Shell-core double-layer gel ball with targeted delivery function as well as preparation method and application of shell-core double-layer gel ball

Abstract

The invention discloses a shell-core double-layer gel ball with a targeted delivery function and a preparation method and application thereof, belongs to the technical field of shell-core gels, and provides the preparation method of the shell-core double-layer gel ball with the targeted delivery function, and the preparation method comprises the following steps: mixing a polysaccharide aqueous solution and a protein aqueous solution in an isovolumetric manner to obtain a composite gel precursor solution, and performing water bath treatment to obtain the shell-core double-layer gel ball with the targeted delivery function. Dropwise adding the thermal copolymerization product into a calcium chloride aqueous solution, curing to obtain a single cross-linked gel ball, immersing the single cross-linked gel ball into a tannic acid aqueous solution, and curing to obtain a shell-core double-layer gel ball. The polysaccharide is used as a core skeleton, and the protein and tannic acid compound is used for filling outer layer pores to form a shell with a compact double-penetrating network structure, so that the barrier property on oxygen and gastrointestinal fluid is remarkably improved, and the storage stability and the in-vivo colonization effect can be improved.

Description

Technical Field

[0001] The present invention belongs to the technical field of core-shell type gels, and particularly relates to a core-shell double-layer gel sphere with a targeted delivery function, a preparation method thereof, and an application thereof. Background Art

[0002] At present, there are various probiotic encapsulation technologies, such as spray drying method, freeze drying method, emulsification method, layer-by-layer encapsulation method, extrusion method, and electrostatic spraying method, etc. Among them, the hydrogel prepared by extrusion has the advantages of simple production method and high nutritional value, and is widely used in fields such as tissue repair and drug delivery. In addition, in terms of different encapsulation materials, natural proteins and polysaccharides are the two main encapsulation materials due to their good biocompatibility, biodegradability, etc. However, protein-based materials will be degraded by pepsin in the body and lose their protective effect. Therefore, polysaccharides have certain potential as probiotic encapsulation materials. However, gels prepared from a single polysaccharide have defects such as low encapsulation rate and easy lysis leading to premature release of probiotics, and the protective effect on probiotics is not ideal enough. Composite gels can effectively improve such problems.

[0003] Existing studies have shown that encapsulating probiotics with prebiotic materials can improve their stress resistance and colonization effect in the body. Low-esterified pectin can form a stable egg-box structure with calcium ions, and has advantages in terms of safety, processability, and cost. And studies have shown that 30% esterified pectin can promote the adhesion of Lactiplantibacillus plantarum to intestinal epithelial cells and has good stability in in vitro digestion. However, the interior of single-ion solidified gels such as pectin and sodium alginate will still be rapidly penetrated by gastric juice, and it is impossible to ensure the same protective effect for strains with different gastric juice tolerances. Summary of the Invention

[0004] To solve the above technical problems, the present invention provides a core-shell double-layer gel sphere with a targeted delivery function, a preparation method thereof, and an application thereof. Using polysaccharides as the core framework and a protein and tannic acid complex to fill the outer pores, a dense double-permeating network structure shell is formed, which significantly improves the barrier properties to oxygen and gastrointestinal fluids, and can improve storage stability and in vivo colonization effect.

[0005] To achieve the above object, the present invention provides a preparation method of a core-shell double-layer gel sphere with a targeted delivery function, comprising the following steps:

[0006] (1) Mix an aqueous polysaccharide solution and an aqueous protein solution in equal volume to obtain a composite gel precursor solution;

[0007] (2) Subject the composite gel precursor solution obtained in step (1) to a water bath treatment to obtain a thermal copolymerization product;

[0008] (3) The hot copolymerization product obtained in step (2) is dropped into an aqueous calcium chloride solution for primary curing to obtain single-crosslinked gel beads.

[0009] (4) The single-crosslinked gel beads obtained in step (3) are immersed in an aqueous tannic acid solution for secondary curing to obtain core-shell double-layer gel beads.

[0010] Preferably, in step (1), the mass concentration of the aqueous polysaccharide solution is 2-6%, and the aqueous polysaccharide solution is prepared from one or more of pectin, sodium alginate, konjac glucomannan, hemicellulose or chitosan.

[0011] Preferably, in step (1), the mass concentration of the aqueous protein solution is 3-10%, and the aqueous protein solution is prepared from one or more of type A gelatin, type B gelatin, fish gelatin, casein or collagen.

[0012] Preferably, in step (2), the temperature of the water bath treatment is 80-100 °C, and the time of the water bath treatment is 2-4 h.

[0013] Preferably, in step (3), the dropping speed is 150-250 μL / min, and the hot copolymerization product is 10-20 cm away from the liquid surface of the aqueous calcium chloride solution during dropping; in step (3), the mass concentration of the aqueous calcium chloride solution is 1-6%.

[0014] Preferably, in step (3), the time of the primary curing is 20-40 min.

[0015] Preferably, in step (4), the mass concentration of the aqueous tannic acid solution is 1-12%.

[0016] Preferably, in step (4), the time of the secondary curing is 20-40 min.

