Preparation method of natto kinase microcapsule stable in stomach environment

Abstract

The application discloses a preparation method of a gastric environment stable natto kinase microcapsule and belongs to the field of food processing. The natto kinase-sodium alginate microcapsule with a three-dimensional network structure is prepared through an electrostatic spraying technology, and then a polyelectrolyte film is formed by chitosan and sodium alginate to coat the microcapsule through electrostatic interaction, so that the natto kinase-sodium alginate-chitosan microcapsule is prepared as a white regular sphere, the particle size is about 367 mu m, and the embedding rate is about 60%. The activity of natto kinase rapidly disappears in an artificial gastric juice, and the natto kinase protected by the natto kinase microcapsule retains 80.41% of the enzyme activity after 2 hours in the artificial gastric juice. The product prepared by the method can avoid the damage of the gastric environment to the activity of natto kinase, so that the natto kinase can smoothly pass through the gastric acid environment and retain a certain enzyme activity, is finally released in the intestinal tract, and the stability of the natto kinase is effectively improved.

Description

TECHNICAL FIELD

[0001] The present application belongs to the field of food processing, and particularly relates to a preparation method of a gastric environment stable nattokinase microcapsule. BACKGROUND

[0002] Cardiovascular disease is the disease with the largest number of deaths in the world, which seriously threatens the health of the public, and is also one of the important factors endangering the health of the elderly.

[0003] Nattokinase is a basic serine protease composed of 275 amino acid residues produced by Bacillus subtilis. Its main functions include anti-thrombosis, antihypertensive, anti-atherosclerosis, lipid-lowering, and anti-platelet, etc. Compared with the currently used drugs for treating cardiovascular embolism, such as urokinase, streptokinase and tissue-type plasminogen activator, nattokinase has the advantages of direct or indirect action on fibrin, rapid and efficient, low cost, safety and non-toxicity, etc. After oral administration, nattokinase must be absorbed by small intestinal epithelial cells through the gastrointestinal tract and transported to the whole body circulation to exert its biological effects. Pepsin and low pH environment can destroy the spatial structure of nattokinase, resulting in loss of its fibrinolytic activity.

[0004] Sodium alginate is a kind of anionic natural polysaccharide extracted from brown algae, which has a large number of active groups, pH sensitivity, biocompatibility, degradability and other characteristics, and is widely used in food processing, biomedical and other fields. Sodium alginate microcapsules have obvious pH sensitivity. When the microcapsules are in a pH<3.4 environment, the carboxylic acid group is in a non-ionic state (-COOH), and the microcapsule molecular chain shrinks, thereby reducing the stimulation of the gastric environment to the core material. When pH>4.4, the carboxyl group is ionized to -COO-, and the increase of negative charge electrostatic repulsion and hydrophilic group leads to the swelling of the polymer chain. The drug or active substance can be mixed with sodium alginate by embedding method, and cross-linked by adding divalent cations or small molecule cross-linking agents. During the cross-linking process, the drug or active substance is wrapped in the microcapsule, thereby forming a drug-loaded microcapsule. However, the leakage of low molecular weight active substances through the pores during the loading process is a bottleneck problem of alginate microcapsules. Chitosan is the product of N-deacetylation of chitin and is also the only cationic polysaccharide in natural polysaccharides. Sodium alginate and chitosan with opposite charges form a pH-sensitive polyelectrolyte complex through ionic interaction, which can reduce the voids of sodium alginate microcapsules.

[0005] A kind of natto kinase enteric soluble capsule and preparation method are disclosed in patent CN115364067A. Among them, sodium alginate is used as the main wall material, natto kinase is used as the content, and the properties of sodium alginate are used to protect natto kinase. The natto kinase is made into a capsule with enteric solubility, and the particle size of the obtained capsule is 2-4mm. Because the particle size is large, it limits its application in food. Chen Jingxin et al. prepared three kinds of natto kinase microcapsules by complex coacervation method, immobilization method and β-cyclodextrin envelope method. Through in vitro digestion simulation experiment, the residual enzyme activity of the three kinds of microcapsules after 2 hours in artificial gastric juice is low, which is 36.49%, 32.37% and 47.85% respectively.

