A probiotic hydrogel and its preparation method and application

Abstract

The present invention belongs to the field of biomedical materials technology, and specifically relates to a probiotic hydrogel and its preparation method and application. The probiotic hydrogel comprises a hydrogel substrate formed by oxidized methylmalonamide hyaluronic acid; and the hydrogel substrate is cross-linked with probiotics and extracellular polysaccharides secreted by the probiotics, wherein the probiotics are Lactobacillus acidophilus. L. acidipiscis 10851 , the extracellular polysaccharide is L. acidipiscis 10851 The probiotic hydrogel of the present invention is suitable for various traumas and surgical wounds, and can stop bleeding, reduce wound infection and promote wound healing.

Description

Technical Field

[0001] The present invention belongs to the technical field of biomedical materials, and in particular relates to a probiotic hydrogel and a preparation method and application thereof. Background Art

[0002] Wound healing is a complex and delicate biological process involving multiple stages, including hemostasis, inflammation, proliferation, and remodeling. Probiotics, as a vital component of the human microbiome, can modulate the host immune system and inhibit harmful inflammatory responses through their metabolites and direct interactions, providing a new therapeutic strategy for wound healing. Probiotic hydrogels, innovative materials that combine the advantages of probiotics and polymer hydrogels, are becoming a research hotspot in the field of wound care. The development of a new probiotic hydrogel specifically for wound healing offers patients improved therapeutic outcomes and quality of life.

[0003] Lactobacillus acidipiscis is a probiotic found in sour fish and sour cheese. It is widely used in the food industry. The identified strains include L. acidipiscis 10851 、 L. acidipiscis 10822 、 L. acidipiscis 10852 Recent research has found L. acidipiscis 10851 It also plays an important role in immune regulation. Therefore, this application uses it in hydrogels through research. Summary of the Invention

[0004] The purpose of the present invention is to provide a probiotic hydrogel that promotes the repair of skin or body tissue wounds and hemostasis. L. acidipiscis 10851 It is cross-linked with EPS-la10851, and after adding a photoinitiator, it is cured by UV light to form a probiotic hydrogel. The resulting probiotic hydrogel has good biocompatibility, adhesion, and the functions of preventing bacterial infection, stopping bleeding and promoting wound healing, and is suitable for various traumas and surgical wounds.

[0005] To achieve the above object, the present invention adopts the following technical solutions:

[0006] In one aspect of the present invention, a probiotic hydrogel is disclosed, comprising a hydrogel matrix formed by oxidized methylmalonamidated hyaluronic acid (OHAMA); and the hydrogel matrix contains cross-linked probiotics and exopolysaccharides secreted by the probiotics, wherein the probiotics are Lactobacillus acidophilus. L. acidipiscis 10851 , the extracellular polysaccharide is L. acidipiscis 10851 Secreted exopolysaccharide EPS-la10851.

[0007] It should be noted that the acid fish lactobacillus used in the present invention L. acidipiscis 10851It has the function of regulating the differentiation of γδT, Treg, TH1 and TH17 cells, inhibiting the production of cytokines IFN-γ and IL-17, promoting the production of cytokines IL-10 and IL-13, and can alleviate the inflammatory response of autoimmune diseases.

[0008] The present invention uses OHAMA as the skeleton of the hydrogel, which has higher stability and adhesion to tissues than methylmalonamide hyaluronic acid (HAMA) and is more conducive to wound healing.

[0009] The present invention adopts OHAMA, L. acidipiscis 10851 The hydrogel is formed using bacteria and the extracellular polysaccharide EPS-1a10851 as raw materials. The C=C double bonds in OHAMA cross-link with the peptide bonds in the bacteria and extracellular polysaccharides, solidifying to form a hydrogel. The resulting hydrogel promotes tissue repair and precisely targets probiotics to wounds, preventing their escape. It also promotes fibroblast proliferation, prevents bacterial infection, accelerates wound healing, and provides hemostasis.

