Preparation method of slow-release acid-resistant nattokinase

Abstract

The invention relates to the technical field of biological medicines, in particular to a preparation method of slow-release acid-resistant nattokinase, which adopts a preparation process that nattokinase is solidified and embedded in a core material with a three-dimensional network structure, and a surface cross-linking agent and a novel slow-release agent are combined to form an outer layer structure. The gastric acid resistance and the slow-release effect of the nattokinase are effectively improved. The preparation process is simple, mild in condition and suitable for industrial production.

Description

Technical Field

[0001] The present invention relates to the field of biomedicine technology, and in particular to a method for preparing a sustained-release and acid-resistant nattokinase, which belongs to an innovative technology in the field of biomedicine and aims to improve the stability, bioavailability and sustained-release effect of nattokinase by introducing novel organic compounds and innovative preparation processes, thereby providing a more effective solution for the prevention and treatment of cardiovascular diseases. Background Art

[0002] Cardiovascular disease is one of the major diseases that seriously threaten human health in today's society, and thrombosis is an important pathogenesis of cardiovascular disease. As a serine protease with strong thrombolytic effect, nattokinase has the advantages of high safety, long half-life, and oral administration. It is a nutrient with great potential for thrombolytic effect. However, nattokinase is unstable to gastric acid and is not easily absorbed after oral administration, resulting in low oral bioavailability, which limits its application in clinical practice.

[0003] At present, commonly used thrombolytic preparations have shortcomings such as short half-life and certain side effects. The development of a new generation of thrombolytic functional foods or drugs has broad market prospects. However, the existing nattokinase preparation process has failed to effectively solve the problems of its easy inactivation under acidic conditions, low bioavailability and lack of intestinal sustained-release function. Therefore, it is urgent to develop a new preparation method to improve the stability and sustained-release effect of nattokinase to meet clinical needs. Summary of the invention

[0004] The present invention provides a method for preparing acid-resistant nattokinase with sustained release, which effectively solves the problems in the prior art by introducing new organic compounds and innovative preparation processes. The method comprises the following steps:

[0005] Preparation of a core carrier: chemically modifying commercial konjac flour to obtain a modified konjac glucomannan with a molecular weight of 8000-12000Da, and mixing the modified konjac glucomannan with deacetylated chitosan in a mass ratio of (1-3):1 to form a core carrier with a network structure, wherein the modified konjac glucomannan is prepared by reacting konjac flour with an acid or a base, followed by acid-base neutralization, washing with salt, and vacuum drying;

[0006] Embedding nattokinase: dissolving nattokinase in phosphate buffer (pH 7.0-7.8) to prepare a nattokinase solution with a concentration of 5-20 mg / mL, slowly adding the nattokinase solution to the core carrier solution of the three-dimensional network structure under the conditions of temperature control at 20-30°C and stirring speed at 150-300 rpm, so that the nattokinase is fully embedded in the network structure gap of the core carrier;

[0007] Preparation of micron-sized particles: The system containing nattokinase is made into micron-sized particles by spray drying or freeze drying. When spray drying, the inlet temperature is 100-150°C and the outlet temperature is 50-80°C. When freeze drying, the freezing temperature is -45°C to -15°C.

[0008] Outer coating: one or more combinations of hydroxypropyl methylcellulose phthalate (HPMCP), polyvinyl phthalate (PVAP), and cellulose acetate trimellitate (CAT) are used as coating materials, and the coating materials are dissolved in an organic solvent or an aqueous dispersion medium to form a coating solution, wherein the organic solvent is ethanol or acetone, and an appropriate amount of a plasticizer such as triethyl citrate is added to the aqueous dispersion medium, and the micron-sized particles are coated with an outer layer to increase the weight of the microspheres by 3%-10%. The coating process is carried out in a fluidized bed or a coating pan at a temperature of 30-50°C. When carried out in a fluidized bed, the spray speed and air flow rate are controlled, and when carried out in a coating pan, the rotation speed and the amount of coating solution sprayed are controlled.

[0009] Furthermore, it also includes adding organic compounds to enhance the stability of the core carrier: 2-methyl-4-(1-imidazolyl)butyric acid (hereinafter referred to as compound A) and 3-dodecylthiophene-2-sulfonic acid (hereinafter referred to as compound B) are added to the core carrier, and the addition amount of compound A and compound B is 1%-10% of the total mass of the core carrier and the compound. Before adding, compound A and compound B are respectively dissolved in an organic solvent, and the organic solvent is ethanol or acetone, and then stirred or ultrasonically treated at a temperature of 35-50°C to fully mix compound A and compound B with the core carrier to form a more stable three-dimensional network structure, wherein the imidazole ring of compound A interacts with the amino group of deacetylated chitosan, the thiophene ring of compound B interacts with the aromatic amino acid residue of nattokinase, and hydrogen bonds are formed between the carboxyl group of compound A and the sulfonic acid group of compound B and the hydroxyl group of polysaccharide;

[0010] Furthermore, the frequency of the ultrasonic treatment is 20-50kHz, the treatment time is 20-60min, the stirring speed is 200-500rpm, and the stirring time is 1-3h.

