Shell-core double-layer gel sphere with targeted delivery function and preparation method and application thereof

Abstract

This invention discloses a core-shell bilayer gel sphere with targeted delivery function, its preparation method, and its application. Belonging to the field of core-shell gel technology, it provides a method for preparing core-shell bilayer gel spheres with targeted delivery function, comprising: mixing equal volumes of polysaccharide aqueous solution and protein aqueous solution to obtain a composite gel precursor solution; water bath treatment to obtain a thermal copolymerization product; dripping the product into a calcium chloride aqueous solution; curing to obtain a single-crosslinked gel sphere; immersing the product in a tannic acid aqueous solution; and curing to obtain a core-shell bilayer gel sphere. This invention uses polysaccharide as the core framework, with protein and tannic acid complexes filling the outer pores to form a dense double-penetrating network structure. This significantly improves the barrier properties against oxygen and gastrointestinal fluids, thereby enhancing storage stability and in vivo colonization.

Description

Technical Field

[0001] This invention belongs to the field of shell-core gel technology, and particularly relates to shell-core bilayer gel spheres with targeted delivery function, their preparation methods and applications. Background Technology

[0002] Currently, various probiotic encapsulation technologies exist, including spray drying, freeze drying, emulsification, layer-by-layer encapsulation, extrusion, and electrostatic spraying. Among these, hydrogels prepared by extrusion have advantages such as simple manufacturing process and high nutritional value, and are widely used in tissue repair and drug delivery. Furthermore, regarding different encapsulation materials, natural proteins and polysaccharides are two major encapsulation materials due to their good biocompatibility and biodegradability. However, protein-based materials are susceptible to degradation by pepsin in vivo, resulting in a loss of protective effect. Therefore, polysaccharides have potential as probiotic encapsulation materials. However, gels prepared from single polysaccharides suffer from low encapsulation rates and are prone to lysis, leading to premature release of probiotics, resulting in insufficient protection. Composite gels can effectively improve these problems.

[0003] Previous studies have shown that prebiotic materials encapsulating probiotics can improve their stress resistance and in vivo colonization. Low-esterified pectin and calcium ions can form a stable eggshell structure, offering advantages in safety, processability, and cost. Furthermore, research indicates that 30% esterified pectin can promote the adhesion of *Lactiplantibacillus plantarum* to intestinal epithelial cells, exhibiting good stability during in vitro digestion. However, the interior of single-ion-cured gels such as pectin and sodium alginate can still be rapidly penetrated by gastric juices, making it impossible to guarantee consistent protective effects for strains with different gastric juice tolerance levels. Summary of the Invention

[0004] To address the aforementioned technical problems, this invention proposes a shell-core bilayer gel sphere with targeted delivery function, its preparation method, and its application. Using polysaccharides as the core framework, protein and tannic acid complexes fill the outer pores, forming a dense double-penetrating network structure shell. This significantly enhances the barrier properties against oxygen and gastrointestinal fluids, thereby improving storage stability and in vivo colonization.

[0005] To achieve the above objectives, the present invention provides a method for preparing core-shell bilayer gel spheres with targeted delivery function, comprising the following steps:

[0006] (1) Mix equal volumes of polysaccharide aqueous solution and protein aqueous solution to obtain composite gel precursor solution;

[0007] (2) The composite gel precursor liquid obtained in step (1) is treated in a water bath to obtain a heat copolymer.

[0008] (3) The thermal copolymer obtained in step (2) is dropped into a calcium chloride aqueous solution and cured for the first time to obtain a single cross-linked gel ball;

[0009] (4) The single cross-linked gel spheres obtained in step (3) are immersed in tannic acid aqueous solution and then cured for the second time to obtain shell-core bilayer gel spheres.

[0010] Preferably, 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 polysaccharides.

[0011] Preferably, 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.

[0012] Preferably, 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.

[0013] Preferably, the dripping rate in step (3) is 150-250 μL / min, and the thermal copolymer is 10-20 cm away from the surface of the calcium chloride aqueous solution during dripping; the mass concentration of the calcium chloride aqueous solution in step (3) is 1-6%.

