A hydrogel containing linear malto-oligosaccharides, and a preparation method and application thereof

Abstract

The application discloses a kind of hydrogel containing linear maltoligosaccharide and its preparation method and application, belong to biological medical material technical field.The linear maltoligosaccharide is introduced into chitosan-gelatin hydrogel body in the application, and the synergistic effect of linear maltoligosaccharide, chitosan and gelatin can significantly enhance the cell migration ability, and experiments show that the promotion rate of the hydrogel of the application to keratinocyte migration is increased by about 15%-25% compared with chitosan-gelatin hydrogel without linear maltoligosaccharide.And the application uses natural polymer chitosan and gelatin as matrix material, and linear maltoligosaccharide is natural oligosaccharide, and all components have good biocompatibility and degradability.CCK-8 cytotoxicity detection results show that the cell survival rate of each concentration group is greater than 90%, and there is no cytotoxicity.The hydrogel of the application can be applied to skin wound repair, cartilage tissue engineering, nerve injury repair and other various tissue regeneration scenarios.

Description

Technical Field

[0001] This invention relates to a hydrogel containing linear maltodextrin, its preparation method and application, belonging to the field of biomedical materials technology. Background Technology

[0002] Skin wound healing is a complex biological process involving multiple stages, including inflammatory response, cell proliferation, cell migration, and tissue remodeling. Cell migration (especially the migration of fibroblasts and epithelial cells) is a crucial step in wound reepithelialization and tissue repair. It is estimated that millions of patients worldwide suffer from chronic, difficult-to-heal wounds (such as diabetic foot ulcers, pressure ulcers, and venous ulcers) each year, placing a heavy burden on patients and healthcare systems. Therefore, developing novel biomedical materials that can effectively promote cell migration and accelerate wound healing has significant clinical and social value.

[0003] Hydrogels are materials with a three-dimensional cross-linked hydrophilic polymer network that can absorb and retain large amounts of water or biological fluids. Their structure is similar to the extracellular matrix (ECM), providing a favorable microenvironment for cell growth and supporting cell proliferation and migration. In recent years, hydrogels based on natural polymers have gained widespread application in tissue engineering and regenerative medicine due to their excellent biocompatibility, biodegradability, and low immunogenicity.

[0004] Currently, hydrogel materials used to promote wound healing and tissue repair are mainly classified into the following categories: Synthetic polymer hydrogels: such as polyethylene glycol (PEG) and polyvinyl alcohol (PVA), although they have good mechanical properties and controllability, they lack cell recognition sites, have insufficient bioactivity, and the degradation products of some synthetic materials may be cytotoxic. Natural polymer hydrogels: such as collagen, gelatin, chitosan, alginate, and hyaluronic acid. Natural polymer materials have good biocompatibility and biodegradability, and contain cell recognition sites, which can support cell adhesion and proliferation. Composite hydrogels: These combine two or more natural / synthetic polymers to complement each other's strengths. Among them, chitosan-gelatin composite hydrogels are one of the most widely studied systems.

[0005] Chitosan is a natural cationic polysaccharide with good biocompatibility, biodegradability, and antibacterial properties. Gelatin is a partially hydrolyzed product of collagen, containing an arginine-glycine-aspartic acid (RGD) sequence, which provides excellent bioactive sites for cell adhesion and migration. The hydrogel formed by combining chitosan and gelatin combines the advantages of both and is widely used as a cell culture scaffold and tissue engineering material. Existing patents related to chitosan-gelatin hydrogels exist, for example: CN121775210A discloses a cell super-infiltrating hydrogel scaffold composed of gelatin microspheres and a chitosan-proanthocyanidin solution; CN116115828A discloses a chitosan-lignin sulfonate-gelatin organic hydrogel and its preparation method; other studies involve methods for preparing gelatin-chitosan hydrogel cell scaffolds.

[0006] Although chitosan-gelatin hydrogels have been widely used in tissue engineering, some shortcomings exist. For example, while existing chitosan-gelatin hydrogels can support cell adhesion and proliferation, their activity in promoting cell migration needs further improvement, making it difficult to meet the clinical needs of rapid tissue repair. Furthermore, most chitosan-gelatin hydrogels serve only as cell scaffolds or drug carriers, lacking functional components that actively promote cell migration. Some studies have attempted to load active ingredients such as growth factors into hydrogels, but these efforts suffer from low loading efficiency, uncontrollable release, and high costs.