[0011] Furthermore, during the spray drying process, the atomizer speed is 10000-20000 rpm, and the air flow rate is 50-100 m 3 / h. During the freeze-drying process, the vacuum degree is 0.01-0.1mbar and the sublimation temperature is -20℃ to 0℃.

[0012] Furthermore, the HPMCP, PVAP and CAT are combined as coating materials in a mass ratio of (1-3):(0.5-2):(0.5-2). When an aqueous dispersion medium is used, the amount of plasticizer added is 1%-5% of the mass of the coating material.

[0013] Furthermore, the present invention also provides a nattokinase preparation having sustained-release and acid-resistant properties prepared by the method.

[0014] Furthermore, the present invention also provides an oral thrombolytic drug, comprising the nattokinase preparation and pharmaceutically acceptable excipients, wherein the excipients include one or more of microcrystalline cellulose, sorbitol, magnesium stearate, polyvinyl pyrrolidone, xanthan gum, sodium alginate, and mannitol, and the content of the nattokinase preparation in the oral thrombolytic drug is 10%-50%.

[0015] Furthermore, the present invention also provides a functional food for cardiovascular health care, comprising a nattokinase preparation and food-acceptable auxiliary materials, wherein the auxiliary materials include one or more of sorbitol, collagen peptide, vitamin E, xylitol, gelatin, starch, and sucrose, and the content of the nattokinase preparation in the functional food is 5%-30%.

[0016] Beneficial technical effects of the invention:

[0017] 1. Innovation in the design of new organic compounds:

[0018] Three new organic compounds, polyethylene glycol-modified chitosan, acrylic acid-modified konjac glucomannan, and hydroxyethyl methacrylate-modified glucan, were developed as carrier materials and sustained-release agents for nattokinase. These innovative material designs have not been reported at home and abroad, and provide a new solution for the stability and sustained release of nattokinase. In the core carrier system, the imidazole ring of organic compound A plays an important role. It can interact with certain amino acid residues of nattokinase (such as basic amino acids such as arginine and lysine) through electrostatic effects or hydrogen bonds. This interaction helps to stabilize the structure of nattokinase and prevent it from undergoing conformational changes in complex environments. The carboxyl group can form an amide bond with the amino group of deacetylated chitosan, further strengthening the cross-linking degree of the carrier and making the entire carrier structure more stable. At the same time, the carboxyl group can also form hydrogen bonds with the hydroxyl group of modified konjac glucomannan to enhance the stability of the carrier. The presence of methyl groups can adjust the spatial structure of the carrier, making the distribution of nattokinase in the carrier more reasonable, which is conducive to improving its stability and sustained-release performance. This reasonable distribution can reduce the interaction between nattokinase molecules, avoid inactivation due to aggregation during storage or transportation, the thiophene ring of organic compound B can interact with the aromatic amino acid residues (such as phenylalanine, tyrosine, etc.) of nattokinase through π-π stacking, and this interaction helps to stabilize the structure of nattokinase and protect its active site. Sulfonic acid group can form hydrogen bonds with the hydroxyl groups of modified konjac glucomannan and deacetylated chitosan, strengthen the binding force between carrier and nattokinase. Meanwhile, the strong acidity of sulfonic acid group enables it to resist the erosion of gastric acid in gastric acid environment, plays the role of protecting carrier and nattokinase. The hydrophobicity of long-chain alkyl can form local hydrophobic regions inside the carrier, is conducive to the stable existence of nattokinase. In the intestinal environment, the existence of this hydrophobic region can affect the release rate of nattokinase, realizes the slow-release function. By adjusting the size and properties of the hydrophobic area, the release rate of nattokinase can be controlled so that it can continue to play a thrombolytic role in the intestine. By precisely controlling the chemical reaction steps, temperature, time, and reactant ratio parameters, the structure and properties of these new organic compounds are effectively regulated, ensuring their excellent performance in the preparation of nattokinase.

[0019] 2. Improve the stability in acidic environment:

[0020] 1. Polyethylene glycol-modified chitosan and acrylic acid-modified konjac glucomannan use specific functional groups to significantly improve their stability in acidic environments. The hydrophilicity and steric hindrance effect of polyethylene glycol reduce the protonation degree of chitosan in acidic environments, and the carboxyl group of acrylic acid enhances the stability of konjac glucomannan in acidic environments, effectively protecting the activity of nattokinase in gastric acid.

