A method for preparing nattokinase enteric-sustained release capsules and sustained release gel balls

Abstract

This invention discloses a method for preparing nattokinase enteric-coated sustained-release capsules and sustained-release gel spheres, belonging to the field of sustained-release microcapsules. This invention improves the selection of wall materials and the preparation process, encapsulating nattokinase using a mixture of food-grade enteric-coating shellac and whey protein isolate as the wall material. After spray drying, food-grade enteric-coated microcapsules are obtained. These microcapsules are then embedded in calcium alginate gel, forming enteric-coated gel spheres through stepwise encapsulation. The gel spheres prepared by this invention are safe for consumption, have a high encapsulation rate, are resistant to gastric acid, can be released into the intestines, are easy to store and transport, and can be mass-produced industrially.

Description

Technical Field

[0001] This invention relates to a method for preparing nattokinase enteric-coated sustained-release capsules and sustained-release gel spheres, belonging to the field of sustained-release microcapsules. Background Technology

[0002] Thrombosis is a key factor leading to stroke and coronary heart disease. With the accelerating aging of society, the number of people suffering from thrombotic diseases is expected to continue to rise, posing a significant threat to the health and medical economy of Chinese residents. Thrombosis is mainly formed by the excessive aggregation of fibrin and platelets and is closely related to various diseases such as stroke, cerebral infarction, and myocardial infarction.

[0003] Nattokinase is a serine protease extracted from natto, possessing significant fibrinolytic activity. It can directly dissolve fibrin in thrombi and activate endogenous plasminogen in the human body, thus gently and continuously promoting blood circulation. Furthermore, studies have shown that nattokinase has various beneficial effects, including assisting in lowering blood pressure, improving atherosclerosis, and anticoagulation. Due to its high efficiency and safety, nattokinase shows broad prospects in the prevention and treatment of thrombotic diseases. However, as a protease, nattokinase faces two major obstacles in oral delivery: first, it is prone to irreversible denaturation and inactivation in the highly acidic environment of the stomach; second, it is easily decomposed by digestive enzymes such as trypsin in the intestine, resulting in very low oral bioavailability, severely limiting its application in functional foods or oral medications. To address these challenges, microencapsulation technology has been widely used for the encapsulation and delivery of nattokinase.

[0004] Existing technologies mainly focus on selecting suitable wall material systems and preparation processes to achieve gastric protection and targeted intestinal release of nattokinase. For wall materials, polysaccharide wall materials such as sodium alginate and chitosan are commonly used. While gel beads or microspheres prepared through ionogel methods have a certain sustained-release effect, they may swell or dissolve prematurely in gastric acid, resulting in incomplete protection of the core material. Furthermore, the processes are usually complex and difficult to scale up. Polyacrylic acid resins (Eudragit series) are synthetic polymer wall materials. These materials have precise pH-dependent solubility characteristics, making them ideal for enteric coatings. However, synthetic materials suffer from poor biocompatibility, high cost, and organic solvent residues, which do not align with the development trend of the green food industry. Protein-based wall materials such as gelatin, soy protein isolate, and whey protein isolate are also options. These materials have good biocompatibility, are biodegradable, and possess nutritional value. Whey protein isolate, in particular, with its excellent emulsifying, film-forming, and nutritional value, is a highly promising microcapsule wall material. However, the protective layer formed by a single protein wall material still lacks sufficient density and stability in the harsh acidic environment of the stomach, which may lead to a "burst release" phenomenon and fail to achieve long-term, high-efficiency protection of enzyme activity. In terms of preparation technology, spray drying is one of the most commonly used techniques for preparing microcapsules due to its continuous operation, low cost, and ease of industrial scale-up. Its core lies in rapidly forming microcapsules by encapsulating the core material within the wall material through atomization and drying.

[0005] Patent CN115364067A uses sodium alginate and sodium carboxymethyl cellulose to encapsulate nattokinase into enteric-coated microcapsules. The process involves adding 0.6g of sodium alginate and 0.06g of sodium carboxymethyl cellulose to 20mL of pure water, followed by 10mL of nattokinase solution. Then, 0.5% CaCl2 solution is added dropwise to obtain the microcapsules. The stock nattokinase solution has an enzyme activity of 85.40 FU / mL, and the encapsulation rate is 82.1%. After two hours in gastric juice and three hours in intestinal juice, the enzyme activity is 17.50 FU / mL. However, this technology suffers from problems such as easy swelling and burst release of the microcapsules, poor stability during storage and transportation, and is not conducive to large-scale industrial production.

