An ultrafast self-repairing hydrogel based on hyaluronic acid and gelatin, and its preparation method and application

Abstract

The present invention discloses an ultrafast self-healing hydrogel based on hyaluronic acid and gelatin, and its preparation method and application, comprising the following steps: (1) dissolving hyaluronic acid in deionized water, adding EDC and NHS, stirring at room temperature, then adding tyramine DMSO solution, reacting at room temperature, dialysis and freeze-drying after the reaction to obtain a hyaluronic acid skeleton; (2) dissolving gelatin in deionized water, adding vanillin ethanol solution for reaction, then adding NaH3BCN solution for reduction, dialysis and freeze-drying to obtain a gelatin skeleton; (3) dissolving the hyaluronic acid skeleton in deionized water, then adding horseradish peroxidase to obtain a hyaluronic acid solution; deionizing the gelatin skeleton solution in deionized water, then adding hydrogen peroxide to obtain a gelatin solution; (4) mixing the hyaluronic acid solution with the gelatin solution to obtain the composite hydrogel. The composite hydrogel is used to coat probiotics, which is beneficial to maintaining the activity of probiotics in a simulated digestive tract environment.

Description

Technical Field

[0001] The present invention relates to the field of gel technology, and in particular to an ultrafast self-repairing hydrogel based on hyaluronic acid and gelatin, a preparation method thereof, and an application thereof for coating probiotics. Background Art

[0002] Intestinal microorganisms are extremely important to human health. Their population and quantity not only affect the integrity of the intestinal epithelium, but also regulate systemic physiological activities such as dietary fiber decomposition and vitamin synthesis. Therefore, it is of great significance to ensure the homeostasis of intestinal microbial populations.

[0003] Supplementing with probiotics is an effective way to increase the abundance of beneficial intestinal bacteria, regulate intestinal function, and promote intestinal homeostasis. However, the complex digestive tract environment, such as the low pH environment of gastric acid, bile salts, oxygen gradients, and shear stress generated by gastrointestinal motility, greatly reduces the survival rate of probiotics taken orally; at the same time, the short intestinal retention time makes it difficult for probiotics to colonize. Therefore, it is of great significance to design and prepare a new distal intestinal sustained-release system to overcome the complex internal environment and short intestinal retention time in the upper digestive tract and ensure the activity and quantity of probiotics during delivery. Summary of the Invention

[0004] In response to the above problems, the present invention proposes an ultrafast self-healing hydrogel based on hyaluronic acid and gelatin, its preparation method and application. The composite hydrogel prepared in this application is not a simple combination of the two; rather, it is combined together through peroxidase to encapsulate probiotics. The technical effect of a relatively high survival rate in gastric acid and bile salts is unexpected. In summary, this application has unexpected technical effects and a certain degree of creativity.

[0005] The technical solutions of the present invention are as follows:

[0006] The first object of the present invention is to provide a method for preparing a composite hydrogel based on hyaluronic acid and gelatin, comprising the following steps:

[0007] (1) preparing a hyaluronic acid skeleton by dissolving hyaluronic acid in deionized water, adding 1-ethyl-(3-dimethylaminopropyl)carbodiimide (EDC) and N-hydroxysuccinimide (NHS), stirring at room temperature, then adding tyramine DMSO solution, reacting at room temperature, dialysis after the reaction, and freeze-drying to obtain a hyaluronic acid skeleton;

[0008] (2) preparing a gelatin skeleton by dissolving gelatin in deionized water, adding vanillin ethanol solution to react, then adding NaH3BCN solution for reduction, dialysis, and freeze-drying to obtain a gelatin skeleton;

[0009] (3) dissolving a hyaluronic acid skeleton in deionized water, and then adding horseradish peroxidase to prepare a hyaluronic acid solution; dissolving a gelatin skeleton in deionized water to form a gelatin skeleton solution, and then adding hydrogen peroxide to prepare a gelatin solution;

[0010] (4) Mixing the hyaluronic acid solution and the gelatin solution to prepare the composite hydrogel.

[0011] In one embodiment of the present invention, in step (1), the mass ratio of EDC to hyaluronic acid is 1:4-6; the mass ratio of NHS to hyaluronic acid is 1:6-8.