[0010] In one embodiment of the present invention, OHAMA is obtained by first modifying hyaluronic acid (HA) with methacrylic anhydride (MA), amidating it, and then precipitating it with anhydrous ethanol. The HAMA is then purified to obtain methylpropionamidated hyaluronic acid (HAMA). The HAMA is then oxidized with NaIO4, purified, and freeze-dried to obtain OHAMA. Specifically, the steps may include:

[0011] (1) Prepare HA solution;

[0012] (2) In an ice-water bath, add the MA solution dropwise to the HA solution while stirring, adjust the pH of the solution to 9-10, and stir to mix thoroughly;

[0013] (3) Add ethanol to the stirred solution, centrifuge, and collect the precipitate to obtain HAMA;

[0014] (4) adding deionized water to dissolve the HAMA precipitated in step (3), followed by dialysis and freeze-drying to obtain purified HAMA;

[0015] (5) The HAMA purified in step (4) was used to prepare a solution, NaIO4 was added, the reaction was stirred, and then ethylene glycol was added to terminate the reaction, the solution was dialyzed with deionized water, and freeze-dried to obtain the OHAMA.

[0016] In one implementation of the present invention, in step (1), the concentration of the HA solution is 1-5 wt%.

[0017] In one implementation of the present invention, in step (2), the mass ratio of HA to MA is 1:(4-5), and the reaction time is 1-6 h.

[0018] In one implementation of the present invention, in step (3), the ethanol concentration is 99.6-100 v / v%.

[0019] In one implementation of the present invention, in step (5), in the reaction solution, the concentration of methylpropionamidated hyaluronic acid is 1-3 wt %, the concentration of NaIO 4 is 0.5-1.5 wt %, and the reaction time is 4-8 h.

[0020] In one implementation of the present invention, in step (5), the amount of ethylene glycol added is 2-4 wt % of the reaction solution.

[0021] It should be noted that the extracellular polysaccharide EPS-1a10851 in the present invention is produced by probiotics L. acidipiscis 10851 secretion, by L. acidipiscis 10851 The culture medium was sterilized by boiling, deproteinized by trichloroacetic acid, precipitated by ethanol, dialyzed, and freeze-dried.

[0022] In one implementation of the present invention, the concentration of trichloroacetic acid is 13-15 wt %, and the concentration of ethanol is 90-100 v / v %.

[0023] In one implementation of the present invention, the dialysate used for dialysis has a specification of 3500 kDa.

[0024] Another aspect of the present invention also provides a method for preparing the probiotic hydrogel, comprising: L. acidipiscis 10851 The culture solutions are mixed to obtain a mixed solution, and then a photoinitiator is added to solidify the solution by ultraviolet irradiation to obtain the probiotic hydrogel.

[0025] In one implementation of the present invention, the concentration or percentage of each substance in the preparation method preferably satisfies at least one of the following conditions (a) to (c):

[0026] (a) In the mixed solution, the mass percentage of OHAMA is 2-5%;

[0027] (b) In the mixed solution, the mass percentage of the exopolysaccharide EPS-1a10851 is 1-5%;

[0028] (c) L. acidipiscis 10851 In the culture medium, L. acidipiscis 10851 The concentration is 10 6 -10 8 CFU.

[0029] The present invention has found that selecting the mass percentage or concentration of the solute in the solution within the above range is beneficial to the gelation reaction.

[0030] In one implementation of the present invention, the L. acidipiscis 10851 The cells were inoculated into sterile PBS to obtain culture medium.

[0031] In one implementation of the present invention, the photoinitiator is lithium phenyl (2,4,6-trimethylbenzoyl) phosphate (LAP); preferably, the photoinitiator is added in the form of a solution with a concentration of 10-20 wt%.

[0032] In one implementation of the present invention, the curing is achieved by irradiating ultraviolet light with a wavelength of 405 nm.

