Gastric acid resistant natto powder and preparation method thereof

Abstract

The invention discloses a preparation method of gastric acid resistant natto powder, which comprises the following steps: firstly, adding salt ions with a certain concentration into a natto kinase fermentation concentrated solution to gather natto kinase into a hydrophobic group, and then continuously adding a high-molecular polymer with a hydrophobic effect to wrap the natto kinase hydrophobic group to obtain the gastric acid resistant natto powder. Forming a stable nattokinase activity protection system, and finally drying to prepare a target product, namely the gastric acid-resistant natto powder. The invention also discloses the gastric acid resistant natto powder prepared by the method. Under the condition of certain salt concentration, nattokinase is gathered to form a hydrophobic group, and the hydrophobic group is wrapped by a hydrophobic high-molecular polymer, so that the damage of pepsin is reduced. The gastric acid resistant natto powder can keep more than 80% of nattokinase activity after simulating a gastric acid environment, and solves the problem of gastric acid inactivation of nattokinase.

Description

Technical Field

[0001] The invention relates to the field of health food, and in particular to gastric acid-resistant natto powder and a preparation method thereof. Background Art

[0002] Nattokinase (NK) is an alkaline serine protease produced by the fermentation of Bacillus subtilis. It is also the main active substance in food natto powder. It has strong fibrinolytic, anti-lipid peroxidation and endothelial protection effects, and can significantly reduce blood viscosity, fibrinogen and other blood rheological indicators in the body; it also has the effects of reducing hypertension and hyperlipidemia, promoting cell apoptosis, and regulating autophagy. NK is a natural, safe and efficient ideal thrombolytic agent or dietary supplement, and has great potential for the prevention and treatment of cardiovascular diseases. After oral administration, NK must be absorbed into the blood by the small intestinal epithelial cells through the gastrointestinal tract and transported to the systemic circulation to exert its biological effects. However, NK is subject to many restrictions when it is absorbed by the human body, such as the degradation of NK by gastric acid and protease, and low mucosal permeability. In addition, under the environmental pressure of high temperature, strong acid and alkali, and high salt ion strength, the structure of NK is easily destroyed, and its activity decreases rapidly or even completely inactivated. Therefore, in order to overcome the stress of free NK on the environment during the production, processing, storage, transportation and consumption of food and medicine, it is necessary to reduce the sensitivity of NK to the environment to maintain NK activity and stability; it is necessary to find a suitable preparation method to improve the bioavailability of nattokinase in the human body.

[0003] Natto powder prepared by liquid fermentation of Bacillus subtilis is widely used due to its high production efficiency and convenient operation. However, natto powder prepared by liquid fermentation, removal of bacteria, concentration and drying of Bacillus subtilis is not resistant to gastric acid. After 10 minutes of artificial gastric juice treatment, the enzyme activity is almost 0, which greatly reduces the effect in vivo. Therefore, the preparation of gastric acid-resistant natto powder has important application value. Summary of the invention

[0004] In order to achieve these purposes and other advantages according to the present invention, on the one hand, a preferred embodiment of the present invention provides a method for preparing gastric acid resistant natto powder, comprising the following steps:

[0005] First, a certain concentration of salt ions is added to the nattokinase fermentation concentrate to make the nattokinase aggregate into hydrophobic groups. This is because the presence of salt ions changes the solution environment of the nattokinase, causing changes in its intermolecular interactions, thereby causing the nattokinase to aggregate into hydrophobic groups.

[0006] Then continue to add the high molecular polymer with hydrophobic effect therein, to wrap the nattokinase hydrophobic group, form a stable nattokinase activity protection system, the formation of this protection system, effectively reduces the destruction of nattokinase by pepsin. Pepsin has very strong activity in a gastric acid environment and can decompose protein, but after the protection treatment of the present invention, nattokinase is wrapped by the high molecular polymer, greatly reducing the contact opportunity with pepsin, thereby improving its stability in a gastric acid environment.

[0007] Finally, the target product, gastric acid-resistant natto powder, is prepared by drying. The drying process can remove excess water, making the product easier to store and use.

[0008] According to a preferred embodiment of the present invention, the salt ions are taken from any one or more of sodium chloride, ammonium sulfate, ammonium chloride, sodium sulfate, potassium chloride, potassium sulfate, calcium chloride or calcium sulfate. These salts can ionize different ions in the solution, and by adjusting the ionic strength and type, the aggregation behavior of nattokinase can be accurately controlled to achieve the best protective effect.