[0012] In one embodiment of the present invention, in step (1), the mass concentration of the tyramine DMSO solution is 1-1.5%.

[0013] In one embodiment of the present invention, in step (1), the amount of tyramine used is 50-70% of the mass of hyaluronic acid.

[0014] In one embodiment of the present invention, in step (1), the stirring time is 10-60 min; the reaction time at room temperature is 12-14 h; and the dialysis is performed using a dialysis bag with a molecular weight cut-off of 8-14 kDa for 1-4 days.

[0015] In one embodiment of the present invention, in step (2), the mass ratio of vanillin to gelatin is 1:1-3; the NaH3BCN solution is prepared by dissolving NaH3BCN in a 1M NaOH aqueous solution; and the mass ratio of NaH3BCN to gelatin is 1:14-16.

[0016] In one embodiment of the present invention, in step (2), the reaction conditions are: reaction at 40° C. for 4-6 hours; and dialysis using a dialysis bag with a molecular weight cutoff of 1000 Da for 1-4 days.

[0017] In one embodiment of the present invention, in step (3), the mass ratio of horseradish peroxidase to hyaluronic acid skeleton is 1:60-350; the concentration of hydrogen peroxide in hydrogen peroxide is 9-11 wt%; and the amount of hydrogen peroxide used is 0.6-0.73% of the volume of the gelatin skeleton solution.

[0018] In one embodiment of the present invention, in step (1) and step (3), ultrasound is used to assist dissolution when dissolving the hyaluronic acid or hyaluronic acid skeleton.

[0019] In one embodiment of the present invention, in step (2) and step (3), when dissolving gelatin or gelatin skeleton, a 40° C. water bath is used to assist dissolution.

[0020] In one embodiment of the present invention, in step (1) and step (2), freeze-drying is performed after rapid freezing with liquid nitrogen.

[0021] In one embodiment of the present invention, in step (4), the mass ratio of the hyaluronic acid skeleton in the hyaluronic acid solution to the gelatin skeleton in the gelatin solution is 1:1-35.

[0022] The second object of the present invention is to provide a composite hydrogel based on hyaluronic acid and gelatin prepared by the above preparation method.

[0023] The third object of the present invention is to provide an application of the above-mentioned composite hydrogel based on hyaluronic acid and gelatin as a carrier for coating probiotics.

[0024] The beneficial technical effects of the present invention are:

[0025] The composite hydrogel of the present invention has a good appearance and morphology, good self-healing properties, and its mesoporous morphology is confirmed by cryo-scanning electron microscopy. In addition, the hydrogel has good cell compatibility and gastric fluid stability through cytotoxicity and gastrointestinal digestion experiments. 600 It can be demonstrated that the hydrogel is beneficial for the release and proliferation of probiotics. BRIEF DESCRIPTION OF THE DRAWINGS

[0026] Figure 1 This is the hydrogen nuclear magnetic resonance spectrum of Example 5.

[0027] Figure 2 This is the infrared spectrum of Example 5.

[0028] Figure 3 This is the appearance of the hydrogel of Example 1-9.

[0029] Figure 4 These are low-temperature scanning electron microscope images of Example 3, Example 5, Example 6, Example 7, and Example 8.

[0030] Figure 5 This is the rheological analysis diagram of Example 5.

[0031] Figure 6 This is a diagram of the gastrointestinal digestion experiment of Example 5.

[0032] Figure 7 This is the live / dead cell staining image of Example 5.

[0033] Figure 8 This is the growth curve diagram of Example 5.

[0034] Figure 9 This is the bile salt resistance graph of Example 5.

[0035] Figure 10 This is the gastric acid resistance diagram of Example 5. DETAILED DESCRIPTION

[0036] In order to enable those skilled in the art to better understand the technical solutions of the present invention, the present invention is further described in detail below with reference to specific embodiments.

[0037] Example 1

[0038] A method for preparing a composite hydrogel based on hyaluronic acid and gelatin comprises the following steps:

[0039] (1) Preparation of gelatin skeleton

[0040] Take 1000 mg of gelatin and put it into a 50 mL volumetric flask. Add 20 mL of deionized water and dissolve it in a 40°C water bath. Add 2 mL of 30% vanillin ethanol solution and react for 4 hours. Then add 200 μL of 32% NaH3BCN solution and continue to react for 4 hours. Then dialyze with a 1000 Da dialysis bag for 3 days and freeze-dry.