[0014] Preferably, the curing time in step (3) is 20 to 40 minutes.

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

[0016] Preferably, the curing time in step (4) is 20 to 40 minutes.

[0017] The present invention also provides shell-core bilayer gel spheres with targeted delivery function prepared by the preparation method.

[0018] The present invention also provides the application of the shell-core bilayer gel spheres prepared by the above preparation method, or the shell-core bilayer gel spheres, 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 bilayer gel spheres of this invention, with targeted delivery function, form a polysaccharide eggshell structure framework through cross-linking of polysaccharides such as pectin or sodium alginate with calcium ions. These frameworks are then immersed in a phenolic acid solution. Utilizing protein-phenolic acid covalent cross-linking, type A gelatin, type B gelatin, fish gelatin, casein, or collagen-like proteins react with tannic acid to generate a dense shell layer within minutes. Phenolic acid does not excessively permeate and does not affect the core encapsulated material. Results show that the core-shell bilayer gel spheres significantly enhance the barrier properties against oxygen and gastrointestinal fluids, improving the storage stability and in vivo colonization of probiotics when encapsulating them. Furthermore, the thermal copolymerization product of pectin and gelatin exhibits higher free radical scavenging ability, while significantly reducing viscosity and gelation temperature, which is beneficial for practical production applications of bacterial encapsulation.

[0021] This invention uses pectin as the core framework and leverages the covalent bonding of tannic acid and gelatin to generate a dense shell in situ. The method is simple, controllable, and highly efficient. The raw materials used are natural and non-toxic, suitable for the sustained release and intestinal-targeted delivery of probiotics or pharmaceuticals, and are not limited by the solubility of the encapsulating substances. The thermal copolymerization reaction enhances the material's antioxidant properties while sterilizing it, and significantly reduces the viscosity and gelation temperature of the composite gel precursor solution, which is beneficial for production operations. Furthermore, it reduces the stress caused by shell shrinkage during secondary cross-linking, preventing premature rupture of the shell-core bilayer gel spheres during digestion and reducing the risk of indigestion due to residual shell layers. Attached Figure Description

[0022] To more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the drawings used in the embodiments will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For those skilled in the art, other drawings can be obtained based on these drawings without creative effort.

[0023] Figure 1 The DPPH free radical scavenging rate of the thermal copolymers prepared in Examples 1-3 and the composite gel precursor solutions prepared in Comparative Examples 1-3 was determined. In the figure, Pec is a 4 mg / mL pectin aqueous solution, Gel is a 4 mg / mL gelatin aqueous solution, 4-6 are the composite gel precursor solutions prepared in Comparative Example 1, 2-4 are the composite gel precursor solutions prepared in Comparative Example 2, 4-4 are the composite gel precursor solutions prepared in Comparative Example 3, 4-6c is the thermal copolymer prepared in Example 1, 2-4c is the thermal copolymer prepared in Example 2, and 4-4c is the thermal copolymer prepared in Example 3. In the figure, a, b, c, and d indicate the significance of the difference.

[0024] Figure 2The ABTS free radical scavenging rate of the thermal copolymers prepared in Examples 1-3 and the composite gel precursor solutions prepared in Comparative Examples 1-3 was determined. In the figure, Pec is a 4 mg / mL pectin aqueous solution, Gel is a 4 mg / mL gelatin aqueous solution, 4-6 are the composite gel precursor solutions prepared in Comparative Example 1, 2-4 are the composite gel precursor solutions prepared in Comparative Example 2, 4-4 are the composite gel precursor solutions prepared in Comparative Example 3, 4-6c is the thermal copolymer prepared in Example 1, 2-4c is the thermal copolymer prepared in Example 2, and 4-4c is the thermal copolymer prepared in Example 3. In the figure, a, b, c, d, and e indicate the significance of the difference.