[0021] 2. Hydroxyethyl methacrylate modified dextran serves as a sustained-release layer, synergistically working with the core carrier material to further enhance nattokinase's ability to resist damage in an acidic environment, ensuring that it remains stable before entering the intestine.

[0022] 3. Enhanced loading and slow-release capabilities:

[0023] 1. The three new organic compounds all have unique structures and functional groups, which enhance the adsorption and loading capacity of nattokinase. The electrostatic interaction between the amino group of chitosan and nattokinase, the esterification reaction between konjac glucomannan and acrylic acid, and the grafting reaction between glucan and hydroxyethyl methacrylate all provide stable binding sites for nattokinase.

[0024] 2. The design of the sustained-release agent achieves the slow release of nattokinase in the intestine. The gradual hydrolysis of the ester group in the hydroxyethyl methacrylate-modified dextran in the intestinal environment and the slow degradation of the cross-linking agent on the surface of the nattokinase microspheres jointly control the release rate of nattokinase, prolong its action time in the body, and improve its bioavailability.

[0025] 4. Optimize the innovation of preparation process:

[0026] 1. The preparation process of solidifying nattokinase and embedding it in the three-dimensional network structure core material, and combining the surface cross-linking agent and the new sustained-release agent to form the outer layer structure, effectively improves the gastric acid resistance and sustained-release effect of nattokinase. This preparation process is simple, mild, and suitable for industrial production.

[0027] 2. By optimizing the preparation process parameters, such as granulation size, stirring temperature and time, and sustained-release agent coating thickness, the performance of nattokinase microspheres was precisely controlled, improving the quality and stability of the product.

[0028] 5. Innovative applications of multidisciplinary research:

[0029] 1. The present invention integrates knowledge and technology of multiple disciplines such as chemical synthesis, material science and biomedicine. The design and synthesis of organic compounds involve chemical principles and reaction engineering, the selection and application of carrier materials and sustained-release agents reflect the innovation of material science, and the stability and sustained-release effect of nattokinase provide new ideas and methods for the development of the biomedicine field.

[0030] 2. This innovative application of multidisciplinary research provides a comprehensive solution to complex biomedical problems and has broad application prospects and promotion value. DETAILED DESCRIPTION

[0031] Embodiment 1:

[0032] (I) Core carrier part

[0033] 1. Modified Konjac Glucomannan and Deacetylated Chitosan

[0034] 1. Structure and properties

[0035] 1. Modified konjac glucomannan is obtained by treating konjac flour with a chemical modification method, and its molecular weight is in the range of 8000-12000Da. This modification process gives it a unique molecular structure, which is rich in hydroxyl groups. These hydroxyl groups have a specific spatial arrangement within and between molecules, providing active sites for subsequent chemical reactions and physical interactions. Deacetylated chitosan is the product of partial deacetylation of chitosan, and there are amino and hydroxyl groups on its molecular chain. The amino group is alkaline and can react with acidic substances, which provides a chemical basis for its potential application in acidic environments; the hydroxyl group can participate in the formation of hydrogen bonds and enhance the interaction between molecules.

[0036] 2. Group action mechanism

[0037] 1. The two are cross-linked to form a three-dimensional network structure, and this process involves multiple interactions. Hydroxyl groups interact with each other through hydrogen bonds to build a stable network connection. This hydrogen bond network plays a supporting and stabilizing role in the entire structure. Hydrogen bonds are also formed between amino groups and hydroxyl groups, further strengthening the stability of the network. The three-dimensional network structure formed by this cross-linking provides an ideal physical support and protective environment for nattokinase. Nattokinase molecules can be embedded in the gaps of the network structure. The network structure effectively limits the freedom of movement of nattokinase molecules, thereby reducing the chance of nattokinase contacting gastric acid in the gastric acid environment, and to a certain extent protecting nattokinase from gastric acid damage. In addition, this three-dimensional network structure has unique advantages in the intestinal environment, which can achieve the slow release of nattokinase, thereby exerting a long-lasting thrombolytic effect.

[0038] 2. Newly added organic compound A (2-methyl-4-(1-imidazolyl)butyric acid)

[0039] 1. Structure and properties

[0040] 1. Its chemical formula is C 10 H 16 O4N2, the molecular structure contains an imidazole ring, a carboxyl group and two methyl groups. The imidazole ring is a heterocyclic structure with aromaticity and alkalinity, which gives the molecule certain chemical activity and the ability to interact with other molecules. The carboxyl group is acidic, which enables it to participate in a variety of chemical reactions, such as reacting with alkaline groups, and also to form hydrogen bonds with other molecules. The methyl groups affect the spatial structure and solubility of the molecule, and their presence changes the overall physical and chemical properties of the molecule.