[0009] According to a preferred embodiment of the present invention, the salt ion is sodium chloride. Sodium chloride is widely available, cheap, and has a good effect in regulating the aggregation of nattokinase, making it a very ideal choice.

[0010] According to a preferred embodiment of the present invention, when the salt ion is sodium chloride, the concentration of sodium chloride is 0.5-5 mol / L.

[0011] When the salt ion is sodium chloride, after a large number of experimental verifications, when the concentration of sodium chloride is 0.5-5mol / L, nattokinase can be effectively aggregated to form a hydrophobic group, while the subsequent reaction and the activity of nattokinase will not be negatively affected. Within this concentration range, the subsequent high molecular polymer encapsulation step can be ensured to proceed smoothly, thereby forming a stable protection system.

[0012] According to a preferred embodiment of the present invention, the high molecular polymer is selected from any one or more of sodium polyacrylate, konjac glucoside, chitosan, polyglutamic acid, polydextrose, chitin, sodium alginate, cyclodextrin, and carboxymethyl cellulose. These high molecular polymers have different structures and properties, but all have hydrophobic effects and can interact with the hydrophobic group of nattokinase to form a stable encapsulation structure.

[0013] According to a preferred embodiment of the present invention, the high molecular polymer is sodium polyacrylate. Sodium polyacrylate has good solubility and stability, and when encapsulating the hydrophobic group of nattokinase, it can form a compact and stable protective structure, effectively improving the tolerance of nattokinase in a gastric acid environment.

[0014] According to a preferred embodiment of the present invention, when the macromolecular polymer is sodium polyacrylate, the concentration of sodium polyacrylate is 1-8% g / 100ml. Within this concentration range, sodium polyacrylate can fully wrap up the nattokinase hydrophobic group, and will not affect the performance or cost of the product because of the excessive concentration.

[0015] On the other hand, a preferred embodiment of the present invention also provides a gastric acid-resistant natto powder prepared according to the preparation method.

[0016] The present invention at least includes the following beneficial effects: the present invention utilizes that under certain salt concentration conditions, nattokinase aggregates to form hydrophobic groups, which are wrapped by hydrophobic high molecular polymers, thereby reducing the damage of pepsin. The gastric acid-resistant natto powder of the present invention can maintain more than 80% of the nattokinase activity after being subjected to a simulated gastric acid environment, thereby solving the problem of gastric acid inactivation of nattokinase.

[0017] Other advantages, objectives and features of the present invention will be embodied in part through the following description, and in part will be understood by those skilled in the art through study and practice of the present invention. BRIEF DESCRIPTION OF THE DRAWINGS

[0018] Figure 1 This is the microstructure of the gastric acid-resistant natto powder prepared in Example 1. DETAILED DESCRIPTION

[0019] The present invention is further described in detail below in conjunction with embodiments so that those skilled in the art can implement the invention with reference to the description.

[0020] The following description is used to disclose the present invention so that those skilled in the art can implement the present invention. The preferred embodiments described below are only examples, and those skilled in the art can think of other obvious variations. The basic principles of the present invention defined in the following description can be applied to other embodiments, variations, improvements, equivalents, and other technical solutions that do not deviate from the spirit and scope of the present invention.

[0021] Example 1

[0022] The present embodiment provides a method for preparing gastric acid-resistant natto powder, comprising the following steps:

[0023] 1. Prepare fermentation liquid: 2% g / 100 ml of soy peptone, 3% glucose, Na 2 HPO 4 12H 2 O 2%, NaH 2 PO 4 ·H2O 0.5%, MgSO 4 7H 2O 0.02%, CaCl 2 0.02%, mixed according to the proportion to prepare the fermentation liquid;

[0024] 2. Fermentation

[0025] Take frozen seeds, thaw them, and inoculate 0.1% of the liquid culture medium in a shake flask. Shake and culture them in a shaking incubator at 180 rpm and 37°C for 16-18 hours to obtain seed liquid. The liquid culture medium contains: 15 g / L glucose, 15 g / L peptone, 10 g / L yeast extract, 5 g / L disodium hydrogen phosphate dodecahydrate, 1 g / L sodium dihydrogen phosphate monohydrate, 0.2 g / L magnesium sulfate, and pH 7.0-7.6.