[0025] Figure 3 The Zeta potential of the thermal copolymers prepared in Examples 1-3 and the composite gel precursor solutions prepared in Comparative Examples 1-3 is measured. In the figure, Pec is a 4 mg / mL pectin aqueous solution, and Gel is a 4 mg / mL gelatin aqueous solution. Figures 4-6 are composite gel precursor solutions prepared in Comparative Example 1, 2-4 are composite gel precursor solutions prepared in Comparative Example 2, 4-4 are composite gel precursor solutions prepared in Comparative Example 3, 4-6c are thermal copolymers prepared in Example 1, 2-4c are thermal copolymers prepared in Example 2, and 4-4c are thermal copolymers prepared in Example 3. In the figure, "ns" indicates no significant difference, and "****" indicates a significant difference.

[0026] Figure 4 The macroscopic viscosity index of the thermal copolymers prepared in Examples 1-3 and the composite gel precursors prepared in Comparative Examples 1-3 was determined. In the figure, 4-6 are the composite gel precursors prepared in Comparative Example 1, 2-4 are the composite gel precursors prepared in Comparative Example 2, 4-4 are the composite gel precursors prepared in Comparative Example 3, 4-6c are the thermal copolymers prepared in Example 1, 2-4c are the thermal copolymers prepared in Example 2, and 4-4c are the thermal copolymers prepared in Example 3.

[0027] Figure 5 The mechanical properties of the composite gel precursor solutions prepared in Comparative Examples 1-3, the core-shell bilayer gels prepared in Comparative Examples 1-3, and the core-shell bilayer gels prepared in Examples 1-3 were determined. In Figure 1, 4-6 represents the composite gel precursor solution prepared in Comparative Example 1, 2-4 represents the composite gel precursor solution prepared in Comparative Example 2, 4-4 represents the composite gel precursor solution prepared in Comparative Example 3, 4-6T represents the core-shell bilayer gel prepared in Comparative Example 1, 2-4T represents the core-shell bilayer gel prepared in Comparative Example 2, 4-4T represents the core-shell bilayer gel prepared in Comparative Example 3, 4-6cT represents the core-shell bilayer gel prepared in Example 1, 2-4cT represents the core-shell bilayer gel prepared in Example 2, and 4-4cT represents the core-shell bilayer gel prepared in Example 3.

[0028] Figure 6 The gastrointestinal digestibility characteristics of the thermal copolymer prepared in Example 4, the composite gel precursor prepared in Comparative Example 4, the core-shell bilayer gel spheres prepared in Comparative Example 4, and the core-shell bilayer gel spheres prepared in Example 4 were determined. In the figure, A represents simulated gastric digestion, B represents simulated intestinal digestion, 3-6c represents the thermal copolymer prepared in Example 4, 3-6 represents the composite gel precursor prepared in Comparative Example 4, 3-6T represents the core-shell bilayer gel spheres prepared in Comparative Example 4, and 3-6cT represents the core-shell bilayer gel spheres prepared in Example 4. Detailed Implementation

[0029] Various exemplary embodiments of the present invention will now be described in detail. This detailed description should not be considered as limiting the present invention, but rather as a more detailed description of certain aspects, features, and embodiments of the present invention.

[0030] It should be understood that the terms described herein are intended only to describe particular embodiments and are not intended to limit the present invention. In addition, for numerical ranges herein, it should be understood that each intermediate value between the upper and lower limits of the range is also specifically disclosed. Each smaller range between any intermediate value within a stated value or stated range and any other stated value or intermediate value within the stated range is also encompassed by the present invention. The upper and lower limits of these smaller ranges may be independently included or excluded within the scope.

[0031] Unless otherwise indicated, all technical and scientific terms used herein have the same meaning as commonly understood by those skilled in the art. Although only preferred methods and materials are described herein, any methods and materials similar or equivalent to those described herein may also be used in the practice 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 associated with the documents. In the event of any conflict with any incorporated document, the contents of this specification shall prevail.

[0032] Various modifications and variations can be made to the specific embodiments described in this specification without departing from the scope or spirit of the invention, as will be apparent to those skilled in the art. Other embodiments derived from this specification will also be apparent to those skilled in the art. This specification and embodiments are merely exemplary.

[0033] The words “include,” “including,” “have,” “contain,” etc. used in this document are open-ended terms, meaning including but not limited to.