[0041] 2. Group action mechanism

[0042] 1. In the core carrier system, the imidazole ring of organic compound A plays an important role. It can interact with certain amino acid residues of nattokinase (such as basic amino acids such as arginine and lysine) through electrostatic interaction or hydrogen bonding. This interaction helps to stabilize the structure of nattokinase and prevent it from undergoing conformational changes in complex environments. The carboxyl group can form an amide bond with the amino group of deacetylated chitosan, further strengthening the cross-linking degree of the carrier and making the entire carrier structure more stable. At the same time, the carboxyl group can also form a hydrogen bond with the hydroxyl group of modified konjac glucomannan to enhance the stability of the carrier. The presence of methyl can adjust the spatial structure of the carrier, making the distribution of nattokinase in the carrier more reasonable, which is conducive to improving its stability and sustained-release performance. This reasonable distribution can reduce the interaction between nattokinase molecules and avoid inactivation due to aggregation during storage or transportation.

[0043] 3. Newly added organic compound B (3-dodecylthiophene-2-sulfonic acid)

[0044] 1. Structure and properties

[0045] 1. Chemical formula is C 12 H 20 O3S, its structure contains a thiophene ring, a sulfonic acid group and a long-chain alkyl group. The thiophene ring has aromaticity and unique electron cloud characteristics, and can participate in interactions such as π-π stacking. The sulfonic acid group has strong acidity and good water solubility, which makes it have special chemical stability in acidic environments. The long-chain alkyl group is hydrophobic, which has an important influence on the behavior of molecules in different environments.

[0046] 2. Group action mechanism

[0047] 1. In the core carrier, the thiophene ring of the organic compound B can interact with the aromatic amino acid residues (such as phenylalanine, tyrosine, etc.) of nattokinase through π-π stacking, and this interaction helps to stabilize the structure of nattokinase and protect its active site. The sulfonic acid group can form hydrogen bonds with the hydroxyl groups of modified konjac glucomannan and deacetylated chitosan, thereby enhancing the binding force between the carrier and nattokinase. At the same time, the strong acidity of the sulfonic acid group enables it to resist the erosion of gastric acid in the gastric acid environment, thus playing a role in protecting the carrier and nattokinase. The hydrophobicity of the long-chain alkyl group can form a local hydrophobic region inside the carrier, which is conducive to the stable existence of nattokinase. In the intestinal environment, the presence of this hydrophobic region can affect the release rate of nattokinase and realize the sustained-release function. By adjusting the size and properties of the hydrophobic region, the release rate of nattokinase can be controlled, so that it can continue to play a thrombolytic role in the intestine.

[0048] (ii) Outer coating

[0049] 1. Hydroxypropyl methylcellulose phthalate (HPMCP), polyvinyl alcohol phthalate (PVAP), cellulose acetate trimellitate (CAT), etc.

[0050] Structure and properties

[0051] 1. These compounds all contain functional groups such as ester groups, which are the chemical basis for their specific properties. Ester groups are relatively stable in acidic environments and are not easily hydrolyzed by gastric acid, giving the coating material tolerance in gastric acid. The hydroxypropyl and methyl groups in HPMCP can affect its solubility and flexibility. The presence of hydroxypropyl groups increases the hydrophilicity of the molecule, allowing the material to interact with water molecules to a certain extent, while the methyl groups affect the spatial structure and flexibility of the molecule. The polyvinyl alcohol part in PVAP gives it a certain hydrophilicity and viscosity, which helps the coating adhere to the surface of the microspheres and form a continuous film layer. The benzene trisaccharide structure of CAT gives it good film-forming properties and acid resistance, and the benzene ring structure increases the rigidity of the molecule, which is conducive to the formation of a stable film structure.

[0052] Group action mechanism

[0053] 1. Ester groups are relatively stable in the gastric acid environment and are not easily hydrolyzed by gastric acid. When used as coating materials, they form a continuous film layer on the surface of the microsphere particles. In gastric acid, this film can prevent gastric acid molecules from contacting the internal nattokinase, acting as a physical barrier. At the same time, the mutual cross-linking between them (possibly through the interaction between ester groups or the synergistic effect with other functional groups) enhances the strength and stability of the membrane, ensuring that it will not easily break in the gastric acid environment, thereby protecting nattokinase. In the intestinal environment, due to the high pH value of the intestine, the properties of these coating materials change, the permeability of the membrane increases, and nattokinase can be slowly released. This pH-responsive release mechanism is achieved by the change in the ionization state or solubility of the functional groups in the coating material under different pH conditions. For example, in an acidic environment, some functional groups in the coating material may remain protonated, making the membrane structure tight; while in the alkaline environment of the intestine, the functional groups are deprotonated, resulting in increased membrane permeability and the release of nattokinase.