[0033] Compared with the prior art, the present invention has the following beneficial effects:

[0034] The probiotic hydrogel of the present invention is loaded with L. acidipiscis 10851 Its secretion products have the effect of inhibiting bacteria and promoting wound healing; EPS-la10851 is used as L. acidipiscis 10851 The hydrogel contains nutrients that maintain the bacteria's high activity within the hydrogel. Using OHAMA as the hydrogel matrix enhances biocompatibility and adhesion, as well as its hemostatic properties. The hydrogel is suitable for various traumatic and surgical wounds, stopping bleeding, reducing wound infection, and promoting wound healing. BRIEF DESCRIPTION OF THE DRAWINGS

[0035] Figure 1 This is an optical photograph of the EH@la probiotic hydrogel according to an embodiment of the present invention;

[0036] Figure 2 The antibacterial test results of the EH@la probiotic hydrogel according to the embodiment of the present invention are shown;

[0037] Figure 3 This is a graph showing the biocompatibility results of the EH@la probiotic hydrogel according to an embodiment of the present invention;

[0038] Figure 4 This is a diagram showing the in vivo wound healing results of the EH@la probiotic hydrogel according to an embodiment of the present invention;

[0039] Figure 5 This is the in vivo hemostatic result of the EH@la probiotic hydrogel according to the embodiment of the present invention. DETAILED DESCRIPTION

[0040] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as those commonly understood by those skilled in the art of the present invention. The terms used in this specification of the present invention are only for the purpose of describing specific embodiments and are not intended to limit the present invention.

[0041] Unless otherwise specified, the reagents, materials, and equipment used in the examples of the present invention are all commercially available; and the experimental methods, unless otherwise specified, are all conventional experimental methods in the art.

[0042] The acid fish Lactobacillus 10851 used in the embodiment of the present invention was purchased from the China Industrial Microorganism Culture Collection Center (CICC), the strain collection number is CICC10851, the Chinese name is acid fish combined Lactobacillus 10851, the Latin name Ligilactobacillus acididipiscis10851 (abbreviation: L. acidipiscis 10851 ).

[0043] The present invention provides a probiotic hydrogel with antibacterial properties and wound healing promoting properties, comprising OHAMA, L. acidipiscis 10851 Bacteria and extracellular polysaccharide EPS-la10851.

[0044] OHAMA is obtained by oxidation of HAMA, and has higher stability and adhesion to tissues than HAMA. Moreover, the C=C double bond in OHAMA can cross-link with the peptide bond in bacteria and extracellular polysaccharides, thus enhancing the stability and adhesion of the hydrogel. L. acidipiscis 10851 Its secretion products have the effect of inhibiting bacteria and promoting wound healing; EPS-la10851 is used as L. acidipiscis 10851 The nutrients in the hydrogel maintain the high activity of the bacteria. The various components cooperate and support each other, improving the performance of the hydrogel and being more conducive to wound healing.

[0045] The present invention also provides a method for preparing the probiotic hydrogel described in the above technical solution, wherein OHAMA, EPS-la10851 and L. acidipiscis 10851 The culture solutions are mixed to obtain a mixed solution, and then a photoinitiator solution is added, and the mixed solution is cured by ultraviolet light irradiation to obtain the probiotic hydrogel.

[0046] Specifically, the method includes the following steps:

[0047] Mix OHAMA and EPS-1a10851 evenly, grind briefly and mix, preferably in a clean bench, and sterilize with UV;

[0048] Will L. acidipiscis 10851 Culture in PBS to obtain a culture medium, preferably at a concentration of 10 6 -10 8 CFU;

[0049] Dissolve the sterilized OHAMA and EPS-la10851 in the probiotics L. acidipiscis 10851The culture medium is fully dissolved, and a photoinitiator LAP solution is added, preferably the concentration of the LAP solution is 10-20 wt %, and the probiotic hydrogel of the present invention is obtained after 405 nm ultraviolet light irradiation.

