Ultrafast self-repairing hydrogel based on hyaluronic acid and gelatin as well as preparation method and application of ultrafast self-repairing hydrogel

Abstract

The invention discloses an ultrafast self-repairing hydrogel based on hyaluronic acid and gelatin and a preparation method and application thereof. The preparation method comprises the following steps: (1) dissolving hyaluronic acid in deionized water, adding EDC and NHS, stirring at room temperature, then adding a tyramine DMSO solution, reacting at room temperature, and dialyzing and freeze-drying after the reaction is finished to prepare a hyaluronic acid skeleton; (2) gelatin is dissolved in deionized water, a vanillin ethanol solution is added for a reaction, then a NaH3BCN solution is added for reduction, dialysis and freeze-drying, and a gelatin skeleton is prepared; (3) dissolving the hyaluronic acid skeleton in deionized water, and then adding horse radish peroxidase to prepare a hyaluronic acid solution; adding the gelatin skeleton solution into deionized water, and then adding hydrogen peroxide to prepare a gelatin solution; and (4) mixing the hyaluronic acid solution with the gelatin solution to prepare the composite hydrogel. The coating material is used for coating the probiotics and is beneficial to activity maintenance of the probiotics in a simulated digestive tract environment.

Description

Technical Field

[0001] The invention relates to the technical field of gel, 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 directly oral probiotics; 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 application proposes an ultrafast self-healing hydrogel based on hyaluronic acid and gelatin, and 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, and 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 has a certain degree of creativity.

[0005] The technical solution of the present invention is 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, 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, dialyzing after the reaction, and freeze-drying to obtain a hyaluronic acid skeleton;

[0008] (2) preparing a gelatin skeleton, dissolving gelatin in deionized water, adding vanillin ethanol solution to react, then adding NaH3BCN solution to reduce, dialyze, and freeze-dry 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 obtain 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 cutoff 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: reacting 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 9-11wt%; 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 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 good appearance and morphology, good self-healing performance, and its mesoporous morphology is proved by cryo-scanning electron microscopy. In addition, the hydrogel has good cell compatibility and gastric juice 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 Examples 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] Fig. 9 This is the bile salt resistance graph of Example 5.

[0035] Fig.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 solution of the present invention, the present invention is further described in detail below in conjunction with 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, dissolve it in a 40°C water bath, add 2 mL of 30% vanillin ethanol solution, react for 4 hours, then add 200 μL of 32% NaH3BCN solution, continue to react for 4 hours, dialyze with a 1000 Da dialysis bag for 3 days, and freeze-dry.

[0041] (2) Preparation of hyaluronic acid skeleton

[0042] Take 500 mg of hyaluronic acid and put it into a 50 mL volumetric flask, add 40 mL of deionized water and ultrasonically dissolve it. After complete dissolution, add 108 mg of EDC and 65 mg of NHS and stir at room temperature for 30 minutes; then add 20 mL of 1.5% tyramine DMSO solution dropwise, react at room temperature for 12 hours, dialyze with 8-14 kDa dialysis bag for 3 days, and freeze-dry.

[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, 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, 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, and 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, 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, 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, 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, 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, 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, and 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, 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, 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, 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, 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, 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, and 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, 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, 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, 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) of 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, 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, 0.4 μL of 0.5% horseradish peroxidase and 0.2 μL of 10% hydrogen peroxide were added respectively, and the mixture was mixed and stirred to prepare a hyaluronic acid-based hydrogel.

[0112] Test method:

[0113] 1. Nuclear magnetic resonance hydrogen spectrum: Deuterated water is used as solvent and the test is carried out on a 400MHz nuclear magnetic resonance spectrometer.

[0114] Figure 1 is the hydrogen nuclear magnetic resonance spectrum of Example 5. Figure 1 It can be seen that compared with the raw materials, the NMR 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 spectrum: Use the Fourier transform infrared spectrometer produced by Platinum Elmer Co., Ltd. and the potassium bromide tablet method. The specific operation is as follows: weigh spectrally pure potassium bromide powder and five samples in different proportions at a ratio of 150:1, put them in a mortar and grind them until the powder size is less than 2.5μm. After grinding, take out the sample and put it into a tablet press to make a transparent or translucent disc of uniform thickness. Take out the sample and test it.

