CN112402624B - Nattokinase sustained-release microsphere and preparation method thereof - Google Patents

Nattokinase sustained-release microsphere and preparation method thereof Download PDF

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CN112402624B
CN112402624B CN202011271511.9A CN202011271511A CN112402624B CN 112402624 B CN112402624 B CN 112402624B CN 202011271511 A CN202011271511 A CN 202011271511A CN 112402624 B CN112402624 B CN 112402624B
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nattokinase
sustained
chitosan
water
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CN112402624A (en
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杨瑞金
张轩
仝艳军
吕小妹
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Suzhou Forsyth Biotechnology Co ltd
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Jiangnan University
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Abstract

The invention discloses a nattokinase sustained-release microsphere and a preparation method thereof, belonging to the technical field of food or medicines. According to the slow release microsphere, chitosan is used as an inner core auxiliary material, and nattokinase is solidified on the inner core through a cross-linking agent, so that the effects of enhancing the stability of enzyme and slow release are achieved. On the basis, mTG enzyme is taken as a cross-linking agent to cross-link casein on the outer layer of the microsphere to form the sandwich-like nattokinase sustained-release microsphere, so that the gastric acid resistance of the nattokinase in the microsphere can be further improved, and the action time and the antithrombotic effect of the nattokinase can be prolonged. The two nattokinase-chitosan microspheres can be used as an oral preparation of nattokinase, can be added into food and beverage as an ingredient, and can also be used for producing nattokinase slow-release microsphere powder or/and concentrated solution. Compared with pure zymoprotein, the nattokinase slow-release microsphere of the invention used as a functional factor additive can improve the shelf life of antithrombotic functional food and the bioavailability of functional factors.

Description

Nattokinase sustained-release microsphere and preparation method thereof
Technical Field
The invention relates to a nattokinase sustained-release microsphere and a preparation method thereof, belonging to the technical field of food or medicines.
Background
In modern social life, blood in human blood vessels is often in a high-viscosity state, thrombus formation in the blood vessels can cause serious cardiovascular diseases such as cerebral infarction, myocardial infarction and the like, particularly great harm is caused to the health of middle-aged and elderly people, 1500 ten thousands of patients with thrombus exist all over the world, potential thrombolytic agents have $ 20 billion in the market, but the current thrombolytic agents such as urokinase, streptokinase, recombinant tissue plasminogen activator and the like have various defects of short half-life period, great side effect, high price, incapability of oral administration and the like, and are difficult to become applicable public medicines. The nattokinase has the advantages of good safety, quick action, low cost, oral administration and the like. The enzyme can be produced by fermentation of Bacillus subtilis. Therefore, the method is used for developing a new generation of thrombolytic agent or health food and has extremely attractive and wide prospect.
Most of the current nattokinase products are processed into concentrated agents for oral administration, most of the nattokinase products are tablets or capsules, the production process is that the nattokinase dry powder obtained by fermentation and extraction is added with auxiliary materials and then directly pressed into tablets or pills or filled into capsules, the manufactured nattokinase preparation is inactivated quickly in the gastric environment, and even if the nattokinase preparation is protected by viscous substances such as chitosan, gelatin, sodium alginate, cyclodextrin and the like, the effective rate of the nattokinase which enters blood after being absorbed through the digestive tract is still low.
Therefore, it is necessary to adopt a more efficient method for protecting the enzymatic activity of nattokinase, and improve the shelf life and the slow release effect in the digestive tract and the absorption efficiency of the product.
Disclosure of Invention
In order to solve the problems, the invention provides a nattokinase sustained-release microsphere and application thereof. The diameter of the slow-release microsphere is 300nm-300 mu m, chitosan is used as a core, natto kinase is crosslinked on the chitosan microsphere, and furthermore, proteins such as cow milk casein and the like are used as a protective agent of the natto kinase and are crosslinked on the surface of the particle (the particle design is shown in figure 1A). The sustained-release microspheres have the advantages of long enzyme activity retention time, gastric acid resistance and wide application prospect.
The invention aims to provide a nattokinase slow-release microsphere, which comprises the step of crosslinking nattokinase to chitosan slow-release microsphere, wherein the diameter of the nattokinase slow-release microsphere is 300nm-300 mu m.
In one embodiment, the diameter is 300nm to 30 μm.
In one embodiment, the cross-linking agent used to cross-link nattokinase to the chitosan microspheres (i.e., cross-linking agent B) is genipin.
In one embodiment, the natto kinase is crosslinked to the chitosan microspheres, and the method comprises the steps of dripping a crosslinking agent onto the chitosan microspheres, dripping natto kinase solution, and reacting for a period of time to obtain the natto kinase slow-release microspheres.
In one embodiment, the concentration of the nattokinase solution is 1-100 mg/ml.
In one embodiment, the nattokinase is produced by fermentation of bacillus subtilis.
In one embodiment, the chitosan microspheres and nattokinase are added in a mass ratio of 5: 1-50: 1.
In one embodiment, the reaction time is from 2h to 10 h.
In one embodiment, the nattokinase is cross-linked to the chitosan microsphere, and specifically comprises the following components: dissolving genipin in 10-90% ethanol solution to prepare a crosslinking agent B with the genipin concentration of 1-10 mg/mL, dropwise adding the crosslinking agent B onto the chitosan microspheres until the chitosan microspheres are wet, dropwise adding a nattokinase solution with the concentration of 5-50mg/mL for crosslinking, reacting for 2-10 h, washing for multiple times, and freeze-drying to obtain the nattokinase sustained-release microspheres.
In one embodiment, the nattokinase slow-release microspheres further comprise protein cross-linked to chitosan microspheres cross-linked with nattokinase to obtain protein-embedded nattokinase slow-release microspheres.
In one embodiment, the protein is casein.
In one embodiment, the protein is further crosslinked using a crosslinking agent (i.e., crosslinker C) that is Transglutaminase (TG).