[0034] The materials used in this invention are sourced as follows: 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); gelatin is type B gelatin derived from pigskin, purchased from Shanghai Yuanye Biotechnology Co., Ltd., item number S22176; tannic acid is purchased from Shanghai Maclean Biochemical Technology Co., Ltd., item number T818845; calcium chloride is purchased from Shanghai Maclean Biochemical Technology Co., Ltd., item number C832203; sodium alginate is purchased from Shanghai Maclean Biochemical Technology Co., Ltd., item number S817374; type A gelatin is purchased from Shanghai Yuanye Biotechnology Co., Ltd., item number S25197; konjac glucomannan is purchased from Hefei Bomei Biotechnology Co., Ltd., item number PK2989; and collagen is purchased from Shanghai Maclean Biochemical Technology Co., Ltd., item number C875812.

[0035] Example 1

[0036] Pectin was mixed with sterile water and stirred at 40°C for 4 hours until completely dissolved. After cooling to room temperature, a 4 mg / mL pectin aqueous solution was obtained. Type B gelatin was mixed with sterile water and stirred at 50°C for 6 hours until completely dissolved. After cooling to room temperature, a 6 mg / mL gelatin aqueous solution was obtained. Tannic acid was mixed with sterile water and dissolved by sonication at room temperature for 2 hours to obtain a 3% (w / w) tannic acid aqueous solution.

[0037] Equal volumes of pectin aqueous solution and gelatin aqueous solution were mixed to obtain a composite gel precursor solution. The solution was treated in a water bath at 90°C for 3 hours to obtain a thermal copolymer (4-6c). The thermal copolymer was added dropwise to a 2% calcium chloride aqueous solution at a rate of 200 μL / min, with the thermal copolymer at a distance of 15 cm from the surface of the calcium chloride aqueous solution. After a first curing of 30 minutes, single-crosslinked gel spheres were obtained. The single-crosslinked gel spheres were immersed in a 3% tannic acid aqueous solution and cured for a second time for 30 minutes to obtain a shell-core bilayer gel sphere (4-6cT).

[0038] Example 2

[0039] Pectin was mixed with sterile water and stirred at 40°C for 4 hours until completely dissolved. After cooling to room temperature, a 2 mg / mL pectin aqueous solution was obtained. Type B gelatin was mixed with sterile water and stirred at 50°C for 6 hours until completely dissolved. After cooling to room temperature, a 4 mg / mL gelatin aqueous solution was obtained. Tannic acid was mixed with sterile water and dissolved by sonication at room temperature for 2 hours to obtain a 3% (w / w) tannic acid aqueous solution.

[0040] Equal volumes of pectin aqueous solution and gelatin aqueous solution were mixed to obtain a composite gel precursor solution. The solution was treated in a water bath at 90°C for 3 hours to obtain a thermal copolymer (2-4c). The thermal copolymer was added dropwise to a 2% calcium chloride aqueous solution at a rate of 200 μL / min, with the thermal copolymer at a distance of 15 cm from the surface of the calcium chloride aqueous solution. After a first curing of 30 minutes, single-crosslinked gel spheres were obtained. The single-crosslinked gel spheres were immersed in a 3% tannic acid aqueous solution and cured for a second time for 30 minutes to obtain a shell-core bilayer gel sphere (2-4cT).

[0041] Example 3

[0042] Pectin was mixed with sterile water and stirred at 40°C for 4 hours until completely dissolved. After cooling to room temperature, a 4 mg / mL pectin aqueous solution was obtained. Type B gelatin was mixed with sterile water and stirred at 50°C for 6 hours until completely dissolved. After cooling to room temperature, a 4 mg / mL gelatin aqueous solution was obtained. Tannic acid was mixed with sterile water and dissolved by sonication at room temperature for 2 hours to obtain a 3% (w / w) tannic acid aqueous solution.

[0043] Equal volumes of pectin aqueous solution and gelatin aqueous solution were mixed to obtain a composite gel precursor solution. The solution was treated in a water bath at 90°C for 3 hours to obtain a thermal copolymer (4-4c). The thermal copolymer was added dropwise to a 2% calcium chloride aqueous solution at a rate of 200 μL / min, with the thermal copolymer at a distance of 15 cm from the surface of the calcium chloride aqueous solution. After a first curing of 30 minutes, single-crosslinked gel spheres were obtained. The single-crosslinked gel spheres were immersed in a 3% tannic acid aqueous solution and cured for a second time for 30 minutes to obtain a shell-core bilayer gel sphere (4-4cT).

