Wound dressing comprising a lactobacillus ferment supernatant hydrogel and method of making same

Abstract

The present application belongs to the field of biomedical materials. Specifically, the present application provides a kind of Lactobacillus fermentum containing fermented supernatant hydrogel wound dressing, the Lactobacillus fermentum chitosan hydrogel includes Lactobacillus fermentum fermented supernatant, chitosan, genipin. In this paper, by using rat scald model and diabetic mouse wound model, the influence of hydrogel on wound healing is explored. The hydrogel wound dressing provided by the present application has excellent antibacterial performance, can accelerate the wound healing of wound dressing, and detects the bacteriostatic effect on escherichia coli and staphylococcus aureus.

Description

TECHNICAL FIELD

[0001] The present application relates to the field of biomedical materials, in particular to a kind of plant lactobacillus ferment supernatant hydrogel wound dressing and preparation method thereof. BACKGROUND

[0002] Wound dressing is a wound dressing product, which is a material for covering sores, wounds or other damages. Wound dressings are mainly divided into passive dressings, interactive dressings and bioactive dressings (occlusive dressings) according to the mechanism of action on the wound. Passive dressings passively cover the wound and absorb exudates, providing limited protection for the wound; interactive dressings have various forms of interaction between the dressing and the wound, such as absorbing exudates and toxic substances, allowing gas exchange to create a good environment for healing, relying on a barrier outer structure to prevent environmental microorganisms from invading, preventing cross-infection of the wound, etc.; the common feature of bioactive dressings is that they can prevent the wound from drying, so they are also called occlusive dressings, and the main materials used are collagen, chitin / chitosan, calcium alginate, etc. This kind of gel has biocompatibility, good water absorption, air permeability, adhesion and antibacterial and hemostatic effects. It can not only absorb wound exudates to ensure adequate drainage, but also retain part of the exudates in the dressing to maintain a local humid environment that simulates the physiological healing of the wound, which is beneficial to the regeneration of wound granulation tissue and epithelial cells and accelerates wound healing. In synthetic dressings, some substances beneficial to wound healing, such as drugs, cytokines, growth factors, etc., can be added to form various dressings such as drug dressings, cytokine dressings and growth factor dressings, thereby enhancing the efficacy of wound dressings in inhibiting bacterial proliferation and promoting wound healing.

[0003] Traditional dressings are mainly dry gauze and oil gauze, which can only play a simple physical shielding role, and are easy to adhere to the wound, causing secondary damage when changing dressings. At the same time, traditional dressings also have the problems of poor air permeability, easy bacterial growth and wound infection, etc. Hydrogel is a synthetic or natural polymer material with a three-dimensional hydrophilic network, with a large number of water molecules as the dispersion medium. With the widespread recognition of the wet healing theory in clinical practice, hydrogel dressings have been widely used.

[0004] Chitin, also known as chitinous, chitinous, chemical name (1, 4)-2-acetamido-2-deoxy-β-D-glucan. It is a natural high molecular weight polysaccharide, which exists in large quantities in marine arthropods such as shrimp, crab shells. The annual biosynthesis of chitin in nature is second only to cellulose, and it is the second largest renewable resource on earth. The product formed by removing the acetyl group on C2 of chitin is called chitosan. Because chitosan has good antibacterial, hemostatic, anti-inflammatory effects, and has good biocompatibility and biodegradability, it has been widely used in medical materials. The hydrogel itself is soft, and can create a suitable moist environment for wound healing by fully contacting the wound. The medical dressing developed by using chitosan hydrogel has the advantages of high softness, comfortable use, etc., and can reduce pain and control bleeding volume, and some hydrogels can also play a role in antibacterial and anti-inflammatory. In addition, in the healing and regeneration of new skin at the wound site, chitosan hydrogel can be degraded by itself, and is more easily absorbed by the body.