[0054] (III) Preparation method

[0055] (I) Preparation of nattokinase microspheres assisted by new organic compounds

[0056] 1. Preparation process

[0057] Step 1: Preparation of a carrier containing a new organic compound

[0058] 1. First, a mixture of modified konjac glucomannan and deacetylated chitosan is prepared according to an established method. During the mixing process, organic compound A (2-methyl-4-(1-imidazolyl)butyric acid) and organic compound B (3-dodecylthiophene-2-sulfonic acid) are respectively dissolved in an appropriate amount of organic solvent (such as ethanol or acetone), and then added to the mixture. Stir evenly to make the organic compounds evenly distributed in the mixture. The interaction between the organic compound and the polysaccharide and chitosan is promoted by heating (such as 40-50°C) or ultrasonic treatment. During the heating process, the thermal motion of the molecules is intensified, which is conducive to the chemical bonding and hydrogen bond formation between the organic compound and the carrier material. Ultrasonic treatment produces a local high temperature and high pressure environment through the cavitation effect, which further promotes the interaction between molecules. In this process, the interactions between the imidazole ring of organic compound A and the amino group of chitosan, and between the thiophene ring of organic compound B and the aromatic amino acid residues of nattokinase are gradually formed. At the same time, the hydrogen bonding between the carboxyl group of organic compound A, the sulfonic acid group of organic compound B and the hydroxyl group of polysaccharide is also strengthened, thus forming a more stable three-dimensional network structure.

[0059] Step 2: Embedding of Nattokinase

[0060] 1. Slowly add the nattokinase solution (nattokinase is dissolved in a suitable buffer to maintain its activity) to the above-mentioned carrier solution containing the new organic compound. Stir evenly so that the nattokinase molecules fully enter the gaps in the network structure. In this process, the embedding process can be optimized by controlling the temperature (such as 25-30°C) and the stirring speed (such as 200-300rpm). The appropriate temperature and stirring speed help the nattokinase molecules to be evenly dispersed and embedded in the network structure of the carrier without affecting its activity. In this process, the interaction between nattokinase and the organic compound further stabilizes its position in the carrier. For example, the electrostatic interaction between the basic amino acids of nattokinase and the imidazole ring of organic compound A, and the π-π stacking effect between the aromatic amino acids of nattokinase and the thiophene ring of organic compound B, all help to fix the position of nattokinase in the carrier and prevent it from detaching from the carrier during subsequent processing.

[0061] Step 3: Preliminary forming and drying

[0062] 1. The mixed system containing nattokinase and the new organic compound is made into micron-sized particles by spray drying or freeze drying to obtain preliminary nattokinase microspheres. During spray drying, the inlet temperature (such as 120-150°C) and the outlet temperature (such as 60-80°C) are controlled to ensure that the activity of nattokinase is not affected and the particles are well formed. During the spray drying process, the solution is atomized into small droplets, and the hot air quickly evaporates the solvent in the droplets to form solid particles of the solute. The appropriate inlet and outlet temperatures can avoid the inactivation of nattokinase due to high temperature, while ensuring that the particles have good morphology and particle size distribution. During freeze drying, the freezing temperature is controlled at -40°C to -20°C, and then sublimation drying is carried out under vacuum conditions. During the freezing process, the water in the solution forms ice crystals, which directly sublimate into water vapor under vacuum conditions to obtain dry microspheres. This method has little effect on the activity of nattokinase and can better maintain its activity. During the drying process, the three-dimensional network structure of the carrier is further fixed, and nattokinase is stably wrapped in it, providing a stable core for the subsequent coating process.

[0063] Step 4: Outer coating

[0064] 1. Place the preliminarily formed nattokinase microspheres in a fluidized bed, and prepare HPMCP, PVAP, CAT, etc. into a coating solution (dissolved in an organic solvent such as ethanol or acetone) in a certain ratio (such as 2:1:1). Spray the coating solution on the surface of the microspheres by spraying, and volatilize the organic solvent under the action of hot air (such as 40-50°C), and the coating material forms a uniform film layer on the surface of the microspheres. Control the spray speed of the coating solution and the air flow rate of the fluidized bed to increase the weight of the microspheres by 5-8%, and complete the outer coating process. In this process, the sprayed coating solution evaporates the organic solvent rapidly under the action of hot air, and the coating material is deposited on the surface of the microspheres to form a tight protective film. Appropriate spray speed and air flow can ensure the uniformity of the coating and the consistency of thickness, avoid the situation where the coating is too thick or too thin, thereby ensuring the stability of the nattokinase microspheres in the gastric acid environment and the sustained release performance in the intestine.

[0065] 2. Advantages

[0066] The introduction of new organic compounds A and B enhances the stability of the inner core carrier and better protects nattokinase. The new organic compounds work synergistically with the various interactions between nattokinase and the carrier to improve the tolerance of nattokinase in complex environments. At the same time, the outer coating further improves the stability of nattokinase in gastric acid, and this preparation method is relatively simple and easy to control, and can be industrialized. In the intestine, nattokinase can be slowly released to exert a long-acting thrombolytic effect. This method combines the advantages of the inner core carrier and the outer coating, guarantees the stability and sustained-release performance of nattokinase from multiple levels, and provides reliable technical support for its application in the field of oral thrombolysis.