[0026] The Bacillus subtilis seed solution is inoculated into the fermentation solution at a ratio of 3% ml / 100 ml, and the fermentation solution is cultured for 20 hours at 30% dissolved oxygen, 35-40° C., and 300-600 rpm;

[0027] 3. Separation: Centrifuge the fermentation liquid to obtain the supernatant, and concentrate it through a filter membrane to obtain the nattokinase fermentation concentrate.

[0028] 4. Determination of Nattokinase Activity:

[0029] (1) Reagent preparation:

[0030] Solution A: Weigh 121.1g of Tris and add water to dissolve it. Use about 42ml of concentrated hydrochloric acid to adjust the pH to 8.0. Finally, dilute to 1L as the mother solution. Take 10ml of the mother solution and dilute to 500ml with water to obtain Solution A.

[0031] Solution B: Weigh 9g of NaCl, add water to dissolve, and make up to 1L.

[0032] 50mg / ml fibrinogen solution: weigh 1g of fibrinogen powder and dissolve it in 20ml of solution A to fully dissolve it.

[0033] 100U / ml thrombin solution: Dissolve 1000U thrombin in 10ml of solution A and allow it to fully dissolve.

[0034] Urokinase solution standard: First, take 2 ml of sterile saline (0.9% W / V) to dissolve the urokinase standard (1240 IU / tube) to make a 620 IU / ml solution. Then take 1 ml from the above solution into another small tube containing 1 ml of saline to make a 310 IU / ml solution, and dilute it into 155 IU / ml, 77.5 IU / ml, 38.75 IU / ml, and 19.375 IU / ml standard urokinase solutions in sequence for use.

[0035] (2) Experimental methods:

[0036] Preparation of fibrin plate: weigh 0.15g agar (0.75%) and 0.1755g NaCl (0.15M), add to a 50ml small conical flask; measure 20ml of solution A to dissolve the agar; stopper the conical flask and place it in a microwave oven to heat and dissolve; when the conical flask cools to 50℃, add 1ml of 50mg / ml fibrinogen solution and 100μl of 100U / ml thrombin to the flask, mix well and quickly pour into a sterilized plate; leave at room temperature for 30min to form a fibrin clot.

[0037] Preparation of the sample solution: Take an appropriate amount of the sample to be tested, add Solution B to dissolve it, and dilute it to a concentration within the range of the standard curve. If the sample is solid, centrifuge it after dissolution and take the supernatant for testing.

[0038] Draw a urokinase standard curve: use a hole puncher to make 7 holes on the fibrin plate and mark them; take 10μl of standard urokinase solution of each concentration into the corresponding hole, place it for 10 minutes and then transfer it to a 37℃ constant temperature incubator for reaction. After 18 hours, take out and measure the two vertical diameters of each dissolution circle; set three parallel groups for the above operation, and take the average value after measurement; then use the common logarithm of the product of the vertical diameters (S) as the horizontal axis and the common logarithm of the standard urokinase concentration as the vertical axis to make a urokinase enzyme activity standard curve.

[0039] Determination of thrombolytic activity of the sample to be tested: Take 10ul of the sample to be tested and load it on the same plate according to the above operation, react in a constant temperature incubator at 37℃, measure the two vertical diameters of the dissolution circle after 18 hours, and calculate the enzyme activity of the sample according to the standard curve method and dilution concentration.

[0040] 5. Add sodium chloride to the fermentation concentrate at a final concentration of 2 mol / L, dissolve it fully, and stir to mix.

[0041] 6. According to the final concentration of sodium polyacrylate 4% g / 100ml and the nattokinase activity 2w IU / ml, the sodium polyacrylate and the fermentation concentrate were fully stirred and mixed at a pH value of 7.0, and finally dried to obtain gastric acid-resistant natto powder.

[0042] Figure 1 This is the microstructure of the gastric acid-resistant natto powder prepared in Example 1.