[0005] In recent years, the application market of chitosan hydrogel products is increasing year by year, and in the production and application process of chitosan hydrogel medical dressings, two major problems are often encountered. (1) Chitosan itself is difficult to dissolve, it is not soluble in water solution, and it can only be dissolved in rare organic acids (acetic acid, lactic acid, etc.) and a few inorganic acids (such as hydrochloric acid), and long time stirring is required during dissolution; (2) The cross-linking of chitosan molecules is mainly catalyzed by two types of cross-linking agents: one is a physical cross-linking agent represented by β-glycerophosphate sodium, and the chitosan hydrogel produced by cross-linking of this cross-linking agent has the problem of poor mechanical properties; the other is a chemical cross-linking agent represented by glutaraldehyde, and the hydrogel produced by cross-linking of this cross-linking agent has good mechanical properties, but the chemical cross-linking agent generally has strong biological toxicity, which will have adverse effects in application.

[0006] To solve the problem of poor solubility of chitosan, the current measures mainly use dilute acetic acid to stir for a long time and ultrasonic to make chitosan dissolve. This method is time-consuming and energy-consuming, and it will introduce acetic acid into the system, which is not conducive to the stability of the hydrogel. Lactobacillus plantarum is a gram-positive, short rod, anaerobic or facultative anaerobic, acid-tolerant, heterofermentative lactobacillus group. Fermentation can produce various organic acids such as acetic acid, succinic acid and lactic acid. The fermentation products of Lactobacillus plantarum have been confirmed to be able to reduce the bacterial load, the number of neutrophils, apoptotic and necrotic cells in the ulcer wound, and effectively promote wound healing. Due to the presence of various organic acids, the fermentation supernatant of Lactobacillus plantarum can promote the dissolution of chitosan. On the other hand, there is sufficient evidence that the skin microbiota is closely related to wound healing. These microorganisms produce bioactive compounds to regulate immune responses, change cell infiltration, and express chemotactic factors in wounds. In recent years, scholars at home and abroad have gradually increased the functional research of probiotics and their metabolites, and found that exosomes from probiotics have great therapeutic potential, which can up-regulate the expression of growth factors, promote angiogenesis, cell migration, proliferation and re-epithelialization process, thereby accelerating wound closure. In addition, it can also reduce the content of scars in the wound by preventing the differentiation of fibroblasts. SUMMARY

[0007] Based on this, the present application uses Lactobacillus plantarum HJ-S2 to extract metabolites through fermentation, and combines them with chitosan hydrogel to prepare a medical hydrogel product with the function of promoting wound healing. Through in vitro and in vivo experiments, it has been shown that it has excellent anti-bacterial and anti-inflammatory abilities, promotes the closure of infected wounds and the regeneration of new tissues, and the present application can open up a new way for the application of probiotics in the clinical management of infected wounds.

[0008] In one aspect, the present application provides a Lactobacillus plantarum chitosan hydrogel, wherein the Lactobacillus plantarum chitosan hydrogel comprises Lactobacillus plantarum fermentation supernatant, chitosan and genipin.

[0009] In some embodiments, the Lactobacillus plantarum fermentation supernatant is prepared by the following method:

[0010] (1) Pick Lactobacillus plantarum single colony and inoculate into MRS medium, incubate at 37℃ overnight on a shaker;

[0011] (2) Take the bacterial solution of step (1) and inoculate into MRS medium for enrichment culture;

[0012] (3) Centrifuge and filter the enrichment culture solution obtained in step (2) to obtain Lactobacillus plantarum supernatant.

[0013] In some embodiments, the Lactobacillus plantarum is Lactobacillus plantarum HJ-S2.

[0014] In some embodiments, the volume-mass ratio of the Lactobacillus plantarum fermentation supernatant, chitosan and genipin is 100 mL:(1-3) g:(0.02-0.1) g.

[0015] In some embodiments, the volume-mass ratio of the Lactobacillus plantarum fermentation supernatant, chitosan and genipin is 100 mL:(1-2) g:(0.025-0.5) g.

[0016] In some embodiments, the volume-mass ratio of the Lactobacillus plantarum fermentation supernatant, chitosan and genipin is 100 mL:2 g:0.05 g.