[0006] In recent years, a new technology for preparing microcapsules by electrostatic spraying method has attracted widespread attention. Electrostatic spraying technology is to use high-voltage electrostatic field to atomize fluid into small droplets, which do directional motion under the combined action of electrostatic field force and external force and are adsorbed on the target. The whole preparation process is simple, the preparation time is short, the electrostatic action effectively reduces the surface tension of the droplets, reduces the droplet size, accelerates the solvent evaporation, provides larger surface area and volume ratio for the release of active substances. In addition, the electrostatic spraying technology is controllable, does not involve high-temperature operation, does not contact organic solvents, can maintain the activity of active substances, and the process is simple. SUMMARY

[0007] [TECHNICAL PROBLEM]

[0008] The present application aims to solve the problems of large particle size of natto kinase microcapsules and low residual enzyme activity, which leads to inactivation of natto kinase in the stomach environment.

[0009] [TECHNICAL SCHEME]

[0010] To solve the above problems, the present application provides a preparation method of natto kinase microcapsules stable in stomach environment. Sodium alginate and chitosan are used to embed natto kinase, which can better protect the activity of the enzyme and prevent it from being affected by the external environment. The specific steps include:

[0011] S1, respectively prepare a sodium alginate aqueous solution with a mass concentration of 1-3wt%, a calcium chloride aqueous solution with a mass concentration of 2-4wt%, and a chitosan acetic acid solution with a mass concentration of 0.5-1.5wt%;

[0012] S2, preparation of natto kinase-sodium alginate mixed solution: mix 5000FU / g natto kinase with the sodium alginate aqueous solution obtained in S1, and stir to mix the natto kinase with the sodium alginate aqueous solution to obtain a natto kinase-sodium alginate mixed solution;

[0013] S3, preparation of the nattokinase-sodium alginate microcapsule: the nattokinase-sodium alginate mixed solution prepared in S2 is sprayed into the calcium chloride solution prepared in S1 by using the electrostatic spraying technology to obtain the nattokinase-sodium alginate microcapsule, and the stirring is continuously carried out at room temperature for 20-40 minutes;

[0014] S4, preparation of the nattokinase-sodium alginate-chitosan microcapsule: the nattokinase-sodium alginate microcapsule prepared in S3 is washed with deionized water for 3-5 times, and then is placed in the chitosan acetic acid solution prepared in S1 to be magnetically stirred and soaked for 0.5-1.5 hours to obtain the nattokinase-sodium alginate-chitosan microcapsule, and the nattokinase-sodium alginate-chitosan microcapsule is washed with deionized water for 3-5 times to remove the excessive chitosan.

[0015] In an embodiment of the present application, the mass of the nattokinase in the nattokinase-sodium alginate mixed solution prepared in S2 accounts for 5% of the mass of the nattokinase-sodium alginate mixed solution.

[0016] In an embodiment of the present application, the parameters of the electrostatic spraying technology used in S3 are as follows: the flow rate is 0.1-0.5 mL / min, the voltage is 15-20 kV, the inner diameter of the needle is 0.04 mm, and the distance between the electrostatic spraying needle and the surface of the calcium chloride solution is 10-15 cm.

[0017] The present application also includes the nattokinase microcapsule prepared by the above method.

[0018] The present application also includes the application of the above nattokinase microcapsule in the field of food processing.

[0019] [Advantages]

[0020] 1. The nattokinase-sodium alginate-chitosan microcapsule is prepared by spraying the nattokinase-sodium alginate solution into the calcium chloride solution by using the electrostatic spraying technology, and then placing it in the chitosan solution, the prepared nattokinase microcapsule which is stable in the stomach environment is a white regular sphere, the average particle size of the finally obtained microcapsule is about 367 μm, and the embedding rate is about 60%.