[0050] In order to further illustrate the present invention, the technical solution provided by the present invention is described in detail below with reference to the accompanying drawings and embodiments, but they should not be construed as limiting the scope of protection of the present invention.

[0051] The following abbreviations EPS-la10851 represent extracellular polysaccharide, OHAMA represent oxidized methylmalonamidated hyaluronic acid, EH represent base material hydrogel, and EH@la represent probiotic hydrogel.

[0052] Example 1

[0053] The present invention provides a probiotic hydrogel EH@la. The preparation method of the probiotic hydrogel is as follows:

[0054] 1) Preparation of OHAMA:

[0055] Prepare a 3wt% hyaluronic acid solution.

[0056] According to the mass ratio of hyaluronic acid to methacrylic anhydride = 1:4.5, methylmalonic anhydride was added dropwise while stirring in an ice-water bath. The pH of the solution was adjusted to 9 with NaOH solution, and the reaction was stirred for 6 hours.

[0057] The stirred solution after the reaction was completed was poured into a 99 v / v% ethanol solution to obtain a precipitate;

[0058] Deionized water was added to re-dissolve the precipitate, and ethanol was removed by dialysis. Purified HAMA powder was obtained by freeze-drying and stored at -20°C in the dark.

[0059] Prepare 1 wt% purified HAMA solution, add 1 wt% NaIO4 and continue stirring for 8 h, then add 3 wt% ethylene glycol to terminate the reaction, dialyze and freeze-dry to obtain OHAMA.

[0060] 2) Preparation of EPS-la10851: Culture L. acidipiscis 10851 ,collect L. acidipiscis 10851 The culture liquid was boiled at 100°C and centrifuged to remove bacterial residues; protein was removed by 14wt% trichloroacetic acid and then centrifuged; exopolysaccharides were precipitated with 99v / v% ethanol and collected after centrifugation; the liquid was reconstituted with sterile deionized water and dialyzed to remove excess trichloroacetic acid; and EPS-la10851 was obtained by freeze-drying.

[0061] 3) Preparation of EH@la probiotic hydrogel: 300 mg EPS-La10851 was dissolved in 10 mL of 10 6 CFUL. acidipiscis 10851 150 mg of OHAMA was added to PBS, followed by 100 μL of 10 wt% LAP solution. After complete dissolution, the solution was irradiated with 405 nm UV light. After 15 minutes of UV irradiation, a probiotic hydrogel was obtained.

[0062] Example 2

[0063] The embodiment of the present invention provides a probiotic hydrogel EH@la. The only difference between the preparation method of the probiotic hydrogel and that of Example 1 is that 200 mg of OHAMA is added in step 3), and the solution is irradiated with ultraviolet light for 10 minutes to obtain the probiotic hydrogel.

[0064] Example 3

[0065] The embodiment of the present invention provides a probiotic hydrogel EH@la. The only difference between the preparation method of the probiotic hydrogel and that of Example 2 is that 300 mg of OHAMA is added in step 3), and the solution is irradiated with ultraviolet light for 5 minutes to obtain the probiotic hydrogel.

[0066] Example 4

[0067] The embodiment of the present invention provides a probiotic hydrogel EH@la. The only difference between the preparation method of the probiotic hydrogel and that of Example 2 is that the concentration of the LAP solution in step 3) is 15 wt %, and the probiotic hydrogel is obtained after the solution is irradiated with ultraviolet light for 2 min.

[0068] Example 5

[0069] The embodiment of the present invention provides a probiotic hydrogel EH@la. The only difference between the preparation method of the probiotic hydrogel and that of Example 2 is that the concentration of the LAP solution in step 3) is 20 wt %, and the probiotic hydrogel is obtained after the solution is irradiated with ultraviolet light for 30 s.

[0070] Comparative Example 1

[0071] 1) Preparation of OHAMA: same as in Example 1.