[0006] Therefore, developing a stable, green process that can simultaneously achieve efficient encapsulation, strong gastric protection, and precise intestinal release of nattokinase microcapsules is a technical problem that urgently needs to be solved in this field. Summary of the Invention

[0007] To simultaneously address multiple issues such as insufficient gastric protection, poor targeting, environmentally unfriendly processes, limited wall material performance, and poor stability during storage and transportation, this invention improves the selection of wall materials and the preparation process. It encapsulates nattokinase using a mixture of food-grade enteric-coated shellac and whey protein isolate as the wall material, followed by spray drying to obtain food-grade enteric-coated microcapsules. These microcapsules are then embedded in calcium alginate gel, forming enteric-coated gel spheres through stepwise encapsulation. The gel spheres prepared by this invention are characterized by being safe for consumption, having a high encapsulation rate, being resistant to gastric acid, allowing for sustained intestinal release, facilitating storage and transportation, and enabling large-scale industrial production.

[0008] The first objective of this invention is to provide a method for preparing enteric-coated microcapsules of nattokinase, comprising the following steps: A mixture of whey protein isolate and nattokinase was mixed with shellac solution at a volume ratio of 0.5 to 2:1 and stored overnight to obtain a whey protein isolate-nattokinase-shellac mixture; the mixture was then spray-dried to obtain nattokinase enteric-coated microcapsules. The whey protein isolate-nattokinase mixture is obtained by mixing whey protein isolate solution and nattokinase solution.

[0009] In one embodiment, the whey protein isolate concentration in the whey protein isolate-nattokinase-shellac mixture is 8-12% w / w, the nattokinase solution concentration is 8-12% w / w, and the shellac solution concentration is 8-12% w / w.

[0010] In one embodiment, whey protein isolate solution and nattokinase solution are mixed at a volume ratio of 4 to 1:1.

[0011] In one embodiment, a whey protein isolate-nattokinase mixture is mixed with a shellac solution at a volume ratio of 0.5 to 2:1.

[0012] In one embodiment, the inlet air temperature for spray drying is 165~175°C.

[0013] In one embodiment, the feed flow rate for spray drying is 10-15 mL / min.

[0014] In one embodiment, the shellac solution is obtained by dissolving shellac in an aqueous solution of ammonium bicarbonate and stirring at 50-60°C for 1-1.5 h.

[0015] A second objective of this invention is to provide nattokinase enteric-coated microcapsules prepared by any of the methods described above.

[0016] The third objective of this invention is to provide an enteric-coated sustained-release nattokinase gel ball, prepared from the above-mentioned enteric-coated nattokinase microcapsules, through the following steps: Nattokinase enteric-coated microcapsules were dispersed in water and mixed with sodium alginate aqueous solution to obtain a suspension; the suspension was then added dropwise to calcium chloride aqueous solution to prepare calcium alginate enteric-coated sustained-release gel spheres.

[0017] In one embodiment, the concentration of nattokinase enteric-coated microcapsules in the suspension is 55-75 g / L, the concentration of sodium alginate aqueous solution is 1.8-2.2% w / w, and the concentration of calcium chloride aqueous solution is 0.08-0.12 mol / L.

[0018] In one embodiment, a constant flow syringe pump is used to drop the suspension into an aqueous calcium chloride solution at a flow rate of 4.5 to 5.5 mL / min.

[0019] In one embodiment, the nattokinase enteric-coated microcapsules are in the form of a powder solid, with a particle size of approximately 20 μm and an encapsulation efficiency of 92.57%. The nattokinase enteric-coated sustained-release gel spheres are spherical solids with a particle size of 1.0~1.5mm and an encapsulation rate of 97.18%.

[0020] A fourth objective of this invention is to provide the application of the above-mentioned nattokinase enteric-coated microcapsules or any of the above-mentioned nattokinase enteric-coated sustained-release gel balls in the food and pharmaceutical fields.

[0021] Beneficial effects of the present invention This invention improves the selection of wall materials and the preparation process. Nattokinase is encapsulated using a mixture of food-grade enteric-coating shellac and whey protein isolate as the wall material, followed by spray drying to obtain food-grade enteric-coated microcapsules. These microcapsules are then embedded in calcium alginate gel, forming enteric-coated gel spheres through stepwise encapsulation. The gel spheres prepared by this invention are characterized by being safe for consumption, having a high encapsulation rate, being resistant to gastric acid, allowing for sustained release into the intestines, facilitating storage and transportation, and enabling large-scale industrial production.