[0017] In another aspect, the present application provides a preparation method of the Lactobacillus plantarum chitosan hydrogel, wherein the method comprises:

[0018] (a) preparing a Lactobacillus plantarum supernatant;

[0019] (b) preparing a chitosan solution; adding chitosan into the Lactobacillus plantarum supernatant prepared in step (a) to prepare a chitosan solution by stirring;

[0020] (c) preparing a genipin solution;

[0021] (d) mixing the chitosan solution prepared in step (b) and the genipin solution prepared in step (c) to obtain the Lactobacillus plantarum chitosan hydrogel.

[0022] In some embodiments, the Lactobacillus plantarum supernatant is prepared by:

[0023] (i) picking a single colony of Lactobacillus plantarum and inoculating it into a MRS medium, and culturing it overnight at 37°C on a shaker; (ii) taking the bacterial solution obtained in step (i) and inoculating it into a MRS medium for enrichment culture; (iii) centrifuging and filtering the enrichment culture solution obtained in step (ii) to obtain the Lactobacillus plantarum supernatant.

[0024] In some embodiments, the chitosan solution is prepared by weighing chitosan and stirring it in the Lactobacillus plantarum fermentation supernatant overnight to obtain a chitosan solution with a mass fraction of 1-3%.

[0025] In some embodiments, the chitosan solution is prepared by weighing chitosan and stirring it in the Lactobacillus plantarum fermentation supernatant overnight to obtain a chitosan solution with a mass fraction of 1-2%.

[0026] In some embodiments, the chitosan solution is prepared by weighing chitosan and stirring it in the Lactobacillus plantarum fermentation supernatant overnight to obtain a chitosan solution with a mass fraction of 2%.

[0027] In some embodiments, the genipin solution is prepared by weighing genipin powder, dissolving in anhydrous ethanol to obtain a genipin solution with a concentration of 2-10 mg / mL.

[0028] In some embodiments, the genipin solution is prepared by weighing genipin powder, dissolving in anhydrous ethanol to obtain a genipin solution with a concentration of 2.5-5 mg / mL.

[0029] In some embodiments, the genipin solution is prepared by weighing genipin powder, dissolving in anhydrous ethanol to obtain a genipin solution with a concentration of 5 mg / mL.

[0030] In some embodiments, the mixed cross-linking is obtained by mixing chitosan solution and genipin solution with a volume ratio of (5-15): 1 uniformly, and then reacting at about 37°C for 20-30 h.

[0031] In some embodiments, the mixed cross-linking is obtained by mixing chitosan solution and genipin solution with a volume ratio of 10: 1 uniformly, and then reacting at 37°C for 24 h.

[0032] Terminology

[0033] Certain embodiments of the present application will now be described in detail with reference to certain embodiments thereof. For the sake of brevity, certain embodiments of the application are highlighted by comparing the present application to prior art. Throughout the present specification and claims, certain terms take on meanings that are commonly used by those in the art. It should be understood that the use of terminology adoptions in the detailed description is for the purpose of describing particular embodiments. It is further noted that the use of certain words or phrases in various places in the specification is not intended to serve limitation for the scope of the application.

[0034] It is to be further understood that, because some of the constituent program components and methods of the present application can be integrated into a single package or program, including a computer- or processor- readable medium, claims, where appropriate, can be brought, or fractional, based on the constituent program components or individual steps that can be present (to the extent appropriate) in one particular implementation of the present application.

[0035] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this application belongs. All patents and publications mentioned herein are incorporated by reference in their entirety.

[0036] In the description of the present specification, the description of the terms "one embodiment", "some embodiments", "an example", "a specific example", or "some examples" and the like means that the specific features, structures, materials or characteristics described in connection with the embodiment or example are included in at least one embodiment or example of the present application. In the present specification, the illustrative description of the above terms does not necessarily refer to the same embodiment or example. Also, the specific features, structures, materials or characteristics described can be combined in any suitable manner in any one or more embodiments or examples. In addition, the person skilled in the art can combine and combine the different embodiments or examples described in the present specification and the features of the different embodiments or examples, without contradiction.

[0037] In the following, all the numbers disclosed herein are approximate, whether or not the word "approximately" or "about" is used. The value of each number can vary by 1%, 2%, 5%, 7%, 8%, 10%, 15%, or 20% or more. Whenever a number having a value of N is disclosed, any number having a value of N + / - 1%, N + / - 2%, N + / - 3%, N + / - 5%, N + / - 7%, N + / - 8%, N + / - 10%, N + / - 15%, or N + / - 20% is explicitly disclosed, where "+" / -" means plus or minus.

