Vitamin k2 complex microcapsule system with gastric protection-intestinal sustained release function and preparation method and application thereof

Abstract

The application discloses a vitamin K2 compound microcapsule system with gastric protection-intestinal sustained release function and a preparation method and application thereof, and relates to the technical field of medicine, in particular to a vitamin K2 compound microcapsule system with gastric protection-intestinal sustained release function, which comprises three layers from inside to outside, namely a core layer, an intermediate layer and an outer layer; the core layer is a zein-shellac hydrophobic core for wrapping vitamin K2; the intermediate layer is a pea protein-pectin electrostatic compound; and the outer layer is a sodium alginate and chitosan double-network gel. The microcapsule system can be closely combined in a gastric acid environment after oral administration, reduces the release of active ingredients in the stomach, then is retained at the intestinal mucosa for a long time, slowly releases the contents, slowly presents the function outside, prolongs the half-life, and improves the effect. Vitamin K2 directly or indirectly participates in and regulates various physiological processes related to health, and plays an important role in maintaining bone health, preventing cardiovascular diseases, protecting nerves, improving metabolism and the like.

Description

TECHNICAL FIELD

[0001] The present application belongs to the technical field of nanocarriers, and particularly relates to a vitamin K2 complex microcapsule system with gastric protection-intestinal sustained release function and a preparation method and application thereof. BACKGROUND

[0002] Vitamin K compounds are a group of fat-soluble vitamins with 2-methyl-1,4-naphthoquinone mother structure found in nature, and are an important family of nutrients. Natural vitamin K includes vitamin K1 and vitamin K2. Among them, vitamin K2 mainly exists in natto, meat and cheese, has a polyisoprene side chain, and is divided into different subtypes according to different numbers of isoprene groups, including tetraene menadione (MK-4), heptaene menadione (MK-7), nonaene menadione (MK-9), etc. MK-7 is a representative component thereof.

[0003] With the continuous in-depth research on vitamin K2, its important influence on normal physiological functions of the human body is gradually receiving more attention. In terms of maintaining bone health, vitamin K2 can promote bone formation by stimulating osteoblast differentiation; maintain normal bone mineralization rate in the skeleton by converting glutamic acid residues of osteocalcin (OC) into γ-carboxyglutamic acid residues; and inhibit bone resorption through its anti-catabolic activity (reducing osteoclast differentiation and inhibiting osteoblast apoptosis). Vitamin K2 as a drug for clinical treatment of osteoporosis can promote bone bud cell growth, inhibit osteoclast production, regulate bone metabolism markers and maintain bone homeostasis, and improve the bone health status of patients.

[0004] In terms of preventing cardiovascular diseases, vitamin K2 can inhibit cardiovascular calcification by activating matrix Gla protein, reduce the risk of arteriosclerosis / arterial calcification and coronary heart disease (CHD). Matrix Gla protein is a natural potent calcification inhibitor that needs to be carboxylated by vitamin K-dependent γ-carboxylase to be active. Supplementing vitamin K2 can maintain the sustained presence of vascular calcification inhibitors in the body.

[0005] In addition, vitamin K2 can participate in the protection of nerves, improvement of metabolism and inhibition of tumor growth, and play an important role in the treatment of related diseases through various mechanisms such as inhibition of apoptosis signaling pathways, reduction of inflammatory response, protection of mitochondrial function, improvement of insulin resistance and promotion of autophagy.

[0006] However, vitamin K2 is a fat-soluble vitamin, which is difficult to be uniformly added into water-soluble products, and its application field is limited. In use, high-quality fat needs to be used to assist the absorption of vitamin K2, so that better absorption and conversion effect can be achieved. In addition, vitamin K2 is sensitive to ultraviolet rays and alkali, and is easily decomposed by light. Vitamin K2 is easily inactivated during long-term storage, and needs to be improved in stability by process. Vitamin K2 is often mixed with calcium, magnesium and other minerals required for calcium metabolism in practical application because of its effect of regulating calcium metabolism. However, vitamin K2 is easily decomposed when it is in contact with these minerals, resulting in unstable content in the product.

[0007] Microencapsulation technology refers to a technology of using a suitable high molecular polymer material as a carrier to coat a certain object (core or inner phase) in a semi-permeable membrane (wall or outer phase) to form a micro-particle dispersion system. The shielding function of the wall or outer phase protects the core or inner phase, and enhances the stability of the product. Subsequently, under the action of certain external stimuli or sustained release mechanism, the function of the object is exhibited outside, thereby achieving the effect of sustained release and prolonged action time.

[0008] Therefore, it is urgent to develop a vitamin K2 complex microcapsule system with gastric protection and intestinal sustained release function. Vitamin K2 can be effectively protected from light, air, pH value and minerals by embedding vitamin K2 using microencapsulation technology, effectively solving the problem of instability of vitamin K2, so as to maintain the effectiveness of the product. At the same time, the microencapsulated vitamin K2 can be dispersed in water, and the use is not limited. The microencapsulated vitamin K2 can be stably used in various products including water-soluble solutions. SUMMARY

[0009] In order to solve the problems proposed in the above background art, the purpose of the present application is to provide a vitamin K2 complex microcapsule system with gastric protection and intestinal sustained release function, and a preparation method and application thereof.

[0010] The vitamin K2 complex microcapsule system with gastric protection and intestinal sustained release function provided by the present application first uses zein-gum hydrophobic core to wrap vitamin K2 as the core layer, which protects vitamin K2 from light, air, pH value and minerals through the shielding effect of the capsule wall. Secondly, the pea protein-pectin electrostatic complex is used as the intermediate layer, which forms a sub-micron complex through electrostatic adsorption. The complex wall material structure of pea protein-pectin is more compact, and has better protection effect on vitamin K2. Finally, the outer layer uses sodium alginate (SA) and chitosan (CS) to form a complex through Ca 2+The cross-linking and hydrogen bonding form a double network gel, which improves the mechanical strength and adhesion, can reach the specific site (intestinal mucosa) aggregation, and achieves the purpose of controlled release. The three-layer composite embedding system of vitamin K2 provided by the application can be closely combined in the gastric acid environment after oral administration, reduce the release of active ingredients in the stomach, then slowly release the contents in the intestinal mucosa, slowly present the function outside, prolong the half-life, and improve the effect. Vitamin K2 directly or indirectly participates in, regulates a variety of and health-related physiological processes, plays an important role in maintaining bone health, preventing cardiovascular diseases, protecting nerves, improving metabolism, and the like, and the corn alcohol soluble protein and the pea protein can also increase nutrition, promote health, and meet various needs of the body.