In one embodiment, the further protein crosslinking is to add a protein solution and a corresponding crosslinking agent into chitosan microspheres crosslinked with nattokinase, stir for reaction for a period of time, centrifuge after the reaction, remove supernatant, and wash to obtain the protein-embedded nattokinase sustained-release microspheres.
In one embodiment, the stirring reaction time is 10min to 4 h.
In one embodiment, the protein is further cross-linked, in particular: dissolving TG enzyme in water to prepare a cross-linking agent C with the concentration of 5-20mg/mL, and dispersing protein in water to prepare a protein solution with the concentration of 10-80 mg/mL; and then adding the protein solution and the cross-linking agent C into the chitosan microspheres cross-linked with the nattokinase, stirring for reaction, centrifuging, discarding supernatant, and washing to obtain the protein-embedded nattokinase sustained-release microspheres.
In one embodiment, the chitosan microspheres are prepared by an emulsion crosslinking method.
In one embodiment, the preparation of chitosan microspheres:
(1) mixing an emulsifier and an organic solvent according to a certain proportion to prepare an oil phase;
(2) adding chitosan into acetic acid solution with volume concentration of 1-3%, magnetically stirring for 1-24 hr to dissolve completely, standing to obtain water phase with chitosan mass concentration of 0.3-3%;
(3) dissolving sodium tripolyphosphate in water to prepare a cross-linking agent A with the concentration of the sodium tripolyphosphate of 3mg/mL-30 mg/mL;
(4) dropwise adding the water phase obtained in the step (2) into the oil phase obtained in the step (1) while stirring according to the volume ratio of the water phase to the oil phase of 1:1-1:10, and continuously stirring for 10min-6h to obtain a W/O type emulsion;
(5) dropwise adding the cross-linking agent A obtained in the step (3) into the emulsion obtained in the step (4) for one time or multiple times under the stirring condition, stirring for 0.5-12h, centrifuging at high speed, removing the upper layer, washing the solid at the lower layer of the precipitate for multiple times, and drying in vacuum to obtain the chitosan microspheres.
In one embodiment, the nattokinase sustained-release microspheres are liquid preparations or dry powder preparations.
Specifically, the preparation method of the nattokinase sustained-release microsphere comprises the following two methods:
the method comprises the following steps: genipin cross-linking, the steps are as follows:
1) mixing an emulsifier and an organic solvent according to the volume ratio of the emulsifier to the organic solvent of 1:100-1:10 to prepare an oil phase, wherein the emulsifier is span-80, tween-80 or diethylene glycol monooleate, and the organic solvent is liquid paraffin or ethyl benzoate;
2) adding chitosan into acetic acid solution with volume concentration of 1-3%, magnetically stirring for 1-24 hr to dissolve completely, standing to obtain water phase with chitosan mass concentration of 0.3-3%;
3) dissolving sodium tripolyphosphate in water to prepare a cross-linking agent A with the concentration of the sodium tripolyphosphate of 3mg/mLl-30 mg/mL;
4) dissolving genipin in 10-90% ethanol solution to obtain crosslinking agent B with genipin concentration of 1-10 mg/mL;
5) dispersing nattokinase in water to prepare aqueous solution with the concentration of 5-50 mg/mL;
6) dropwise adding the water phase obtained in the step 2) into the oil phase obtained in the step 1) while stirring according to the volume ratio of the water phase to the oil phase of 1:1-1:10, and continuously stirring for 10min-6h to obtain the W/O type emulsion.
7) Dropwise adding the cross-linking agent A obtained in the step 3) into the emulsion obtained in the step 6) once or for multiple times under the stirring condition, stirring for 0.5-12h, centrifuging at high speed, removing the upper layer, washing the solid at the lower layer of the precipitate with one or two solvents of petroleum ether, acetone, isopropanol, ethanol and chloroform for 2-3 times respectively, and drying in vacuum to obtain the chitosan microspheres with white powder appearance.
8) Dripping the cross-linking agent B obtained in the step 4) on the microspheres obtained in the step 7) until the microspheres are wetted, dripping the nattokinase solution obtained in the step 5) for cross-linking, washing for multiple times after reacting for 0.5-10h, and freeze-drying to obtain the chitosan-nattokinase sustained-release microspheres.
The second method comprises the following steps: genipin-TG enzyme crosslinking, the steps are as follows:
1) mixing an emulsifier and an organic solvent according to the volume ratio of the emulsifier to the organic solvent of 1:100-1:10 to prepare an oil phase, wherein the emulsifier is span-80, tween-80 or diethylene glycol monooleate, and the organic solvent is liquid paraffin or ethyl benzoate;
2) adding chitosan into acetic acid solution with volume concentration of 1-3%, magnetically stirring for 1-24 hr to dissolve completely, standing to obtain water phase with chitosan mass concentration of 0.3-3%;
3) dissolving sodium tripolyphosphate in water to prepare a cross-linking agent A with the concentration of the sodium tripolyphosphate of 3mg/mL-30 mg/mL;
4) dissolving genipin in 10-90% ethanol solution to obtain crosslinking agent B with genipin concentration of 1-10 mg/mL, and dissolving TG enzyme in water to obtain crosslinking agent C with concentration of 5-15 mg/mL;
5) dispersing nattokinase in water to prepare aqueous solution with the concentration of 5-50 mg/mL; dispersing cow milk casein in water to prepare 20-70mg/mL aqueous solution;
6) dropwise adding the water phase obtained in the step 2) into the oil phase obtained in the step 1) while stirring according to the volume ratio of the water phase to the oil phase of 1:1-1:10, and continuously stirring for 10min-6h to obtain W/O type emulsion;
7) dropwise adding the cross-linking agent A obtained in the step 3) into the emulsion obtained in the step 6) once or for multiple times under the stirring condition, stirring for 0.5-12h, centrifuging at a high speed, removing the upper layer, washing the solid at the lower layer of the precipitate with one or two solvents of petroleum ether, acetone, isopropanol, ethanol and chloroform for 2-3 times respectively, and drying in vacuum to obtain chitosan microspheres with white powder appearance;
8) adding the nattokinase solution and the cross-linking agent B into the chitosan microspheres obtained in the step 7), adding water, stirring for reaction, and centrifuging to obtain precipitates;
9) washing the precipitate obtained in the step 8) for many times, adding a casein solution and a cross-linking agent C, and stirring for reaction;
10) centrifuging the reaction solution and removing the supernatant; and (4) washing the microspheres with water, and freeze-drying to obtain the casein-embedded nattokinase sustained-release microspheres.