[0038] The chitosan used in the present application does not have a requirement for molecular weight, and the greater the molecular weight, the stronger the gel capacity. In some embodiments, the chitosan described in the present application has a molecular weight of 30-100 million, and a degree of deacetylation of 80%-99%. In some embodiments, the chitosan described in the present application has a molecular weight of 60-100 million, and a degree of deacetylation of 85%-90%. BRIEF DESCRIPTION OF DRAWINGS

[0039] Figure 1 The diameter of the inhibition zone.

[0040] Figure 2 The healing condition of the rat burn wound.

[0041] Figure 3 The healing condition of the rat burn model - skin section (HE staining).

[0042] Figure 4 The healing condition of the rat burn model - skin section (Masson staining).

[0043] Figure 5 The appearance of the hydrogel containing HJ-S2 metabolites.

[0044] Figure 6 The healing condition of the diabetic mouse wound.

[0045] Figure 7Wound healing rate for diabetic mice.

[0046] Figure 8 Wound healing for diabetic mice - skin section (Masson staining). DETAILED DESCRIPTION

[0047] In order to make the objectives, technical solutions, and advantages of the present application clearer, further detailed descriptions of the present application are provided below in conjunction with embodiments. The specific embodiments described herein are merely intended to explain the present application and should not be used to form any limitation on the present application. In addition, descriptions of well-known structures and techniques are omitted in the following description to avoid unnecessary confusion of the concepts of the present disclosure. Such structures and techniques are also described in many publications.

[0048] The reagents used in the present application can be purchased from the market or can be prepared by the methods described in the present application. Preparation Example 1: Fermentation of Lactobacillus plantarum HJ-S2

[0049] (1) A single colony of HJ-S2 was picked and inoculated into a test tube containing 10 mL of MRS medium, and incubated at 37°C with a shaking speed of 180 rpm overnight. (The strain HJ-S2 was deposited with the China General Microbiological Culture Collection Center on May 7, 2019, and the deposit number is CGMCC No. 17720.)

[0050] (2) 2 mL of the bacterial solution in the test tube was inoculated into a conical flask containing 100 mL of MRS medium, and placed in a shaking incubator at 37°C with a shaking speed of 180 rpm for 24 hours.

[0051] Preparation Example 2: Extraction of Fermentation Supernatant

[0052] The bacterial solution after enrichment culture was centrifuged using a high-speed centrifuge. The centrifugation conditions were a speed of 9500 rpm for 30 min. After centrifugation, the supernatant was retained, and the centrifugation step was repeated once. The supernatant was filtered twice using a 0.22 μm sterile filter membrane to obtain HJ-S2 fermentation supernatant free of bacterial cells.

[0053] Preparation Example 3: Preparation of HJ-S2 Hydrogel

[0054] (1) 2 g of chitosan was weighed and magnetically stirred in 100 mL of HJ-S2 fermentation supernatant overnight to obtain a chitosan solution A with a mass fraction of 2%.

[0055] (2) 0.05 g of genipin powder was dissolved in 10 mL of anhydrous ethanol and ultrasonicated for 30 min to obtain a genipin solution A with a concentration of 5 mg / mL.

[0056] (3) Take 10 mL of the above-obtained chitosan solution A, add 1 mL of the genipin solution A, invert to mix, and then centrifuge at a speed of 2000 rpm for 5 min. Discard the precipitate, take the supernatant, add to a culture dish, and react at 37°C for 24 h to obtain a genipin-crosslinked chitosan hydrogel containing HJ-S2 metabolically active substances (HJ-S2 hydrogel A). The appearance is as shown in Figure 5

[0057] Preparation Example 4: Preparation of HJ-S2 hydrogel

[0058] (1) Weigh 1 g of chitosan, and magnetically stir in 100 mL of HJ-S2 fermentation supernatant overnight to obtain a chitosan solution B with a mass fraction of 1%.