[0041] (2) Preparation of hyaluronic acid skeleton

[0042] 500 mg of hyaluronic acid was placed in a 50 mL volumetric flask, and 40 mL of deionized water was added for ultrasonic dissolution. After complete dissolution, 108 mg of EDC and 65 mg of NHS were added and stirred at room temperature for 30 minutes. Then, 20 mL of 1.5% tyramine DMSO solution was added dropwise. After reacting at room temperature for 12 hours, the mixture was dialyzed using an 8-14 kDa dialysis bag for 3 days and freeze-dried.

[0043] (3) Preparation of composite hydrogel

[0044] 0.8 mg of hyaluronic acid skeleton was placed in a 1.5 mL centrifuge tube, 150 μL of deionized water was added to dissolve it, and 2 μL of 0.5% horseradish peroxidase was added to prepare a hyaluronic acid solution. 4 mg of gelatin skeleton was placed in a 1.5 mL centrifuge tube, 150 μL of deionized water was added to dissolve it, and 1 μL of 10% hydrogen peroxide was added to prepare a gelatin solution.

[0045] The hyaluronic acid solution and the gelatin solution are quickly mixed together to obtain the composite hydrogel.

[0046] Example 2

[0047] A method for preparing a composite hydrogel based on hyaluronic acid and gelatin comprises the following steps:

[0048] (1) Same as Example 1;

[0049] (2) Same as Example 1;

[0050] (3) Preparation of composite hydrogel

[0051] 0.8 mg of hyaluronic acid skeleton was placed in a 1.5 mL centrifuge tube, 150 μL of deionized water was added to dissolve it, and then 2 μL of 0.5% horseradish peroxidase was added to prepare a hyaluronic acid solution. 16 mg of gelatin skeleton was placed in a 1.5 mL centrifuge tube, 150 μL of deionized water was added to dissolve it, and 1 μL of 10% hydrogen peroxide was added to prepare a gelatin solution.

[0052] The hyaluronic acid solution and the gelatin solution are quickly mixed together to obtain the composite hydrogel.

[0053] Example 3

[0054] A method for preparing a composite hydrogel based on hyaluronic acid and gelatin comprises the following steps:

[0055] (1) Same as Example 1;

[0056] (2) Same as Example 1;

[0057] (3) Preparation of composite hydrogel

[0058] 0.8 mg of hyaluronic acid skeleton was placed in a 1.5 mL centrifuge tube, 150 μL of deionized water was added to dissolve it, and 2 μL of 0.5% horseradish peroxidase was added to prepare a hyaluronic acid solution. 28 mg of gelatin skeleton was placed in a 1.5 mL centrifuge tube, 150 μL of deionized water was added to dissolve it, and 1 μL of 10% hydrogen peroxide was added to prepare a gelatin solution.

[0059] The hyaluronic acid solution and the gelatin solution are quickly mixed together to obtain the composite hydrogel.

[0060] Example 4

[0061] A method for preparing a composite hydrogel based on hyaluronic acid and gelatin comprises the following steps:

[0062] (1) Same as Example 1;

[0063] (2) Same as Example 1;

[0064] (3) Preparation of composite hydrogel

[0065] 2 mg of hyaluronic acid skeleton was placed in a 1.5 mL centrifuge tube, 150 μL of deionized water was added to dissolve it, and 2 μL of 0.5% horseradish peroxidase was added to prepare a hyaluronic acid solution. 4 mg of gelatin skeleton was placed in a 1.5 mL centrifuge tube, 150 μL of deionized water was added to dissolve it, and 1 μL of 10% hydrogen peroxide was added to prepare a gelatin solution.

[0066] The hyaluronic acid solution and the gelatin solution are quickly mixed together to obtain the composite hydrogel.