[0022] Specifically: The nattokinase enteric-coated sustained-release gel spheres are spherical solids with a particle size of 1.0~1.5 mm and an encapsulation rate of 97.18%. After 2 hours of in vitro digestion in simulated gastric juice, 94.89% of the enzyme activity is still retained. The nattokinase is stably and continuously released in simulated intestinal juice for 6 hours, with a release amount of over 50% and a total release time of over 10 hours. Attached Figure Description

[0023] Figure 1 The effect of protein wall materials on encapsulation efficiency and enzyme activity.

[0024] Figure 2 The effect of wall material concentration on encapsulation efficiency and enzyme activity.

[0025] Figure 3 The effect of the ratio of protein wall material to core material on encapsulation efficiency and enzyme activity.

[0026] Figure 4 Optimize the shellac addition ratio.

[0027] Figure 5 Encapsulation efficiency, drug loading, and enzyme activity retention of nattokinase enteric-coated sustained-release gel spheres prepared at different concentrations were determined.

[0028] Figure 6 To simulate the digestion results of gastrointestinal fluids for nattokinase, microcapsules, and gel balls.

[0029] Figure 7 Results on the thermal stability of nattokinase, microcapsules, and gel beads.

[0030] Figure 8 Results on pH stability of nattokinase, microcapsules, and gel spheres.

[0031] Figure 9 To assess the stability of nattokinase, microcapsules, and gel spheres during storage at 4°C.

[0032] Figure 10 To assess the stability of nattokinase, microcapsules, and gel balls during storage at 25°C. Detailed Implementation

[0033] The preferred embodiments of the present invention are described below. It should be understood that the embodiments are for better explanation of the present invention and are not intended to limit the present invention.

[0034] Test method: I. Nattokinase Enzyme Activity Assay Method: The method follows the group standard T / CQAP 2001-2022, using the agarose fibrin plate method. The assay method is as follows: (1) Preparation of fibrin plates: 1) Take 40 mL of 15 g / L agarose solution, incubate in a 50°C water bath for 30 min, add 3.0 mL of 1~2 U thrombin while stirring, and mix immediately for 10 s. 2) Take 40 mL of 1.5 mg / mL fibrinogen solution and place it in a 100 mL beaker. Heat it in a 37°C water bath for 5 min. Add agarose and thrombin solution, mix immediately for 10 s, pour quickly into a culture dish, and place horizontally at room temperature for 1 h. 3) Draw a circle with the same diameter as the bottom of the petri dish on paper. Draw a smaller circle with a diameter of 24 mm in the center of the first circle. Place the prepared plate on the smaller circle as the center point. Use a 3 mm diameter punch to make vertical holes in the fibrin plate. 4) The number of holes required is the sum of the standard series number and the number of samples (including parallel samples). The holes should be 15 mm apart to prevent the fusion rings from crossing and affecting the measured diameter results. (2) Preparation of standard curve: Take urokinase standard, add the required PBS buffer to dissolve it, and prepare a urokinase standard solution of 1000 IU / mL. Prepare a series of gradients according to 1000, 800, 600, 500, 400, 300, 200, 100, 50, and 25 IU / mL. Take 10 μL into a plate with wells and incubate at 37℃ for 18 h. Take out the plate and measure the diameter of the lysis zone immediately. Plot a regression curve with the logarithm of the lysis zone area as the abscissa and the logarithm of the concentration as the ordinate to obtain the corresponding regression equation. (3) Sample determination: Weigh an appropriate amount of sample into a suitable volumetric flask. The sample volume should be such that the final spotting concentration is approximately 200–400 IU / mL. Accurately pipette 10 μL of the sample solution and spot it onto the flask. Perform 1–2 parallel samples and accurately label the sample numbers. After spotting, cover the petri dish and place it in a 37°C incubator for 18 hours. Measure the melting zone and substitute it into the standard curve to determine the enzyme activity. II. Determination of encapsulation efficiency and enzyme activity retention: Accurately weighed samples were dispersed in deionized water and magnetically stirred at low speed for 1 hour to release nattokinase on the surface. Subsequently, the mixture was centrifuged at 6500 g for 8 minutes. The precipitate was washed and dispersed in water, repeated twice, and then sonicated at 4°C. The activity of this fraction was determined. N 1), This value represents the content of encapsulated nattokinase. The total enzyme activity of the sample was determined by direct sonication of the unwashed sample. N 2).