[0011] In order to achieve the above purpose, the technical scheme adopted by the application is as follows: on the one hand, the application provides a microcapsule system, which comprises three layers, and the core layer, the intermediate layer and the outer layer are sequentially arranged from inside to outside.

[0012] The core layer is a zein-shellac hydrophobic core, the intermediate layer is a pea protein-pectin electrostatic complex, and the outer layer is a sodium alginate (SA) and chitosan (CS) double network gel.

[0013] On the other hand, the application provides an application of the microcapsule system as described above as a vitamin K2 embedding system.

[0014] On the other hand, the application provides a vitamin K2 composite microcapsule system with a gastric protection-intestinal sustained release function, which comprises three layers, and the core layer, the intermediate layer and the outer layer are sequentially arranged from inside to outside.

[0015] The core layer is a zein-shellac hydrophobic core wrapped with vitamin K2, the intermediate layer is a pea protein-pectin electrostatic complex, and the outer layer is a sodium alginate (SA) and chitosan (CS) double network gel.

[0016] Further, according to the total mass percentage of 100%, the following raw material components are included: vitamin K2 is 5% to 20%, zein is 5% to 20%, shellac is 5% to 15%, pea protein is 5% to 20%, low-ester pectin is 5% to 15%, sodium alginate is 10% to 30%, calcium chloride (CaCl2) is 5% to 15%, and chitosan is 5% to 15%.

[0017] Further, the vitamin K2 is in the MK-7 configuration.

[0018] On the other hand, the application provides a preparation method of the vitamin K2 composite microcapsule system with a gastric protection-intestinal sustained release function as described above, which comprises the following steps:

[0019] (1) corn alcohol-soluble protein is fully dissolved in a solvent to obtain a corn alcohol-soluble protein solution, vitamin K2 is added to the corn alcohol-soluble protein solution to obtain a mixed solution, water is then added to the mixed solution, and a rotary evaporator is used to emulsify and evaporate to obtain a rotary evaporation emulsion, shellac is dissolved in water to obtain a shellac aqueous solution, the rotary evaporation emulsion is then added to the shellac aqueous solution, and the pH of the system is adjusted to 2.5-3.5, and the nanoscale corn alcohol-soluble protein-based vitamin K2 oil microcapsule core is obtained after continuous stirring for a period of time;

[0020] (2) pea protein is dissolved in water to obtain a pea protein aqueous solution, the pH of the pea protein aqueous solution is adjusted to 3-4.5, the nanoscale corn alcohol-soluble protein-based vitamin K2 oil microcapsule core prepared in step (1) is added to the pea protein aqueous solution with a pH of 3-4.5, and low ester pectin is added after stirring to obtain a uniform mixture, and a microjet homogenizer is then used to homogenize to obtain a micro-nanoscale double-layer composite structure vitamin K2 microcapsule;

[0021] (3) sodium alginate is dissolved in water to obtain a sodium alginate aqueous solution, the pH of the sodium alginate aqueous solution is adjusted to 3-4.5, the micro-nanoscale double-layer composite structure vitamin K2 microcapsule prepared in step (2) is added to the sodium alginate aqueous solution with a pH of 3-4.5, and stirring is performed to obtain an intermediate mixture, calcium chloride and chitosan are dissolved in water to obtain a chitosan aqueous solution of calcium chloride 2, and the intermediate mixture is then extruded into the stirring chitosan aqueous solution of calcium chloride using a vibrating nozzle to obtain a uniform three-layer protective structure vitamin K2 oil particle, i.e., a vitamin K2 composite microcapsule system with a gastric protection-intestinal sustained release function.

[0022] Further, the solvent in step (1) is selected from an ethanol aqueous solution with a volume percentage of 60%-80%;

[0023] In step (1), the emulsification pressure is 600-1000 bar, and the emulsification frequency is 1-5 times;

[0024] In step (1), the rotary evaporation is carried out at 65-75°C under reduced pressure, and the rotary evaporation time is 5-15 min;

[0025] In step (1), the continuous stirring time is 30-50 min.

[0026] Further, in step (2), the homogenization pressure is 10,000-15,000 psi, and the homogenization frequency is 1-5 times.

[0027] Further, in step (3), the frequency of the vibrating nozzle is 800-1000 Hz.

[0028] In still another aspect, the present application provides a use of the vitamin K2 complex microcapsule system with gastric protection-intestinal sustained release function as described above in any of the aspects in the preparation of a drug for maintaining bone health, preventing cardiovascular diseases, protecting nerves, and improving metabolism.

[0029] Compared with the prior art, the present application has the following beneficial effects:

[0030] (1) The zein has strong hydrophobicity, and the shellac has strong adhesion and strong plasticity, and the combination of the two improves the mechanical properties, stability and emulsifying properties of the zein, improves the biocompatibility of the shellac, and has excellent properties of both. In addition, the combination of the hydrophobicity of the zein and the pH sensitivity of the shellac can realize zero release in the stomach and target release in the intestine.

[0031] (2) Compared with soybean protein, pea protein has good solubility and stirring stability, but the pea protein has low surface charge, few disulfide bonds, and low mechanical strength. Pectin can interact with pea protein through non-covalent interaction, stabilize the protein gel network, improve flexibility and ductility, and achieve the effect of high active substance retention, with a higher retention rate than the arabic gum system. The pea protein-pectin electrostatic complex can also control release with pH changes. In the stomach acid environment (pH 2.0-3.5), the positively charged pea protein is tightly combined with the negatively charged pectin, and the pores are shrunk. In the intestine (pH 6.5-7.5), the charge of the pea protein is reversed, and the complex is dissociated to release the contents.

[0032] (3) Chitosan and sodium alginate are both natural polysaccharides, and have good biocompatibility. There are a large number of carboxyl and hydroxyl functional groups in sodium alginate, and chitosan is a positively charged polymer compound. The coacervate formed by chitosan and sodium alginate can well protect the core material from oxidation. The positive charge of chitosan enables it to have electrostatic adsorption with the negatively charged cell surface or targeting ligand. By utilizing the affinity of chitosan to mucosa and the selective performance to special parts, the tissue targeting of the microcapsule can be improved, and the residence time of the microcapsule in the target site can be prolonged.

[0033] (4) The microcapsule system provided by the present application has low toxicity of degradation products, and is a good carrier for active molecules. As a drug carrier, it has a high encapsulation rate, can remain stable and not easily broken in the body, ensures the effective loading and in vivo release of vitamin K2, increases nutrition and promotes health, and is not easy to cause inflammatory reaction.