[0044] Example 4

[0045] Pectin was mixed with sterile water and stirred at 40°C for 4 hours until completely dissolved. After cooling to room temperature, a 3 mg / mL pectin aqueous solution was obtained. Type B gelatin was mixed with sterile water and stirred at 50°C for 6 hours until completely dissolved. After cooling to room temperature, a 6 mg / mL gelatin aqueous solution was obtained. Tannic acid was mixed with sterile water and dissolved by sonication at room temperature for 2 hours to obtain a 3% (w / w) tannic acid aqueous solution.

[0046] Equal volumes of pectin aqueous solution and gelatin aqueous solution were mixed to obtain a composite gel precursor solution. The solution was treated in a water bath at 90°C for 3 hours to obtain a thermal copolymer (3-6c). The thermal copolymer was added dropwise to a 2% calcium chloride aqueous solution at a rate of 200 μL / min, with the thermal copolymer at a distance of 15 cm from the surface of the calcium chloride aqueous solution. After a first curing of 30 minutes, single-crosslinked gel spheres were obtained. The single-crosslinked gel spheres were immersed in a 3% tannic acid aqueous solution and cured for a second time for 30 minutes to obtain a core-shell bilayer gel sphere (3-6cT).

[0047] Example 5

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

[0049] Equal volumes of sodium alginate aqueous solution and gelatin aqueous solution were mixed to obtain a composite gel precursor solution. The solution was treated in a water bath at 90°C for 3 hours to obtain a thermal copolymer. The thermal copolymer was then dropped into a 2% calcium chloride aqueous solution at a rate of 200 μL / min, with the thermal copolymer at a distance of 15 cm from the surface of the calcium chloride aqueous solution. After a first curing of 30 minutes, single-crosslinked gel spheres were obtained. The single-crosslinked gel spheres were then immersed in a 3% tannic acid aqueous solution and cured for a second time for 30 minutes to obtain a core-shell bilayer gel sphere.

[0050] Example 6

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

[0052] Konjac glucomannan aqueous solution and collagen aqueous solution were mixed in equal volumes to obtain a composite gel precursor solution. The mixture was treated in a water bath at 90℃ for 3 hours to obtain a thermal copolymer. The thermal copolymer was dropped into a 2% calcium chloride aqueous solution at a rate of 200 μL / min, with the thermal copolymer at a distance of 15 cm from the surface of the calcium chloride aqueous solution. The first curing was carried out for 30 minutes to obtain a single-crosslinked gel sphere. The single-crosslinked gel sphere was immersed in a 3% tannic acid aqueous solution and then cured for 30 minutes to obtain a shell-core bilayer gel sphere.

[0053] Example 7

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

[0055] Equal volumes of pectin aqueous solution and gelatin aqueous solution were mixed to obtain a composite gel precursor solution. The solution was treated in a water bath at 80°C for 4 hours to obtain a thermal copolymer. The thermal copolymer was dropped into a 1% calcium chloride aqueous solution at a rate of 150 μL / min, with the thermal copolymer at a distance of 20 cm from the surface of the calcium chloride aqueous solution. After a first curing of 20 min, single-crosslinked gel spheres were obtained. The single-crosslinked gel spheres were immersed in a 1% tannic acid aqueous solution and cured for a second time for 20 min to obtain a shell-core bilayer gel sphere.

[0056] Example 8

[0057] Pectin was mixed with sterile water and stirred at 40°C for 4 hours until completely dissolved. After cooling to room temperature, a 6 mg / mL pectin aqueous solution was obtained. Type B gelatin was mixed with sterile water and stirred at 50°C for 6 hours until completely dissolved. After cooling to room temperature, a 1 mg / mL gelatin aqueous solution was obtained. Tannic acid was mixed with sterile water and dissolved by sonication at room temperature for 2 hours to obtain a 12% (w / w) tannic acid aqueous solution.