[0017] The present invention also provides core-shell double-layer gel beads with a targeted delivery function prepared by the preparation method.

[0018] The present invention also provides the application of the core-shell double-layer gel beads prepared by the preparation method or the core-shell double-layer gel beads in the preparation of probiotic targeted delivery capsules.

[0019] Compared with the prior art, the present invention has the following advantages and technical effects:

[0020] The core-shell double-layer gel beads with targeted delivery function in the present invention form a polysaccharide eggbox structure skeleton through ionic crosslinking of polysaccharides such as pectin or sodium alginate with calcium ions, and then are immersed in a phenolic acid solution. Protein-phenolic acid covalent crosslinking is utilized. Protein substances such as type A gelatin, type B gelatin, fish gelatin, casein or collagen react with tannic acid to generate a dense shell layer within several minutes. The phenolic acid will not penetrate excessively and does not affect the core-embedded substances. The results show that the oxygen and gastrointestinal fluid barrier properties of the core-shell double-layer gel beads are significantly improved. When the core-shell double-layer gel beads encapsulate probiotics, the storage stability and in vivo colonization effect of the probiotics can be enhanced. In addition, the thermocopolymerization product of the mixture of pectin and gelatin has higher free radical scavenging ability, and at the same time, the viscosity and gel temperature are significantly reduced, which is beneficial to the actual production application of bacteria encapsulation.

[0021] The present invention uses pectin as the core skeleton and in-situ generates a dense shell layer through the covalent binding of tannic acid and gelatin. The method is simple and controllable, and the production efficiency is high. The raw materials used are natural and non-toxic, suitable for the slow release and intestinal targeted delivery of materials such as probiotics or drugs, and are not restricted by the solubility of the encapsulated substances. The thermocopolymerization reaction sterilizes while improving the antioxidant effect of the material, and significantly reduces the viscosity and gel temperature of the composite gel precursor solution, which is beneficial to production operations. In addition, it can reduce the stress caused by the shrinkage of the shell layer during secondary crosslinking, avoid the premature rupture of the core-shell double-layer gel beads during digestion, and also reduce the risk of indigestion caused by the residual shell layer. Brief Description of the Drawings

[0022] In order to more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the following will briefly introduce the drawings required to be used in the embodiments. Obviously, the drawings in the following description are only some embodiments of the present invention. For those of ordinary skill in the art, without creative efforts, other drawings can be obtained based on these drawings.

[0023] Figure 1 For the determination of the DPPH free radical scavenging rate of the thermocopolymerization products prepared in Examples 1 to 3 and the composite gel precursor solutions prepared in Comparative Examples 1 to 3, in the figure, Pec is an aqueous pectin solution with a concentration of 4 mg / mL, Gel is an aqueous gelatin solution with a concentration of 4 mg / mL, 4-6 in the figure is the composite gel precursor solution prepared in Comparative Example 1, 2-4 in the figure is the composite gel precursor solution prepared in Comparative Example 2, 4-4 in the figure is the composite gel precursor solution prepared in Comparative Example 3, 4-6c in the figure is the thermocopolymerization product prepared in Example 1, 2-4c in the figure is the thermocopolymerization product prepared in Example 2, 4-4c in the figure is the thermocopolymerization product prepared in Example 3, and a, b, c, and d in the figure represent the significance of differences;

[0024] Figure 2Determination of the ABTS radical scavenging rate of the thermal copolymerization products prepared in Examples 1-3 and the composite gel precursor solutions prepared in Comparative Examples 1-3. In the figure, Pec is an aqueous pectin solution with a concentration of 4 mg / mL, Gel is an aqueous gelatin solution with a concentration of 4 mg / mL, 4-6 in the figure is the composite gel precursor solution prepared in Comparative Example 1, 2-4 in the figure is the composite gel precursor solution prepared in Comparative Example 2, 4-4 in the figure is the composite gel precursor solution prepared in Comparative Example 3, 4-6c in the figure is the thermal copolymerization product prepared in Example 1, 2-4c in the figure is the thermal copolymerization product prepared in Example 2, 4-4c in the figure is the thermal copolymerization product prepared in Example 3, and a, b, c, d, and e in the figure represent the significance of differences;

[0025] Figure 3 Determination of the Zeta potential of the thermal copolymerization products prepared in Examples 1-3 and the composite gel precursor solutions prepared in Comparative Examples 1-3. In the figure, Pec is an aqueous pectin solution with a concentration of 4 mg / mL, Gel is an aqueous gelatin solution with a concentration of 4 mg / mL, 4-6 in the figure is the composite gel precursor solution prepared in Comparative Example 1, 2-4 in the figure is the composite gel precursor solution prepared in Comparative Example 2, 4-4 in the figure is the composite gel precursor solution prepared in Comparative Example 3, 4-6c in the figure is the thermal copolymerization product prepared in Example 1, 2-4c in the figure is the thermal copolymerization product prepared in Example 2, 4-4c in the figure is the thermal copolymerization product prepared in Example 3, "ns" in the figure indicates no significant difference, and "****" indicates a significant difference;

