol-gel method to form a uniform gel layer; E) After drying and calcination, a carrier loaded with a Cu-Zn-Ti ternary catalyst is obtained; (2) Preparation of microbial carriers loaded with complex enzymes: a) immersing the microbial carrier in a compound enzyme solution in which flavor protease and neutral protease are compounded in a mass ratio of 1:1; b) coating the surface of the carrier to form a protective layer and fix the complex enzyme; (3) Enzymatic hydrolysis reaction and product separation: Sturgeon surimi is added into the raw material input layer of the microbial decomposition tank, and enters the enzymatic reaction layer through the input port, and then the carrier loaded with the Cu-Zn-Ti catalyst and the microbial carrier loaded with the composite enzyme are loaded into the enzymatic reaction layer, the temperature is 25-50°C, the pH is 6.8-7.2, and the stirring speed is 100-200rpm. The reaction product is filtered through the product collection layer to obtain a clear liquid, and the sturgeon protein peptide is obtained after drying.

2. The method for preparing a sturgeon protein peptide for enhancing immunity according to claim 1, characterized in that: The microbial decomposition tank in step (3) comprises a raw material input layer, an enzymatic hydrolysis reaction layer and a product collection layer. The raw material input layer is located at the top of the tank body and is provided with a raw material input port. The enzymatic hydrolysis reaction layer is located below the raw material input layer and is loaded with a Cu-Zn-Ti ternary catalyst carrier and a microbial carrier. A stirring device and a ventilation system are provided. The product collection layer is located below the enzymatic hydrolysis reaction layer.

3. The method for preparing a sturgeon protein peptide for enhancing immunity according to claim 1, characterized in that: The enzymatic reaction layer is filled with a Cu-Zn-Ti ternary catalyst carrier and a microbial carrier. The Cu-Zn-Ti ternary catalyst carrier is a porous ceramic carrier with a filling weight of 1-10 parts, and the microbial carrier loaded with the complex enzyme is a porous ceramic carrier or a polymer material carrier with a filling weight of 50-100 parts.

4. The method for preparing a sturgeon protein peptide for enhancing immunity according to claim 2, characterized in that: The composite microorganism is fixed on a microorganism carrier, and the composite microorganism is a mixture of Bacillus natto and Bifidobacterium in a mass ratio of 2:

1.

5. The method for preparing a sturgeon protein peptide for enhancing immunity according to claim 2, characterized in that: The microbial carrier is coated with a sodium alginate coating layer or a chitosan coating layer, the coating thickness is 1-10 microns, and the added amount of sodium alginate or chitosan is 2 parts by weight.

6. The method for preparing a sturgeon protein peptide for enhancing immunity according to claim 1, characterized in that: The surfactant in step (1) is hexadecyltrimethylammonium bromide with a concentration of 0.5-3.0 wt %.

7. The method for preparing a sturgeon protein peptide for enhancing immunity according to claim 1, characterized in that: The hydrothermal reaction conditions of step (1) include: hydrothermal reaction at 135° C. for 18 hours.

8. The method for preparing a sturgeon protein peptide for enhancing immunity according to claim 1, characterized in that: The calcination temperature in step (1) is 500° C. and the calcination time is 2-5 hours.

9. The method for preparing a sturgeon protein peptide for enhancing immunity according to claim 1, characterized in that: In the step (1), the Cu-Zn-Ti ternary catalyst is fixed on the outermost layer of the microbial carrier by a sol-gel method.