[0059] (2) Take 0.05 g of genipin powder, dissolve in 10 mL of anhydrous ethanol, and ultrasonicate for 30 min to obtain a genipin solution A with a concentration of 5 mg / mL.

[0060] (3) Take 10 mL of the above-obtained chitosan solution B, add 1 mL of the genipin solution A, invert to mix, and then centrifuge at a speed of 2000 rpm for 5 min. Discard the precipitate, take the supernatant, add to a culture dish, and react at 37°C for 24 h to obtain HJ-S2 hydrogel B.

[0061] Preparation Example 5: Preparation of HJ-S2 hydrogel

[0062] (1) Weigh 2 g of chitosan, and magnetically stir in 100 mL of HJ-S2 fermentation supernatant overnight to obtain a chitosan solution A with a mass fraction of 2%.

[0063] (2) Take 0.025 g of genipin powder, dissolve in 10 mL of anhydrous ethanol, and ultrasonicate for 30 min to obtain a genipin solution B with a concentration of 2.5 mg / mL.

[0064] (3) Take 10 mL of the chitosan solution A, add 1 mL of the genipin solution B, invert to mix, and then centrifuge at a speed of 2000 rpm for 5 min. Discard the precipitate, take the supernatant, add to a culture dish, and react at 37°C for 24 h to obtain HJ-S2 hydrogel C.

[0065] Preparation Example 6: Preparation of HJ-S2 hydrogel

[0066] (1) Weigh 1 g of chitosan, and magnetically stir in 100 mL of HJ-S2 fermentation supernatant overnight to obtain a chitosan solution B with a mass fraction of 1%.

[0067] ​(2) Take 0.025 g of genipin powder, dissolve in 10 mL of anhydrous ethanol, and ultrasonic for 30 min to obtain a genipin solution B with a concentration of 2.5 mg / mL.

[0068] (3) Take 10 mL of chitosan solution B, add 1 mL of genipin solution B, invert to mix, and then centrifuge under the condition of 2000 rpm for 5 min. Discard the precipitate, take the supernatant, add to a culture dish, and react at 37℃ for 24 h to obtain HJ-S2 hydrogel D.

[0069] Test Example 1: Detection of antibacterial activity of hydrogel

[0070] (1) Pick single colonies of E. coli and S. aureus, and place in a test tube containing 10 mL of LB medium, and culture on a shaker. The culture condition is 37℃, 180 rpm, and the culture time is 16 h.

[0071] (2) Prepare 50 mL of LB solid medium 2 bottles, sterilize in a high-pressure sterilization pot at 121℃ for 20 min, and when cooled to about 55℃, add 0.5 mL of E. coli and S. aureus bacterial solution to each bottle, shake to mix, and then pour into a plate. After the plate cools and solidifies, punch 4 holes with a diameter of 10 mm on each plate, and add ① 100 μL of 2% chitosan aqueous solution, ② 100 μL of HJ-S2 hydrogel precursor solution, ③ 100 μL of HJ-S2 fermentation supernatant, and ④ 100 μL of pure water to each hole, and then add 10 μL of 5 mg / mL genipin solution to each hole. Culture at 37℃ for 24 h, and measure the diameter of the inhibition zone. The results are shown in Figure 1 , which shows that the hydrogel precursor solution has a better antibacterial effect. Figure 1 , 1: chitosan aqueous solution; 2: HJ-S2 hydrogel; 3: HJ-S2 fermentation supernatant; 4: pure water.

[0072] Preparation method of E. coli and S. aureus bacterial solution: pick single colonies of E. coli and S. aureus, and place in a test tube containing 10 mL of LB medium, and culture on a shaker. The culture condition is 37℃, 180 rpm, and the culture time is 16 h.

[0073] Preparation method of 2% chitosan aqueous solution ①: take 2 g of chitosan, dissolve in 100 mL of 1% acetic acid to obtain.

[0074] Preparation method of HJ-S2 hydrogel precursor solution ②: take 2 g of chitosan, dissolve in 100 mL of HJ-S2 fermentation supernatant to obtain.

[0075] Preparation method of 5 mg / mL genipin solution: take 0.05 g of genipin powder, dissolve in 10 mL of anhydrous ethanol, and ultrasonic for 30 min to obtain.