[0072] 2) Preparation of EPS-1a10851: Same as Example 1.

[0073] 3) Preparation of probiotic hydrogel: 300 mg of EPS-La10851 was dissolved in 10 mL of PBS, followed by 300 mg of OHAMA and finally 100 μL of 10% LAP solution. After complete dissolution, the solution was irradiated with 405 nm UV light. After irradiation for 30 seconds, the hydrogel matrix EH was obtained.

[0074] Effect verification:

[0075] 1. Comparison of the gelling time of hydrogels in Examples 1-5 is shown in Table 1. It can be seen that the amount of photoinitiator and OHAMA both have an impact on the gelling time. Figure 1 Shown is a light yellow hydrogel.

[0076] Table 1 Comparison of gelation time of probiotic hydrogels in Examples 1-5

[0077]

[0078] 2. Conventional antibacterial test: using bacterial OD 600 The antibacterial effects of the hydrogel of Example 5 and the EH hydrogel of Comparative Example 1 were detected by absorbance value.

[0079] Two types of bacterial strains were selected for testing, namely Gram-positive bacteria and Gram-negative bacteria. The hydrogel was incubated with each bacterial strain for 24 h, and then the bacterial solution was taken to detect OD 600 The absorbance value was recorded as A1, and the control group without adding hydrogel had its absorbance value recorded as A0.

[0080] The calculation formula of the antibacterial rate is: A0-A1 / A0×100%

[0081] The test results are as follows Figure 2 As shown in Example 5, the EH@la probiotic hydrogel has an inhibition rate of 68% and 85% against Escherichia coli and Staphylococcus aureus respectively. E.coli and S.aureus The antibacterial rates were 25% and 26% respectively; the results showed that the hydrogels of Comparative Example 1 and Example 5 had antibacterial effects, but the addition of probiotics L. acidipiscis 10851 After that, the antibacterial effect was significantly improved.

[0082] 3. Biocompatibility testing

[0083] CCK8 method: The hydrogels of Comparative Example 1 and Example 5 were immersed in complete cell culture medium (DMEMF12 + 10% FBS + 1% PS) at 37°C and incubated on a shaker at 37°C and 110 rpm for 24 h. The extraction medium was then removed and filtered through a 0.22 μm filter. c ) is complete cell culture medium without extract (DMEM F12+10% FBS+1% PS); blank group (A b) contains only complete culture medium (DMEM F12 + 10% FBS + 1% PS) and does not contain 3T3 cells. 8,000 3T3 cells were seeded into each well of a 96-well plate and placed in a 5% CO2, 37°C constant temperature incubator for 24 hours, and the cell status was observed. The culture medium in the wells was aspirated, and then 100 μL of the extract of different samples, the control group, and the blank group were added to the 96-well plate. Four parallel samples were set up for each group and incubated in a 5% CO2, 37°C constant temperature incubator for 24 hours; after the incubation period, 10 μL of CCK8 solution was added to each well and incubated at 37°C for 1 hour; after the incubation period, the absorbance at 450nm was measured using a microplate reader. The following formula was used to calculate:

[0084] Cell viability(%)=[(A s -A b ) / (A c -A b )]*100

[0085] Note: A s is the absorbance of the experimental group; A b is the absorbance of the blank group; A c The absorbance of the control group

[0086] Cell live-death staining method: 3T3 cells 3×10 5 The cells were cultured in 6-well plates with the hydrogel extracts of each group, and the complete culture medium was replaced with the cells at 37°C with 5% CO2 for 24 h. After the culture, Calcein-AM / PI staining solution was added and incubated at 37°C for 30 min. The staining was photographed under a fluorescence microscope.