In one embodiment, the ethanol solution in the step 4) has a concentration of 30% (v/v) ethanol and the concentration of the cross-linking agent B is 3 mg/mL. If crosslinker C is present, its concentration may be 10 mg/mL.
In one embodiment, the step 5) of dispersing nattokinase in water is to prepare an aqueous solution with the concentration of 10 mg/mL. If a bovine milk casein solution is used, the concentration may be 50 mg/mL.
In one embodiment, the step 8): taking 5g of the chitosan microspheres obtained in the step 7), adding 40mL of nattokinase solution and 5mL of cross-linking agent B, adding water to 50mL, stirring at 37 ℃ for reaction for 8h, and centrifuging to obtain precipitates.
In one embodiment, the step 9): the precipitate obtained in step 8) was washed with water several times, 20mL of casein solution and 5mL of crosslinking agent C were added, and the reaction was stirred at 37 ℃ for 4 hours.
In one embodiment, the step (10): the reaction solution was centrifuged at 1000g for 5min to discard the supernatant. Washing the microsphere with water for 2-3 times, and freeze drying to obtain casein-embedded nattokinase sustained-release microsphere in blue powder.
In one embodiment, the drug loading rate of the nattokinase is 1000-50000 mu g/g microspheres. Optionally, the activity of the nattokinase is 1000-100000 IU/g of microspheres.
The second purpose of the invention is to provide a liquid preparation, which contains the nattokinase slow-release microspheres.
The third purpose of the invention is to provide a solid preparation, which contains the nattokinase slow-release microspheres.
The preparation can be an oral preparation, and the oral preparation contains the nattokinase slow-release microspheres. Can be used for preventing or treating thrombotic cardiovascular diseases.
In one embodiment, the oral preparation takes the nattokinase sustained-release microspheres with effective dose as the main active ingredient, and can be prepared into any preparation formulation suitable for clinical use, such as tablets, capsules, granules, powder, liquid preparations, enemas and the like, by adding conventional pharmaceutical excipients, flavoring agents, disintegrating agents, preservatives, lubricants, wetting agents, binding agents, solvents, thickening agents, solubilizing agents and the like according to a certain preparation process.
The fifth purpose of the invention is to provide a health food which contains the nattokinase slow-release microspheres.
The invention has the following beneficial effects:
(1) according to the slow release microsphere, chitosan is used as an inner core auxiliary material, and nattokinase is solidified on the inner core through a cross-linking agent, so that the effects of enhancing the stability and slow release of enzyme are achieved; the nattokinase-chitosan microspheres solidified by the genipin have better stability in acid gastric juice, can be gradually slowly released in gastrointestinal tracts and keep thrombolytic activity; on the basis, mTG enzyme is taken as a cross-linking agent to cross-link casein on the outer layer of the microsphere to form the sandwich-like nattokinase sustained-release microsphere, so that the gastric acid resistance of the nattokinase in the microsphere can be further improved, and the action time and the antithrombotic effect of the nattokinase can be prolonged; in summary, compared with the existing nattokinase preparation, on one hand, the nattokinase slow-release microsphere prepared by the invention is fixed on the microsphere through chemical crosslinking, so that the stability of the nattokinase slow-release microsphere is greatly enhanced while the activity is maintained; on the other hand, the nattokinase sustained-release microspheres have acid resistance after being orally taken, can be gradually and slowly released and absorbed in the alimentary canal to enter blood circulation and keep thrombolytic activity, prolongs the effective action time of the nattokinase in the body and improves the antithrombotic effect.
(2) The nattokinase slow-release microspheres can be used as an oral preparation of nattokinase and used for preventing or treating thrombotic cardiovascular diseases; can also be used as ingredient to be added into food and beverage to produce health food with antithrombotic function; also can be used as independent powder or/concentrated solution to produce natto kinase/functional factor food additive; the preparation method can also be used for preparing powder or/and concentrated solution to be used as a controlled release independent feeding bag of health-care food with an antithrombotic function, the powder or/and concentrated solution is combined with a food main body to form natto kinase controlled release antithrombotic function food, and a consumer opens a release device (such as a button or a knob) to release natto kinase microsphere powder or/and concentrated solution in the controlled release bag before taking the natto kinase controlled release functional food, and the natto kinase microsphere powder or/and concentrated solution is mixed with the food main body (such as milk, fruit juice and other beverages) and then is eaten; compared with pure zymoprotein, the nattokinase slow-release microsphere of the invention used as a functional factor additive can improve the shelf life of antithrombotic functional food and the bioavailability of functional factors.
Drawings
FIG. 1: a is a design schematic diagram; b is the picture of the nattokinase slow release microsphere of the embodiment 2 and the embodiment 4; c is a phase contrast microscope photograph of the chitosan-nattokinase-casein slow release microspheres; d is a scanning electron microscope photo of the chitosan microspheres; e is a scanning electron microscope picture of the chitosan-nattokinase sustained-release microspheres; f is a scanning electron microscope picture of the chitosan-nattokinase-casein sustained-release microspheres.
FIG. 2 is the comparison of the drug loading capacity and gastric acid resistance of chitosan-nattokinase sustained-release microspheres prepared under different conditions.
FIG. 3 shows the enzyme activity retention rate of the sustained-release granules after being treated by gastric juice for different time.
FIG. 4 shows the enzyme activity retention and release of the sustained release granule in intestinal juice.