[0116] Figure 2 It is the infrared spectrum of Example 5. Figure 2 It can be seen that the infrared absorption peak of the composite hydrogel is inconsistent with that of the skeleton, which suggests that new substances are generated.

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

[0118] Figure 3 The following 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 the sample cross section. The hyaluronic acid-gelatin composite hydrogel was freeze-dried, then frozen and broken by liquid nitrogen, the fracture surface was gold-sprayed, and then transferred to the scanning electron microscope sample stage for photography and observation.

[0120] Figure 4 The low temperature scanning electron microscope images of Example 3, Example 5, Example 6, Example 7 and Example 8 are shown in FIG. 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 (Discovery HR-2) with a diameter of 20 mm was used to conduct 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 scan 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 strains were 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 It can be seen that Example 5 is similar to the human body modulus and has good self-healing 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 salt, 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 gastrointestinal digestion experiment diagram 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 test: Caco-2 cells were used for in vitro viability test. Cells were treated with composite hydrogel (20 mg / mL), gelatin skeleton (20 mg / mL), hyaluronic acid skeleton (20 mg / mL) and control culture medium, incubated for 24 hours, and cell activity was detected using a live / dead staining kit.

[0126] Figure 7 This is the live / dead cell staining diagram of Example 5. Figure 7 It can be seen that the hydrogel in Example 5 has a larger green portion representing living cells and a larger red portion representing dead cells, which indicates that the hydrogel has good cell compatibility.

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

[0128] Figure 8 is the growth curve of Example 5, wherein the deionized water is replaced by the concentrated bacterial suspension. Figure 8 As can be seen, the OD measured by the hydrogel encapsulating probiotics 600It is basically similar to free bacteria, and it is speculated that the composite hydrogel has bacterial compatibility.

[0129] 9. Bile salt resistance experiment: After the implanted lactobacillus was passaged twice, the cells were 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 hydrogel, hyaluronic acid-based hydrogel and composite hydrogel were prepared by 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 content of probiotics was determined.

[0130] Fig. 9 is the bile salt resistance diagram of Example 5, from Fig. 9 It can be seen that compared with free probiotics, the activity of probiotics encapsulated by hyaluronic acid-based hydrogel and composite hydrogel after bile salt still reached a higher level, much higher than gelatin-based hydrogel. It is speculated that the hydrogel plays a protective role on probiotics.

[0131] 10. Gastric acid resistance experiment: After the implanted lactobacilli were passaged twice, they were 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 hydrogel, hyaluronic acid-based hydrogel and composite hydrogel were prepared by 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 content of probiotics was determined.

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

[0133] In summary, the method of the present invention can obtain a composite hydrogel with relatively good overall properties, maintaining the appearance of traditional hydrogels. In addition, the encapsulated probiotics have higher activity after passing through gastric acid and bile salts, which protects the probiotics. The use of this set of technology can produce a hydrogel carrier that is beneficial for coating plant lactobacillus, which has a good market prospect.

[0134] The embodiments provided above are not intended to limit the scope of the present invention, and the steps described are not intended to limit the execution order thereof. Those skilled in the art may make obvious improvements to the present invention in combination with existing common knowledge, which also 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) preparing a hyaluronic acid skeleton, dissolving hyaluronic acid in deionized water, adding EDC and 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; (2) preparing a gelatin skeleton, dissolving gelatin in deionized water, adding vanillin ethanol solution to react, then adding NaH3BCN solution, dialyzing, and freeze-drying to obtain a gelatin skeleton; (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; (4) Mixing the hyaluronic acid solution and the gelatin solution to obtain the composite hydrogel.

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, characterized in that: 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, characterized in that: 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; the mass ratio of NaH3BCN to gelatin is 1:14-16.

6. The preparation method according to claim 1, characterized in that: 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.

7. The preparation method according to claim 1, characterized in that: 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. The preparation method according to claim 1, characterized in that: 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.

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

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

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