[0034] (5) Adopt innovative process: three-step method of anti-solvent precipitation-high pressure microfluidization-vibration co-extrusion, anti-solvent precipitation does not need complex equipment and tedious operation steps, easy to realize; high pressure microfluidization homogenizer can realize efficient dispersion and homogenization treatment, compared with traditional homogenization technology, can realize product particle size distribution narrower, higher stability, more not easy to appear precipitation or stratification phenomenon; vibration co-extrusion can make the material more closely in the extrusion process, improve the forming quality. The three-step method combines the advantages of each technology, making the whole preparation process more efficient and controllable. BRIEF DESCRIPTION OF DRAWINGS

[0035] Figure 1 The vitamin K2 retention rate result graph of the vitamin K2 composite microcapsule system with gastric protection-intestinal sustained release function prepared by the present application examples 1-4 and comparative examples 1-6 after 15min, 60min and 120min in simulated gastric juice;

[0036] Figure 2 The vitamin K2 retention rate result graph of the vitamin K2 composite microcapsule system with gastric protection-intestinal sustained release function prepared by the present application examples 1-4 and comparative examples 1-6 after 15min, 60min and 120min in simulated intestinal juice. DETAILED DESCRIPTION

[0037] In order to better understand the content of the present application, the content of the present application is further explained in combination with specific implementation methods below, but the protection content of the present application is not limited to the following examples.

[0038] Example 1

[0039] The vitamin K2 composite microcapsule system with gastric protection-intestinal sustained release function includes three layers, from inside to outside, the core layer, the intermediate layer and the outer layer;

[0040] The core layer is a zein-shellac hydrophobic core wrapped with vitamin K2, the intermediate layer is a pea protein-pectin electrostatic complex, and the outer layer is a sodium alginate (SA) and chitosan (CS) double network gel;

[0041] According to 100% of the total mass percentage of raw materials, the following raw material components are included: vitamin K2 is 20%, zein is 10%, shellac is 10%, pea protein is 10%, low ester pectin is 10%, sodium alginate is 20%, CaCl2 is 10%, and chitosan is 10%.

[0042] The preparation method of the vitamin K2 composite microcapsule system with gastric protection-intestinal sustained release function includes the following steps:

[0043] (1) 10% zein was dissolved in 100 mL of 70% volume fraction of ethanol aqueous solution, and then stirred for 0.5 h to obtain a zein solution, 20% vitamin K2 was added to the zein solution and stirred for 0.5 h to obtain a mixed solution, then 30 mL of deionized water was added to the mixed solution, and emulsified 4 times using a high-pressure homogenizer at a pressure of 800 bar, and then 70°C reduced pressure rotary evaporation for 10 min to obtain a rotary evaporation emulsion, 10% shellac was dissolved in 50 mL of water to obtain a shellac aqueous solution, then the rotary evaporation emulsion was added to the shellac aqueous solution, and the pH of the system was adjusted to 3.5 using a hydrochloric acid solution, and then stirred for 40 min to obtain a nanoscale zein-based vitamin K2 oil microcapsule core;

[0044] (2) 10% pea protein was dissolved in 100 mL of water to obtain a pea protein aqueous solution, the pH of the pea protein aqueous solution was adjusted to 3.5, the nanoscale zein-based vitamin K2 oil microcapsule core prepared in step (1) was added to the pea protein aqueous solution with a pH of 3.5 and stirred uniformly, then 10% low ester pectin was added, and then homogenized 3 times using a micro-jet homogenizer at a pressure of 15000 psi to obtain a micro-nanoscale double-layer composite structure vitamin K2 microcapsule;

[0045] (3) 20% sodium alginate was dissolved in 100 mL of water to obtain a sodium alginate aqueous solution, the pH of the sodium alginate aqueous solution was adjusted to 3.5, the micro-nanoscale double-layer composite structure vitamin K2 microcapsule prepared in step (2) was added to the sodium alginate aqueous solution with a pH of 3.5, and stirred for 0.5 h to obtain an intermediate mixture, 10% CaCl2 and 10% chitosan were dissolved in 50 mL of water to obtain a chitosan aqueous solution of CaCl2, and then the intermediate mixture was extruded into the stirring chitosan aqueous solution of CaCl2 using a vibrating nozzle at a frequency of 1000 Hz to obtain a vitamin K2 composite microcapsule system with gastric protection-intestinal sustained release function.

[0046] Example 2

[0047] The vitamin K2 composite microcapsule system with gastric protection-intestinal sustained release function comprises three layers from inside to outside, which are core layer, intermediate layer and outer layer;

[0048] The core layer is a zein-shellac hydrophobic core wrapped with vitamin K2, the intermediate layer is a pea protein-pectin electrostatic composite, and the outer layer is a sodium alginate (SA) and chitosan (CS) double network gel;

[0049] According to 100% of the total mass percentage of raw materials, the following raw material components are included: vitamin K2 is 20%, zein is 7%, shellac is 13%, pea protein is 15%, low ester pectin is 5%, sodium alginate is 30%, CaCl2 is 5%, and chitosan is 5%.

[0050] A method for preparing a vitamin K2 composite microcapsule system with gastric protection and intestinal sustained-release functions, comprising the following steps:

[0051] (1) Dissolve 7% zein in 100 mL of a 60% volume fraction aqueous ethanol solution, then stir for 0.5 h to obtain a zein solution, add 20% vitamin K2 to the zein solution, stir for 0.5 h to obtain a mixed solution, then add 30 mL of deionized water to the mixed solution, emulsify once using a high-pressure homogenizer at a pressure of 1000 bar, and then perform rotary evaporation at 70°C for 15 min to obtain a rotary evaporation emulsion, dissolve 13% shellac in 50 mL of water to obtain a shellac aqueous solution, then add the rotary evaporation emulsion to the shellac aqueous solution, and adjust the pH of the system to 2.5 using a hydrochloric acid solution, and continue to stir for 50 min to obtain a nanoscale zein-based vitamin K2 oil microcapsule core;

[0052] (2) Dissolve 15% pea protein in 100 mL of water to obtain a pea protein aqueous solution, adjust the pH of the pea protein aqueous solution to 4.5, add the nanoscale zein-based vitamin K2 oil microcapsule core prepared in step (1) to the pea protein aqueous solution with a pH of 4.5 and stir until uniform, then add 5% low-ester pectin, and then use a micro-jet homogenizer to homogenize 5 times at a pressure of 13000 psi to obtain a vitamin K2 microcapsule with a micro-nanoscale double-layer composite structure;

[0053] (3) Dissolve 30% sodium alginate in 100 mL of water to obtain a sodium alginate aqueous solution, adjust the pH of the sodium alginate aqueous solution to 4.5, add the vitamin K2 microcapsule with a micro-nanoscale double-layer composite structure prepared in step (2) to the sodium alginate aqueous solution with a pH of 4.5, and stir for 0.5 h to obtain an intermediate mixture, dissolve 5% CaCl2 and 5% chitosan in 50 mL of water to obtain a chitosan aqueous solution of CaCl2, and then use a vibrating nozzle to extrude the intermediate mixture into the stirring chitosan aqueous solution of CaCl2 at a frequency of 900 Hz to obtain a vitamin K2 composite microcapsule system with gastric protection and intestinal sustained-release functions.