[0067] Example 5

[0068] A method for preparing a composite hydrogel based on hyaluronic acid and gelatin comprises the following steps:

[0069] (1) Same as Example 1;

[0070] (2) Same as Example 1;

[0071] (3) Preparation of composite hydrogel

[0072] 2 mg of hyaluronic acid skeleton was placed in a 1.5 mL centrifuge tube, 150 μL of deionized water was added to dissolve it, and then 2 μL of 0.5% horseradish peroxidase was added to prepare a hyaluronic acid solution. 16 mg of gelatin skeleton was placed in a 1.5 mL centrifuge tube, 150 μL of deionized water was added to dissolve it, and 1 μL of 10% hydrogen peroxide was added to prepare a gelatin solution.

[0073] The hyaluronic acid solution and the gelatin solution are quickly mixed together to obtain the composite hydrogel.

[0074] Example 6

[0075] A method for preparing a composite hydrogel based on hyaluronic acid and gelatin comprises the following steps:

[0076] (1) Same as Example 1;

[0077] (2) Same as Example 1;

[0078] (3) Preparation of composite hydrogel

[0079] 2 mg of hyaluronic acid skeleton was placed in a 1.5 mL centrifuge tube, 150 μL of deionized water was added to dissolve it, and 2 μL of 0.5% horseradish peroxidase was added to prepare a hyaluronic acid solution. 28 mg of gelatin skeleton was placed in a 1.5 mL centrifuge tube, 150 μL of deionized water was added to dissolve it, and 1 μL of 10% hydrogen peroxide was added to prepare a gelatin solution.

[0080] The hyaluronic acid solution and the gelatin solution are quickly mixed together to obtain the composite hydrogel.

[0081] Example 7

[0082] A method for preparing a composite hydrogel based on hyaluronic acid and gelatin comprises the following steps:

[0083] (1) Same as Example 1;

[0084] (2) Same as Example 1;

[0085] (3) Preparation of composite hydrogel

[0086] 3.2 mg of hyaluronic acid skeleton was placed in a 1.5 mL centrifuge tube, 150 μL of deionized water was added to dissolve it, and then 2 μL of 0.5% horseradish peroxidase was added to prepare a hyaluronic acid solution. 4 mg of gelatin skeleton was placed in a 1.5 mL centrifuge tube, 150 μL of deionized water was added to dissolve it, and 1 μL of 10% hydrogen peroxide was added to prepare a gelatin solution.

[0087] The hyaluronic acid solution and the gelatin solution are quickly mixed together to obtain the composite hydrogel.

[0088] Example 8

[0089] A method for preparing a composite hydrogel based on hyaluronic acid and gelatin comprises the following steps:

[0090] (1) Same as Example 1;

[0091] (2) Same as Example 1;

[0092] (3) Preparation of composite hydrogel

[0093] 3.2 mg of hyaluronic acid skeleton was placed in a 1.5 mL centrifuge tube, 150 μL of deionized water was added to dissolve it, and then 2 μL of 0.5% horseradish peroxidase was added to prepare a hyaluronic acid solution. 16 mg of gelatin skeleton was placed in a 1.5 mL centrifuge tube, 150 μL of deionized water was added to dissolve it, and 1 μL of 10% hydrogen peroxide was added to prepare a gelatin solution.

[0094] The hyaluronic acid solution and the gelatin solution are quickly mixed together to obtain the composite hydrogel.

[0095] Example 9

[0096] A method for preparing a composite hydrogel based on hyaluronic acid and gelatin comprises the following steps:

[0097] (1) Same as Example 1;

[0098] (2) Same as Example 1;

[0099] (3) Preparation of composite hydrogel

[0100] 3.2 mg of hyaluronic acid skeleton was placed in a 1.5 mL centrifuge tube, 150 μL of deionized water was added to dissolve it, and 2 μL of 0.5% horseradish peroxidase was added to prepare a hyaluronic acid solution. 28 mg of gelatin skeleton was placed in a 1.5 mL centrifuge tube, 150 μL of deionized water was added to dissolve it, and 1 μL of 10% hydrogen peroxide was added to prepare a gelatin solution.

[0101] The hyaluronic acid solution and the gelatin solution are quickly mixed together to obtain the composite hydrogel.