[0067] (II) Preparation of coating after blending of new organic compound and nattokinase

[0068] 1. Preparation process

[0069] Step 1: Blending of new organic compound and nattokinase

[0070] 1. Dissolve organic compound A (2-methyl-4-(1-imidazolyl)butyric acid) and organic compound B (3-dodecylthiophene-2-sulfonic acid) in a buffer solution containing a protective agent (such as mannitol). Then mix the two solutions with the nattokinase solution in a certain proportion (such as the molar ratio of organic compounds A, B and nattokinase is 1:1:2), and stir evenly at a low temperature (such as 4-10°C) to fully combine the organic compounds with nattokinase. The protective agent mannitol can prevent nattokinase from being inactivated due to excessive local concentration or environmental changes during subsequent treatment. During the blending process, a stable interaction is formed between the imidazole ring of organic compound A and the basic amino acid residues of nattokinase, and between the thiophene ring of organic compound B and the aromatic amino acid residues of nattokinase. At the same time, there is also a certain interaction between the carboxyl group of organic compound A, the sulfonic acid group of organic compound B and the water molecules or other components in the buffer solution, making the system more stable. Low-temperature stirring helps to reduce the thermal motion of molecules, avoid the inactivation of nattokinase due to vigorous stirring or high temperature, and is conducive to the formation of specific interactions between organic compounds and nattokinase.

[0071] Step 2: Carrier preparation and embedding of nattokinase-new organic compound complex

[0072] 1. Prepare the three-dimensional network structure carrier of modified konjac glucomannan and deacetylated chitosan (method as before). Slowly add the nattokinase-new organic compound complex solution into the carrier solution, and under gentle stirring (such as 100-200rpm) and room temperature, make the complex uniformly dispersed and embedded in the network structure of the carrier. In this process, the interaction between the complex and the carrier is further strengthened, and the chemical bonding and hydrogen bonding between the new organic compound and the carrier make the fixation of nattokinase in the carrier more firmly. Gentle stirring conditions can prevent the destruction of nattokinase and the carrier structure, ensure that the complex can be uniformly dispersed in the carrier, and form a stable combination between nattokinase and the carrier, and improve the stability of nattokinase in the carrier.

[0073] Step 3: Coating and molding

[0074] 1. The carrier particles containing the nattokinase-new organic compound complex are made into microspheres of suitable size (diameter of 0.15-0.4um) by dripping or extrusion, and then the microspheres are placed in a coating pan and coated with mixed coating materials such as HPMCP, PVAP, CAT (dissolved in an aqueous dispersion medium, and an appropriate amount of plasticizer such as triethyl citrate is added). By rotating the coating pan (rotation speed such as 20-30rpm) and controlling the spraying amount of the coating liquid, the coating is evenly covered on the surface of the microspheres to form nattokinase microspheres with sustained release and acid-resistant functions. During the coating process, the coating material in the aqueous dispersion medium is gradually deposited on the surface of the microspheres to form a protective film. The addition of plasticizers makes the coating have better flexibility and stability, further enhancing the tolerance of the nattokinase microspheres in the gastric acid environment and the sustained release performance in the intestine. This method uses an aqueous dispersion medium for coating, reduces the use of organic solvents, is more environmentally friendly, and can better control the quality and uniformity of the coating, improve the quality stability of the product, and is conducive to industrial production.

[0075] 2. Advantages

[0076] This method first allows the new organic compound to fully combine with nattokinase to improve the stability of nattokinase in a targeted manner. The specific interaction between the new organic compound and nattokinase can better protect the active site of nattokinase. Then, the dual protection of the carrier and the coating is used to further enhance the tolerance of nattokinase in the gastric acid environment and the sustained release performance in the intestine. The use of aqueous dispersion medium for coating during the preparation process reduces the use of organic solvents, is more environmentally friendly, and has stable product quality, which can be used to produce oral thrombolytic preparations. This method comprehensively improves the performance of nattokinase microspheres through sophisticated step design and material selection, from the protection of nattokinase, the construction of the carrier to the application of the coating, and provides a promising preparation route for the development of oral thrombolytic drugs.

[0077] Example 2

[0078] 1. Material Preparation

[0079] 1. Raw materials: commercial konjac flour, chitosan, nattokinase fermentation extract, 2-methyl-4-(1-imidazolyl)butyric acid (organic compound A), 3-dodecylthiophene-2-sulfonic acid (organic compound B), hydroxypropyl methylcellulose phthalate (HPMCP), polyvinyl phthalate (PVAP), cellulose acetate trimellitate (CAT), anhydrous ethanol, acetone, mannitol, etc.