[0021] 2. The activity of the nattokinase rapidly disappears in the artificial gastric juice, and the nattokinase protected by the nattokinase microcapsule retains 80.41% of the enzyme activity after 2 hours in the artificial gastric juice, which can effectively avoid the damage of the stomach environment to the activity of the nattokinase and effectively improve the stability of the nattokinase.

[0022] 3. The nattokinase-sodium alginate-chitosan microcapsule can be applied in the field of food processing as a functional food. BRIEF DESCRIPTION OF DRAWINGS

[0023] Figure 1 The microstructure picture of the nattokinase-sodium alginate-chitosan microcapsule prepared in Example 1.

[0024] Figure 2 Fourier infrared spectrum of the nattokinase-sodium alginate-chitosan microcapsule prepared in Example 1;

[0025] Figure 3 In vitro simulated digestion experiment results of the nattokinase-sodium alginate-chitosan microcapsule prepared in Example 1 and free nattokinase;

[0026] Figure 4 Residual enzyme activity of the nattokinase-sodium alginate-chitosan microcapsule prepared in Example 1 and free nattokinase in the stomach contents of mice at different times;

[0027] Figure 5 Residual enzyme activity of the nattokinase-sodium alginate-chitosan microcapsule prepared in Example 1 and free nattokinase in the contents of the front part of the cecum of mice at different times;

[0028] Figure 6 Residual enzyme activity of the nattokinase-sodium alginate-chitosan microcapsule prepared in Example 1 and free nattokinase in the contents of the rear part of the cecum of mice at different times;

[0029] Figure 7 In vitro simulated digestion experiment results of the nattokinase-sodium alginate-chitosan microcapsule and the nattokinase-sodium alginate-chitosan microcapsule;

[0030] Figure 8 Macroscopic comparison chart of the nattokinase-sodium alginate-chitosan microcapsule and the nattokinase-sodium alginate-chitosan microcapsule;

[0031] Figure 9 In vitro simulated digestion experiment results of the nattokinase-sodium alginate microcapsule and the nattokinase-sodium alginate-chitosan microcapsule. DETAILED DESCRIPTION

[0032] Example 1

[0033] The preparation method of the nattokinase-sodium alginate-chitosan microcapsule is realized according to the following steps:

[0034] S1, respectively prepare a 2wt% sodium alginate aqueous solution, a 3wt% calcium chloride aqueous solution, and a 1wt% chitosan acetic acid solution;

[0035] S2, Preparation of Natto Kinase-Sodium Alginate Mixed Solution: 5000 FU / g of natto kinase was mixed with the sodium alginate aqueous solution obtained in S1 to obtain a natto kinase-sodium alginate mixed solution by stirring at room temperature, wherein the mass of natto kinase accounted for 5% of the mass of the natto kinase-sodium alginate mixed solution.

[0036] S3, Preparation of Natto Kinase-Sodium Alginate Microcapsules: The natto kinase-sodium alginate mixed solution obtained in S2 was sprayed into the calcium chloride solution obtained in S1 using an electrostatic spraying technique to obtain natto kinase-sodium alginate microcapsules, which were continuously stirred at room temperature for 30 minutes. The parameters of the electrostatic spraying technique were as follows: flow rate 0.2 mL / min, voltage 17.5 kV, inner diameter of needle 0.04 mm, and distance between the electrostatic spraying needle and the surface of the calcium chloride solution 12 cm.

[0037] S4, Preparation of Natto Kinase-Sodium Alginate-Chitosan Microcapsules: The natto kinase-sodium alginate microcapsules obtained in S3 were washed with deionized water for 4 times, and then were placed in the chitosan acetic acid solution obtained in S1 for magnetic stirring and immersion for 1 hour to obtain natto kinase-sodium alginate-chitosan microcapsules, which were washed with deionized water for 4 times to remove excess chitosan.