[0058] Equal volumes of pectin aqueous solution and gelatin aqueous solution were mixed to obtain a composite gel precursor solution. The solution was treated in a water bath at 100°C for 2 hours to obtain a thermal copolymer. The thermal copolymer was dropped into a 6% calcium chloride aqueous solution at a rate of 250 μL / min, with the thermal copolymer at a distance of 10 cm from the surface of the calcium chloride aqueous solution. After a first curing of 40 min, single-crosslinked gel spheres were obtained. The single-crosslinked gel spheres were immersed in a 12% tannic acid aqueous solution and cured for a second time for 40 min to obtain a shell-core bilayer gel sphere.

[0059] Comparative Example 1

[0060] Pectin was mixed with sterile water and stirred at 40°C for 4 hours until completely dissolved. After cooling to room temperature, a 4 mg / mL pectin aqueous solution was obtained. Type B gelatin was mixed with sterile water and stirred at 50°C for 6 hours until completely dissolved. After cooling to room temperature, a 6 mg / mL gelatin aqueous solution was obtained.

[0061] Pectin aqueous solution and gelatin aqueous solution are mixed in equal volumes to obtain composite gel precursor solution (4-6). The composite gel precursor solution is added dropwise to a 2% calcium chloride aqueous solution at a rate of 200 μL / min. When adding the composite gel precursor solution, it is 15 cm away from the surface of the calcium chloride aqueous solution. After the first curing is 30 min, a single cross-linked gel ball is obtained. The single cross-linked gel ball is immersed in a 3% tannic acid aqueous solution and cured for a second time for 30 min to obtain a shell-core bilayer gel ball (4-6T).

[0062] Comparative Example 2

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

[0064] Pectin aqueous solution and gelatin aqueous solution are mixed in equal volumes to obtain composite gel precursor solution (2-4). The composite gel precursor solution is added dropwise to a 2% calcium chloride aqueous solution at a rate of 200 μL / min. When adding the composite gel precursor solution, it is 15 cm away from the surface of the calcium chloride aqueous solution. After the first curing is 30 min, a single cross-linked gel sphere is obtained. The single cross-linked gel sphere is immersed in a 3% tannic acid aqueous solution and cured for a second time for 30 min to obtain a shell-core bilayer gel sphere (2-4T).

[0065] Comparative Example 3

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

[0067] Pectin aqueous solution and gelatin aqueous solution are mixed in equal volumes to obtain composite gel precursor solution (4-4). The composite gel precursor solution is added dropwise to a 2% calcium chloride aqueous solution at a rate of 200 μL / min. When adding the composite gel precursor solution, it is 15 cm away from the surface of the calcium chloride aqueous solution. After the first curing is 30 min, a single cross-linked gel sphere is obtained. The single cross-linked gel sphere is immersed in a 3% tannic acid aqueous solution and cured for a second time for 30 min to obtain a shell-core bilayer gel sphere (4-4T).

[0068] Comparative Example 4

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

[0070] Pectin aqueous solution and gelatin aqueous solution are mixed in equal volumes to obtain composite gel precursor solution (3-6). The composite gel precursor solution is added dropwise to a 2% calcium chloride aqueous solution at a rate of 200 μL / min. When adding the composite gel precursor solution, it is 15 cm away from the surface of the calcium chloride aqueous solution. After the first curing is 30 min, a single cross-linked gel ball is obtained. The single cross-linked gel ball is immersed in a 3% tannic acid aqueous solution and cured for a second time for 30 min to obtain a shell-core bilayer gel ball (3-6T).

[0071] Experimental Example

[0072] I. Determination of antioxidant capacity.

[0073] The antioxidant activity of 4 mg / mL pectin aqueous solution (Pec), 4 mg / mL gelatin aqueous solution (Gel), the thermal copolymers 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 4-4c) diluted 10-fold was determined using the DPPH kit (catalog number A153-1-1, Nanjing Jiancheng) and the ABTS kit (catalog number A015-2-1, Nanjing Jiancheng) according to the instructions.