FIG. 5 shows the effect of the black tail experiment in mice. Wherein:
a is a slow release mode of chitosan-nattokinase-casein slow release microspheres in design;
b is the black tail condition of each group of mice. B1: normal mouse photos; b2: 7-day photographs of the thrombus control group; b3: 7-day photos of natto kinase group; b4: 7-day photographs of the particle groups; b5: the black tail length of the thrombus control group was varied with time; b6: the length of the black tail of the nattokinase group changes along with time; b7: the black tail length of the particle group varied with time.
And C is a tail histological section micrograph of the black-tail mouse. C1, C2, C3: normal mice; c4: thrombus is visible in the blood vessel cavity at the position 6cm away from the tail end of the thrombus control group; c5: thrombus is visible in the vascular cavity of the thrombus control group 3cm away from the tail end of the tail part, and edema, inflammation and necrosis are visible in tissues around the blood vessel; c6: the natto kinase group is 6cm away from the tail end, the vascular wall is complete, and thrombus in the cavity is partially dissolved (#); c7: the natto kinase group is 3cm away from the tail end, and thrombus is visible in the vascular cavity; c8: the particle group is 6cm away from the tail end of the tail part, and no thrombus is seen in the blood vessel; c9: the group of particles was 3cm from the tail end, and a small amount of residue after thrombolysis was observed in the blood vessel.
FIG. 6 shows the enzyme activity retention rates of the sustained-release granules during storage under different conditions.
A: drying at 25 ℃; b: drying at 4 ℃; c: a water phase at 25 ℃; d: aqueous phase at 4 ℃.
Detailed description of the preferred embodiments
Example 1: chitosan-nattokinase slow-release microsphere
Preparation of chitosan-nattokinase slow-release microsphere
1) Mixing an emulsifier and an organic solvent according to the ratio of the emulsifier: mixing organic solvents in a volume ratio of 1:50 to prepare an oil phase, wherein the used emulsifier is span-80, and the used organic solvent is liquid paraffin;
2) adding chitosan into acetic acid solution with volume concentration of 3%, magnetically stirring for 2h to fully dissolve the chitosan, standing to prepare water phase with chitosan mass concentration of 0.5%;
3) dissolving sodium tripolyphosphate in water to prepare a cross-linking agent A with the concentration of the sodium tripolyphosphate being 10 mg/ml;
4) dissolving genipin in 30% ethanol solution to obtain crosslinking agent B with genipin concentration of 3 mg/ml;
5) dispersing nattokinase in water to prepare an aqueous solution with the concentration of 50 mg/ml;
6) dropwise adding the water phase obtained in the step 2) into the oil phase obtained in the step 1) while stirring according to the volume ratio of the water phase to the oil phase of 1:2, and continuously stirring for 2 hours to obtain a W/O type emulsion;
7) dropwise adding 10ml of the cross-linking agent A obtained in the step 3) into 1L of the emulsion obtained in the step 6) at one time under the stirring condition, stirring for 1h, centrifuging at a high speed, removing the upper layer, washing the solid at the lower layer of the precipitate with petroleum ether and ethanol for 2 times respectively, and drying in vacuum to obtain chitosan microspheres with white powder appearance;
8) 5g of microspheres obtained in the step 7) are taken, 5ml of cross-linking agent B obtained in the step 4) is dripped, 10ml of nattokinase solution obtained in the step 5) is added for cross-linking, the mixture is stirred at 37 ℃ for reaction for 30min and then is washed for 3 times, and the nattokinase sustained-release microspheres are obtained by freeze drying and are in blue powder shape.
(II) characterization of microsphere morphology
The microsphere is sprayed with gold and observed under a scanning electron microscope, and the result is shown in figure 1, wherein the diameter of the microsphere is mainly distributed between 1 and 5 mu m.
Example 2
Optimization of preparation conditions of chitosan-nattokinase sustained-release microspheres
1) Mixing an emulsifier and an organic solvent according to the ratio of the emulsifier: mixing organic solvents in a volume ratio of 1:50 to prepare an oil phase, wherein the used emulsifier is span-80, and the used organic solvent is liquid paraffin;
2) adding chitosan into acetic acid solution with volume concentration of 3%, magnetically stirring for 2h to fully dissolve the chitosan, standing to prepare water phase with chitosan mass concentration of 0.5%;
3) dissolving sodium tripolyphosphate in water to prepare a cross-linking agent A with the concentration of the sodium tripolyphosphate being 10 mg/ml;
4) dissolving genipin in 30% ethanol solution to obtain crosslinking agent B with genipin concentration of 3 mg/ml;
5) dispersing nattokinase in water to prepare an aqueous solution with the concentration of 50 mg/ml;
6) dropwise adding the water phase obtained in the step 2) into the oil phase obtained in the step 1) while stirring according to the volume ratio of the water phase to the oil phase of 1:3, and continuously stirring for 10min-6h to obtain a W/O type emulsion;
7) dropwise adding the cross-linking agent A obtained in the step 3) into the emulsion obtained in the step 6) at intervals of 30min under the stirring condition, stirring for 2h, centrifuging at a high speed, removing the upper layer, washing the precipitated lower layer of solid with petroleum ether for 2 times respectively, and drying in vacuum to obtain chitosan microspheres with white powder appearance;
8) dividing the microspheres obtained in the step 7) into a plurality of parts of 1 g. Respectively dropwise adding 1ml of the cross-linking agent B obtained in the step 4) and 0.4 ml, 0.8 ml, 1.2 ml, 1.6 ml and 2.0ml of the nattokinase solution obtained in the step 5) and adding purified water to 10ml for cross-linking, stirring at 37 ℃ for reaction for 1h, washing with water for 3 times, and freeze-drying to obtain the nattokinase sustained-release microspheres which are blue powder.