[0035] The encapsulation efficiency (EE), enzyme loading (LC), and enzyme activity retention (Ret) of the sample are calculated using the following formulas:

[0036] in, N 1 and N 2 represents the encapsulated enzyme activity and total enzyme activity, respectively; m NK and m T These represent the actual input mass of nattokinase and the total mass of the encapsulation system, respectively. E before and E after These represent enzyme activity per unit mass before and after encapsulation, respectively.

[0037] Raw materials used in the examples: Nattokinase: Provided by the State Key Laboratory of Food Science and Resources, Jiangnan University (Wuxi, Jiangsu Province, China).

[0038] Bovine thrombin (1000 U / g) and bovine fibrinogen were purchased from Shenyang Baiying Biotechnology Co., Ltd. Urokinase was purchased from the National Institutes for Food and Drug Control. Whey protein isolate, soy protein isolate, and shellac were purchased from Shanghai Yuanye Biotechnology Co., Ltd. Pea protein isolate was purchased from Shandong Jianyuan Biotechnology Co., Ltd. Dipotassium hydrogen phosphate, potassium dihydrogen phosphate, sodium chloride, trisodium citrate, gelatin, calcium chloride, ammonium bicarbonate, etc., were purchased in analytical grade from Sinopharm Chemical Reagent Co., Ltd. Pepsin and trypsin were purchased from Shanghai Myriel Biochemical Technology Co., Ltd.

[0039] Example 1 A method for preparing enteric-coated microcapsules of nattokinase, comprising the following steps: (1) Shellac was dissolved in a 2.1% (w / w) ammonium bicarbonate aqueous solution and stirred at 50°C for 1 h to prepare a shellac solution with a shellac concentration of 10% (w / w). Whey protein isolate was dissolved in water to obtain a 10% (w / w) whey protein isolate solution; Nattokinase was dissolved in water to obtain a 10% (w / w) nattokinase solution; (2) Mix whey protein isolate solution and nattokinase solution at a volume ratio of 1:1 to obtain whey protein isolate-nattokinase mixture; The whey protein isolate-nattokinase mixture and shellac solution were mixed at a volume ratio of 1:1 and stirred at 200 rpm for 30 min to obtain the whey protein isolate-nattokinase-shellac mixture. (3) Spray drying of whey protein isolate-nattokinase-shellac mixture to obtain nattokinase enteric-coated microcapsules; spray drying conditions: inlet air temperature = 170 ± 5 ℃; outlet air temperature = 70 ± 5 ℃; feed flow rate = 13 mL / min.

[0040] The enzyme activity of the spray-dried microcapsules was detected using the agarose fibrin plate method, and their encapsulation efficiency and enzyme activity retention rate were tested. Under optimal conditions, the encapsulation efficiency reached 92.57%, and the enzyme activity retention rate reached 93.31%.

[0041] Example 2 Based on Example 1, the types of protein wall materials in step (1) were changed to whey protein isolate, whey protein, gelatin, soy protein isolate, and pea protein isolate, while the remaining steps remained the same. The encapsulation rate and enzyme activity retention rate were then tested.

[0042] Example 3 Based on Example 1, the concentrations of whey protein isolate, nattokinase and shellac in step (1) were changed to 5%, 7%, 10%, 15% and 20%, respectively, while the remaining steps remained the same. The encapsulation rate and enzyme activity retention rate were then tested.

[0043] Example 4 Based on Example 1, the ratio of whey protein isolate and nattokinase in step (2) was changed to 4:1, 2:1, 1:1, 1:2, and 1:4, while the remaining steps remained the same. The encapsulation rate and enzyme activity retention rate were then tested.

[0044] Example 5 Based on Example 1, the inlet air temperature of spray drying in step (3) was changed to 140℃, 150℃, 160℃, 170℃ and 180℃ respectively, while the other steps remained the same. The encapsulation rate, enzyme activity retention rate and moisture content were tested.