[0038] Comparative Example 1

[0039] S1, Preparation of Sodium Alginate Aqueous Solution, Calcium Chloride Aqueous Solution and Chitosan Acetic Acid Solution: 2wt% of sodium alginate aqueous solution, 3wt% of calcium chloride aqueous solution and 1wt% of chitosan acetic acid solution were prepared, respectively.

[0040] S2, Preparation of Sodium Alginate-Chitosan Microcapsules: The sodium alginate solution obtained in S1 was sprayed into the calcium chloride solution using an electrostatic spraying technique to obtain sodium alginate microcapsules, which were continuously stirred at room temperature for 30 minutes. The parameters of the electrostatic spraying technique were as follows: flow rate 0.2 mL / min, voltage 17.5 kV, inner diameter of needle 0.04 mm, and distance between the electrostatic spraying needle and the surface of the calcium chloride solution 12 cm. After the sodium alginate microcapsules were washed with deionized water for 4 times, they were placed in the chitosan acetic acid solution obtained in S1 for magnetic stirring and immersion for 1 hour to obtain sodium alginate-chitosan microcapsules, which were washed with deionized water for 4 times to remove excess chitosan.

[0041] The microstructure of nattokinase-sodium alginate-chitosan microcapsules was observed using a cold field emission scanning electron microscope (Hitachi SU8010, Japan). First, the nattokinase-sodium alginate-chitosan microcapsules were placed on a stage, and then the stage containing the microcapsules was immersed in liquid nitrogen for approximately 3-5 minutes to rapidly freeze the microcapsules. After placing the stage in a vacuum chamber, the protruding portion was cut off. The nattokinase-sodium alginate-chitosan microcapsules were sublimated at -90°C for 20 minutes, and then sputtered with gold for 120 seconds. Observation was then performed at 10kV and 300x magnification. Figure 1 As shown: Nattokinase-sodium alginate-chitosan microcapsules are white, regular spheres with a polyelectrolyte membrane on the surface.

[0042] Fourier transform infrared spectroscopy was used to analyze nattokinase-sodium alginate-chitosan microcapsules (Example 1), sodium alginate-chitosan microcapsules (Comparative Example 1), and nattokinase samples. Figure 2 As shown, in the infrared spectrum of nattokinase, 3221.86 cm⁻¹ -1 The absorption peak at 1408.30 cm⁻¹ is due to the stretching vibration of the OH group. -1 The nearby peaks were designated as crystal absorption bands. In the infrared spectrum of sodium alginate-chitosan microcapsules, the sugar rings on the polysaccharide are visible at 3425.14 cm⁻¹. -1 The OH bending vibration at 1700 cm⁻¹ is a characteristic peak for sodium alginate and chitosan. -1 A series of absorption peaks related to the vibration of the C=O group appeared at 1607.82 cm⁻¹. -1 The characteristic peak of the amide I band was observed at 1300 cm⁻¹. -1 The broad peak at 3446.91 cm⁻¹ is caused by the absorption of the CN stretching vibration belonging to the amide III band, a unique characteristic peak of chitosan. In the infrared spectrum of nattokinase-sodium alginate-chitosan microcapsules, the -OH group is located at 3446.91 cm⁻¹. -1 The stretching vibration at 2930 cm⁻¹ (characteristic peak of sodium alginate). The stretching vibration of CH in the CH₂ group at 2930 cm⁻¹. -1 At point C=C, the symmetrical tensile vibration occurs at 1637 cm. -1 The absorption peak is at 616.00 cm⁻¹. -1 The absorption peak at 1100 cm⁻¹ is due to the stretching vibration of the C-Cl bond. -1 The displacement observed at this point may be due to COOH present on the surface of the microcapsules. - and NH3 + Interionic interactions between functional groups.