[0074] The results are as follows Figure 1 As shown, 4 mg / mL pectin aqueous solution (Pec) and 4 mg / mL gelatin aqueous solution (Gel) had low DPPH free radical scavenging rates. The antioxidant activity of the composite gel precursor solutions (4-6, 2-4 and 4-4) prepared in Examples 1-3 was slightly improved. The thermal copolymers (4-6c, 2-4c and 4-4c) prepared in Examples 1-3 significantly improved the DPPH free radical scavenging ability, and the effect was positively correlated with the concentration. The thermal copolymer (4-6c) prepared in Example 1 had the strongest DPPH free radical scavenging ability.

[0075] like Figure 2 As shown, the 4 mg / mL pectin aqueous solution (Pec) has low antioxidant activity, while the 4 mg / mL gelatin aqueous solution (Gel) has ABTS free radical scavenging ability. The ABTS free radical scavenging ability of the composite gel precursor solutions (4-6, 2-4, and 4-4) prepared in Comparative Examples 1-3 increases with the increase of gelatin aqueous solution concentration. The thermal copolymers (4-6c, 2-4c, and 4-4c) prepared in Examples 1-3 significantly improve the ABTS free radical scavenging ability, and the thermal copolymer (4-6c) prepared in Example 1 has the strongest ABTS free radical scavenging ability.

[0076] II. Potential and Particle Size.

[0077] The Zeta potential of 4 mg / mL pectin aqueous solution (Pec), 4 mg / mL gelatin aqueous solution (Gel), the thermal copolymers 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) diluted 10-fold was determined using a Malvern particle size analyzer (ZetasizerNano ZS90, UK). The equilibration temperature was 25°C, the equilibration time was 120 s, the sample loading volume was 850 μL, and the pH value was also measured.

[0078] like Figure 3As shown, at 25°C, the absolute value of the Zeta potential of a 4 mg / mL pectin aqueous solution (Pec) is higher than that of a 4 mg / mL gelatin aqueous 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 pectin and gelatin aqueous solutions. The thermal copolymers obtained in Examples 1-3 (4-6c, 2-4c, and 4-4c) show a slight decrease in the absolute value of the Zeta potential, indicating that the ratio of pectin to gelatin has a relatively small effect on the microparticle double layer.

[0079] III. Macroscopic viscosity index.

[0080] The macroscopic viscosity index of the thermal copolymers (4-6c, 2-4c, and 4-4c) prepared in Examples 1-3 and the composite gel precursors (4-6, 2-4, and 4-4) prepared in Comparative Examples 1-3 was determined using an optical microrheometer (Rheolaser Master, France). The experimental temperature range was 28-34°C, the sample loading volume was 30 mL, and the detection began after 2 hours of temperature equilibration.

[0081] like Figure 4 As shown, the macroscopic viscosity index of the composite gel precursor liquids (4-6, 2-4 and 4-4) prepared in Comparative Examples 1 to 3 increased with decreasing temperature. The composite gel precursor liquid (4-4) prepared in Comparative Example 3 and the composite gel precursor liquid (4-6) prepared in Comparative Example 1 transformed from a viscous liquid to an elastic solid state at 28.0℃ and 28.5℃, respectively. The results indicate that the viscosity and gel temperature of the thermal copolymer products (4-6c, 2-4c and 4-4c) prepared in Examples 1 to 3 are significantly lower than those of the composite gel precursor liquids (4-6, 2-4 and 4-4) prepared in Comparative Examples 1 to 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 bilayer gels (4-6T, 2-4T, and 4-4T) prepared in Comparative Examples 1–3, and the core-shell bilayer gels (4-6cT, 2-4cT, and 4-4cT) prepared in Examples 1–3 were determined using a texture analyzer (SMS TA.XTplus, UK).