As shown in figure 2, when the ratio of the nattokinase to the chitosan microspheres reaches 8:100 and the reaction time reaches 8 hours, the influence of further increasing the addition amount of the nattokinase or prolonging the reaction time on the drug loading rate is small. The chitosan microspheres with the diameter of 2 mu m are loaded with nattokinase and then have the best gastric acid resistance effect, and the drug loading rate has little influence on the gastric acid resistance effect under the diameter condition.
The drug loading rate, the encapsulation rate and the enzyme activity retention rate in gastric acid are comprehensively considered, chitosan microspheres with the diameter of 2 mu m are selected, and the optimal condition is that the crosslinking reaction is carried out for 8 hours at the ratio of 8mg of nattokinase to 100mg of chitosan. The chitosan-nattokinase slow-release microsphere prepared under the condition has the drug loading rate of 2.93 percent and the encapsulation rate of 36.6 percent, the enzyme activity retention rate in gastric juice within 2 hours is 80.0 percent, and the enzyme activity retention rate within 4 hours is 67.6 percent. By analyzing the gastric acid resistance of the chitosan-nattokinase sustained-release microspheres obtained under various conditions, we think that the gastric acid resistance effect is difficult to further improve through condition optimization, which is probably related to the fact that the nattokinase loaded on the particles is directly contacted with gastric acid. And a protein protective layer is added on the outer layer of the particle, so that a better enzyme activity protection effect can be expected.
The microspheres produced under the above conditions were designated as particles 1(p1), as shown in FIG. 1B.
Example 3: preparation of chitosan-nattokinase slow-release microsphere
1) Mixing an emulsifier and an organic solvent according to the ratio of the emulsifier: mixing organic solvents in a volume ratio of 1:50 to prepare an oil phase, wherein the used emulsifier is span-80, and the used organic solvent is liquid paraffin;
2) adding chitosan into acetic acid solution with volume concentration of 3%, magnetically stirring for 2h to fully dissolve the chitosan, standing to prepare water phase with mass concentration of chitosan of 3%;
3) dissolving sodium tripolyphosphate in water to prepare a cross-linking agent A with the concentration of the sodium tripolyphosphate being 30 mg/ml;
4) dissolving genipin in 90% ethanol solution to obtain crosslinking agent B with genipin concentration of 10 mg/ml;
5) dispersing nattokinase in water to prepare an aqueous solution with the concentration of 50 mg/ml;
6) dropwise adding the water phase obtained in the step 2) into the oil phase obtained in the step 1) while stirring according to the volume ratio of the water phase to the oil phase of 1:3, and continuously stirring for 30min to obtain a W/O type emulsion;
7) dropwise adding the cross-linking agent A obtained in the step 3) into the emulsion obtained in the step 6) under the stirring condition, stirring for 2h, centrifuging at a high speed, removing the upper layer, washing the solid at the lower layer of the precipitate with petroleum ether for 2 times, and drying in vacuum to obtain chitosan microspheres with white powder appearance;
8) taking 5g of the microspheres obtained in the step 7), dropwise adding 2ml of the cross-linking agent B obtained in the step 4, adding 8ml of the nattokinase solution obtained in the step 5), adding purified water to 50ml for cross-linking, stirring at 37 ℃ for reaction for 15min, then washing for 3 times, and freeze-drying to obtain the nattokinase sustained-release microspheres which are blue powder.
Example 4: preparation of casein-embedded nattokinase sustained-release microspheres (chitosan-nattokinase-casein sustained-release microspheres)
Preparation of casein embedded nattokinase slow release microsphere
1) Mixing an emulsifier and an organic solvent according to the ratio of the emulsifier: mixing organic solvents in a volume ratio of 1:50 to prepare an oil phase, wherein the used emulsifier is span-80, and the used organic solvent is liquid paraffin;
2) adding chitosan into acetic acid solution with volume concentration of 3%, magnetically stirring for 2h to fully dissolve the chitosan, standing to prepare water phase with chitosan mass concentration of 0.5%;
3) dissolving sodium tripolyphosphate in water to prepare a cross-linking agent A with the concentration of the sodium tripolyphosphate being 10 mg/ml;
4) dissolving genipin in 30% ethanol solution to obtain crosslinking agent B with genipin concentration of 3mg/ml, and dissolving TG enzyme in water to obtain crosslinking agent C with concentration of 10 mg/ml;
5) dispersing nattokinase in water to prepare an aqueous solution with the concentration of 50 mg/ml; dispersing cow milk casein in water to prepare 50mg/ml water solution;
6) dropwise adding the water phase in the step 2 into the oil phase obtained in the step 1) while stirring according to the volume ratio of the water phase to the oil phase of 1:5, and continuously stirring for 2 hours to obtain a W/O type emulsion;
7) dropwise adding the cross-linking agent A obtained in the step 3) into the emulsion obtained in the step 6) at intervals of 30min under the stirring condition, stirring for 4h, centrifuging at a high speed, removing an upper layer, washing a solid at a lower layer of a precipitate with petroleum ether and ethanol for 2 times respectively, and drying in vacuum to obtain the chitosan microspheres with white powder appearance;
8) taking 5g of the chitosan microspheres obtained in the step 7), adding 8ml of nattokinase solution and 5ml of cross-linking agent B, adding water to 50ml, stirring at 37 ℃ for reacting for 8 hours, and centrifuging to obtain precipitates;
9) washing the precipitate obtained in the step 8) with water for 3 times, adding 20ml of casein solution and 5ml of cross-linking agent C, and stirring for 1h at 37 ℃;
10) the reaction solution was centrifuged at 1000g for 5min to discard the supernatant. Washing the microsphere with water for 2-3 times, and freeze drying to obtain casein-embedded nattokinase sustained-release microsphere in blue powder.
The microspheres produced according to the above procedure were designated as particles 2(p2), as shown in FIG. 1B.
(II) Effect detection
1. Microsphere morphology characterization
Observing the microsphere after metal spraying under a scanning electron microscope
The microspheres obtained by adding sodium tripolyphosphate twice had smaller diameters than those obtained by adding sodium tripolyphosphate all at once in example 1.