[0043] The nattokinase-sodium alginate-chitosan microcapsules prepared in Example 1 were respectively placed in artificial gastric juice at 37°C and 180 r / min for 2 hours. The nattokinase-sodium alginate-chitosan microcapsules were filtered out, washed with deionized water for 3 times, and then placed in artificial intestinal juice at 37°C and 180 r / min for 3 hours. The enzyme activity in the artificial intestinal juice was measured. Meanwhile, the nattokinase stock solution was placed in artificial gastric juice at 37°C and 180 r / min for 2 hours. The enzyme activity in the artificial gastric juice was measured. The results are shown in Table 1. Figure 3 As shown in Table 1, the activity of free nattokinase in artificial gastric juice rapidly disappeared. The enzyme activity was reduced by 90% in 0.5 hour, and finally completely inactivated in 1 hour. The nattokinase-sodium alginate-chitosan microcapsules effectively protected nattokinase. After 2 hours in artificial gastric juice, 80.41% of the enzyme activity was retained. After 3 hours in artificial intestinal juice, 24.22% of the enzyme activity was retained. The nattokinase-sodium alginate-chitosan microcapsules can avoid the destruction of the gastric environment to the activity of nattokinase, and effectively improve the stability of nattokinase.

[0044] Twenty-four mice were divided into two groups, and respectively gavaged with free nattokinase and the nattokinase-sodium alginate-chitosan microcapsules prepared in Example 1. Three mice without gavage were used as blank controls. Three mice in each group were euthanized at different time intervals. The intestinal tract of the mice was divided into two parts at the cecum. The stomach and the contents of the two parts of the intestinal tract were respectively mixed with 1 mL of normal saline, and after ultrasonic treatment, the supernatant was centrifuged to measure the enzyme activity. The enzyme activity of the stomach and intestinal contents of the mice without gavage was used as a control. The enzyme activity of the stomach and intestinal contents of the mice with gavage was measured, and the control value was subtracted. The residual rate of the enzyme activity of the stomach and intestinal contents of the mice in the two groups was determined. The results are shown in Table 2. Figure 4 5 As shown in Table 2, after 10 minutes of gavage, the residual enzyme activity of the free nattokinase group in the stomach contents was only about 15%, while the nattokinase-sodium alginate-chitosan microcapsule group still had 90% of the residual enzyme activity. After 2 hours, the free nattokinase group had almost no activity. The nattokinase-sodium alginate-chitosan microcapsule group still had 50.69% of the residual enzyme activity. The residual enzyme activity of the free nattokinase group in the contents of the intestinal tract before the cecum was between 5% and 10%. The residual enzyme activity of the nattokinase-sodium alginate-chitosan microcapsule group increased to 30% within 0-2 hours, and gradually decreased to 11% at 6 hours. The residual enzyme activity of the two groups in the contents of the intestinal tract after the cecum varied between 0% and 15%. The nattokinase-sodium alginate-chitosan microcapsules protected nattokinase during the passage through the stomach, and produced higher enzyme activity compared with free nattokinase.

[0045] Comparative Example 2

[0046] S1, respectively prepare a 2wt% sodium alginate aqueous solution and a 3wt% calcium chloride aqueous solution; ​

[0047] S2, preparation of natto kinase-sodium alginate mixed solution: 5000 FU / g natto kinase was mixed with the sodium alginate aqueous solution obtained in step S1, and stirring was performed at room temperature to mix the natto kinase and the sodium alginate aqueous solution to obtain a natto kinase-sodium alginate mixed solution, wherein the mass of the natto kinase accounted for 5% of the mass of the natto kinase-sodium alginate mixed solution;

[0048] S3, preparation of natto kinase-sodium alginate microcapsules: the natto kinase-sodium alginate mixed solution described in step S2 was sprayed into the calcium chloride solution obtained in step S1 using an electrostatic spraying technique to obtain natto kinase-sodium alginate microcapsules, and stirring was continuously performed at room temperature for 30 minutes; the parameters of the electrostatic spraying technique were as follows: flow rate was 0.2 mL / min, voltage was 17.5 kV, inner diameter of needle was 0.04 mm, and the distance between the electrostatic spraying needle and the surface of the calcium chloride solution was 12 cm.