[0007] Linear maltodextrins (LMAOs) are linear oligosaccharides composed of 3-10 glucose units linked by α-1,4-glycosidic bonds. They possess excellent processing adaptability and unique physiological effects, making them a promising sugar source. However, current research on the application of LAOs in promoting cell migration is extremely limited, and no technical solutions for incorporating LAOs into hydrogel systems to promote cell migration have been reported domestically or internationally. Summary of the Invention

[0008] To address the shortcomings of existing technologies, this invention provides a chitosan-gelatin hydrogel containing linear maltodextrin, its preparation method, and its applications. This invention loads linear maltodextrin into a chitosan-gelatin hydrogel network, utilizing the synergistic effect of the three components to significantly enhance the hydrogel's ability to promote cell migration, thus providing a novel functional biomaterial for tissue repair and wound healing.

[0009] This invention is achieved through the following technical solution: The first objective of this invention is to provide a hydrogel containing linear maltodextrin, the hydrogel comprising a gel network constructed with chitosan and gelatin as a matrix, and linear maltodextrin loaded in the gel network.

[0010] In one embodiment of the present invention, the linear maltodextrin is one or more of linear maltotriose, linear maltotetraose, linear maltopentose, linear maltohexaose, and linear maltoheptaose.

[0011] In one embodiment of the present invention, the linear maltodextrin is preferably linear maltopentose.

[0012] In one embodiment of the present invention, the amount of linear maltodextrin added is 4% to 20% of the total mass of chitosan and gelatin.

[0013] In one embodiment of the present invention, the amount of linear maltodextrin added is preferably 10% to 20% of the total mass of chitosan and gelatin.

[0014] In one embodiment of the present invention, the mass ratio of chitosan to gelatin is 1:2 to 1:20.

[0015] In one embodiment of the present invention, the mass ratio of chitosan to gelatin is preferably 1:2 to 1:10.

[0016] In one embodiment of the present invention, the linear maltopentose is prepared by the following method: S1. Prepare starch milk or maltodextrin milk as the reaction substrate, add linear maltopentose generating enzyme, then adjust the pH of the reaction system to 5.5~7.0, heat to liquefy, and after liquefaction, cool down to carry out saccharification reaction to obtain the enzymatic hydrolysis reaction solution. S2. After cooling the enzymatic hydrolysis reaction solution obtained in step S1, yeast is added to carry out the reaction to obtain an oligosaccharide syrup containing linear maltopentose. S3. The oligosaccharide syrup containing linear maltopentose is decolorized, filtered, and treated with anion and cation exchange resins to obtain the maltopentose.

[0017] In one embodiment of the present invention, the concentration of the starch milk or maltodextrin milk is 10%~30% w / w; the starch is selected from one or more of tapioca starch, ordinary corn starch, waxy corn starch, potato starch, rice starch and wheat starch.

[0018] In one embodiment of the present invention, the amino acid sequence of the linear maltopentose generating enzyme is shown in SEQ ID NO.1; the amount added is 25~100 U / g dry substrate.

[0019] In one embodiment of the present invention, the liquefaction is a liquefaction reaction at 80~100°C for 5~30 min.

[0020] In one embodiment of the present invention, the saccharification reaction is carried out at a temperature of 50-70°C for 24-96 h.

[0021] In one embodiment of the present invention, in step S2, the temperature after cooling is 30~40°C.

[0022] In one embodiment of the present invention, the amount of yeast added is 1% to 5% of the mass of starch milk or maltodextrin milk; the reaction time for adding yeast is 10 to 40 h.

[0023] In one embodiment of the present invention, the linear maltopentose is specifically prepared by the following method: (1) Prepare 10%~30% corn starch milk according to the mass ratio and keep it at 60°C for 15 min; add linear maltopentose generating enzyme according to the enzyme dosage of 25~100 U / g dry substrate, adjust the pH to 5.0~7.0, adjust the reaction temperature to 80~100°C and liquefy for 5~30 min, and then immediately cool it down to 50~70°C and carry out the enzymatic hydrolysis reaction for 24~96 h to obtain the enzymatic hydrolysis reaction solution; (2) After inactivating the enzyme in the enzymatic hydrolysis reaction solution obtained in step (1), cool it to 30~40°C, add 1%~5% active yeast powder of starch milk or maltodextrin milk and continue the reaction for 10~40 h to obtain oligosaccharide syrup containing linear maltopentose. (3) Add activated carbon to the oligosaccharide syrup in step (2), decolorize at 40~60℃ for 10~60 min, then centrifuge and filter to remove yeast cells and activated carbon. The decolorized oligosaccharide syrup is then passed through anion and cation exchange resin to remove the anions and cations.