[0102] Comparative Example 1

[0103] A method for preparing a gelatin-based hydrogel comprises the following steps:

[0104] (1) Same as step (1) in Example 1;

[0105] (2) Preparation of gelatin-based hydrogel

[0106] 24 mg of gelatin skeleton was placed in a 1.5 mL centrifuge tube, 300 μL of deionized water was added to dissolve it, 3 μL of 0.5% horseradish peroxidase and 1.5 μL of 10% hydrogen peroxide were added respectively, and the mixture was mixed and stirred to prepare a gelatin-based hydrogel.

[0107] Comparative Example 2

[0108] A method for preparing a hyaluronic acid-based hydrogel comprises the following steps:

[0109] (1) Same as Example 1 (2)

[0110] (2) Preparation of hyaluronic acid-based hydrogel

[0111] 3 mg of hyaluronic acid skeleton was placed in a 1.5 mL centrifuge tube, 300 μL of deionized water was added to dissolve it, 0.4 μL of 0.5% horseradish peroxidase and 0.2 μL of 10% hydrogen peroxide were added respectively, and the mixture was mixed to prepare a hyaluronic acid-based hydrogel.

[0112] Test method:

[0113] 1. H NMR spectrum: Deuterated water was used as solvent and the test was carried out on a 400 MHz NMR spectrometer.

[0114] Figure 1 It is the nuclear magnetic resonance hydrogen spectrum of Example 5. Figure 1 It can be seen that compared with the raw materials, the nuclear magnetic resonance spectra of the hyaluronic acid skeleton and the gelatin skeleton contain characteristic peaks of vanillin and tyramine, which indicates that the skeleton modification is successful.

[0115] 2. Infrared spectroscopy: A Fourier transform infrared spectrometer manufactured by Platinum Elmer Co., Ltd. was used, and the potassium bromide tablet method was employed. The specific operation was as follows: spectrally pure potassium bromide powder and five samples in different proportions were weighed in a ratio of 150:1, respectively, and ground in a mortar until the powder size was less than 2.5 μm. After grinding, the sample was removed and placed in a tablet press to produce a transparent or translucent disc of uniform thickness. The sample was removed and tested.

[0116] Figure 2 It is the infrared spectrum of embodiment 5. Figure 2 It can be seen that the infrared absorption peaks of the composite hydrogel are inconsistent with those of the skeleton, and it is speculated that new substances are generated.

[0117] 3. Hydrogel appearance: After gelation, the photo was taken using a light box (HP60-6LG).

[0118] Figure 3 This is the appearance of the hydrogel of Example 1-9. Figure 3 It can be seen that the composite hydrogels prepared in the embodiments of the present invention are all in gel state and have no obvious difference in appearance from traditional hydrogels.

[0119] 4. Cryogenic Scanning Electron Microscopy (Cryo-SEM, SU8010 / PP3010T, Hitachi / Quorum): Cryo-SEM was used to study the morphology of sample cross-sections. The hyaluronic acid-gelatin composite hydrogel was freeze-dried, then frozen and fractured with liquid nitrogen. The fracture surface was gold-sprayed and transferred to the SEM sample stage for imaging and observation.

[0120] Figure 4 The following are low-temperature scanning electron microscope images of Example 3, Example 5, Example 6, Example 7, and Example 8. Figure 4 It can be seen that Examples 3, 5, 6, 7 and 8 all have porous structures, and the pore sizes are different due to different proportions.

[0121] 5. Rheological properties analysis: A rheometer with a diameter of 20 mm (Discovery HR-2) was used to perform rheological studies on 400 μL of hydrogel. The hydrogel sample was placed between parallel plates, and the storage modulus (G′) and loss modulus (G″) were measured under the conditions of strain of 0.1-1000%, frequency of 0.1-100 Hz, angular frequency range of 1-100 rad / s, and shear rate range of 0.1 / s-100 / s. In order to analyze the viscoelastic region of the hydrogel, a time sweep experiment was performed with constant strain and frequency of 1% and 1 Hz, respectively. At the same time, in order to further study the self-healing properties of the material, high (500%) and low (1%) alternating amplitude oscillatory strain was performed at a frequency of 1 Hz, with each step lasting 120 s.

[0122] Figure 5 This is the rheological analysis diagram of Example 5. Figure 5 As can be seen from the figure, Example 5 has a modulus similar to that of the human body and has good self-repairing properties, and is presumably suitable for use as an oral carrier.