2. Method for measuring enzyme activity and thrombolytic effect
The activity of nattokinase was measured by agarose-fibrinogen plate method using urokinase as a control, and the procedure was as follows:
1) thrombin, plasminogen was dissolved in phosphate buffer (pH 7.4), thrombin concentration 0.1mg/mL, plasminogen concentration 0.5 mg/mL.
Mixing agarose and distilled water, heating to 60 deg.C for dissolving completely, mixing with thrombin/plasminogen solution at a ratio of 1:1, pouring into a culture dish, and cooling to obtain the final product. Punching holes with the aperture of about 2mm on the flat plate at intervals of more than 3 cm.
2) Urokinase was prepared as a 1mg/mL standard solution in phosphate buffer (pH 7.4), 1, 2, 4, 8, 16. mu.L of urokinase was added to each well of the plate, and the plate was incubated at 37 ℃ for 12 hours before measuring the diameter of the lysis ring around each well with a vernier caliper. The results show that the diameter of the lysocoil is linearly related to the logarithm of the enzyme activity.
3) Diluting the sample to be detected with phosphate buffer solution, adjusting pH to 7.4, adding 10 μ L of the sample into each well, adding urokinase standard solution into the other well as a control, and measuring the diameter of the soluble ring after a certain time.
3. Evaluation of Nattokinase sustained Release granule Effect
1) In vitro experiments:
directly dispersing nattokinase or microspheres by PBS or mouse gastric juice, stirring at 37 ℃, absorbing reaction suspension at regular intervals, diluting and neutralizing to be neutral, ultrasonically homogenizing, adding onto a fiber egg white plate, standing at 37 ℃ for 18h, and measuring the diameter of a soluble ring to obtain a nattokinase gastric juice resistant activity curve.
The detection result (figure 3) shows that the nattokinase is almost completely inactivated and irreversible in the free state in gastric juice within 15 minutes, after being crosslinked to the chitosan microsphere through genipin (particle 1), the activity of the nattokinase in gastric juice is remarkably improved, and the enzyme activity is kept to 67.8% after 4 hours. The stability of nattokinase in gastric acid can be obviously improved by directly crosslinking nattokinase and casein by using TG, but only a small amount of nattokinase is protected because a complete protective layer cannot be formed, and most of activity of a crosslinking substance is still lost within 30 minutes. And an outer protective shell formed by cross-linking casein (particle 2) on the microsphere by using TG enzyme can further enhance the gastric acid resistance of the natto kinase embedded in the microsphere. The enzyme activity of the compound can be kept at 88.3 percent after 4 hours in gastric juice, which is obviously higher than that of the particle 1. This result demonstrates that the protective effect of casein on enzymes depends on the structure of the sandwich-like particles formed.
After treating the gastric juice for 2h, pellet 1 and pellet 2 were collected by centrifugation and further digested with intestinal juice. After digestion for different periods of time, the pellet and supernatant were collected by centrifugation and the thrombolytic activity of the pellet and supernatant was determined separately.
The results show that the reduction rate of the residual enzyme activity in the precipitate of the granule 1 is obviously higher than that of the granule 2 after the granule 1 is digested by intestinal juice. The residual enzyme activity after 3 hours is 67.6 percent, and after 6 hours, the residual enzyme activity is 31.0 percent. The slow release of granule 2 is divided into two stages, during the first 3 hours, the casein protective layer is gradually hydrolyzed by trypsin, and only a small amount of nattokinase is released. Thereafter, nattokinase was gradually released (fig. 4). This result shows that granule 2 has a better sustained release function than granule 1, which is associated with the outer layer of granule 2 having a casein protective layer. The granule 2 can be used as oral administration dosage form of nattokinase, and has effects of prolonging drug action time and reducing administration times.
2) Animal experiments:
the antithrombotic effect of the samples was evaluated using the rat black tail model (as shown in fig. 5).
The specific method comprises the following steps:
the model is made by feeding mice at room temperature, freely ingesting drinking water, injecting carrageenan 50mg/kg, forming black tail 24h after injection, the average black tail length is 3.5cm, and the mice are used as thrombus black tail model. Mice were divided into 3 groups, thrombus control group, nattokinase group, and granule group.
From 24 hours after injection, the nattokinase group was fed with nattokinase at 10 mg/kg/day, and the granule group was fed with casein-embedded nattokinase sustained-release microspheres (granules 2) at 350 mg/kg/day.
The length of the black tail section of the tail of the mouse in the thrombus group is gradually increased, one mouse in the group is dead 14 days after carrageenan injection, the tail of all the other mice is broken, and the average length of the residual section is 2.3 +/-0.8 cm.
The black tail of the natto kinase group mice grows slowly and is continuously fed for 13 days (14 days after carrageenan injection), the black tail breaks off, and the average length of the residual section is 3.8 +/-1.1 cm
The black tail length of the granule group did not increase or slightly regress, the feeding was continued for 13 days (14 days after carrageenan injection), the black tail section fell off, and the average length of the residual section of the tail was 6.1 + -1.7 cm.
The above three groups of mice were sectioned at 3cm and 6cm from the tail end, respectively, 5 days after carrageenan injection, fixed, observed under a light mirror after HE staining, and the tail of a normal mouse was used as a control (fig. 5). The observation result shows that four subcutaneous blood vessels are arranged at the tail part of the normal mouse, and after 5 days of carrageenan injection, plaques which are uniformly stained in the blood vessels of the mice of the thrombus control group are thrombus, and tissues around the blood vessels are edema and ulcerated. The tail thrombus of the natto kinase group mouse is not full of blood vessels; the granular group thrombus mostly dissolved, leaving only a small amount of plaque in the vessel.