[0026] Figure 4 Determination of the macroscopic viscosity index of the thermal copolymerization products prepared in Examples 1-3 and the composite gel precursor solutions prepared in Comparative Examples 1-3. 4-6 in the figure is the composite gel precursor solution prepared in Comparative Example 1, 2-4 in the figure is the composite gel precursor solution prepared in Comparative Example 2, 4-4 in the figure is the composite gel precursor solution prepared in Comparative Example 3, 4-6c in the figure is the thermal copolymerization product prepared in Example 1, 2-4c in the figure is the thermal copolymerization product prepared in Example 2, 4-4c in the figure is the thermal copolymerization product prepared in Example 3;

[0027] Figure 5 Determination of the mechanical properties of the composite gel precursor solutions prepared in Comparative Examples 1-3, the core-shell double-layer gels prepared in Comparative Examples 1-3, and the core-shell double-layer gels prepared in Examples 1-3. 4-6 in the figure is the composite gel precursor solution prepared in Comparative Example 1, 2-4 in the figure is the composite gel precursor solution prepared in Comparative Example 2, 4-4 in the figure is the composite gel precursor solution prepared in Comparative Example 3, 4-6T in the figure is the core-shell double-layer gel prepared in Comparative Example 1, 2-4T in the figure is the core-shell double-layer gel prepared in Comparative Example 2, 4-4T in the figure is the core-shell double-layer gel prepared in Comparative Example 3, 4-6cT in the figure is the core-shell double-layer gel prepared in Example 1, 2-4cT in the figure is the core-shell double-layer gel prepared in Example 2, 4-4cT in the figure is the core-shell double-layer gel prepared in Example 3;

[0028] Figure 6 Determination of the gastrointestinal digestion characteristics of the thermocopolymerization product prepared in Example 4, the composite gel precursor solution prepared in Comparative Example 4, the core-shell double-layer gel beads prepared in Comparative Example 4, and the core-shell double-layer gel beads prepared in Example 4. Among them, A is simulated gastric juice digestion, B is simulated intestinal juice digestion. In the figure, 3-6c is the thermocopolymerization product prepared in Example 4, 3-6 in the figure is the composite gel precursor solution prepared in Comparative Example 4, 3-6T in the figure is the core-shell double-layer gel beads prepared in Comparative Example 4, and 3-6cT in the figure is the core-shell double-layer gel beads prepared in Example 4. Detailed implementation manners

[0029] Now, various exemplary implementation manners of the present invention will be described in detail. This detailed description should not be regarded as a limitation of the present invention, but should be understood as a more detailed description of certain aspects, characteristics, and implementation schemes of the present invention.

[0030] It should be understood that the terms described in the present invention are only for describing specific implementation manners and are not used to limit the present invention. Additionally, for the numerical ranges in the present invention, it should be understood that each intermediate value between the upper and lower limits of the range is also specifically disclosed. Each intermediate value within any stated value or stated range, as well as each smaller range between any other stated value or intermediate value within the stated range, is also included in the present invention. The upper and lower limits of these smaller ranges can be independently included or excluded from the range.

[0031] Unless otherwise specified, all technical and scientific terms used herein have the same meaning as commonly understood by those of ordinary skill in the art to which the present invention pertains. Although the present invention only describes preferred methods and materials, any methods and materials similar or equivalent to those described herein can also be used in the implementation or testing of the present invention. All documents mentioned in this specification are incorporated by reference to disclose and describe the methods and / or materials related to the documents. In case of conflict with any incorporated document, the content of this specification shall prevail.

[0032] Without departing from the scope or spirit of the present invention, various improvements and changes can be made to the specific implementation manners of the specification of the present invention, which are obvious to those skilled in the art. Other implementation manners obtained from the specification of the present invention are obvious to those skilled in the art. The specification and examples of the present invention are only exemplary.

[0033] Regarding the terms "comprising", "including", "having", "containing", etc. used herein, they are all open-ended terms, meaning including but not limited to.

[0034] Sources of materials used in the present invention: The pectin is food-grade apple peel pectin with an esterification degree of 30±3%, purchased from Yantai Andre Pectin Co., Ltd. (galacturonic acid ≥65.0%, Shandong, China); the gelatin is type B gelatin sourced from pigskin, purchased from Shanghai Yuanye Bio-Technology Co., Ltd., product number S22176; tannic acid is purchased from Shanghai Macklin Biochemical Co., Ltd., product number T818845; calcium chloride is purchased from Shanghai Macklin Biochemical Co., Ltd., product number C832203; sodium alginate is purchased from Shanghai Macklin Biochemical Co., Ltd., product number S817374; type A gelatin is purchased from Shanghai Yuanye Bio-Technology Co., Ltd., product number S25197; konjac glucomannan is purchased from Hefei Bomei Bio-Technology Co., Ltd., product number PK2989; collagen is purchased from Shanghai Macklin Biochemical Co., Ltd., product number C875812;

[0035] Example 1

[0036] The pectin is mixed with sterile water and stirred at 40°C for 4 h until completely dissolved, then cooled to room temperature to obtain a 4 mg / mL aqueous pectin solution. The type B gelatin is mixed with sterile water and stirred at 50°C for 6 h until completely dissolved, then cooled to room temperature to obtain a 6 mg / mL aqueous gelatin solution. The tannic acid is mixed with sterile water and dissolved by ultrasonic treatment at room temperature for 2 h to obtain a 3% (by mass) aqueous tannic acid solution.