[0080] 2. Instruments and equipment: colloid mill, spray dryer, fluidized bed coater, rotary evaporator, enzyme activity tester, pH meter, constant temperature shaker, etc.

[0081] 2. Preparation process

[0082] 1. Preparation of modified konjac glucomannan: 20 parts of konjac flour and 0.8 mol / L HCl were stirred at room temperature for 13 hours, then acid-base neutralization was performed, the salt was washed with water, and vacuum drying was performed to obtain modified konjac glucomannan with a molecular weight of 8000-12000Da.

[0083] 2. Preparation of a carrier containing a new organic compound: Modified konjac glucomannan and deacetylated chitosan were mixed in a mass ratio of 2:1, and appropriate amounts of ethanol-soluble organic compound A and acetone-soluble organic compound B were added (the amounts of organic compounds A and B added were 5% of the total mass, respectively). The mixture was ultrasonically treated at 45°C for 30 min, and then stirred at 40°C for 2 h to form a carrier solution containing a new organic compound.

[0084] 3. Embedding of Nattokinase: Dissolve the nattokinase fermentation extract in phosphate buffer (pH 7.4) to prepare a nattokinase solution with a concentration of 10 mg / mL. Slowly add the nattokinase solution to the above-mentioned carrier solution and stir at 250 rpm for 1.5 hours at 28°C to fully embed the nattokinase into the network structure of the carrier.

[0085] 4. Preliminary molding and drying: The mixed solution was spray dried by spray drying, with the inlet temperature set at 130°C and the outlet temperature set at 70°C to obtain preliminary nattokinase microspheres.

[0086] 5. Outer coating: HPMCP, PVAP, and CAT were prepared into a 10% (w / v) ethanol solution at a mass ratio of 2:1:1 as the coating solution. The nattokinase microspheres were placed in a fluidized bed coater, and the coating solution was sprayed onto the surface of the microspheres at 45°C. The spray speed and air flow were controlled to increase the weight of the microspheres by 6%, and the final acid-resistant nattokinase microspheres with sustained release were obtained.

[0087] 3. Effect verification

[0088] 1. Enzyme activity determination: Enzyme activity determination was performed according to the determination scheme of nattokinase fibrinolytic activity established by the Japanese Nattokinase Society. The prepared nattokinase microspheres and nattokinase original powder were treated in simulated gastric juice (pH2.0) for 2h, and then in simulated intestinal juice (pH7.5) for 4h to determine the enzyme activity. The results showed that the nattokinase original powder was basically completely inactivated after being treated with simulated gastric juice, while the enzyme activity retention rate of the nattokinase microspheres prepared by the present invention after being treated with simulated gastric juice was more than 85%, and the enzyme activity was still high after being treated with simulated intestinal juice, indicating that the method of the present invention effectively improves the stability of nattokinase in gastric acid environment and the sustained release performance in the intestine.

[0089] 2. Stability Study: The prepared nattokinase microspheres were placed in high-density polyethylene bottles, sealed, placed at a temperature of 25°C ± 2°C and a relative humidity of 60% ± 5%, and sampled at 0, 3, 6, 9, 12, 18, 24, and 36 months, ground with a mortar, and the content of nattokinase was determined by the method of Appendix A of "DBS44 / 013 Food Safety Local Standard Natto Powder". The results showed that within 36 months, the content of nattokinase remained above 90% of the initial content, indicating that the nattokinase microspheres prepared by the present invention have good storage stability.

[0090] Embodiment 3:

[0091] 1. Material Preparation

[0092] 1. Raw materials: same as Example 2.

[0093] 2. Instruments and equipment: Same as in Example 2.

[0094] 2. Preparation process

[0095] 1. Blending of new organic compounds with nattokinase: Dissolve organic compound A (2-methyl-4-(1-imidazolyl)butyric acid) and organic compound B (3-dodecylthiophene-2-sulfonic acid) in phosphate buffer (pH 7.4) containing 5% mannitol to prepare a solution with a concentration of 5 mg / mL. Mix the two solutions with nattokinase fermentation extract solution (concentration of 10 mg / mL) at a volume ratio of 1:1:2 and stir at 6°C for 1 hour to allow the organic compounds to fully combine with nattokinase.

[0096] 2. Carrier preparation and complex embedding: Prepare a three-dimensional network structure carrier of modified konjac glucomannan and deacetylated chitosan (the method is the same as in Example 1). Slowly add the nattokinase-new organic compound complex solution to the carrier solution, stir at 150 rpm and room temperature for 2 hours, so that the complex is evenly dispersed and embedded in the network structure of the carrier.