[0049] Comparative Example 3

[0050] S1, respectively prepare a sodium alginate aqueous solution with a mass concentration of 2 wt%, a calcium chloride aqueous solution with a mass concentration of 3 wt%, and a chitosan acetic acid solution with a mass concentration of 1 wt%;

[0051] S2, preparation of natto kinase-sodium alginate mixed solution: 5000 FU / g natto kinase was mixed with the sodium alginate aqueous solution obtained in step S1, and stirring was performed at room temperature to mix the natto kinase and the sodium alginate aqueous solution to obtain a natto kinase-sodium alginate mixed solution, wherein the mass of the natto kinase accounted for 5% of the mass of the natto kinase-sodium alginate mixed solution;

[0052] S3, preparation of natto kinase-sodium alginate microcapsules: the natto kinase-sodium alginate mixed solution described in step S2 was sprayed into the calcium chloride solution obtained in step S1 using an electrostatic spraying technique to obtain natto kinase-sodium alginate microcapsules, and stirring was continuously performed at room temperature for 30 minutes; the parameters of the electrostatic spraying technique were as follows: flow rate was 0.2 mL / min, voltage was 17.5 kV, inner diameter of needle was 0.04 mm, and the distance between the electrostatic spraying needle and the surface of the calcium chloride solution was 12 cm.

[0053] S4, preparation of natto kinase-sodium alginate-chitosan capsules: after the natto kinase-sodium alginate capsules were washed with deionized water for 4 times, they were placed in the chitosan acetic acid solution obtained in S1 and magnetically stirred for 1 hour to obtain natto kinase-sodium alginate-chitosan capsules, and then washed with deionized water for 4 times to remove excess chitosan

[0054] The nattokinase-sodium alginate-chitosan microcapsules and the nattokinase-sodium alginate microcapsules prepared in Comparative Example 2 and Example 1 were respectively placed in an artificial gastric juice at 37°C and 180 r / min for 2 hours. The nattokinase-sodium alginate-chitosan microcapsules and the nattokinase-sodium alginate microcapsules were filtered out and washed with deionized water for 3 times, and then were placed in an artificial intestinal juice at 37°C and 180 r / min for 3 hours. The enzyme activity in the artificial intestinal juice was measured. Meanwhile, the nattokinase stock solution was placed in an artificial gastric juice at 37°C and 180 r / min for 2 hours, and the enzyme activity in the artificial gastric juice was measured. The results are shown in Table 2. Figure 7 As shown in Table 2, the activity of free nattokinase rapidly disappeared in the artificial gastric juice, and the enzyme activity was reduced by 90% in 0.5 hour, and finally completely inactivated in 1 hour. The nattokinase-sodium alginate-chitosan microcapsules and the nattokinase-sodium alginate microcapsules could effectively protect the nattokinase, and the residual enzyme activities were 73.48% and 80.41% respectively after 2 hours in the artificial gastric juice, and the residual enzyme activities were 27.11% and 24.22% respectively after 3 hours in the artificial intestinal juice. The nattokinase-sodium alginate-chitosan microcapsules had a better protection effect on nattokinase than the nattokinase-sodium alginate-chitosan microcapsules in the gastric environment.

[0055] The macroscopic comparison of the nattokinase-sodium alginate-chitosan microcapsules and the nattokinase-sodium alginate-chitosan microcapsules is shown in Table 3. Figure 8 As shown in Table 3, the particle size of the nattokinase-sodium alginate-chitosan microcapsules was about 2-4 mm, and the particle size of the nattokinase-sodium alginate-chitosan microcapsules was about 367 μm. It can be seen that the electrostatic spraying technology can greatly reduce the particle size of the nattokinase-sodium alginate-chitosan microcapsules, and the microcapsules have a spherical shape, uniform and round, and good dispersibility, which is beneficial to the application in the food processing system.