Example 5: casein embedded nattokinase slow release microsphere
1) Mixing an emulsifier and an organic solvent according to the ratio of the emulsifier: mixing organic solvents in a volume ratio of 1:50 to prepare an oil phase, wherein the used emulsifier is span-80, and the used organic solvent is liquid paraffin;
2) adding chitosan into acetic acid solution with volume concentration of 1%, magnetically stirring for 2h to fully dissolve the chitosan, standing to prepare water phase with chitosan mass concentration of 0.3%;
3) dissolving sodium tripolyphosphate in water to prepare a cross-linking agent A with the concentration of the sodium tripolyphosphate being 1 mg/ml;
4) dissolving genipin in 10% ethanol solution to obtain crosslinking agent B with genipin concentration of 1mg/ml, and dissolving TG enzyme in water to obtain crosslinking agent C with concentration of 10 mg/ml;
5) dispersing nattokinase in water to prepare aqueous solution with the concentration of 10 mg/ml; dispersing cow milk casein in water to prepare 10mg/ml water solution;
6) dropwise adding the water phase in the step 2 into the oil phase obtained in the step 1) while stirring according to the volume ratio of the water phase to the oil phase of 1:2, and continuously stirring for 2 hours to obtain a W/O type emulsion;
7) dropwise adding the cross-linking agent A obtained in the step 3) into the emulsion obtained in the step 6) under the stirring condition, stirring for 6h, centrifuging at a high speed, removing the upper layer, washing the solid at the lower layer of the precipitate with petroleum ether and ethanol for 2 times respectively, and drying in vacuum to obtain chitosan microspheres with white powder appearance;
8) taking 2g of the chitosan microspheres obtained in the step 7), adding 40ml of nattokinase solution and 5ml of cross-linking agent B, stirring and reacting for 1h at 37 ℃, and centrifuging to take precipitates;
9) washing the precipitate obtained in the step 8) with water for 3 times, adding 20ml of casein solution and 5ml of cross-linking agent C, and stirring for 1h at 37 ℃;
10) the reaction solution was centrifuged at 1000g for 5min to discard the supernatant. Washing the microsphere with water for 2-3 times, and freeze drying to obtain casein-embedded nattokinase sustained-release microsphere in blue powder.
Example 6: casein embedded nattokinase slow release microsphere
1) Mixing an emulsifier and an organic solvent according to the ratio of the emulsifier: mixing organic solvents in a volume ratio of 1:100 to prepare an oil phase, wherein the used emulsifier is span-80, and the used organic solvent is liquid paraffin;
2) adding chitosan into acetic acid solution with volume concentration of 3%, magnetically stirring for 2h to fully dissolve the chitosan, standing to prepare water phase with chitosan mass concentration of 1%;
3) dissolving sodium tripolyphosphate in water to prepare a cross-linking agent A with the concentration of the sodium tripolyphosphate being 1 mg/ml;
4) dissolving genipin in 10% ethanol solution to obtain crosslinking agent B with genipin concentration of 5mg/ml, and dissolving TG enzyme in water to obtain crosslinking agent C with concentration of 10 mg/ml;
5) dispersing nattokinase in water to prepare an aqueous solution with the concentration of 25 mg/ml; dispersing cow milk casein in water to prepare 25mg/ml water solution;
6) dropwise adding the water phase in the step 2 into the oil phase obtained in the step 1) while stirring according to the volume ratio of the water phase to the oil phase of 1:5, and continuously stirring for 2 hours to obtain a W/O type emulsion;
7) dropwise adding the cross-linking agent A obtained in the step 3) into the emulsion obtained in the step 6) under the stirring condition, stirring for 6h, centrifuging at a high speed, removing the upper layer, washing the solid at the lower layer of the precipitate with petroleum ether and ethanol for 2 times respectively, and drying in vacuum to obtain chitosan microspheres with white powder appearance;
8) taking 5g of the chitosan microspheres obtained in the step 7), adding 10ml of nattokinase solution and 2ml of cross-linking agent B, stirring and reacting for 1h at 37 ℃, and centrifuging to take precipitates;
9) washing the precipitate obtained in the step 8) with water for 3 times, adding 40ml of casein solution and 5ml of cross-linking agent C, and stirring for 1h at 37 ℃;
10) the reaction solution was centrifuged at 1000g for 5min to discard the supernatant. Washing the microsphere with water for 2-3 times, and freeze drying to obtain casein-embedded nattokinase sustained-release microsphere in blue powder.
Example 7: nattokinase slow release microspheres for improving storage stability of nattokinase
Free nattokinase, nattokinase chitosan sustained-release microspheres (example 3) and casein-embedded nattokinase sustained-release microspheres (example 4) are respectively stored in dry and water phase environments at 4 ℃ and 25 ℃, and the change of the enzyme activity along with time is observed.
The results show that free nattokinase has poor stability and retains only about 45% of thrombolytic activity after 60 days even when refrigerated at 4 ℃ under dry conditions. The nattokinase sustained-release microspheres without casein protection have good stability under dry conditions, but are gradually inactivated in an aqueous phase environment. The casein-protected nattokinase sustained-release microspheres have good storage stability in both dry and aqueous environments (figure 6).
The same stability experiment is carried out on the nattokinase chitosan slow-release microspheres and casein-embedded nattokinase slow-release microspheres obtained in other embodiments, and the effect of obviously improving the storage stability is also shown.
Example 8 application of Nattokinase sustained-release microspheres in tempering acidic fermented milk beverage
Application of nattokinase sustained-release microspheres in blending acidic fermented milk beverage
1. Homogenizing, cooling sterilized fresh milk to 45 deg.C, inoculating Lactobacillus bulgaricus and Streptococcus thermophilus at ratio of 1: 1.
2. Fermenting at 43 deg.C for about 6 hr until fresh milk coagulates into a whole gel.
3. Cooling to 25 deg.C, adding the slow-release microsphere of crosslinked casein prepared in example 4, 3.5g/L nattokinase chitosan, and 1g/L TG enzyme, stirring, and refrigerating at 4 deg.C.
Second, effect detection
1. Stability of dispersion
The mixture is refrigerated at 4 ℃ for 21 days, and no obvious precipitate is observed.