[0087] Figure 3 The results showed that the 3T3 cell viability of the hydrogels in Comparative Example 1 and Example 5, measured using the aforementioned method, was above 85%. Live / dead staining revealed that the hydrogel extract had no effect on cell viability, indicating that the hydrogels in Comparative Example 1 and Example 5 had good cytocompatibility. The 3T3 cell viability of the EH@la probiotic hydrogel in Example 5, as measured by CCK8, reached 143%, demonstrating that the EH@la probiotic hydrogel promotes cell proliferation.

[0088] 4. Probiotic hydrogel promoted wound healing experiment: BALB / c mice were shaved on the back and a wound was created. 20 μL of 1×10 8 CFU / mL S.aureusThe suspension was applied to the wound. The hydrogels from Comparative Example 1 and Example 5 were used to completely coat the wound surface; mice without hydrogel were used as the control group. Finally, all groups were treated with a layer of transparent 3M™ Tegaderm™ to protect the wound surface. After recovery from anesthesia, the animals were monitored for any discomfort and provided with water and feed. The hydrogel was changed daily, and body weight changes and wound healing were recorded.

[0089] The test results are as follows Figure 4 As shown, compared with the EH hydrogel and 3M™ Tegaderm™ transparent dressing group of Example 5 EH@la probiotic hydrogel and the control group 1 EH hydrogel and 3M™ Tegaderm™ transparent dressing, all mice had wound infection on the third day, but on the fifth day, the infection in the blank control group worsened, while the wound infection in the EH hydrogel and EH@la probiotic hydrogel disappeared, indicating that both hydrogels have antibacterial effects; and on the tenth day, the wound healing rate of the control group was 72%, the wound healing rate of the EH hydrogel group reached 84%, and the wound healing rate of the EH@la probiotic hydrogel group reached 97%. The EH@la probiotic hydrogel group has a more significant ability to promote wound healing.

[0090] 5. Hemostasis experiment with EH@la probiotic hydrogel: BALB / c mice were kept alive, their thoracic cavities opened, their livers squeezed out, and a 6 mm wound was incised in the livers using a scalpel. Upon initial bleeding, 100 mg of the probiotic hydrogel from Example 5 was applied to the bleeding site. Filter paper was placed underneath the livers. The bleeding and hemostasis times were recorded, and the weight of the filter paper before and after bleeding was weighed and subtracted to determine the amount of bleeding. A blank control group received no treatment.

[0091] The test results are as follows Figure 5 As shown, the amount of bleeding in the blank control group was 0.215 g, and the amount of bleeding in the EH@la probiotic hydrogel group was 0.0449 g. In Example 5, the amount of bleeding in the EH@la probiotic hydrogel group was significantly reduced compared with the blank control group; the blank control group successfully stopped bleeding in 120 s, and the EH@la probiotic hydrogel successfully stopped bleeding 20 s after applying the wound. The results showed that the EH@la probiotic hydrogel had good hemostatic function.

[0092] The technical features of the above-mentioned embodiments can be combined arbitrarily. In order to make the description concise, not all possible combinations of the technical features in the above-mentioned embodiments are described. However, as long as there is no contradiction in the combination of these technical features, they should be considered to be within the scope of this specification.

[0093] The above-described embodiments merely illustrate several implementations of the present invention, and while their descriptions are relatively specific and detailed, they should not be construed as limiting the scope of the patent. It should be noted that a person skilled in the art would be able to make numerous variations and improvements without departing from the spirit of the present invention, all of which fall within the scope of protection of the present invention. Therefore, the scope of protection of the patent for this invention shall be determined by the appended claims.