[0037] The aqueous pectin solution and the aqueous gelatin solution are mixed in equal volumes to obtain a composite gel precursor solution, which is treated in a 90°C water bath for 3 h to obtain a thermal copolymerization product (4-6c). The thermal copolymerization product is dropped into a 2% (by mass) aqueous calcium chloride solution at a rate of 200 μL / min. When dropping, the thermal copolymerization product is 15 cm away from the liquid surface of the aqueous calcium chloride solution. The first curing is carried out for 30 min to obtain a single cross-linked gel sphere. The single cross-linked gel sphere is immersed in a 3% (by mass) aqueous tannic acid solution, and the second curing is carried out for 30 min to obtain a core-shell double-layer gel sphere (4-6cT).

[0038] Example 2

[0039] The pectin is mixed with sterile water and stirred at 40°C for 4 h until completely dissolved, then cooled to room temperature to obtain a 2 mg / mL aqueous pectin solution. The type B gelatin is mixed with sterile water and stirred at 50°C for 6 h until completely dissolved, then cooled to room temperature to obtain a 4 mg / mL aqueous gelatin solution. The tannic acid is mixed with sterile water and dissolved by ultrasonic treatment at room temperature for 2 h to obtain a 3% (by mass) aqueous tannic acid solution.

[0040] The aqueous pectin solution and the aqueous gelatin solution were mixed in equal volumes to obtain a composite gel precursor solution, which was treated in a water bath at 90 °C for 3 h to obtain a thermal copolymerization product (2-4c). The thermal copolymerization product was dropped into an aqueous calcium chloride solution with a mass concentration of 2% at a rate of 200 μL / min. When dropping, the thermal copolymerization product was 15 cm away from the liquid surface of the aqueous calcium chloride solution. The first curing was carried out for 30 min to obtain a single-crosslinked gel sphere. The single-crosslinked gel sphere was immersed in an aqueous tannic acid solution with a mass concentration of 3%, and the second curing was carried out for 30 min to obtain a core-shell double-layer gel sphere (2-4cT).

[0041] Example 3

[0042] Pectin was mixed with sterile water and stirred at 40 °C for 4 h until completely dissolved, and then cooled to room temperature to obtain an aqueous pectin solution with a concentration of 4 mg / mL. Type B gelatin was mixed with sterile water and stirred at 50 °C for 6 h until completely dissolved, and then cooled to room temperature to obtain an aqueous gelatin solution with a concentration of 4 mg / mL. Tannic acid was mixed with sterile water and dissolved by ultrasonic wave at room temperature for 2 h to obtain an aqueous tannic acid solution with a mass concentration of 3%.

[0043] The aqueous pectin solution and the aqueous gelatin solution were mixed in equal volumes to obtain a composite gel precursor solution, which was treated in a water bath at 90 °C for 3 h to obtain a thermal copolymerization product (4-4c). The thermal copolymerization product was dropped into an aqueous calcium chloride solution with a mass concentration of 2% at a rate of 200 μL / min. When dropping, the thermal copolymerization product was 15 cm away from the liquid surface of the aqueous calcium chloride solution. The first curing was carried out for 30 min to obtain a single-crosslinked gel sphere. The single-crosslinked gel sphere was immersed in an aqueous tannic acid solution with a mass concentration of 3%, and the second curing was carried out for 30 min to obtain a core-shell double-layer gel sphere (4-4cT).

[0044] Example 4

[0045] Pectin was mixed with sterile water and stirred at 40 °C for 4 h until completely dissolved, and then cooled to room temperature to obtain an aqueous pectin solution with a concentration of 3 mg / mL. Type B gelatin was mixed with sterile water and stirred at 50 °C for 6 h until completely dissolved, and then cooled to room temperature to obtain an aqueous gelatin solution with a concentration of 6 mg / mL. Tannic acid was mixed with sterile water and dissolved by ultrasonic wave at room temperature for 2 h to obtain an aqueous tannic acid solution with a mass concentration of 3%.

[0046] The aqueous pectin solution and the aqueous gelatin solution were mixed in equal volumes to obtain a composite gel precursor solution, which was treated in a water bath at 90 °C for 3 h to obtain a thermal copolymerization product (3-6c). The thermal copolymerization product was dropped into an aqueous calcium chloride solution with a mass concentration of 2% at a rate of 200 μL / min. When dropping, the thermal copolymerization product was 15 cm away from the liquid surface of the aqueous calcium chloride solution. The first curing was carried out for 30 min to obtain a single-crosslinked gel sphere. The single-crosslinked gel sphere was immersed in an aqueous tannic acid solution with a mass concentration of 3%, and the second curing was carried out for 30 min to obtain a core-shell double-layer gel sphere (3-6cT).