[0054] Example 3

[0055] The vitamin K2 composite microcapsule system with gastric protection and intestinal sustained-release functions comprises three layers, which are, from inside to outside, a core layer, an intermediate layer, and an outer layer;

[0056] The core layer is a zein-shellac hydrophobic core wrapped with vitamin K2, the intermediate layer is a pea protein-pectin electrostatic composite, and the outer layer is a sodium alginate (SA) and chitosan (CS) double-network gel;

[0057] The following raw material components are included, according to 100% of the total mass of the raw materials: 15% vitamin K2, 20% zein, 5% shellac, 5% pea protein, 15% low-ester pectin, 10% sodium alginate, 15% CaCl2, and 15% chitosan.

[0058] A method for preparing a vitamin K2 composite microcapsule system with gastric protection and intestinal sustained-release functions includes the following steps:

[0059] (1) Dissolve 20% zein in 100 mL of an 80% volume percentage aqueous ethanol solution, then stir for 0.5 h to obtain a zein solution. Add 15% vitamin K2 to the zein solution and stir for 0.5 h to obtain a mixed solution. Then add 30 mL of deionized water to the mixed solution, emulsify three times using a high-pressure homogenizer at a pressure of 900 bar, and then perform rotary evaporation at 70°C for 5 min to obtain a rotary evaporation emulsion. Dissolve 5% shellac in 50 mL of water to obtain a shellac aqueous solution. Then add the rotary evaporation emulsion to the shellac aqueous solution, and use a hydrochloric acid solution to adjust the pH of the system to 3. Continue stirring for 30 min to obtain a nanoscale zein-based vitamin K2 oil microcapsule inner core.

[0060] (2) Dissolve 5% pea protein in 100 mL of water to obtain a pea protein aqueous solution. Adjust the pH of the pea protein aqueous solution to 3. Add the nanoscale zein-based vitamin K2 oil microcapsule inner core prepared in step (1) to the pea protein aqueous solution with a pH of 3 and stir until uniform. Then add 15% low-ester pectin. Subsequently, use a micro-jet homogenizer to homogenize once at a pressure of 10,000 psi to obtain a micro-nanoscale double-layer composite structure vitamin K2 microcapsule.

[0061] (3) Dissolve 10% sodium alginate in 100 mL of water to obtain a sodium alginate aqueous solution. Adjust the pH of the sodium alginate aqueous solution to 3. Add the micro-nanoscale double-layer composite structure vitamin K2 microcapsule prepared in step (2) to the sodium alginate aqueous solution with a pH of 3 and stir for 0.5 h to obtain an intermediate mixture. Dissolve 15% CaCl2 and 15% chitosan in 50 mL of water to obtain a chitosan aqueous solution of CaCl2. Then use a vibrating nozzle to extrude the intermediate mixture into the stirring chitosan aqueous solution of CaCl2 at a frequency of 800 Hz to obtain a vitamin K2 composite microcapsule system with gastric protection and intestinal sustained-release functions.

[0062] Example 4

[0063] The vitamin K2 composite microcapsule system with gastric protection and intestinal sustained-release functions includes three layers, which are, from inside to outside, a core layer, an intermediate layer, and an outer layer.

[0064] The core layer is a corn alcohol-soluble protein-shellac hydrophobic core for wrapping vitamin K2, the middle layer is a pea protein-pectin electrostatic complex, and the outer layer is a sodium alginate (SA) and chitosan (CS) double network gel;

[0065] According to 100% of the total mass percentage of raw materials, the following raw material components are included: 5% vitamin K2, 5% corn alcohol-soluble protein, 15% shellac, 20% pea protein, 12% low-ester pectin, 25% sodium alginate, 6% CaCl2, and 12% chitosan.

[0066] The preparation method of the vitamin K2 composite microcapsule system with gastric protection-intestinal sustained release function includes the following steps:

[0067] (1) Dissolve 5% corn alcohol-soluble protein in 100 mL of a 75% volume percentage aqueous ethanol solution, then stir for 0.5 h to obtain a corn alcohol-soluble protein solution. Add 5% vitamin K2 to the corn alcohol-soluble protein solution and stir for 0.5 h to obtain a mixed solution. Then add 30 mL of deionized water to the mixed solution, emulsify 5 times using a high-pressure homogenizer at a pressure of 600 bar, and then perform rotary evaporation at 70°C for 8 min to obtain a rotary evaporation emulsion. Dissolve 15% shellac in 50 mL of water to obtain a shellac aqueous solution. Then add the rotary evaporation emulsion to the shellac aqueous solution, and use a hydrochloric acid solution to adjust the pH of the system to 3.5. Continue stirring for 45 min to obtain a nanoscale corn alcohol-soluble protein-based vitamin K2 oil microcapsule inner core.

[0068] (2) Dissolve 20% pea protein in 100 mL of water to obtain a pea protein aqueous solution. Adjust the pH of the pea protein aqueous solution to 4. Add the nanoscale corn alcohol-soluble protein-based vitamin K2 oil microcapsule inner core prepared in step (1) to the pea protein aqueous solution with a pH of 4 and stir until uniform. Then add 12% low-ester pectin. Subsequently, use a micro-jet homogenizer to homogenize at a pressure of 12000 psi for 2 times to obtain a micro-nanoscale double-layer composite structure vitamin K2 microcapsule.

[0069] (3) Dissolve 25% sodium alginate in 100 mL of water to obtain a sodium alginate aqueous solution. Adjust the pH of the sodium alginate aqueous solution to 4. Add the micro-nanoscale double-layer composite structure vitamin K2 microcapsule prepared in step (2) to the sodium alginate aqueous solution with a pH of 4 and stir for 0.5 h to obtain an intermediate mixture. Dissolve 6% CaCl2 and 12% chitosan in 50 mL of water to obtain a chitosan aqueous solution of CaCl2. Then use a vibrating nozzle to extrude the intermediate mixture into the stirring chitosan aqueous solution of CaCl2 at a frequency of 950 Hz to obtain a vitamin K2 composite microcapsule system with gastric protection-intestinal sustained release function.