[0043] Example 2

[0044] The present embodiment provides a method for preparing gastric acid-resistant natto powder, comprising the following steps:

[0045] 1. Prepare fermentation liquid: Soybean peptone 1% g / 100ml, glucose 2%, Na 2 HPO 4 12H 2 O 1%, NaH2 PO 4 ·H2O 0.1%, MgSO 4 7H 2 O 0.01%, CaCl 2 0.01%, mixed according to the proportion to prepare the fermentation liquid;

[0046] 2. Fermentation

[0047] Take frozen seeds, thaw them, and inoculate 0.1% of the liquid culture medium in a shake flask. Shake and culture them in a shaking incubator at 180 rpm and 37°C for 16-18 hours to obtain seed liquid. The liquid culture medium contains: 15 g / L glucose, 15 g / L peptone, 10 g / L yeast extract, 5 g / L disodium hydrogen phosphate dodecahydrate, 1 g / L sodium dihydrogen phosphate monohydrate, 0.2 g / L magnesium sulfate, and pH 7.0-7.6.

[0048] The Bacillus subtilis seed solution is inoculated into the fermentation solution at a ratio of 1% ml / 100 ml, and cultured at 30% dissolved oxygen, 35-40° C., and 300-600 rpm for 18 hours to obtain the fermentation stock solution;

[0049] 3. Separation: The fermentation liquid is centrifuged to obtain the supernatant, and then concentrated through a filter membrane to obtain the fermentation concentrate.

[0050] 4. Determination of Nattokinase Activity:

[0051] (1) Reagent preparation:

[0052] Solution A: Weigh 121.1g of Tris and add water to dissolve it. Use about 42ml of concentrated hydrochloric acid to adjust the pH to 8.0. Finally, dilute to 1L as the mother solution. Take 10ml of the mother solution and dilute to 500ml with water to obtain Solution A.

[0053] Solution B: Weigh 9g of NaCl, add water to dissolve, and make up to 1L.

[0054] 50mg / ml fibrinogen solution: weigh 1g of fibrinogen powder and dissolve it in 20ml of solution A to fully dissolve it.

[0055] 100U / ml thrombin solution: Dissolve 1000U thrombin in 10ml of solution A and allow it to fully dissolve.

[0056] Urokinase solution standard: First, take 2 ml of sterile saline (0.9% W / V) to dissolve the urokinase standard (1240 IU / tube) to make a 620 IU / ml solution. Then take 1 ml from the above solution into another small tube containing 1 ml of saline to make a 310 IU / ml solution, and dilute it into 155 IU / ml, 77.5 IU / ml, 38.75 IU / ml, and 19.375 IU / ml standard urokinase solutions in sequence for use.

[0057] (2) Experimental methods:

[0058] Preparation of fibrin plate: weigh 0.15g agar (0.75%) and 0.1755g NaCl (0.15M), add to a 50ml small conical flask; measure 20ml of solution A to dissolve the agar; stopper the conical flask and place it in a microwave oven to heat and dissolve; when the conical flask cools to 50℃, add 1ml of 50mg / ml fibrinogen solution and 100μl of 100U / ml thrombin to the flask, mix well and quickly pour into a sterilized plate; leave at room temperature for 30min to form a fibrin clot.

[0059] Preparation of the sample solution: Take an appropriate amount of the sample to be tested, add Solution B to dissolve it, and dilute it to a concentration within the range of the standard curve. If the sample is solid, centrifuge it after dissolution and take the supernatant for testing.

[0060] Draw a urokinase standard curve: use a hole puncher to make 7 holes on the fibrin plate and mark them; take 10μl of standard urokinase solution of each concentration into the corresponding hole, place it for 10 minutes and then transfer it to a 37℃ constant temperature incubator for reaction. After 18 hours, take out and measure the two vertical diameters of each dissolution circle; set three parallel groups for the above operation, and take the average value after measurement; then use the common logarithm of the product of the vertical diameters (S) as the horizontal axis and the common logarithm of the standard urokinase concentration as the vertical axis to make a urokinase enzyme activity standard curve.

[0061] Determination of thrombolytic activity of the sample to be tested: Take 10ul of the sample to be tested and load it on the same plate according to the above operation, react in a constant temperature incubator at 37℃, measure the two vertical diameters of the dissolution circle after 18 hours, and calculate the enzyme activity of the sample according to the standard curve method and dilution concentration.

[0062] 5. Add sodium chloride to the fermentation concentrate at a final concentration of 0.5 mol / L, dissolve it fully, and stir to mix.

[0063] 6. According to the final concentration of sodium polyacrylate 1% g / 100ml and the nattokinase activity 1wIU / ml, the sodium polyacrylate and the fermentation concentrate were fully stirred and mixed at a pH value of 7.0, and finally dried to obtain gastric acid-resistant natto powder.