[0047] Example 5

[0048] Sodium alginate was mixed with sterile water and stirred at 40 °C for 4 h until completely dissolved. After cooling to room temperature, an aqueous sodium alginate solution with a concentration of 4 mg / mL was obtained. Type A gelatin was mixed with sterile water and stirred at 50 °C for 6 h until completely dissolved. After cooling to room temperature, an aqueous gelatin solution with a concentration of 6 mg / mL was obtained. Tannic acid was mixed with sterile water and dissolved by ultrasonic treatment at room temperature for 2 h to obtain an aqueous tannic acid solution with a mass concentration of 3%.

[0049] The aqueous sodium alginate solution and the aqueous gelatin solution were mixed in equal volumes to obtain a composite gel precursor solution, which was treated in a water bath at 90 °C for 3 h to obtain a thermal copolymerization product. The thermal copolymerization product was dropped into an aqueous calcium chloride solution with a mass concentration of 2% at a rate of 200 μL / min. When dropping, the thermal copolymerization product was 15 cm away from the liquid surface of the aqueous calcium chloride solution. The first curing was carried out for 30 min to obtain single-crosslinked gel beads. The single-crosslinked gel beads were immersed in an aqueous tannic acid solution with a mass concentration of 3%, and the second curing was carried out for 30 min to obtain core-shell double-layer gel beads.

[0050] Example 6

[0051] Konjac glucomannan was mixed with sterile water and stirred at 40 °C for 4 h until completely dissolved. After cooling to room temperature, an aqueous konjac glucomannan solution with a concentration of 4 mg / mL was obtained. Collagen was mixed with sterile water and stirred at 50 °C for 6 h until completely dissolved. After cooling to room temperature, an aqueous collagen solution with a concentration of 6 mg / mL was obtained. Tannic acid was mixed with sterile water and dissolved by ultrasonic treatment at room temperature for 2 h to obtain an aqueous tannic acid solution with a mass concentration of 3%.

[0052] The aqueous konjac glucomannan solution and the aqueous collagen solution were mixed in equal volumes to obtain a composite gel precursor solution, which was treated in a water bath at 90 °C for 3 h to obtain a thermal copolymerization product. The thermal copolymerization product was dropped into an aqueous calcium chloride solution with a mass concentration of 2% at a rate of 200 μL / min. When dropping, the thermal copolymerization product was 15 cm away from the liquid surface of the aqueous calcium chloride solution. The first curing was carried out for 30 min to obtain single-crosslinked gel beads. The single-crosslinked gel beads were immersed in an aqueous tannic acid solution with a mass concentration of 3%, and the second curing was carried out for 30 min to obtain core-shell double-layer gel beads.

[0053] Example 7

[0054] Pectin was mixed with sterile water and stirred at 40 °C for 4 h until completely dissolved. After cooling to room temperature, an aqueous pectin solution with a concentration of 2 mg / mL was obtained. Type B gelatin was mixed with sterile water and stirred at 50 °C for 6 h until completely dissolved. After cooling to room temperature, an aqueous gelatin solution with a concentration of 3 mg / mL was obtained. Tannic acid was mixed with sterile water and dissolved by ultrasonic treatment at room temperature for 2 h to obtain an aqueous tannic acid solution with a mass concentration of 1%.

[0055] The pectin aqueous solution and the gelatin aqueous solution are mixed in equal volumes to obtain a composite gel precursor solution, which is treated in a water bath at 80 °C for 4 h to obtain a thermal copolymerization product. The thermal copolymerization product is dropped into an aqueous calcium chloride solution with a mass concentration of 1% at a rate of 150 μL / min. When dropping, the thermal copolymerization product is 20 cm away from the liquid surface of the aqueous calcium chloride solution, and is first cured for 20 min to obtain a single-crosslinked gel sphere. The single-crosslinked gel sphere is immersed in an aqueous tannic acid solution with a mass concentration of 1%, and is secondarily cured for 20 min to obtain a core-shell double-layer gel sphere.

[0056] Example 8

[0057] Pectin is mixed with sterile water and stirred at 40 °C for 4 h until completely dissolved, and then cooled to room temperature to obtain a pectin aqueous solution with a concentration of 6 mg / mL. Type B gelatin is mixed with sterile water and stirred at 50 °C for 6 h until completely dissolved, and then cooled to room temperature to obtain a gelatin aqueous solution with a concentration of 1 mg / mL. Tannic acid is mixed with sterile water and dissolved by ultrasonic wave at room temperature for 2 h to obtain an aqueous tannic acid solution with a mass concentration of 12%.

[0058] The pectin aqueous solution and the gelatin aqueous solution are mixed in equal volumes to obtain a composite gel precursor solution, which is treated in a water bath at 100 °C for 2 h to obtain a thermal copolymerization product. The thermal copolymerization product is dropped into an aqueous calcium chloride solution with a mass concentration of 6% at a rate of 250 μL / min. When dropping, the thermal copolymerization product is 10 cm away from the liquid surface of the aqueous calcium chloride solution, and is first cured for 40 min to obtain a single-crosslinked gel sphere. The single-crosslinked gel sphere is immersed in an aqueous tannic acid solution with a mass concentration of 12%, and is secondarily cured for 40 min to obtain a core-shell double-layer gel sphere.