[0076] Test Example 2: Evaluation of the effect of the hydrogel on the healing of a degree III burn wound in rats

[0077] (1) Adult male SD rats were injected intraperitoneally with 10% chloral hydrate solution 0.5 mL / 100 g of body weight, and were shaved under light anesthesia. A stainless steel cylinder with a diameter of 2.5 cm was boiled in boiling water for 20 min, and the temperature was kept constant. The cylinder was removed and immediately attached to the back of the rat in the shaved area for 15 s to create a degree III burn model.

[0078] (2) Administration began 8 h after modeling. The administration groups were HJ-S2 hydrogel A (hereinafter referred to as the hydrogel group), 2% chitosan aqueous solution (hereinafter referred to as the chitosan group), and HJ-S2 fermentation supernatant (hereinafter referred to as the supernatant group). The test samples were administered locally to the burn, and the negative control group was administered physiological saline, once a day, 500 μL each time, for 28 consecutive days. The scab area and the state of healing after scab shedding were observed during this period.

[0079] (3) After 28 days of continuous administration, one rat from each group was sacrificed by intraperitoneal injection of an overdose of chloral hydrate. The local skin of the wound was cut with surgical scissors, and was fixed in a tissue fixative for more than 3 days. The paraffin-embedded sections were subjected to histopathological examination.

[0080] The results are shown in Figure 2 (Effect of the hydrogel on the healing of a burn wound in rats), Figure 3 (Histological sections of the skin of a rat burn model (HE staining)), Figure 4 (Histological sections of the skin of a rat burn model (Masson staining)). Figure 2 It was shown that the hydrogel group and the fermentation liquid group had a significant improvement in wound healing and the rate of scab shedding compared to the control group. Figure 3 It was shown that the hydrogel group and the fermentation liquid group had good tissue regeneration, with closely arranged cells and good compactness. The chitosan hydrogel group had loosely arranged cells, and the recovery was worse than the control group. The cells in the control group were loosely arranged, and a small amount of inflammatory cells were still present. Figure 4 It was shown that the hydrogel group had a large number of collagen fibers arranged in an orderly manner. The chitosan group had extremely disordered and sparse collagen fibers, and there was tissue cavitation. The regeneration was not ideal. The fermentation liquid group had relatively orderly arranged collagen fibers. The control group had sparse collagen fibers, and there was a small amount of tissue cavitation.

[0081] Test Example 3: Evaluation of the effect of the hydrogel on the healing of a skin wound in diabetic db / db mice

[0082] (1) Adult male db / db mice were intraperitoneally injected with 0.6 mL / 100 g of 10% chloral hydrate solution. The backs of the mice were then depilated under light anesthesia. Four symmetrical circular holes were punched into the depilated area on the back of each mouse using a 0.6 cm diameter biopsy puncture to establish a diabetic mouse skin trauma model.

[0083] (2) Drug administration began 8 hours after modeling. The drug-administered groups included HJ-S2 hydrogel A (hereinafter referred to as the hydrogel group), 2% chitosan aqueous solution (hereinafter referred to as the chitosan group), HJ-S2 fermentation supernatant (hereinafter referred to as the supernatant group), and human epidermal growth factor gel (hereinafter referred to as the positive control group). The test samples were administered to the scald site, while the negative control group was given normal saline twice daily, 100 μL each time, for 14 consecutive days. During this period, the scab area and scab healing status were observed.

[0084] (3) On the 14th day, one mouse was taken from each group and killed by cervical dislocation. The skin tissue from the back wound was cut with surgical scissors, fixed on filter paper, fixed in tissue fixative for more than 3 days, and embedded in paraffin for sectioning and histopathological examination.