[0064] Example 3

[0065] The present embodiment provides a method for preparing gastric acid-resistant natto powder, comprising the following steps:

[0066] 1. Prepare fermentation liquid: 3% g / 100ml of soy peptone, 5% glucose, Na 2 HPO 4 12H 2 O 2%, NaH 2 PO 4 ·H2O 1%, MgSO 4 7H 2 O 0.05%, CaCl 2 0.02%, mixed according to the proportion to prepare the fermentation liquid;

[0067] 2. Fermentation

[0068] Take frozen seeds, thaw them, and inoculate 0.1% of the liquid culture medium in a shake flask. Shake and culture them in a shaking incubator at 180 rpm and 37°C for 16-18 hours to obtain seed liquid. The liquid culture medium contains: 15 g / L glucose, 15 g / L peptone, 10 g / L yeast extract, 5 g / L disodium hydrogen phosphate dodecahydrate, 1 g / L sodium dihydrogen phosphate monohydrate, 0.2 g / L magnesium sulfate, and pH 7.0-7.6.

[0069] The Bacillus subtilis seed solution is inoculated into the fermentation solution at a ratio of 5% ml / 100 ml, and cultured at 30% dissolved oxygen, 35-40° C., and 300-600 rpm for 24 hours to obtain the fermentation stock solution;

[0070] 3. Separation: The fermentation liquid is centrifuged to obtain the supernatant, and then concentrated through a filter membrane to obtain the fermentation concentrate.

[0071] 4. Determination of Nattokinase Activity:

[0072] (1) Reagent preparation:

[0073] Solution A: Weigh 121.1g of Tris and add water to dissolve it. Use about 42ml of concentrated hydrochloric acid to adjust the pH to 8.0. Finally, dilute to 1L as the mother solution. Take 10ml of the mother solution and dilute to 500ml with water to obtain Solution A.

[0074] Solution B: Weigh 9g of NaCl, add water to dissolve, and make up to 1L.

[0075] 50mg / ml fibrinogen solution: weigh 1g of fibrinogen powder and dissolve it in 20ml of solution A to fully dissolve it.

[0076] 100U / ml thrombin solution: Dissolve 1000U thrombin in 10ml of solution A and allow it to fully dissolve.

[0077] Urokinase solution standard: First, take 2 ml of sterile saline (0.9% W / V) to dissolve the urokinase standard (1240 IU / tube) to make a 620 IU / ml solution. Then take 1 ml from the above solution into another small tube containing 1 ml of saline to make a 310 IU / ml solution, and dilute it into 155 IU / ml, 77.5 IU / ml, 38.75 IU / ml, and 19.375 IU / ml standard urokinase solutions in sequence for use.

[0078] (2) Experimental methods:

[0079] Preparation of fibrin plate: weigh 0.15g agar (0.75%) and 0.1755g NaCl (0.15M), add to a 50ml small conical flask; measure 20ml of solution A to dissolve the agar; stopper the conical flask and place it in a microwave oven to heat and dissolve; when the conical flask cools to 50℃, add 1ml of 50mg / ml fibrinogen solution and 100μl of 100U / ml thrombin to the flask, mix well and quickly pour into a sterilized plate; leave at room temperature for 30min to form a fibrin clot.

[0080] Preparation of the sample solution: Take an appropriate amount of the sample to be tested, add Solution B to dissolve it, and dilute it to a concentration within the range of the standard curve. If the sample is solid, centrifuge it after dissolution and take the supernatant for testing.

[0081] Draw a urokinase standard curve: use a hole puncher to make 7 holes on the fibrin plate and mark them; take 10μl of standard urokinase solution of each concentration into the corresponding hole, place it for 10 minutes and then transfer it to a 37℃ constant temperature incubator for reaction. After 18 hours, take out and measure the two vertical diameters of each dissolution circle; set three parallel groups for the above operation, and take the average value after measurement; then use the common logarithm of the product of the vertical diameters (S) as the horizontal axis and the common logarithm of the standard urokinase concentration as the vertical axis to make a urokinase enzyme activity standard curve.

[0082] Determination of thrombolytic activity of the sample to be tested: Take 10ul of the sample to be tested and load it on the same plate according to the above operation, react in a constant temperature incubator at 37℃, measure the two vertical diameters of the dissolution circle after 18 hours, and calculate the enzyme activity of the sample according to the standard curve method and dilution concentration.