[0045] As shown in Table 1, while a low inlet air temperature can maintain a high enzyme activity retention rate, it significantly reduces the encapsulation efficiency and increases the moisture content. This is because the lower temperature results in a slower evaporation rate and insufficient drying. Excessively high temperatures not only cause heat loss of nattokinase during spray drying but also lead to cracks on the microcapsule surface, exposing the core material. Based on comprehensive analysis, 165℃~175℃ is selected as the inlet air temperature for spray drying.

[0046] Table 1. Effect of inlet air temperature on microcapsules

[0047] Example 6 Based on Example 1, the feed flow rate of spray drying in step (3) was changed to 7 mL / min, 10 mL / min, 13 mL / min and 16 mL / min respectively, while the other steps remained the same. The encapsulation rate, enzyme activity retention rate and moisture content were tested.

[0048] As shown in Table 2, a lower feed rate reduces the moisture content of the microcapsules but also causes excessive heat loss. Under high temperatures, the microcapsules become more brittle, and surface cracks lead to a decrease in encapsulation efficiency. An excessively fast feed rate directly results in a lower outlet air temperature, leading to incomplete drying of the microcapsules. In summary, a feed flow rate of 13 mL / min is most suitable, with an outlet air temperature of 70 ± 5℃.

[0049] Table 2 Effect of feed flow rate on microcapsules

[0050] Example 7 Based on Example 1, the volume ratio of whey protein isolate-nattokinase mixture to shellac solution in step (2) was changed to 5:1, 2:1, 1:1, 0.5:1 and 0.2:1, while the other steps remained the same. The average particle size, moisture content, encapsulation rate and enzyme activity retention rate were tested.

[0051] As shown in Table 3, the average particle size of the microcapsules was 20 μm and the water content was less than 4%.

[0052] Table 3. Average particle size and moisture content of microcapsules

[0053] Note: Different letters indicate significant differences. P <0.05) The results of the nattokinase enteric-coated microcapsules prepared in Examples 1-7 are as follows: Figure 1 , Figure 2 , Figure 3 , Figure 4 As shown in the figure. The results indicate that using 10% (w / w) whey protein isolate and 10% (w / w) shellac as the composite wall material resulted in the highest encapsulation efficiency and the best enzyme activity retention rate, which were 92.57% and 93.31%, respectively.

[0054] Example 8 A method for preparing enteric-coated sustained-release nattokinase gel beads, comprising the following steps: (1) Weigh 1, 3, 5, 7 and 10 g of microcapsules (prepared in Example 1) and disperse them evenly in 50 mL of deionized water; mix each dispersion with 50 mL of sodium alginate aqueous solution (2%, w / w) to obtain suspensions with mass concentrations of 1%, 3%, 5%, 7% and 10% (w / v) of microcapsules in sodium alginate. (2) The suspension was added dropwise to 200 mL of calcium chloride aqueous solution (0.1 mol / L) using a constant flow syringe pump (flow rate: 5 mL / min) to prepare calcium alginate enteric-coated sustained-release gel balls. The encapsulation efficiency, drug loading, enzyme activity retention rate, average particle size and moisture content were tested. The result of the optimized ratio is as follows Figure 5 As shown in the figure. The results indicate that the gel spheres obtained when the mass concentration of microcapsules in sodium alginate is 7% (w / v) are optimal, with the highest encapsulation efficiency of 97.18%.

[0055] As shown in Table 4, when the mass concentration is 7% (w / v), the average particle size of the gel spheres is 1.0~1.5 mm, and the moisture content is less than 7%.

[0056] Table 4. Average particle size and moisture content of gel spheres

[0057] Note: Different letters indicate significant differences. P <0.05) Example 9 The nattokinase enteric-coated microcapsules obtained in Example 1 and the nattokinase enteric-coated sustained-release gel balls obtained in Example 8 were tested for their performance, with calcium alginate gel balls containing only nattokinase as a control.

[0058] 1. Simulates gastrointestinal digestion Nattokinase, microcapsules, and gel beads were placed in simulated gastric juice (SGF, containing 200 U / mL porcine pepsin, 0.2% NaCl, 0.08 mol / L HCl, pH adjusted to 1.20) and digested at 37°C for 30, 60, 90, and 120 minutes, respectively. The pH of the mixture was then adjusted to neutral with NaOH and centrifuged at 6500 g for 5 minutes. The precipitate was washed with water and resuspended in 30 mL of water, then sonicated in an ice bath to fully release NK cells. Fibrinolytic activity was measured, and the retention rate of enzyme activity over time during gastric acid treatment was calculated by comparing with undigested samples. Dynamic curves of residual enzyme activity in the samples were plotted.