[0070] Comparative Example 1

[0071] A vitamin K2 complex microcapsule system with gastric protection and intestinal sustained release function consists of two layers, from the inside out: a core layer and an outer layer.

[0072] The core layer consists of a zein-shellac hydrophobic core encapsulating vitamin K2, while the outer layer is a pea protein-pectin electrostatic complex.

[0073] Based on 100% of the total mass of raw materials, it includes the following raw material components: 20% vitamin K2, 20% zein, 20% shellac, 20% pea protein, and 20% low-ester pectin.

[0074] Preparation method of vitamin K2 complex microcapsule system with gastric protection and intestinal sustained release function: including the following steps:

[0075] (1) Dissolve 20% zein in 100 mL of 70% ethanol aqueous solution and stir for 0.5 h to obtain zein solution. Add 20% vitamin K2 to zein solution and stir for 0.5 h to obtain mixed solution. Then add 30 mL of deionized water to mixed solution. Emulsify 4 times at 800 bar using a high pressure homogenizer. Then evaporate under reduced pressure at 70 °C for 10 min to obtain rotary emulsion. Dissolve 20% shellac in 50 mL of water to obtain shellac aqueous solution. Then add rotary emulsion to shellac aqueous solution and adjust the pH of the system to 3.5 with hydrochloric acid solution. Continue stirring for 40 min to obtain nano-sized zein-based vitamin K2 oil microcapsule core.

[0076] (2) Dissolve 20% pea protein in 100mL of water to obtain pea protein aqueous solution. Adjust the pH of the pea protein aqueous solution to 3.5. Add the core of the nano-sized zein-based vitamin K2 oil microcapsule prepared in step (1) to the pea protein aqueous solution and stir evenly. Then add 20% low-ester pectin. Then homogenize 3 times with a microfluidic homogenizer at a pressure of 15000psi to obtain a vitamin K2 composite microcapsule system with gastric protection and intestinal sustained release function.

[0077] Comparative Example 2

[0078] A vitamin K2 complex microcapsule system with gastric protection and intestinal sustained release function consists of two layers, from the inside out: a core layer and an outer layer.

[0079] The core layer consists of a zein-shellac hydrophobic core encapsulating vitamin K2, and the outer layer is a double network gel of sodium alginate (SA) and chitosan (CS).

[0080] Based on 100% of the total mass of raw materials, the following raw material components are included: Vitamin K2 20%, zein 15%, shellac 15%, sodium alginate 23%, CaCl2 13%, and chitosan 14%.

[0081] Preparation method of vitamin K2 complex microcapsule system with gastric protection and intestinal sustained release function: including the following steps:

[0082] (1) Dissolve 15% zein in 100 mL of 70% ethanol aqueous solution and stir for 0.5 h to obtain zein solution. Add 20% vitamin K2 to zein solution and stir for 0.5 h to obtain mixed solution. Then add 30 mL of deionized water to mixed solution. Emulsify 4 times at 800 bar using a high pressure homogenizer. Then evaporate under reduced pressure at 70 °C for 10 min to obtain rotary emulsion. Dissolve 15% shellac in 50 mL of water to obtain shellac aqueous solution. Then add rotary emulsion to shellac aqueous solution and adjust the pH of the system to 3.5 with hydrochloric acid solution. Continue stirring for 40 min to obtain nano-sized zein-based vitamin K2 oil microcapsule core.

[0083] (2) Dissolve 23% sodium alginate in 100 mL of water to obtain sodium alginate aqueous solution. Adjust the pH of the sodium alginate aqueous solution to 3.5. Add the core of the nano-sized zein-based vitamin K2 oil microcapsules prepared in step (1) to the sodium alginate aqueous solution with pH 3.5 and stir for 0.5 h to obtain intermediate mixture. Dissolve 13% CaCl2 and 14% chitosan in 50 mL of water and stir evenly to obtain chitosan aqueous solution of CaCl2. Then use a vibrating nozzle to squeeze the intermediate mixture into the chitosan aqueous solution of CaCl2 at a frequency of 1000 Hz to obtain a vitamin K2 composite microcapsule system with gastric protection and intestinal sustained release function.

[0084] Comparative Example 3

[0085] A vitamin K2 complex microcapsule system with gastric protection and intestinal sustained release function consists of two layers, from the inside out: a core layer and an outer layer.

[0086] The core layer is a pea protein-pectin electrostatic complex, and the outer layer is a double network gel of sodium alginate (SA) and chitosan (CS).

[0087] Based on 100% of the total mass of raw materials, the following raw material components are included: 20% vitamin K2, 15% pea protein, 15% low-ester pectin, 23% sodium alginate, 13% CaCl2, and 14% chitosan.

[0088] A method for preparing a vitamin K2 composite microcapsule system with gastric protection and intestinal sustained-release functions, comprising the following steps:

[0089] (1) Dissolve 15% pea protein in 100 mL of a 70% volume percentage ethanol aqueous solution to obtain a pea protein solution, adjust the pH of the pea protein solution to 3.5, add 20% vitamin K2 to the pea protein solution with a pH of 3.5, stir for 0.5 h to obtain a mixed solution, then add 15% low-ester pectin, and then use a microjet homogenizer to homogenize 3 times at a pressure of 15000 psi to obtain a nanoscale pea protein-based vitamin K2 oil microcapsule core;

[0090] (2) Dissolve 23% sodium alginate in 100 mL of water to obtain a sodium alginate aqueous solution, adjust the pH of the sodium alginate aqueous solution to 3.5, add the nanoscale pea protein-based vitamin K2 oil microcapsule core prepared in step (1) to the sodium alginate aqueous solution with a pH of 3.5, and stir for 0.5 h to obtain an intermediate mixed solution, dissolve 13% CaCl2 and 14% chitosan in 50 mL of water to obtain a chitosan aqueous solution of CaCl2, then use a vibrating nozzle to extrude the intermediate mixed solution into the stirring chitosan aqueous solution of CaCl2 at a frequency of 1000 Hz to obtain a vitamin K2 composite microcapsule system with gastric protection and intestinal sustained-release functions.

[0091] Comparative Example 4

[0092] A vitamin K2 composite microcapsule system with gastric protection and intestinal sustained-release functions comprises a layer, i.e., a zein-shellac hydrophobic core wrapped with vitamin K2.

[0093] According to 100% of the total mass percentage of raw materials, the following raw material components are included: 50% vitamin K2, 25% zein, and 25% shellac.