2. Thrombolytic activity
The slow release effect of the nattokinase chitosan microspheres is evaluated by taking free enzyme (0.1g/L) with the same amount as the total amount of the nattokinase as a control.
The detection result shows that the nattokinase has an explosion release process in the first 24 hours, releases about 30 percent of the total enzyme activity, slowly releases the enzyme activity in the following 21 days, and the enzyme activity of the milk liquid is 146U/ml after the milk liquid is refrigerated for 21 days. Adding 200U/ml free nattokinase, and refrigerating for 21 days until the enzyme activity of the milk liquid is only 31U/ml.
Although the present invention has been described with reference to the preferred embodiments, it should be understood that various changes and modifications can be made therein by those skilled in the art without departing from the spirit and scope of the invention as defined in the appended claims.

Claims (7)

1. A preparation method of nattokinase sustained-release microspheres is characterized by comprising the following steps:
1) mixing an emulsifier and an organic solvent according to the volume ratio of the emulsifier to the organic solvent of 1:100-1:10 to prepare an oil phase, wherein the emulsifier is span-80, and the organic solvent is liquid paraffin;
2) adding chitosan into 1-3 vol% acetic acid solution, stirring to dissolve completely, standing to obtain water phase with chitosan mass concentration of 0.3-3%;
3) dissolving sodium tripolyphosphate in water to prepare a cross-linking agent A with the concentration of the sodium tripolyphosphate of 3mg/mL-30 mg/mL;
4) dissolving genipin in 10-90% ethanol solution to obtain crosslinking agent B with genipin concentration of 1-10 mg/mL;
5) dispersing nattokinase in water to prepare aqueous solution with the concentration of 5-50 mg/mL;
6) dropwise adding the water phase obtained in the step 2) into the oil phase obtained in the step 1) while stirring according to the volume ratio of the water phase to the oil phase of 1:1-1:10, and continuously stirring for 10min-6h to obtain W/O type emulsion;
7) dropwise adding the cross-linking agent A obtained in the step 3) into the emulsion obtained in the step 6) once or for multiple times under the stirring condition, stirring for 0.5-12h, centrifuging at a high speed, removing the upper layer, washing the solid at the lower layer of the precipitate with one or two solvents of petroleum ether, acetone, isopropanol, ethanol and chloroform for 2-3 times respectively, and drying in vacuum to obtain chitosan microspheres;
8) dripping the crosslinking agent B obtained in the step 4) on the microsphere with the diameter of 2 mu m obtained in the step 7) until the microsphere is wetted, dripping the nattokinase solution obtained in the step 5) to perform crosslinking reaction for 8 hours according to the ratio of 8mg of nattokinase to 100mg of chitosan, washing the microspheres for multiple times by water, and performing freeze drying to obtain the chitosan-nattokinase slow-release microsphere.
2. A preparation method of nattokinase sustained-release microspheres is characterized by comprising the following steps:
1) mixing an emulsifier and an organic solvent according to the volume ratio of the emulsifier to the organic solvent of 1:100-1:50 to prepare an oil phase, wherein the emulsifier is span-80, and the organic solvent is liquid paraffin;
2) adding chitosan into 1-3 vol% acetic acid solution, stirring to dissolve completely, standing to obtain water phase with chitosan mass concentration of 0.3-3%;
3) dissolving sodium tripolyphosphate in water to prepare a cross-linking agent A with the concentration of the sodium tripolyphosphate of 3mg/mL-30 mg/mL;
4) dissolving genipin in 10-90% ethanol solution to obtain crosslinking agent B with genipin concentration of 1mg/mL-10mg/mL, and dissolving TG enzyme in water to obtain crosslinking agent C with concentration of 10 mg/mL;
5) dispersing nattokinase in water to prepare aqueous solution with the concentration of 10-50 mg/mL; dispersing cow milk casein in water to prepare 10-50mg/mL aqueous solution;
6) dropwise adding the water phase obtained in the step 2) into the oil phase obtained in the step 1) while stirring according to the volume ratio of the water phase to the oil phase of 1:2-1:5, and continuously stirring for 2 hours to obtain a W/O type emulsion;
7) dropwise adding the cross-linking agent A obtained in the step 3) into the emulsion obtained in the step 6) once or for multiple times under the stirring condition, stirring, centrifuging at a high speed, removing the upper layer, washing the precipitated lower-layer solid for 2-3 times by using one or two solvents of petroleum ether, acetone, isopropanol, ethanol and chloroform respectively, and drying in vacuum to obtain chitosan microspheres;
8) taking 2-5g of the chitosan microspheres obtained in the step 7), adding 8-40ml of nattokinase solution and 2-5ml of cross-linking agent B, adding water to 50ml, stirring and reacting at 37 ℃ for 1-8h, and centrifuging to obtain precipitates;
9) washing the precipitate obtained in the step 8) for multiple times, adding 20-40ml of casein solution and 5ml of cross-linking agent C, and stirring for 1h at 37 ℃;
10) centrifuging the reaction solution and removing the supernatant; and (4) washing the microspheres with water, and freeze-drying to obtain the casein-embedded nattokinase sustained-release microspheres.
3. Nattokinase sustained-release microspheres, characterized in that the nattokinase sustained-release microspheres are prepared by the method of claim 1 or 2.
4. Use of the nattokinase sustained-release microspheres described in claim 3 or prepared by the method described in claim 1 or 2 for preparing any one of the following: liquid preparation and solid preparation.
5. A liquid preparation or a solid preparation, which contains the nattokinase sustained-release microspheres described in claim 3 or the nattokinase sustained-release microspheres prepared by the method described in claim 1 or 2.
6. Use of the nattokinase sustained-release microspheres described in claim 3 or the nattokinase sustained-release microspheres prepared by the method described in claim 1 or 2 in preparation of medicines.
7. A pharmaceutical preparation comprising the nattokinase sustained-release microspheres according to claim 3.
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