[0059] Comparative Example 1

[0060] Pectin is mixed with sterile water and stirred at 40 °C for 4 h until completely dissolved, and then cooled to room temperature to obtain a pectin aqueous solution with a concentration of 4 mg / mL. Type B gelatin is mixed with sterile water and stirred at 50 °C for 6 h until completely dissolved, and then cooled to room temperature to obtain a gelatin aqueous solution with a concentration of 6 mg / mL.

[0061] The pectin aqueous solution and the gelatin aqueous solution are mixed in equal volumes to obtain a composite gel precursor solution (4-6). The composite gel precursor solution is dropped into an aqueous calcium chloride solution with a mass concentration of 2% at a rate of 200 μL / min. When dropping, the composite gel precursor solution is 15 cm away from the liquid surface of the aqueous calcium chloride solution, and is first cured for 30 min to obtain a single-crosslinked gel sphere. The single-crosslinked gel sphere is immersed in an aqueous tannic acid solution with a mass concentration of 3%, and is secondarily cured for 30 min to obtain a core-shell double-layer gel sphere (4-6T).

[0062] Comparative Example 2

[0063] Pectin was mixed with sterile water and stirred at 40 °C for 4 h until completely dissolved. After cooling to room temperature, a pectin aqueous solution with a concentration of 2 mg / mL was obtained. Gelatin type B was mixed with sterile water and stirred at 50 °C for 6 h until completely dissolved. After cooling to room temperature, a gelatin aqueous solution with a concentration of 4 mg / mL was obtained.

[0064] The pectin aqueous solution and the gelatin aqueous solution were mixed in equal volumes to obtain a composite gel precursor solution (2-4). The composite gel precursor solution was dropped into a calcium chloride aqueous solution with a mass concentration of 2% at a rate of 200 μL / min. When dropping, the composite gel precursor solution was 15 cm away from the liquid surface of the calcium chloride aqueous solution. The first curing was carried out for 30 min to obtain a single cross-linked gel sphere. The single cross-linked gel sphere was immersed in a tannic acid aqueous solution with a mass concentration of 3%, and the second curing was carried out for 30 min to obtain a core-shell double-layer gel sphere (2-4T).

[0065] Comparative Example 3

[0066] Pectin was mixed with sterile water and stirred at 40 °C for 4 h until completely dissolved. After cooling to room temperature, a pectin aqueous solution with a concentration of 4 mg / mL was obtained. Gelatin type B was mixed with sterile water and stirred at 50 °C for 6 h until completely dissolved. After cooling to room temperature, a gelatin aqueous solution with a concentration of 4 mg / mL was obtained.

[0067] The pectin aqueous solution and the gelatin aqueous solution were mixed in equal volumes to obtain a composite gel precursor solution (4-4). The composite gel precursor solution was dropped into a calcium chloride aqueous solution with a mass concentration of 2% at a rate of 200 μL / min. When dropping, the composite gel precursor solution was 15 cm away from the liquid surface of the calcium chloride aqueous solution. The first curing was carried out for 30 min to obtain a single cross-linked gel sphere. The single cross-linked gel sphere was immersed in a tannic acid aqueous solution with a mass concentration of 3%, and the second curing was carried out for 30 min to obtain a core-shell double-layer gel sphere (4-4T).

[0068] Comparative Example 4

[0069] Pectin was mixed with sterile water and stirred at 40 °C for 4 h until completely dissolved. After cooling to room temperature, a pectin aqueous solution with a concentration of 3 mg / mL was obtained. Gelatin type B was mixed with sterile water and stirred at 50 °C for 6 h until completely dissolved. After cooling to room temperature, a gelatin aqueous solution with a concentration of 6 mg / mL was obtained.

[0070] The pectin aqueous solution and the gelatin aqueous solution were mixed in equal volumes to obtain a composite gel precursor solution (3-6). The composite gel precursor solution was dropped into a calcium chloride aqueous solution with a mass concentration of 2% at a rate of 200 μL / min. When dropping, the composite gel precursor solution was 15 cm away from the liquid surface of the calcium chloride aqueous solution. The first curing was carried out for 30 min to obtain a single cross-linked gel sphere. The single cross-linked gel sphere was immersed in a tannic acid aqueous solution with a mass concentration of 3%, and the second curing was carried out for 30 min to obtain a core-shell double-layer gel sphere (3-6T).

[0071] Experimental Example

[0072] 1. Determination of antioxidant capacity.

[0073] The antioxidant activities of 4 mg / mL pectin aqueous solution (Pec), 4 mg / mL gelatin aqueous solution (Gel), the thermo-copolymerization products prepared in Examples 1-3 (4-6c, 2-4c, and 4-4c), and the composite gel precursor solutions prepared in Comparative Examples 1-3 (4-6, 2-4, and 4-4) after being diluted 10 times were measured using a DPPH kit (product number A153-1-1, Nanjing Jiancheng) and an ABTS kit (product number A015-2-1, Nanjing Jiancheng) according to the steps in the instruction manual.