[0083] 5. Add sodium chloride to the fermentation concentrate at a final concentration of 5 mol / L, dissolve it fully, and stir to mix.

[0084] 6. According to the final concentration of sodium polyacrylate 8% g / 100ml and the nattokinase activity 3w IU / ml, the sodium polyacrylate and the fermentation concentrate were fully stirred and mixed at a pH value of 7.0, and finally dried to obtain gastric acid-resistant natto powder.

[0085] Comparative Example 1

[0086] The preparation method is the same as that of Example 1, except that step 6 does not contain sodium polyacrylate.

[0087] Comparative Example 2

[0088] The preparation method is the same as that of Example 1, except that step 5 is omitted, i.e., sodium chloride is not contained.

[0089] Comparative Example 3

[0090] The preparation method is the same as that of Example 1, except that sodium chloride and sodium polyacrylate are not contained, and ordinary natto powder is directly dried to obtain the natto powder.

[0091] Comparative Example 4

[0092] The preparation method is the same as that of Example 1, except that step 6 does not contain sodium polyacrylate but is replaced by 4% γ-polyglutamic acid.

[0093] Comparative Example 5

[0094] The preparation method is the same as that of Example 1, except that step 5 is omitted and step 6 does not contain sodium polyacrylate but is replaced by 4% γ-polyglutamic acid.

[0095] Comparative Example 6

[0096] The preparation method is the same as that of Example 1, except that step 6 does not contain sodium polyacrylate but is replaced with 1% chitosan.

[0097] Comparative Example 7

[0098] The preparation method is the same as that of Example 1, except that step 6 does not contain sodium polyacrylate and is replaced by 5% β-cyclodextrin.

[0099] Comparative Example 8

[0100] The preparation method is the same as that of Example 1, except that step 6 does not contain sodium polyacrylate but is replaced with 1% sodium alginate.

[0101] Comparative Example 9

[0102] The preparation method is the same as that of Example 1, except that step 6 does not contain sodium polyacrylate and is replaced by 5% sodium carboxymethyl cellulose.

[0103] Comparative Example 10

[0104] The preparation method is the same as that of Example 1, except that step 6 does not contain sodium polyacrylate but is replaced with 2% konjac glucoside.

[0105] Comparative Example 11

[0106] The preparation method is the same as that of Example 1, except that the fermentation concentrate obtained in step 3 is purified by composite gel filtration chromatography and cross-linked agarose ion chromatography to obtain a pure product of nattokinase with a purity greater than or equal to 95%. Step 6 does not contain sodium polyacrylate.

[0107] Comparative Example 12

[0108] The preparation method is the same as that of Comparative Example 11, except that step 5 is not included.

[0109] The gastric acid-resistant natto powder and common natto powder prepared in Examples 1-3 and Comparative Examples 1-12 were prepared to have the same activity and placed in artificial gastric juice at 37°C and 180 r / min for 2 hours, and then the nattokinase activity was determined according to step 4 in Example 1. The results are as follows:

[0110] Table 1 Activity comparison before and after artificial gastric juice treatment

[0111]

[0112]

[0113] The results are shown in the table above: the activity of ordinary natto powder in the three groups of comparative examples in artificial gastric juice almost completely disappeared after 2 hours; the gastric acid-resistant natto powder in the groups of Examples 1-3 can effectively protect nattokinase, and the residual enzyme activity in artificial gastric juice after 2 hours is the highest, which is 82.60%, 83.23% and 82.44% respectively; the residual enzyme activity of natto powder in the groups of Comparative Examples 1 and 2 is 18.12% and 68.71%, both lower than that in the group of Example 1, indicating that both sodium chloride and sodium polyacrylate can enhance the protection of nattokinase in the gastric juice environment, but the protective effect of sodium polyacrylate is more prominent, and the combination of the two has a more significant effect; the residual enzyme activity of natto powder in the group of Comparative Example 4 is 64.29%, which is lower than 82.60% in the group of Example 1, and the residual enzyme activity of natto powder in the group of Comparative Example 5 is 40.58%, which is lower than 68.71% in the group of Comparative Example 2, indicating that the protective effect of sodium polyacrylate is better than that of γ-polyglutamic acid. The residual enzyme activity of natto powder in groups 6-10 of comparative examples was lower than 82.60% of that in group 1 of embodiment, indicating that the protective effect of the combination of sodium chloride and sodium polyacrylate was optimal. The residual enzyme activity of nattokinase in group 11 of comparative example was 10.23%, which was higher than that in group 12 of comparative example, indicating that the stability of nattokinase can be improved under conditions above a certain salt concentration, and nattokinase is also suitable for purification process.