[0094] A method for preparing a vitamin K2 composite microcapsule system with gastric protection and intestinal sustained-release functions, comprising the following steps:

[0095] Dissolve 25% zein in 100 mL of a 70% volume percentage ethanol aqueous solution, then stir for 0.5 h to obtain a zein solution, add 50% vitamin K2 to the zein solution, stir for 0.5 h to obtain a mixed solution, then add 30 mL of deionized water to the mixed solution, emulsify 4 times using a high-pressure homogenizer at a pressure of 800 bar, and then perform rotary evaporation at 70°C for 10 min to obtain a rotary evaporation emulsion, dissolve 25% shellac in 50 mL of water to obtain a shellac aqueous solution, then add the rotary evaporation emulsion to the shellac aqueous solution, and use a hydrochloric acid solution to adjust the pH of the system to 3.5, continue to stir for 40 min to obtain a vitamin K2 composite microcapsule system with gastric protection and intestinal sustained-release functions.

[0096] Comparative Example 5

[0097] The vitamin K2 complex microcapsule system with gastric protection and intestinal sustained-release function comprises one layer, i.e., a vitamin K2 wrapped by a pea protein-pectin electrostatic complex;

[0098] The following raw material components are included according to 100% of the total mass percentage of the raw materials: 50% of vitamin K2, 25% of pea protein, and 25% of low-ester pectin.

[0099] The preparation method of the vitamin K2 complex microcapsule system with gastric protection and intestinal sustained-release function includes the following steps:

[0100] 25% of pea protein is dissolved in 100 mL of water to obtain a pea protein aqueous solution, the pH of the pea protein aqueous solution is adjusted to 3.5, 50% of vitamin K2 is added to 100 mL of the pea protein aqueous solution with a pH of 3.5 and stirred uniformly, then 25% of low-ester pectin is added, and then the micro-jet homogenizer is used for homogenization three times at a pressure of 15000 psi to obtain the vitamin K2 complex microcapsule system with gastric protection and intestinal sustained-release function.

[0101] Comparative Example 6

[0102] The vitamin K2 complex microcapsule system with gastric protection and intestinal sustained-release function comprises one layer, i.e., a vitamin K2 wrapped by a sodium alginate (SA) and chitosan (CS) double-network gel;

[0103] The following raw material components are included according to 100% of the total mass percentage of the raw materials: 35% of vitamin K2, 35% of sodium alginate, 15% of CaCl2, and 15% of chitosan.

[0104] The preparation method of the vitamin K2 complex microcapsule system with gastric protection and intestinal sustained-release function includes the following steps:

[0105] 35% of sodium alginate is dissolved in 100 mL of water to obtain a sodium alginate aqueous solution, the pH of the sodium alginate aqueous solution is adjusted to 3.5, 35% of vitamin K2 is added to 100 mL of the sodium alginate aqueous solution with a pH of 3.5, and stirring is performed for 0.5 h to obtain an intermediate mixture, 15% of CaCl2 and 15% of chitosan are dissolved in 50 mL of water to obtain a chitosan solution of CaCl2, and then the intermediate mixture is extruded into the stirring chitosan solution of CaCl2 using a vibration nozzle at a frequency of 1000 Hz to obtain the vitamin K2 complex microcapsule system with gastric protection and intestinal sustained-release function.

[0106] Test Example 1: Detection of the embedding rate of the vitamin K2 complex microcapsule system with gastric protection and intestinal sustained-release function

[0107] The embedding rate of vitamin K2 in the vitamin K2 complex microcapsule system with gastric protection and intestinal sustained release function prepared in Examples 1 to 4 and Comparative Examples 1 to 6 was detected by solvent extraction method. The calculation formula of the embedding rate was: (the content of vitamin K2 embedded in the microcapsule system / the total content of vitamin K2 in the microcapsule system) * 100%, and the total content of vitamin K2 in the microcapsule system = the content of vitamin K2 on the surface of the microcapsule system + the content of vitamin K2 embedded in the microcapsule system. The detection results are shown in Table 1.

[0108] Measurement of the content of vitamin K2 on the surface of the microcapsule system: an appropriate amount of microcapsule system was weighed on filter paper, the sample on the filter paper was washed by soaking with anhydrous ethanol, the filtrate was collected, and the content of vitamin K2 was detected by HPLC method.

[0109] Measurement of the content of vitamin K2 embedded in the microcapsule system: the microcapsule system washed by anhydrous ethanol was transferred to a stoppered conical flask, anhydrous ethanol was added, and after ultrasonic treatment and centrifugation, the supernatant was taken, and the content of vitamin K2 was detected by HPLC method.

[0110] Table 1: Detection results of the embedding rate of the vitamin K2 complex microcapsule system with gastric protection and intestinal sustained release function

[0111]

[0112]

[0113] As can be seen from the results in Table 1, the embedding rates of the vitamin K2 complex microcapsule system with gastric protection and intestinal sustained release function provided in Examples 1 to 4 are all above 99%, which are higher than those in Comparative Examples 1 to 6, and the embedding rate of Example 4 reaches 99.8%. The vitamin K2 complex microcapsule system with gastric protection and intestinal sustained release function provided in the application can provide better protection for vitamin K2, and improve the utilization rate and stability of vitamin K2.

[0114] Test Example 2: Measurement of particle size, PDI and Zeta potential of the vitamin K2 complex microcapsule system with gastric protection and intestinal sustained release function

[0115] An appropriate amount of the vitamin K2 complex microcapsule system with gastric protection and intestinal sustained release function prepared in Examples 1 to 4 and Comparative Examples 1 to 6 was respectively prepared into a solution with a concentration of 0.1 mg / mL by using ethanol, and the particle size distribution, PDI and Zeta potential thereof were detected by using a Zetasizer Pro Malven electric potential particle size analyzer. The sample was repeatedly measured for 3 times, and the measurement results were shown as average values. The measurement results are shown in Table 2.