[0056] The nattokinase-sodium alginate-chitosan microcapsules and the nattokinase-sodium alginate microcapsules prepared in Comparative Example 2 and Example 1 were respectively placed in an artificial gastric juice at 37°C and 180 r / min for 2 hours. The nattokinase-sodium alginate-chitosan microcapsules and the nattokinase-sodium alginate microcapsules were filtered out and washed with deionized water for 3 times, and then were placed in an artificial intestinal juice at 37°C and 180 r / min for 3 hours. The enzyme activity in the artificial intestinal juice was measured. Meanwhile, the nattokinase stock solution was placed in an artificial gastric juice at 37°C and 180 r / min for 2 hours, and the enzyme activity in the artificial gastric juice was measured. The results are shown in Table 2. Figure 9As shown: Natto kinase-alginic acid sodium-chitosan microcapsules and natto kinase-alginic acid sodium microcapsules can effectively protect natto kinase, after 2 hours in artificial gastric juice, the remaining enzyme activity was 80.41% and 74.30% respectively, after 3 hours in artificial intestinal juice, the remaining enzyme activity was 24.22% and 30.73% respectively. Natto kinase-alginic acid sodium-chitosan microcapsules in the stomach environment on the protection effect of natto kinase is better than natto kinase-alginic acid sodium microcapsules, the remaining enzyme activity increased by 6%. Natto kinase-alginic acid sodium-chitosan microcapsules can avoid the destruction of the stomach environment on the activity of natto kinase, effectively improve the stability of natto kinase.

[0057] The above provided examples are not intended to limit the scope of the present application, and the described steps are not intended to limit the order of execution. Those skilled in the art make obvious improvements to the present application in combination with existing common knowledge, which also falls within the protection scope defined by the claims of the present application.

Claims

1. A method for preparing nattokinase microcapsules, characterized in that, Includes the following steps: S1. Solution Preparation: Prepare sodium alginate aqueous solution, calcium chloride aqueous solution, and chitosan acetate solution respectively; the mass concentration of sodium alginate aqueous solution is 2wt%, the mass concentration of calcium chloride aqueous solution is 3wt%, and the mass concentration of chitosan acetate solution is 1wt%. S2. Preparation of Nattokinase-Sodium Alginate Mixed Solution: 5000 FU / g nattokinase was mixed and stirred with the sodium alginate aqueous solution obtained in S1 to obtain a nattokinase-sodium alginate mixed solution; the nattokinase in the nattokinase-sodium alginate mixed solution accounted for 5% of the total mass of the nattokinase-sodium alginate mixed solution. S3. Preparation of Nattokinase-Sodium Alginate Microcapsules: The nattokinase-sodium alginate mixed solution described in S2 was sprayed into the calcium chloride solution obtained in S1 using electrostatic spraying technology to obtain nattokinase-sodium alginate microcapsules. The mixture was stirred continuously for 20-40 minutes. The specific parameters for using electrostatic spraying technology were: flow rate of 0.1-0.5 mL / min, voltage of 15-20 kV, needle inner diameter of 0.04 mm, and distance between the electrostatic spraying needle and the surface of the calcium chloride solution of 10-15 cm. Preparation of S4 Nattokinase-Sodium Alginate-Chitosan Microcapsules: After washing the nattokinase-sodium alginate microcapsules obtained in S3 with deionized water 3-5 times, place them in the chitosan acetate solution obtained in S1 and stir magnetically for 0.5-1.5 hours to obtain nattokinase-sodium alginate-chitosan microcapsules. Finally, wash them with deionized water 3-5 times.

2. A nattokinase microcapsule prepared according to the method described in claim 1.

3. A nattokinase microcapsule according to claim 2, characterized in that, The particle size is 367 μm.

4. The application of the nattokinase microcapsule according to claim 2 in the field of food processing.

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