[0074] The results are as Figure 1 shown. The scavenging rates of 4 mg / mL pectin aqueous solution (Pec) and 4 mg / mL gelatin aqueous solution (Gel) on DPPH radicals are relatively low. The antioxidant effect of the composite gel precursor solutions prepared in Comparative Examples 1-3 (4-6, 2-4, and 4-4) is slightly improved. The thermo-copolymerization products prepared in Examples 1-3 (4-6c, 2-4c, and 4-4c) significantly enhance the scavenging ability of DPPH radicals, and the effect is positively correlated with the concentration. The thermo-copolymerization product (4-6c) prepared in Example 1 has the strongest scavenging ability of DPPH radicals.

[0075] As Figure 2 shown, the antioxidant activity of 4 mg / mL pectin aqueous solution (Pec) is low. 4 mg / mL gelatin aqueous solution (Gel) has the ability to scavenge ABTS radicals. The ABTS radical scavenging ability of the composite gel precursor solutions prepared in Comparative Examples 1-3 (4-6, 2-4, and 4-4) increases with the increase in the concentration of the gelatin aqueous solution. The thermo-copolymerization products prepared in Examples 1-3 (4-6c, 2-4c, and 4-4c) significantly enhance the ABTS radical scavenging ability. The thermo-copolymerization product (4-6c) prepared in Example 1 has the strongest ABTS radical scavenging ability.

[0076] 2. Potential and particle size.

[0077] The Zeta potential of 4 mg / mL pectin aqueous solution (Pec), 4 mg / mL gelatin aqueous solution (Gel), the thermo-copolymerization products prepared in Examples 1-3 (4-6c, 2-4c, and 4-4c), and the composite gel precursor solutions prepared in Comparative Examples 1-3 (4-6, 2-4, and 4-4) after being diluted 10 times was measured using a Malvern particle size analyzer (Zetasizer Nano ZS90, UK). The equilibrium temperature was 25 °C, the equilibrium time was 120 s, the sample volume was 850 μL, and the pH value was also measured.

[0078] As Figure 3As shown in the figure, at 25 °C, the absolute value of the Zeta potential of a 4 mg / mL aqueous pectin solution (Pec) is higher than that of a 4 mg / mL aqueous gelatin solution (Gel). The Zeta potentials of the composite gel precursor solutions (4-6, 2-4, and 4-4) prepared in Comparative Examples 1-3 are between those of the aqueous pectin solution and the aqueous gelatin solution. The thermal copolymerization products (4-6c, 2-4c, and 4-4c) prepared in Examples 1-3 slightly reduced the absolute value of the Zeta potential, and the ratio of pectin to gelatin had a small effect on the electric double layer of the microparticles.

[0079] III. Macroscopic viscosity index.

[0080] The macroscopic viscosity indices of the thermal copolymerization products (4-6c, 2-4c, and 4-4c) prepared in Examples 1-3 and the composite gel precursor solutions (4-6, 2-4, and 4-4) prepared in Comparative Examples 1-3 were measured using an optical microrheometer (Rheolaser Master, France). The experimental temperature range was 28-34 °C, the sample volume was 30 mL, and the detection started after 2 h of temperature equilibration.

[0081] As Figure 4 shown, the macroscopic viscosity indices of the composite gel precursor solutions (4-6, 2-4, and 4-4) prepared in Comparative Examples 1-3 increased with the decrease in temperature. The composite gel precursor solution (4-4) prepared in Comparative Example 3 and the composite gel precursor solution (4-6) prepared in Comparative Example 1 changed from a viscous liquid state to an elastic solid state at 28.0 °C and 28.5 °C, respectively. The results show that the viscosities and gelation temperatures of the thermal copolymerization products (4-6c, 2-4c, and 4-4c) prepared in Examples 1-3 are significantly lower than those of the composite gel precursor solutions (4-6, 2-4, and 4-4) prepared in Comparative Examples 1-3.

[0082] IV. Texture analysis.

[0083] The mechanical properties of the composite gel precursor solutions (4-6, 2-4, and 4-4) prepared in Comparative Examples 1-3, the core-shell double-layer gels (4-6T, 2-4T, 4-4T) prepared in Comparative Examples 1-3, and the core-shell double-layer gels (4-6cT, 2-4cT, and 4-4cT) prepared in Examples 1-3 were measured using a texture analyzer (SMS TA.XTplus, UK).

[0084] The results are as Figure 5As shown, the mechanically enhanced double-layered core-shell gels obtained by secondary cross-linking, namely the double-layered core-shell gels (4-6T, 2-4T, 4-4T) prepared in Comparative Examples 1 to 3, have significantly improved mechanical properties, preventing the premature disintegration of the double-layered core-shell gels under gastrointestinal peristalsis. The products obtained after thermal copolymerization are the double-layered core-shell gels (4-6cT, 2-4cT, 4-4cT) prepared in Examples 1 to 3. The double-layered core-shell gel of Example 2 (2-4cT) with a low pectin concentration shrank during secondary cross-linking, and the increased network density led to an increase in hardness. Increasing the pectin concentration can prevent the collapse and shrinkage of the egg-box structure framework during secondary cross-linking. The compressive modulus of the double-layered core-shell gel of Example 1 (4-6cT) decreased, but its volume shrinkage rate was lower than that of other samples with secondary cross-linking. This indicates that the protein denaturation network formed by the 6 mg / mL gelatin aqueous solution under the action of tannic acid is dense enough to effectively reduce further shrinkage and water loss inside the composite gel.