Claims

1. A probiotic hydrogel, characterized in that: The invention comprises a hydrogel matrix formed by oxidizing methylmalonamide hyaluronic acid; and the hydrogel matrix is ​​cross-linked with probiotics and extracellular polysaccharides secreted by the probiotics, wherein the probiotics are Lactobacillus acidophilus. L. acidipiscis 10851 , the extracellular polysaccharide is L. acidipiscis 10851 secreted exopolysaccharide EPS-la10851; The preparation of the oxidized methylpropionamidated hyaluronic acid comprises the following steps: (1) Preparing hyaluronic acid solution; (2) In an ice-water bath, add the methacrylic anhydride solution dropwise to the hyaluronic acid solution while stirring, adjust the pH of the solution to 9-10, and stir to mix thoroughly; (3) adding ethanol to the stirred solution, centrifuging, and collecting the precipitate to obtain methylpropionamidated hyaluronic acid; (4) adding deionized water to dissolve the methylpropionamide hyaluronic acid precipitated in step (3), and then dialyzing and freeze-drying to obtain purified methylpropionamide hyaluronic acid; (5) preparing a solution of the methylpropionamide hyaluronic acid purified in step (4), adding NaIO4, stirring the reaction, then adding ethylene glycol to terminate the reaction, dialyzing with deionized water, and freeze-drying to obtain the oxidized methylpropionamide hyaluronic acid; The probiotic hydrogel satisfies at least one of the following conditions A) to E): A) In step (1), the concentration of the hyaluronic acid solution is 1-5wt%; B) In step (2), the mass ratio of hyaluronic acid to methacrylic anhydride is 1:(4-5), and the reaction time is 1-6 h; C) In step (3), the ethanol concentration is 99.6-100 v / v%; D) In ​​step (5), in the reaction solution, the concentration of methylpropionamidated hyaluronic acid is 1-3 wt %, the concentration of NaIO4 is 0.5-1.5 wt %, and the reaction time is 4-8 h; E) In step (5), the amount of ethylene glycol added is 2-4 wt% of the reaction solution.

2. The probiotic hydrogel according to claim 1, characterized in that: The preparation of the exopolysaccharide EPS-1a10851 comprises the following steps: (1) Cultivation L. acidipiscis 10851 , collect the culture solution, boil it at 100℃ and then centrifuge it to remove the bacteria and their residues to obtain the culture supernatant; (2) Use trichloroacetic acid to remove proteins from the culture supernatant; (3) Add ethanol, incubate at 4°C overnight, centrifuge, and collect the precipitated extracellular polysaccharides; (4) Redissolve the exopolysaccharide with deionized water and dialyze to remove excess trichloroacetic acid; (5) freeze-drying to obtain the extracellular polysaccharide EPS-la10851.

3. The probiotic hydrogel according to claim 2, characterized in that: The concentration of the trichloroacetic acid is 13-15 wt %, and the concentration of the ethanol is 90-100 v / v %.

4. The method for preparing the probiotic hydrogel according to any one of claims 1 to 3, wherein: Oxidized methylpropionamidated hyaluronic acid, EPS-la10851 and L. acidipiscis 10851 The culture solutions are mixed to obtain a mixed solution, and then a photoinitiator is added to solidify the solution by ultraviolet irradiation to obtain the probiotic hydrogel.

5. The method for preparing the probiotic hydrogel according to claim 4, wherein: Satisfy at least one of the following conditions (a)-(c): (a) In the mixed solution, the mass percentage of oxidized methylpropionamidated hyaluronic acid is 2-5%; (b) In the mixed solution, the mass percentage of the exopolysaccharide EPS-1a10851 is 1-5%; (c) L. acidipiscis 10851 In the culture medium, L. acidipiscis 10851 The concentration is 10 6 -10 8 CFU.

6. The method for preparing the probiotic hydrogel according to claim 4, wherein: described L. acidipiscis 10851 The cells were inoculated into sterile PBS to obtain culture medium.

7. The method for preparing the probiotic hydrogel according to claim 4, wherein: The photoinitiator is phenyl (2,4,6-trimethylbenzoyl) lithium phosphate.

8. Use of the probiotic hydrogel according to any one of claims 1 to 3 or the probiotic hydrogel prepared by the preparation method according to any one of claims 4 to 7 in skin or body tissue wound repair and hemostasis.

Images