[0123] 6. Gastrointestinal digestion experiment: Hydrogels were prepared using rhodamine aqueous solution, and the same mass of hydrogels were immersed in simulated gastric fluid (SGF) and simulated intestinal fluid (SIF), respectively. Each 100 mL of simulated gastric fluid (SGF) contained 1 g of pepsin and 0.9 g of NaCl, and the pH was adjusted to 1.2 with 1.0 M HCl. Each 100 mL of simulated intestinal fluid (SIF) contained 1 g of pancreatic enzyme, 0.68 g of KH2PO4, and 1 g of bile salts, and the pH was adjusted to 7.0 with 1.0 M NaOH. Photos were taken every 30 minutes. The process of in vitro simulated continuous digestion of the hydrogel from SGF to SIF was also recorded. The hydrogel was placed in SGF and incubated at 37°C for 150 minutes, and then transferred to SIF for incubation. During this period, photos were taken to record the degradation of the hydrogel during the continuous digestion process.

[0124] Figure 6 This is a diagram of the gastrointestinal digestion experiment of Example 5. Figure 6 It can be seen that the hydrogel formed in Example 5 can maintain its intact morphology in simulated gastric fluid, but will degrade in simulated intestinal fluid. It is speculated that the hydrogel has good stability in gastric acid, but can be completely degraded in the intestinal environment.

[0125] 7. Cytotoxicity Assay: In vitro viability assays were performed using Caco-2 cells. Cells were treated with the composite hydrogel (20 mg / mL), gelatin scaffold (20 mg / mL), hyaluronic acid scaffold (20 mg / mL), and control culture medium. The cells were incubated for 24 hours, and cell viability was detected using a live / dead staining kit.

[0126] Figure 7 This is the live / dead cell staining diagram of Example 5. Figure 7 As can be seen from the figure, the hydrogel in Example 5 has more green parts representing living cells and more red parts representing dead cells, which suggests that the hydrogel has good cell compatibility.

[0127] 8. Growth curve: After the implanted lactobacilli were passaged twice, centrifuged at 3000 rpm for 5 minutes, resuspended in deionized water, and 300 μL of bacterial suspension was used to replace deionized water to form a gel. 2 mL of artificial intestinal fluid and 400 μL of hyaluronidase were then used to degrade the composite hydrogel until it was completely degraded. The digested composite hydrogel, gelatin skeleton, hyaluronic acid skeleton, and control intestinal fluid were added to 20 mL of culture medium, respectively, and cultured at 37°C with shaking. The OD was measured every hour. 600 until the growth stabilizes.

[0128] Figure 8 The growth curve of Example 5 is shown in FIG. 5 , wherein deionized water is replaced by concentrated bacterial suspension. Figure 8 As can be seen, the OD measured by the hydrogel encapsulating probiotics 600The results were basically similar to those of free bacteria, suggesting that the composite hydrogel was bacterially compatible.

[0129] 9. Bile salt resistance experiment: After the implanted lactobacilli were passaged twice, centrifuged at 3000 rpm for 5 min, and resuspended in deionized water to obtain a bacterial suspension. The bacterial suspension was used instead of deionized water to form a gel. Gelatin-based hydrogels, hyaluronic acid-based hydrogels, and composite hydrogels were prepared using the methods of Comparative Example 1, Comparative Example 2, and Example 5, respectively. Free bacteria were used as the control group. After incubation in bile salts for 0 h, 2 h, and 4 h, the probiotic content was measured.

[0130] Figure 9 is the bile salt resistance diagram of Example 5, from Figure 9 As can be seen in the figure, compared with free probiotics, the activity of probiotics encapsulated in hyaluronic acid-based hydrogels and composite hydrogels remained high after exposure to bile salts, far exceeding that of gelatin-based hydrogels. It is speculated that the hydrogels play a protective role for probiotics.

[0131] 10. Gastric acid resistance experiment: The implanted lactobacilli were passaged twice, centrifuged at 3000 rpm for 5 min, and resuspended in deionized water to obtain a bacterial suspension. The bacterial suspension was used instead of deionized water to form a gel. Gelatin-based hydrogels, hyaluronic acid-based hydrogels, and composite hydrogels were prepared using the methods of Comparative Example 1, Comparative Example 2, and Example 5, respectively. Free bacteria were used as the control group. After incubation in gastric acid for 0 h, 1 h, and 2 h, the probiotic content was determined.