[0024] The second objective of this invention is to provide a method for preparing the hydrogel, comprising the following steps: preparing chitosan solution, gelatin solution and linear maltodextrin solution respectively; mixing chitosan solution, gelatin solution and linear maltodextrin solution in proportion and stirring evenly to obtain a mixed solution; and allowing the mixed solution to stand to prepare the hydrogel.

[0025] In one embodiment of the present invention, the chitosan solution is prepared by dissolving chitosan in an aqueous acetic acid solution.

[0026] In one embodiment of the present invention, the concentration of chitosan in the chitosan solution is 1~10g / 100mL.

[0027] In one embodiment of the present invention, the volume fraction of acetic acid in the aqueous acetic acid solution is 0.5% to 2%.

[0028] In one embodiment of the present invention, the gelatin solution is obtained by dissolving gelatin in water.

[0029] In one embodiment of the present invention, the concentration of gelatin in the gelatin solution is 10~30g / 100mL.

[0030] In one embodiment of the present invention, the linear maltodextrin solution is obtained by dissolving linear maltodextrin in water.

[0031] In one embodiment of the present invention, the concentration of linear maltodextrin in the linear maltodextrin solution is 20~60g / 100mL.

[0032] In one embodiment of the present invention, chitosan solution, gelatin solution and linear maltodextrin solution are mixed in a volume ratio of 8~12:8~12:1.

[0033] In one embodiment of the present invention, the mixed solution is placed at 20~30°C and allowed to stand for 15~30 hours.

[0034] A third objective of this invention is to provide the application of the hydrogel in the preparation of products for skin wound repair, cartilage tissue engineering, and nerve injury repair.

[0035] The beneficial effects of this invention are: (1) For the first time, linear maltodextrin was introduced into the chitosan-gelatin hydrogel system: After searching domestic and foreign patents and literature, there are currently no reports on linear maltodextrin and chitosan-gelatin composite hydrogels. This invention enriches the types of functional hydrogels and opens up new avenues for the application of linear maltodextrin in the field of biomedical materials.

[0036] (2) Significantly enhances cell migration ability: The synergistic effect of linear malt oligosaccharide, chitosan and gelatin shows that the hydrogel of this invention promotes keratinocyte migration by about 15% to 25% more than the chitosan-gelatin hydrogel without linear malt oligosaccharide.

[0037] (3) Good biocompatibility: This invention uses natural high molecular weight chitosan and gelatin as matrix materials, and linear maltodextrin as a natural oligosaccharide. All components have good biocompatibility and degradability. CCK-8 cytotoxicity test results showed that the cell survival rate of each concentration group was greater than 90%, and there was no cytotoxicity.

[0038] (4) The preparation process is simple and the conditions are mild: the preparation method of the present invention is simple to operate and the reaction conditions are mild (20-60℃, no high temperature and high pressure required), which is suitable for large-scale production and has good prospects for industrial application.

[0039] (5) Wide range of applications: The hydrogel of this invention can be applied to various tissue regeneration scenarios such as skin wound repair, cartilage tissue engineering, and nerve injury repair. Attached Figure Description

[0040] To more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the drawings used in the description of the embodiments or the prior art will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For those skilled in the art, other drawings can be obtained based on these drawings without creative effort.

[0041] Figure 1 Scanning electron microscopy image of a hydrogel containing linear maltodextrin; Figure 2 The results of the antibacterial experiment on Escherichia coli and Staphylococcus aureus; Figure 3 These are the results of the hydrogel cytotoxicity experiment; Figure 4 The results are from a hydrogel cell scratch assay. Detailed Implementation

[0042] The present invention will be further illustrated below with specific examples. These embodiments are for illustrative purposes only and are not intended to limit the scope of the invention. Furthermore, after reading the teachings of this invention, those skilled in the art can make various alterations or modifications to the invention, and these equivalent forms also fall within the scope defined by the appended claims.