[0085] The results are as follows Figure 6 (Effect of hydrogel on wound healing in diabetic mice), Figure 7 (Effect of hydrogel on wound healing rate in diabetic mice), Figure 8 (Wound healing status of diabetic mice - skin sections (Masson staining)) shown. Figure 6 The results showed that the wound healing in the hydrogel group and fermentation liquid group was significantly improved, and the wounds were almost completely healed on the 14th day. Figure 7 The changes in wound healing rates of each group within 14 days after modeling were statistically analyzed. The overall trend of wound healing rates was hydrogel group > fermentation liquid group > positive control group > model group > chitosan group. Figure 8 The hydrogel group, fermentation liquid group, and positive control group showed good tissue healing, with inflammatory cells largely eliminated and abundant, densely arranged collagen fibers. In the chitosan group, collagen fibers were sparsely arranged and disordered, but inflammatory cells were still visible. In the model group, collagen fibers were sparsely arranged, but a large number of inflammatory cells were still present.

[0086] The methods of the present invention have been described through preferred embodiments. It is apparent that those skilled in the art will be able to modify or appropriately alter and combine the methods and applications described herein within the scope, spirit, and spirit of the present invention to implement and apply the technology of the present invention. Those skilled in the art may refer to the disclosure herein and appropriately modify the process parameters to achieve the desired effect. It is particularly important to note that all similar substitutions and modifications apparent to those skilled in the art are considered encompassed by the present invention.

Claims

1. A Lactobacillus plantarum chitosan hydrogel, characterized in that The Lactobacillus plantarum chitosan hydrogel comprises Lactobacillus plantarum fermentation supernatant, chitosan, and genipin. The plant lactobacillus fermentation supernatant is prepared by the following method: (1) Pick a single colony of Lactobacillus plantarum, inoculate it into MRS medium, and culture it in a shaking incubator at 37°C overnight; (2) Take the bacterial solution from step (1) and inoculate it into a container containing MRS medium for enrichment culture; (3) centrifuging and filtering the enriched culture fluid obtained in step (2) to obtain a Lactobacillus plantarum supernatant; The Lactobacillus plantarum is Lactobacillus plantarum HJ-S2; The volume mass ratio of the Lactobacillus plantarum fermentation supernatant, chitosan and genipin is: 100 mL﹕(1-3) g﹕(0.02-0.1) g.

2. The Lactobacillus plantarum chitosan hydrogel according to claim 1, wherein The volume mass ratio of the Lactobacillus plantarum fermentation supernatant, chitosan and genipin is: 100 mL﹕2 g﹕0.05 g.

3. A method for preparing the Lactobacillus plantarum chitosan hydrogel according to any one of claims 1 or 2, characterized in that: The method comprises: (a) preparing a Lactobacillus plantarum supernatant; (b) preparing a chitosan solution; adding chitosan to the Lactobacillus plantarum supernatant prepared in step (a) and stirring to prepare a chitosan solution; (c) preparing a genipin solution; (d) The chitosan solution prepared in step (b) and the genipin solution prepared in step (c) are mixed and cross-linked to obtain a Lactobacillus plantarum chitosan hydrogel.

4. The preparation method according to claim 3, characterized in that The preparation of the Lactobacillus plantarum supernatant is carried out by: (i) picking a single colony of Lactobacillus plantarum, inoculating it into MRS medium, and culturing it overnight at 37°C on a shaking platform; (ii) taking the bacterial solution from step (i), inoculating it into MRS medium for enrichment culture; (iii) centrifuging and filtering the enriched culture obtained in step (ii) to obtain a supernatant of Lactobacillus plantarum.

5. The preparation method according to claim 3, characterized in that The chitosan solution is prepared by weighing chitosan and magnetically stirring the chitosan in a Lactobacillus plantarum fermentation supernatant overnight to obtain a chitosan solution with a mass fraction of 1-3%.

6. The preparation method according to claim 3, characterized in that The genipin solution is prepared by weighing genipin powder and dissolving the powder in anhydrous ethanol to obtain a genipin solution with a concentration of 2 to 10 mg / mL.

7. The preparation method according to claim 3, characterized in that The mixed cross-linking is obtained by uniformly mixing a chitosan solution and a genipin solution in a volume ratio of (5-15) : 1, and reacting the mixture at 37°C for 20-30 hours.

8. The preparation method according to claim 7, characterized in that The mixed cross-linking is obtained by uniformly mixing a chitosan solution and a genipin solution at a volume ratio of 10:1, and reacting the mixture at 37° C. for 24 hours.

Images