[0132] Figure 10 is the gastric acid resistance diagram of Example 5, from Figure 10 It can be seen that compared with free probiotics, the activity of probiotics encapsulated in the composite hydrogel still reached a high level after passing through gastric acid, which was much higher than that of gelatin-based hydrogel and hyaluronic acid-based hydrogel. It is speculated that the hydrogel alone did not play a protective role, while the composite hydrogel played a protective role for the probiotics.

[0133] In summary, the method of the present invention can produce a composite hydrogel with relatively good overall properties while maintaining the appearance of traditional hydrogels. Furthermore, the encapsulated probiotics exhibit high activity after exposure to gastric acid and bile salts, providing protection for the probiotics. This technology can be used to produce hydrogel carriers that are beneficial for encapsulating Lactobacillus plantarum, demonstrating promising market prospects.

[0134] The embodiments provided above are not intended to limit the scope of the present invention, nor are the steps described to limit their execution order. Any obvious improvements to the present invention made by those skilled in the art in combination with existing common knowledge shall fall within the scope of protection defined by the claims of the present invention.

Claims

1. A method for preparing a composite hydrogel based on hyaluronic acid and gelatin, characterized in that: The preparation method comprises the following steps: (1) Preparation of hyaluronic acid skeleton: dissolve hyaluronic acid in deionized water, add EDC and NHS, stir at room temperature, then add tyramine DMSO solution, react at room temperature, dialyze and freeze-dry after the reaction to obtain hyaluronic acid skeleton; (2) Preparation of gelatin skeleton: gelatin was dissolved in deionized water, and vanillin ethanol solution was added to react, followed by addition of NaH3BCN solution, dialyzed, and freeze-dried to obtain gelatin skeleton; (3) dissolving the hyaluronic acid skeleton in deionized water, and then adding horseradish peroxidase to prepare a hyaluronic acid solution; dissolving the gelatin skeleton in deionized water to form a gelatin skeleton solution, and then adding hydrogen peroxide to prepare a gelatin solution; (4) mixing the hyaluronic acid solution and the gelatin solution to prepare the composite hydrogel; The mass ratio of the hyaluronic acid skeleton in the hyaluronic acid solution to the gelatin skeleton in the gelatin solution is 1:1-35.

2. The preparation method according to claim 1, characterized in that In step (1), the mass ratio of EDC to hyaluronic acid is 1:4-6; the mass ratio of NHS to hyaluronic acid is 1:6-8.

3. The preparation method according to claim 1, wherein In step (1), the mass concentration of the tyramine DMSO solution is 1-1.5%; the amount of tyramine used is 50-70% of the mass of the hyaluronic acid.

4. The preparation method according to claim 1, wherein In step (1), the stirring time is 10-60 min; the reaction time at room temperature is 12-14 h; and the dialysis is performed using a dialysis bag with a molecular weight cutoff of 8-14 kDa for 1-4 days.

5. The preparation method according to claim 1, characterized in that In step (2), the mass ratio of vanillin to gelatin is 1:1-3; the NaH3BCN solution is prepared by dissolving NaH3BCN in a 1M NaOH aqueous solution; and the mass ratio of NaH3BCN to gelatin is 1:14-16.

6. The preparation method according to claim 1, wherein In step (2), the reaction conditions are: reaction at 40°C for 4-6 h; dialysis using a dialysis bag with a molecular weight cutoff of 1000 Da for 1-4 days.

7. The preparation method according to claim 1, wherein In step (3), the mass ratio of horseradish peroxidase to hyaluronic acid skeleton is 1:60-350; the concentration of hydrogen peroxide in hydrogen peroxide is 9-11wt%; and the amount of hydrogen peroxide used is 0.6-0.73% of the volume of the gelatin skeleton solution.

8. A composite hydrogel based on hyaluronic acid and gelatin prepared by the preparation method according to any one of claims 1 to 7.

9. An application of the composite hydrogel based on hyaluronic acid and gelatin according to claim 8, characterized in that: Used as a carrier to encapsulate probiotics.

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