[0059] Release kinase, microcapsules, and gel beads were placed in simulated intestinal fluid (SIF, containing 500 U / mL trypsin, 0.68% potassium dihydrogen phosphate, 0.2 mol / L sodium hydroxide, pH adjusted to 6.80) to investigate their release kinetics. After 2 hours of gastric digestion, the mixture was combined with 150 mL of intestinal fluid and digested at 37°C for 60, 120, 180, and 240 minutes, respectively. The reaction solution was centrifuged at 6500 g for 5 minutes, and the supernatant was collected to determine its fibrinolytic activity to evaluate NK release efficiency. Simultaneously, compared with samples not digested in intestinal fluid, the retention rate of enzyme activity over time during intestinal fluid treatment was calculated, and dynamic curves of residual enzyme activity were plotted. A blank control group was set up in the experiment: simulated gastric juice and simulated intestinal juice were mixed at a ratio of 1:5 (volume ratio) (without adding any sample) to subtract the inherent fibrinolytic activity of the digestive enzyme itself. The blank value was subtracted from the sample measurement value to obtain the actual activity data derived from nattokinase.

[0060] The results are as follows Figure 6As shown in the figure, the residual activity changes of naked nattokinase, microcapsules, and gel spheres during simulated digestion reflect the depletion of nattokinase in the stomach and its subsequent release into the intestine. Unencapsulated nattokinase is inactivated within 30 minutes in gastric juice, while microencapsulation technology provides effective protection.

[0061] Two hours later, the microcapsules still retained 92.57% activity, indicating that the composite wall material provided significant protection. The gel spheres retained 94.89% activity, demonstrating that the two-step encapsulation significantly improved resistance to gastric acid. In the intestinal stage, shellac dissolution induced pH-dependent release of nattokinase. The release process of the microcapsules was completed within 4 hours, while the gel spheres maintained stable release for 6 hours, with a release rate exceeding 50%, and a total release time exceeding 10 hours.

[0062] 2. Thermal stability The thermal stability of nattokinase, enteric-coated microcapsules, and enteric-coated sustained-release gel beads was tested.

[0063] 0.5 g of naked nattokinase, microcapsules, and gel spheres were dispersed in 10 mL of deionized water and treated at different temperatures (37, 40, 45, 50, 55, and 60 °C) for 1 hour. Enzyme activity was measured after heat treatment. The fibrinolytic activity of the sample incubated at 37 °C was taken as 100%, and the relative enzyme activity retention rate (expressed as a percentage of this normalized value) of each high-temperature treated sample was calculated.

[0064] The results are as follows Figure 7 As shown, the optimal catalytic temperature for nattokinase is 37°C, at which its enzyme activity reaches its peak. When the temperature exceeds this optimal value, the enzyme structure denatures, leading to a gradual loss of fibrinolytic activity. The fibrinolytic activity of naked NK cells decreases significantly above 45°C and is completely inactivated at 60°C. Microencapsulation improves thermal stability, which is attributed to the heat tolerance of the whey protein matrix and the shellac's reduction of wall material solubility, thereby limiting water permeation. At 60°C, the microcapsules still retain 12.87% of the enzyme activity, while the gel spheres retain 34.13% of the activity.

[0065] 3. pH stability The pH stability of nattokinase, enteric microcapsules, and enteric sustained-release gel beads was tested.

[0066] 0.5 g of naked nattokinase, microcapsules, and gel spheres were dispersed in 10 mL of deionized water at pH values ​​of 1, 3, 5, 7, 8, and 9, respectively. After incubation at 37°C for 1 hour, their fibrinolytic activity was measured. The maximum activity of the sample after incubation in deionized water at pH 8 for 1 hour was taken as 100%, and the relative enzyme activity retention rate (expressed as a percentage of this normalized value) was calculated for each pH condition.

[0067] The results are as follows Figure 8As shown, nattokinase maintains relatively stable and high catalytic activity under weakly alkaline or neutral conditions, but its activity is partially impaired below pH 5 and completely inactivated below pH 3. Under pH 7 and 9 conditions, the fibrinolytic activity of all samples remained essentially intact. In acidic environments (pH < 5), the residual enzyme activity of the microcapsules was significantly higher than that of the unencapsulated nattokinase, and the residual enzyme activity of the gel spheres was very high. These results indicate that constructing a robust barrier effectively protects nattokinase from harsh acidic environments, thereby maximizing its enzyme activity.