[0114] Sodium polyacrylate is a food additive. It is a high molecular compound with both hydrophilic and hydrophobic groups. It can enhance the bonding force between proteins and form a dense and stable system. Under a certain concentration of salt ions, nattokinase aggregates to form hydrophobic groups. Then, a high molecular polymer with hydrophobic effect is added to wrap the hydrophobic groups of nattokinase, forming a more stable nattokinase activity protection system, thereby reducing the damage of pepsin and improving the effect of resisting gastric acid.

[0115] Thrombolytic function assay

[0116] 70 mice were randomly divided into 7 groups, each with 10 mice, namely blank control group, model group, Example 1 group, and Comparative Examples 1 to 4 groups. The mice were starved for 8 hours before the formal experiment.

[0117] On the first day of the experiment, the other 6 groups of mice except the blank group were intraperitoneally injected with 300 mg / kg of carrageenan to induce tail thrombosis. At the same time, the mice in Example 1 group and Comparative Examples 1-4 groups were gavaged with 20,000 IU / kg of the corresponding natto powder. The blank control group and the model group were gavaged with equal volumes of normal saline. The gavage was continued for 7 days. Free drinking water, the temperature was 20-25°C. After stopping the gavage for 1 day, the total tail length of the mice and the length of the tail with thrombosis were counted. The results showed that the average length of the tail thrombus in the model group was 6.46 cm, proving that the model was successful.

[0118] Table 2 Comparison of thrombolytic efficacy of natto powder in each group

[0119]

[0120]

[0121] The mean lengths of the tail thrombi in Example 1 and Comparative Examples 1-4 were 1.02 cm, 3.31 cm, 2.04 cm, 4.57 cm and 2.35 cm, respectively. Compared with the comparative examples, the gastric acid-resistant natto powder of the present invention can significantly enhance the active protection of nattokinase in gastric juice, and can be smoothly absorbed into the intestine and into the blood to exert its anti-thrombotic effect.

[0122] Although the embodiments of the present invention have been disclosed as above, they are not limited to the applications listed in the specification and the implementation modes. They can be fully applied to various fields suitable for the present invention. For those familiar with the art, additional modifications can be easily implemented. Therefore, without departing from the general concept defined by the claims and the scope of equivalents, the present invention is not limited to the specific details and the embodiments shown and described herein.

Claims

1. A method for preparing gastric acid-resistant natto powder, characterized in that: The following steps are involved: First, a certain concentration of salt ions is added to the nattokinase fermentation concentrate to make the nattokinase aggregate into hydrophobic groups, and then a high molecular polymer with hydrophobic effect is added thereto to wrap the nattokinase hydrophobic groups to form a stable nattokinase activity protection system, and finally the target product, gastric acid-resistant natto powder, is prepared by drying.

2. The method for preparing gastric acid-resistant natto powder according to claim 1, characterized in that: The salt ions are selected from any one or more of sodium chloride, ammonium sulfate, ammonium chloride, sodium sulfate, potassium chloride, potassium sulfate, calcium chloride or calcium sulfate.

3. The method for preparing gastric acid-resistant natto powder according to claim 2, characterized in that: The salt ion is sodium chloride.

4. The method for preparing gastric acid-resistant natto powder according to claim 2, characterized in that: When the salt ion is sodium chloride, the concentration of sodium chloride is 0.5-5 mol / L.

5. The method for preparing gastric acid-resistant natto powder according to claim 1, characterized in that: The high molecular polymer is selected from any one or more of sodium polyacrylate, konjac glucoside, chitosan, polyglutamic acid, polydextrose, chitin, sodium alginate, cyclodextrin and carboxymethyl cellulose.

6. The method for preparing gastric acid-resistant natto powder according to claim 5, characterized in that: The high molecular polymer is sodium polyacrylate.

7. The method for preparing gastric acid-resistant natto powder according to claim 6, characterized in that: When the high molecular polymer is sodium polyacrylate, the concentration of sodium polyacrylate is 1-8% g / 100 ml.

8. Gastric acid-resistant natto powder prepared according to the preparation method described in any one of claims 1 to 7.

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