[0116] Table 2: Measurement results of particle size, PDI and Zeta potential

[0117] Particle size PDI Zeta potential Example 1 405.7 0.129 -13.47 Example 2 408.9 0.125 -13.56 Example 3 417.2 0.133 -13.20 Example 4 421.4 0.147 -12.80 Comparative Example 1 506.2 0.214 -12.52 Comparative Example 2 513.8 0.218 -12.45 Comparative Example 3 518.5 0.217 -12.58 Comparative Example 4 595.4 0.246 -11.50 Comparative Example 5 589.8 0.237 -12.02 Comparative Example 6 592.2 0.241 -11.80

[0118] PDI (polydispersity index) is an index for measuring the width of particle size distribution, and the smaller the value is, the narrower the particle size distribution is, i.e. the more uniform the particle size is. As can be seen from the results in Table 2, the PDI value of the vitamin K2 composite microcapsule system with gastric protection-intestinal sustained release function provided by Examples 1-4 is less than 0.2, indicating that the particle size distribution of the system is very uniform, and the system has good stability and dispersibility. Zeta potential is an important index for measuring the surface charge of particles, and it reflects the charge state and stability of particles in solution. Nanoparticles with higher absolute value of Zeta potential are relatively more stable. The absolute value of Zeta potential of the vitamin K2 composite microcapsule system with gastric protection-intestinal sustained release function provided by Examples 1-4 is greater than that of Comparative Examples 1-6, indicating that the particle size distribution of the system is more uniform, and the system is not easily attracted to each other to form a large amount of agglomeration, and has good stability and dispersibility.

[0119] Test Example 3: Stability detection

[0120] The stability of the vitamin K2 composite microcapsule system with gastric protection-intestinal sustained release function prepared by Examples 1-4 and Comparative Examples 1-6 was evaluated after being placed under different conditions for 90 days. The appearance, odor, embedding rate, retention rate, and reconstitution were evaluated.

[0121] The appearance and odor were tested by sensory evaluation method.

[0122] The detection method of embedding rate is shown in Test Example 1.

[0123] Retention rate detection: The content of vitamin K2 before embedding was set as the initial content C1 of vitamin K2. After the vitamin K2 composite microcapsule system with gastric protection-intestinal sustained release function was placed under different conditions for 90 days, the total content of vitamin K2 in the sample was determined, which was the content C2 of vitamin K2 in the vitamin K2 composite microcapsule system with gastric protection-intestinal sustained release function after being stored for a period of time, and the retention rate of vitamin K2 = C2 / C1 x 100%. The determination of the total content of vitamin K2: The microcapsule system was transferred to a stoppered conical flask, anhydrous ethanol was added, and the supernatant was taken after ultrasonic and centrifugal treatment. The content of vitamin K2 was determined by HPLC method.

[0124] Reconstitution: 10 g of sample was taken, added into 100 mL of water at room temperature, and stirred with a glass rod for 5 minutes, and the appearance was observed.

[0125] 1) The vitamin K2 composite microcapsule system with gastric protection-intestinal sustained release function prepared by Examples 1-4 and Comparative Examples 1-6 was filled into sodium-calcium glass bottles and sealed, and the stability test was carried out under the condition of temperature 37℃ and humidity 75%, and the test results are shown in Table 3.

[0126] Table 3 Stability test results under the condition of temperature 37℃ and humidity 75%

[0127]

[0128]

[0129] From the results in Table 3, it can be seen that the appearance, odor, and solubility of the vitamin K2 composite microcapsule system prepared in Examples 1-4 having the gastric protection-intestinal sustained release function did not change after 90 days, the embedding rate and retention rate of vitamin K2 slightly decreased but was not significant, and was still above 98%, and was better than that of Comparative Examples 1-6, indicating that the physical properties of the three-layer microcapsule system provided by the present application remained stable for 90 days, had a good protection effect on vitamin K2, and maintained good solubility and dispersibility.

[0130] 2) The vitamin K2 composite microcapsule system prepared in Examples 1-4 and Comparative Examples 1-6 having the gastric protection-intestinal sustained release function was filled into an oxygen-filled glass bottle and sealed for stability testing, and the test results are shown in Table 4.

[0131] Table 4 Stability test results under oxygen-containing conditions

[0132]

[0133]

[0134] From the results in Table 4, it can be seen that the appearance, odor, and solubility of the vitamin K2 composite microcapsule system prepared in Examples 1-4 having the gastric protection-intestinal sustained release function did not change after 90 days, the embedding rate and retention rate of vitamin K2 slightly decreased but was not significant, and was still above 98%, and was better than that of Comparative Examples 1-6, indicating that the physical properties of the three-layer microcapsule system provided by the present application remained stable for 90 days, had a good protection effect on vitamin K2, and maintained good solubility and dispersibility.

[0135] 3) The vitamin K2 composite microcapsule system prepared in Examples 1-4 and Comparative Examples 1-6 having the gastric protection-intestinal sustained release function was filled into a glass bottle and sealed for stability testing under a 4500 Lx light intensity, and the test results are shown in Table 5.

[0136] Table 5 Stability test results under a 4500 Lx light intensity

[0137]

[0138]

[0139] From the results in Table 5, it can be seen that the appearance, odor, and solubility of the vitamin K2 composite microcapsule system prepared in Examples 1-4 having the gastric protection-intestinal sustained release function did not change after 90 days, the vitamin K2 embedding rate and retention rate slightly decreased but was not significant, and were all better than those of Comparative Examples 1-6, indicating that the physical properties of the three-layer microcapsule system provided by the present application remained stable for 90 days, had a good protection effect on vitamin K2, could avoid damage to vitamin K2 by light, and maintained good solubility and dispersibility.

[0140] 4) The vitamin K2 composite microcapsule system prepared in Examples 1-4 and Comparative Examples 1-6 having the gastric protection-intestinal sustained release function was sealed in a glass bottle and placed in a constant temperature oven at 60°C for stability testing, and the test results are shown in Table 6.

[0141] Table 6 Stability test results under constant temperature conditions at 60°C

[0142]

[0143]

[0144] From the results in Table 6, it can be seen that the appearance, odor, and solubility of the vitamin K2 composite microcapsule system prepared in Examples 1-4 having the gastric protection-intestinal sustained release function did not change after 90 days, the vitamin K2 embedding rate and retention rate slightly decreased but was not significant, and were all better than those of Comparative Examples 1-6, indicating that the physical properties of the three-layer microcapsule system provided by the present application remained stable for 90 days, had a good protection effect on vitamin K2, could avoid damage to vitamin K2 by high temperature, and maintained good solubility and dispersibility.