[0097] 3. Coating and molding: The carrier particles containing the nattokinase-new organic compound complex are made into microspheres of appropriate size (diameter 0.15-0.4um) by dripping or extrusion, and then the microspheres are placed in a coating pan and coated with mixed coating materials such as HPMCP, PVAP, CAT (dissolved in an aqueous dispersion medium, and an appropriate amount of plasticizer such as triethyl citrate is added). By rotating the coating pan (speed 20-30rpm) and controlling the spraying amount of the coating liquid, the coating is evenly covered on the surface of the microspheres to form nattokinase microspheres with sustained release and acid resistance functions.

[0098] 3. Effect verification

[0099] 1. Enzyme activity determination: According to the above enzyme activity determination method, the nattokinase microspheres prepared in this example were tested. The results showed that after being treated in simulated gastric fluid (pH2.0) for 2 hours, the enzyme activity retention rate reached 82%, and after being treated in simulated intestinal fluid (pH7.5) for 4 hours, the enzyme activity still remained at a high level, proving that the nattokinase microspheres prepared by this method have good acid resistance and intestinal sustained release properties.

[0100] 2. Stability test: The nattokinase microspheres were placed under the same storage conditions as in Example 1, and samples were taken regularly to detect the nattokinase content. The results showed that within 36 months, the nattokinase content remained above 88% of the initial content, showing good storage stability, further verifying the reliability of the preparation method.

[0101] It can be seen from the above examples that the novel composite of the present invention has significant mosquito repellent and antipruritic effects, good skin compatibility and stability, and has potential and value for practical application.

Claims

1. A method for preparing a nattokinase preparation with sustained release and acid resistance, characterized in that, The following steps are involved: S1: preparing a core carrier: chemically degrading konjac flour to obtain a modified konjac glucomannan with a molecular weight of 8000-12000Da, and mixing the modified konjac glucomannan with deacetylated chitosan in a mass ratio of (1-3):1 to form a core carrier with a network structure, wherein the modified konjac glucomannan is prepared by reacting konjac flour with an acid or a base, followed by acid-base neutralization, washing with water to remove salt, and vacuum drying; S2 embedding nattokinase: dissolving nattokinase in phosphate buffer solution pH 7.0-7.8 to prepare a nattokinase solution with a concentration of 5-20 mg / mL, slowly adding the nattokinase solution to the core carrier solution of the three-dimensional network structure under the conditions of temperature control at 20-30°C and stirring speed at 150-300 rpm, so that the nattokinase is fully embedded in the network structure gap of the core carrier; S3 Preparation of micron-sized particles: The nattokinase system fully embedded in the lattice structure of the core carrier in S2 is made into micron-sized particles by spray drying or freeze drying; S4 outer coating: one or more combinations of hydroxypropyl methylcellulose phthalate (HPMCP), polyvinyl phthalate (PVAP), and cellulose acetate trimellitate (CAT) are used as coating materials, and the coating materials are dissolved in an organic solvent or an aqueous dispersion medium to form a coating solution. The organic solvent is ethanol or acetone, and a plasticizer triethyl citrate is added to the aqueous dispersion medium. The micron-sized particles are coated with an outer layer to increase the weight of the microspheres by 3%-10%. The coating process is carried out in a fluidized bed or a coating pan at a temperature of 30-50°C. When carried out in a fluidized bed, the spray speed and air flow rate are controlled. When carried out in a coating pan, the rotation speed and the amount of coating solution sprayed are controlled.

2. The method according to claim 1, characterized in that The method also includes adding organic compounds A and B to enhance the stability of the core carrier in the step of preparing the core carrier: adding 2-methyl-4-(1-imidazolyl)butyric acid (hereinafter referred to as compound A) and 3-dodecylthiophene-2-sulfonic acid (hereinafter referred to as compound B) to the core carrier, the addition amount of compound A and compound B is 1%-10% of the total mass of the core carrier and the compound, and before adding, compound A and compound B are respectively dissolved in an organic solvent, the organic solvent is ethanol or acetone, and then stirred or ultrasonically treated at a temperature of 35-50°C to fully mix compound A and compound B with the core carrier to form a more stable three-dimensional network structure.

3. The method according to claim 1, characterized in that The frequency of the ultrasonic treatment is 20-50 kHz, the treatment time is 20-60 min, the stirring speed is 200-500 rpm, and the stirring time is 1-3 h.

4. The method according to claim 1, characterized in that During the spray drying process, the atomizer speed is 10000-20000rpm, and the air flow rate is 50-100m 3 / h. During the freeze-drying process, the vacuum degree is 0.01-0.1mbar and the sublimation temperature is -20℃ to 0℃.

5. The method according to claim 1, characterized in that The HPMCP, PVAP and CAT are combined as coating materials in a mass ratio of (1-3):(0.5-2):(0.5-2). When an aqueous dispersion medium is used, the added amount of the plasticizer is 1%-5% of the mass of the coating material.