[0068] 4. Storage stability The storage stability of nattokinase, enteric-coated microcapsules, and enteric-coated sustained-release gel beads was tested.

[0069] Nattokinase, microcapsules, and gel spheres were stored in dry environments at 4°C and 25°C, respectively. Samples were taken at different time points during the 60-day storage period, and their fibrinolytic activity was determined using fibrin plates. The relative enzyme activity at each time point was calculated with the initial enzyme activity of each sample as 100%.

[0070] The results are as follows: Figure 9 , Figure 10 As shown, free nattokinase exhibits relatively poor stability. After 60 days of refrigeration at 4°C, it retains approximately 82.37% of its thrombolytic activity, while at room temperature (25°C), it retains approximately 79.67% of its thrombolytic activity. Microcapsules with primary encapsulation show some protection compared to free nattokinase under both 4°C and 25°C storage conditions. However, double-layer encapsulated gel spheres demonstrate a more significant protective effect than the former two, exhibiting higher enzyme activity retention and stronger stability within the same storage period. After 60 days of storage, the enzyme activity loss of nattokinase in the double-layer encapsulation system is controlled within 5%, indicating that the double-layer encapsulation structure can form a denser and more stable protective network, significantly improving its long-term storage stability under both room temperature and refrigeration conditions, and is more conducive to maintaining activity during actual production, transportation, and storage.

[0071] Although the present invention has been disclosed above with reference to preferred embodiments, it is not intended to limit the present invention. Anyone skilled in the art can make various modifications and alterations without departing from the spirit and scope of the present invention. Therefore, the scope of protection of the present invention should be determined by the claims.

Claims

1. A method for preparing enteric-coated microcapsules of nattokinase, characterized in that, The steps are as follows: A mixture of whey protein isolate and nattokinase was mixed with shellac solution at a volume ratio of 0.5 to 2:1 and stored overnight to obtain a whey protein isolate-nattokinase-shellac mixture; the mixture was then spray-dried to obtain nattokinase enteric-coated microcapsules. The whey protein isolate-nattokinase mixture is obtained by mixing whey protein isolate solution and nattokinase solution.

2. The method according to claim 1, characterized in that, The concentrations of whey protein isolate, nattokinase, and shellac in the whey protein isolate-nattokinase-shellac mixture were 8-12% w / w, 8-12% w / w, and 8-12% w / w, respectively.

3. The method according to claim 1, characterized in that, Whey protein isolate solution and nattokinase solution are mixed at a volume ratio of 4 to 1:1; whey protein isolate-nattokinase mixed solution is mixed with shellac solution at a volume ratio of 0.5 to 2:

1.

4. The method according to claim 1, characterized in that, The inlet air temperature for spray drying is 165~175℃.

5. The method according to claim 1, characterized in that, Shellac solution is obtained by dissolving shellac in an aqueous solution of ammonium bicarbonate and stirring at 50-60°C for 1-1.5 h.

6. Nattokinase enteric-coated microcapsules prepared by the method according to any one of claims 1 to 5.

7. A nattokinase enteric-coated sustained-release gel ball, characterized in that, The nattokinase enteric-coated microcapsules described in claim 6 are prepared by the following steps: Nattokinase enteric-coated microcapsules were dispersed in water and mixed with sodium alginate aqueous solution to obtain a suspension; the suspension was then added dropwise to calcium chloride aqueous solution to prepare calcium alginate enteric-coated sustained-release gel spheres.

8. The nattokinase enteric-coated sustained-release gel ball according to claim 7, characterized in that, The concentration of nattokinase enteric-coated microcapsules in the suspension was 55–75 g / L, the concentration of sodium alginate aqueous solution was 1.8–2.2 w / w, and the concentration of calcium chloride aqueous solution was 0.08–0.12 mol / L.

9. The nattokinase enteric-coated sustained-release gel ball according to claim 7, characterized in that, The suspension was dripped into the calcium chloride aqueous solution using a constant flow syringe pump.

10. The application of the nattokinase enteric-coated microcapsules of claim 6 or the nattokinase enteric-coated sustained-release gel balls of any one of claims 7 to 9 in the food and pharmaceutical fields.

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