[0145] Test Example 4: In vitro digestion simulation

[0146] 0.5 g of the vitamin K2 composite microcapsule system prepared in Examples 1-4 and Comparative Examples 1-6 having the gastric protection-intestinal sustained release function was added to 10 mL of simulated gastric fluid or simulated intestinal fluid, respectively. The composition of the simulated gastric fluid and the simulated intestinal fluid is shown in Table 7. 1 mg of 1000 U / mg pepsin was added to the simulated gastric fluid and the pH was adjusted to 2.1 with hydrochloric acid, and 1 mg of 1000 U / mg complex enzyme (trypsin and amylase, mass ratio of trypsin to amylase 1:1) was added to the simulated intestinal fluid and the pH was adjusted to 7.0. The retention rate of vitamin K2 was then measured at 15 min, 60 min, and 120 min, respectively. The results of the retention rate of vitamin K2 in the simulated gastric fluid are shown in Table 8, and the results of the retention rate of vitamin K2 in the simulated intestinal fluid are shown in Table 9. Figure 1 Figure 2

[0147] Table 7 Composition of simulated gastric fluid and simulated intestinal fluid​​

[0148] Ingredients Simulated gastric fluid Simulated intestinal fluid 0.5 mol / L KCI / mL 6.9 6.8 0.5 mol / L KH2PO4 / mL 0.9 0.8 1 mol / L NaHCO3 / mL 12.5 42.5 2 mol / L NaCI / mL 11.8 9.6 0.15 mol / L MgCl2 / mL 0.4 1.1 0.5 mol / L (NH4)2CO3 / mL 0.5 /

[0149] from Figure 1 , Figure 2 The results show that the vitamin K2 retention rate of the vitamin K2 composite microcapsule systems with gastric protection and intestinal sustained-release functions prepared in Examples 1-4 was higher than that of Comparative Examples 1-6 in simulated gastric juice. The high retention rate of vitamin K2 in simulated gastric juice indicates that it can retain its activity for as long as possible in the human stomach. However, the vitamin K2 retention rate of the vitamin K2 composite microcapsule systems with gastric protection and intestinal sustained-release functions prepared in Examples 1-4 was lower than that of Comparative Examples 1-6 in simulated intestinal juice. The low retention rate of vitamin K2 in simulated intestinal juice indicates that it promotes absorption in the intestine. Vitamin K2 is a fat-soluble vitamin and is easily destroyed by gastric acid and digestive enzymes in the stomach. The three-layer microcapsule system for encapsulating vitamin K2 provided by this invention can tightly bind in the acidic environment of the stomach, reducing the release of the active ingredient in gastric juice, and then releasing it in the intestine, thus improving the therapeutic effect. The three-layer microcapsule system for vitamin K2 utilizes a layered structure to effectively protect vitamin K2 from structural damage in the gastric environment, achieving high retention of vitamin K2 in the stomach and high release in the intestine.

[0150] The above description is only a specific embodiment of the present invention and not all embodiments. Any equivalent modifications made by those skilled in the art to the technical solutions of the present invention by reading the present invention specification are covered by the claims of the present invention.

Claims

1. An application of a microencapsulation system as a vitamin K2 encapsulation system, the microencapsulation system comprising three layers, from the inside out being a core layer, a middle layer and an outer layer; the core layer being a zein-shellac hydrophobic core, the middle layer being a pea protein-low-ester pectin electrostatic complex, and the outer layer being a sodium alginate and chitosan dual-network gel.

2. A vitamin K2 complex microcapsule system with gastric protection and intestinal sustained-release function, characterized in that, It consists of three layers, from the inside out: the core layer, the middle layer, and the outer layer. The core layer is a vitamin K2 encapsulated in a zein-shellac hydrophobic core, the middle layer is a pea protein-low ester pectin electrostatic complex, and the outer layer is a sodium alginate and chitosan dual-network gel.

3. The vitamin K2 complex microcapsule system with gastric protection and intestinal sustained-release function according to claim 2, characterized in that, Based on 100% of the total mass of raw materials, the following raw material components are included: Vitamin K2 5%~20%, zein 5%~20%, shellac 5%~15%, pea protein 5%~20%, low-ester pectin 5%~15%, sodium alginate 10%~30%, calcium chloride 5%~15%, and chitosan 5%~15%.

4. The method for preparing the vitamin K2 complex microcapsule system with gastric protection and intestinal sustained-release function as described in any one of claims 2 to 3, characterized in that, Includes the following steps: (1) Dissolve zein in a solvent to obtain a zein solution. Add vitamin K2 to the zein solution and stir until homogeneous to obtain a mixed solution. Then add water to the mixed solution, emulsify using a high-pressure homogenizer, and then evaporate to obtain a rotary emulsion. Dissolve shellac in water to obtain a shellac aqueous solution. Then add the rotary emulsion to the shellac aqueous solution and adjust the pH of the system to 2.5~3.

5. Continue stirring for a period of time to obtain the core of nano-sized zein-based vitamin K2 oil microcapsules. (2) Dissolve pea protein in water to obtain pea protein aqueous solution, adjust the pH of pea protein aqueous solution to 3~4.5, add the core of nano-sized zein-based vitamin K2 oil microcapsules prepared in step (1) to pea protein aqueous solution with pH 3~4.5 and stir evenly, then add low-ester pectin, and then homogenize using a microfluidic homogenizer to obtain vitamin K2 microcapsules with micro-nano-sized bilayer composite structure; (3) Sodium alginate is dissolved in water to obtain sodium alginate aqueous solution. The pH of sodium alginate aqueous solution is adjusted to 3~4.

5. The vitamin K2 microcapsules with micro-nano-scale bilayer composite structure prepared in step (2) are added to sodium alginate aqueous solution with pH 3~4.5 and stirred evenly to obtain intermediate mixture. Calcium chloride and chitosan are dissolved in water and stirred evenly to obtain calcium chloride chitosan aqueous solution. Then, the intermediate mixture is squeezed into the stirred calcium chloride chitosan aqueous solution using a vibrating nozzle to obtain uniform vitamin K2 oil particles with a three-layer protective structure, that is, vitamin K2 composite microcapsule system with gastric protection and intestinal sustained release function.

5. The preparation method according to claim 4, characterized in that, The solvent mentioned in step (1) is selected from an aqueous ethanol solution with a volume percentage of 60% to 80%; The emulsification pressure in step (1) is 600~1000 bar, and the number of emulsifications is 1~5 times; The rotary evaporation in step (1) is carried out at 65~75℃ under reduced pressure, and the rotary evaporation time is 5~15 min; The stirring time mentioned in step (1) is 30~50 min.

6. The preparation method according to claim 4, characterized in that, The homogenization pressure in step (2) is 10,000 to 15,000 psi, and the number of homogenization cycles is 1 to 5.

7. The preparation method according to claim 4, characterized in that, The frequency of the vibrating nozzle in step (3) is 800~1000 Hz.

8. The use of the vitamin K2 complex microcapsule system with gastric protection and intestinal sustained release function as described in any one of claims 2 to 3 in the preparation of drugs for maintaining bone health, preventing cardiovascular disease, protecting nerves, and improving metabolism.

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