[0043] Source of raw materials The linear maltopentose and linear maltotetraose used in the examples were prepared in the laboratory, and the preparation method is as follows: Method for preparing linear maltopentose: Step (1): Prepare 250 g of 20% (w / w) corn starch milk with deionized water and incubate at 60°C for 15 min. Add linear maltopentose generating enzyme at an enzyme dosage of 50 U / g dry substrate, adjust the pH to 6.0, adjust the reaction temperature to 90°C, liquefy for 15 min, and then immediately cool down to 60°C for 96 h of enzymatic hydrolysis. Step (2): After boiling the solution obtained in step (1) for 30 min to inactivate the enzyme, cool it to 35°C, add 2% active yeast powder and continue the reaction for 24 h; Step (3): Add 1.0% activated carbon to the sugar solution in step (2), decolorize at 50℃ for 30 min, then centrifuge and filter to remove yeast cells and activated carbon, and obtain linear maltopentose with a transmittance of more than 99.5%. Step (4): The decolorized sugar solution from step (3) is passed through anion and cation exchange resin to remove the anions and cations and reduce the conductivity to below 10 μS / cm; the distribution of small molecule sugars in the sugar solution is analyzed by ion chromatography, and the proportion of linear maltopentose is 80.67%.

[0044] Method for preparing linear maltotetrasaccharide: Step (1): Prepare 250 g of 30% (w / w) maltodextrin with deionized water, heat to 90°C and keep warm for 1 h. Allow the maltodextrin to dissolve completely, cool to 50°C and adjust the pH to 7.0; add linear maltotetrasaccharide generating enzyme at an enzyme dosage of 40 U / g dry substrate, stir and hydrolyze at 50°C for 20 h to obtain maltotetrasaccharide enzyme hydrolysate; then inoculate with 2% yeast, grow and ferment at 30°C for 24 h, centrifuge and filter to remove yeast cells, and retain the supernatant; Step (2): Add 1.0% activated carbon to the sugar solution in step (1), decolorize at 50℃ for 30 min, then centrifuge and filter to remove yeast cells and activated carbon, and obtain linear maltotetrasaccharide with a transmittance of more than 99.5%. Step (3): The decolorized sugar solution from step (2) is passed through anion and cation exchange resin to remove the anions and cations and reduce the conductivity to below 10 μS / cm; the distribution of small molecule sugars in the sugar solution is analyzed by ion chromatography, and the proportion of linear maltodextrose is 93.24%.

[0045] The amino acid sequence of the above-mentioned linear maltopentose synthase is shown in SEQ ID NO.1: AAPFNGTMMQYFEWYLPDDGTLWTKVANEANNLSSLGITALWLPPAYKGTSRSDVGYGVYDLYDLGEFNQKGTVRTKYGTKAQYLQAIQAAHAAGMQVYADVVFDHKGGADGTEWVDAVEVNPSDRNQEISGTYQIQAYTKFDFPGRGNTYSSFKWRWYHFDGVDWDESRKLSRIYKFRGIGKAWDWEVDTENGNYDYLMYADLDMDHPEVVTELKNWGKWYVNTTNIDGFRLDAVKHIKFSFFPDWLSYVRSQTGKPLFTVGEYWSYDINKLHNYITKTNGTMSLFDAPLHNKFYTASKSGGAFDMRTLMTNTLMKDQPTLAVTFVDNHDTEPGQALQSWVDPWFKPLAYAFILTRQEGYPGVFYGDYYGIPQYNIPSLKSKIDPLLIARRDYAYGTQHDYLDHSDIIGWTREGVTEKPGSGLAALITDGPGGSKWMYVGKQHAGKVFYDLTGNRSDTVTITSDGWGEFKVNGGSVSVWVPRKTTVSTITRPITTRPWTGEFVRWTEPRLVAWP The amino acid sequence of the above-mentioned linear maltooligosaccharide-forming enzyme is shown in SEQ ID NO.2: MSHILRAAVLAAVLLPFPALADQAGKSPAGVRYHGGDEIILQGFHWNVVREAPNDWYNILRQQASTIAADGFSAIWMPVPWRDFSSWTDGGKSGGGEGYFWHDFNKNGRYGSD AQLRQAAGALGGAGVKVLYDVVPNHMNRGYPDKEINLPAGQGFWRNDCADPGNYPNDCDDGDRFIGGESDLNTGHPQIYGMFRDELANLRSGYGAGGFRFDFVRGYAPERVDSW MSDSADSSFCVGELWKGPSEYPSWDWRNTASWQQIIKDWSDRAKCPVFDFALKERMQNGSVADWKHGLNGNPDPRWREVAVTFVDNHDTGYSPGQNGGQHHWALQDGLIRQAY AYILTSPGTPVVYWSHMYDWGYGDFIRQLIQVRRTAGVRADSAISFHSGYSGLVATVSGSQQTLVVALNSDLANPGQVASGSFSEAVNASNGQVRVWRSGSGDGGGNDGGEGGL Chitosan (degree of deacetylation ≥90%) was purchased from Shanghai Titan Technology Co., Ltd.