[0084] The results are as follows Figure 5As shown, the core-shell bilayer gels with secondary crosslinking, namely the core-shell bilayer gels (4-6T, 2-4T, 4-4T) prepared in Comparative Examples 1-3, exhibit significantly improved mechanical properties, preventing premature disintegration under stress during gastrointestinal peristalsis. The products obtained after thermal copolymerization are the core-shell bilayer gels (4-6cT, 2-4cT, 4-4cT) prepared in Examples 1-3. The core-shell bilayer gel (2-4cT) of Example 2 with a low pectin concentration shrinks during secondary crosslinking, and the increased network density leads to increased hardness. Increasing the pectin concentration can prevent the egg-box structure skeleton from collapsing and shrinking during secondary crosslinking. The core-shell bilayer gel (4-6cT) of Example 1 has a lower compressive modulus, but its volume shrinkage rate is lower than other secondary crosslinked samples. This indicates that the protein denaturation network of 6 mg / mL gelatin aqueous solution under the action of tannic acid is sufficiently dense, effectively reducing further shrinkage and water loss inside the composite gel.

[0085] V. In vitro simulated digestion.

[0086] The thermal copolymer (3-6c) prepared in Example 4, the composite gel precursor liquid (3-6) prepared in Comparative Example 4, the shell-core bilayer gel spheres (3-6T) prepared in Comparative Example 4, and the shell-core bilayer gel spheres (3-6cT) prepared in Example 4 were placed in simulated gastric buffer solution and simulated intestinal buffer solution, respectively, and digested in a shaker at 37°C and 120 rpm for 8 hours. The samples were photographed and recorded every 1 hour using a stereomicroscope.

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

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

[0089] The results are as follows Figure 6 As shown, Figure 6 In section A, gastric juice digestion is simulated. Figure 6In Example B, which simulates intestinal digestion, the composite gel precursor solution (3-6) prepared in Comparative Example 4 swelled within the first 2 hours of intestinal digestion, followed by rapid dissolution and disintegration. The thermal copolymer product (3-6c) prepared in Example 4 was completely dispersed after 4 hours of intestinal digestion, exhibiting the fastest digestion rate. The shell-core bilayer gel spheres (3-6cT) prepared in Example 4 and the shell-core bilayer gel spheres (3-6T) prepared in Comparative Example 4 swelled and increased in volume after 3 hours, but still maintained their intact shape. Furthermore, the shell-core bilayer gel spheres (3-6cT) prepared in Example 4 were uniformly dispersed after 8 hours of digestion, with a lower risk of shell residue, while the shell-core bilayer gel spheres (3-6T) prepared in Comparative Example 4, which had not undergone thermal copolymerization, still had some undigested material.

[0090] The embodiments described above are merely preferred embodiments of the present invention and are not intended to limit the scope of the present invention. Various modifications and improvements made by those skilled in the art to the technical solutions of the present invention without departing from the spirit of the present invention should fall within the protection scope defined by the claims of the present invention.

Claims

1. A method for preparing core-shell bilayer gel spheres with targeted delivery function, characterized in that, Includes the following steps: (1) Mix equal volumes of polysaccharide aqueous solution and protein aqueous solution to obtain composite gel precursor solution; (2) The composite gel precursor solution obtained in step (1) is treated in a water bath to obtain a heat copolymer. (3) The thermal copolymer obtained in step (2) is dropped into an aqueous solution of calcium chloride and cured for the first time to obtain single cross-linked gel spheres; (4) The single cross-linked gel spheres obtained in step (3) are immersed in tannic acid aqueous solution and cured for the second time to obtain shell-core bilayer gel spheres; The mass concentration of the polysaccharide aqueous solution in step (1) is 2-6%, and the polysaccharide aqueous solution is prepared from pectin; The mass concentration of the protein aqueous solution in step (1) is 3-10%, and the protein aqueous solution is prepared from type B gelatin; The temperature of the water bath treatment in step (2) is 80~100℃, and the time of the water bath treatment is 2~4h.

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

3. The preparation method according to claim 1, characterized in that, In step (3), the first curing time is 20~40 min.

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

5. The preparation method according to claim 1, characterized in that, The curing time in step (4) is 20~40 min.

6. The core-shell bilayer gel spheres with targeted delivery function prepared by the preparation method according to any one of claims 1 to 5.

7. The application of the shell-core bilayer gel spheres prepared by the preparation method according to any one of claims 1 to 5 or the shell-core bilayer gel spheres according to claim 6 in the preparation of probiotic targeted delivery capsules.

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