[0085] V. In vitro simulated digestion.

[0086] The thermally copolymerized product (3-6c) prepared in Example 4, the composite gel precursor solution (3-6) prepared in Comparative Example 4, the double-layered core-shell gel beads (3-6T) prepared in Comparative Example 4, and the double-layered core-shell gel beads (3-6cT) prepared in Example 4 were separately placed into simulated gastric buffer solution and simulated intestinal buffer solution, and digested in a shaker at 37 °C and 120 rpm for 8 h. Stereomicroscopic microscopy was used to take pictures and record every 1 h.

[0087] Preparation of simulated gastric buffer solution (SGF): Weigh 2.8 g of NaCl, 514.4 mg of KCl, 2.1 g of NaHCO3, 225.0 mg of KH2PO4, 20.3 mg of MgCl2·6H2O, and 78.6 mg of (NH4)2CO3, dissolve them in 1000 mL of distilled water, and adjust the pH to 2.5 with 0.1 M HCl.

[0088] Preparation of simulated intestinal buffer solution (SIF): Weigh 2.2 g of NaCl, 507.0 mg of KCl, 7.1 g of NaHCO3, 108.9 mg of KH2PO4, and 67.1 mg of MgCl2·6H2O, dissolve them in 1000 mL of distilled water, and adjust the pH to 7.5.

[0089] The results are as Figure 6 shown, Figure 6 where A is digestion in simulated gastric juice, Figure 6In B, it is the digestion in simulated intestinal fluid. The composite gel precursor solution (3-6) prepared in Comparative Example 4 swelled first within the first 2 h of intestinal digestion and then rapidly eroded and disintegrated. The thermocopolymerization product (3-6c) prepared in Example 4 was completely dispersed after 4 h of intestinal digestion, and the digestion rate was the fastest. The core-shell double-layer gel beads (3-6cT) prepared in Example 4 and the core-shell double-layer gel beads (3-6T) prepared in Comparative Example 4 swelled in volume at 3 h but still maintained their complete shape. Moreover, the core-shell double-layer gel beads (3-6cT) prepared in Example 4 were uniformly dispersed at 8 h of digestion, and the risk of residual shell layer was lower, while some of the core-shell double-layer gel beads (3-6T) prepared in Comparative Example 4 without thermocopolymerization reaction were still not digested.

[0090] The above-described embodiments are only descriptions of the preferred modes of the present invention and do not limit the scope of the present invention. Without departing from the design spirit of the present invention, various deformations and improvements made by those of ordinary skill in the art to the technical solutions of the present invention shall fall within the protection scope determined by the claims of the present invention.

Claims

1. A method for preparing a core-shell double-layer gel sphere with targeted delivery function, characterized in that: The following steps are involved: (1) mixing equal volumes of a polysaccharide aqueous solution and a protein aqueous solution to obtain a composite gel precursor solution; (2) treating the composite gel precursor solution obtained in step (1) in a water bath to obtain a thermal copolymerization product; (3) the thermal copolymerization product obtained in step (2) is dropped into a calcium chloride aqueous solution for first solidification to obtain single cross-linked gel spheres; (4) The single cross-linked gel sphere obtained in step (3) is immersed in a tannic acid aqueous solution and solidified for a second time to obtain a core-shell double-layer gel sphere.

2. The preparation method according to claim 1, characterized in that: The mass concentration of the polysaccharide aqueous solution in step (1) is 2-6%, and the polysaccharide aqueous solution is prepared from one or more of pectin, sodium alginate, konjac glucomannan, hemicellulose or amino polysaccharide.

3. The preparation method according to claim 1, characterized in that: The mass concentration of the protein aqueous solution in step (1) is 3-10%, and the protein aqueous solution is prepared from one or more of type A gelatin, type B gelatin, fish gelatin, casein or collagen.

4. The preparation method according to claim 1, characterized in that: The temperature of the water bath treatment in step (2) is 80-100° C., and the time of the water bath treatment is 2-4 hours.

5. The preparation method according to claim 1, characterized in that: The dripping speed in step (3) is 150-250 μL / min, and the thermal copolymerization product is 10-20 cm away from the surface of the calcium chloride aqueous solution during the dripping; the mass concentration of the calcium chloride aqueous solution in step (3) is 1-6%.

6. The preparation method according to claim 1, characterized in that: The first curing time in step (3) is 20 to 40 minutes.

7. The preparation method according to claim 1, characterized in that: The mass concentration of the tannic acid aqueous solution in step (4) is 1 to 12%.

8. The preparation method according to claim 1, characterized in that: The second curing time in step (4) is 20 to 40 minutes.

9. The core-shell double-layer gel sphere with targeted delivery function prepared by the preparation method according to any one of claims 1 to 8.

10. Use of the core-shell double-layer gel sphere prepared by the preparation method according to any one of claims 1 to 8 or the core-shell double-layer gel sphere according to claim 9 in preparing probiotic targeted delivery capsules.

Images