[0046] The gelatin was purchased from Sinopharm Chemical Reagent Co., Ltd.

[0047] Detection method: Methods for detecting linear maltodextrins: Product composition analysis: Using G1-G6 standards as quantitative and qualitative standards, the monosaccharide, disaccharide, and oligosaccharide components in the products were detected using HPAEC-PAD. The analytical conditions were as follows: A Thermo Scientific CarboPacPA 200 column was used, with 0.10 M NaOH and 0.50 M NaAc solution as the mobile phase, a flow rate of 0.5 mL / min, a column temperature of 35℃, and an injection volume of 25 μL. The percentage of each monosaccharide component's total conversion to the substrate in G1-G6 was calculated as follows:

[0048] Characterization of the hydrogel: The obtained hydrogel was freeze-dried and then observed by scanning electron microscopy (SEM).

[0049] Specifically, the present invention provides the following solution: The present invention first provides a hydrogel containing linear maltodextrin, the hydrogel comprising a gel network constructed with chitosan and gelatin as a matrix, and linear maltodextrin loaded in the gel network.

[0050] In this invention, the linear maltodextrin is one or more of linear maltotriose, linear maltotetraose, linear maltopentose, linear maltohexaose, and linear maltoheptaose. Linear maltopentose is preferred.

[0051] In this invention, the amount of linear maltodextrin added is 4% to 20% of the total mass of chitosan and gelatin. Preferably, it is 10% to 20% of the total mass of chitosan and gelatin.

[0052] In this invention, the mass ratio of chitosan to gelatin is 1:2 to 1:20, preferably 1:2 to 1:10.

[0053] In this invention, the linear maltopentose is prepared by the following method: S1. Prepare starch milk or maltodextrin milk as the reaction substrate, add linear maltopentose generating enzyme, then adjust the pH of the reaction system to 5.5~7.0, heat to liquefy, and after liquefaction, cool down to carry out saccharification reaction to obtain the enzymatic hydrolysis reaction solution. S2. After cooling the enzymatic hydrolysis reaction solution obtained in step S1, yeast is added to carry out the reaction to obtain an oligosaccharide syrup containing linear maltopentose. S3. The oligosaccharide syrup containing linear maltopentose is decolorized, filtered, and treated with anion and cation exchange resins to obtain the maltopentose.

[0054] In this invention, the concentration of the starch milk or maltodextrin milk is 10%~30% w / w; the starch is selected from one or more of tapioca starch, ordinary corn starch, waxy corn starch, potato starch, rice starch and wheat starch.

[0055] In this invention, the amino acid sequence of the linear maltopentose generating enzyme is shown in SEQ ID NO.1; the amount added is 25~100 U / g dry substrate.

[0056] In this invention, the liquefaction is carried out at 80~100°C for 5~30 min.

[0057] In this invention, the saccharification reaction is carried out at a temperature of 50-70°C for 24-96 h.

[0058] In this invention, in step S2, the temperature after cooling is 30~40°C.

[0059] In this invention, the amount of yeast added is 1% to 5% of the mass of starch milk or maltodextrin milk; the reaction time for adding yeast is 10 to 40 hours.

[0060] In this invention, the linear maltopentose is specifically prepared by the following method: (1) Prepare 10%~30% corn starch milk according to the mass ratio and keep it at 60°C for 15 min; add linear maltopentose generating enzyme according to the enzyme dosage of 25~100 U / g dry substrate, adjust the pH to 5.0~7.0, adjust the reaction temperature to 80~100°C and liquefy for 5~30 min, and then immediately cool it down to 50~70°C and carry out the enzymatic hydrolysis reaction for 24~96 h to obtain the enzymatic hydrolysis reaction solution; (2) After inactivating the enzyme in the enzymatic hydrolysis reaction solution obtained in step (1), cool it to 30~40°C, add 1%~5% active yeast powder of starch milk or maltodextrin milk and continue the reaction for 10~40 h to obtain oligosaccharide syrup containing linear maltopentose. (3) Add activated carbon to the oligosaccharide syrup in step (2), decolorize at 40~60℃ for 10~60 min, then centrifuge and filter to remove yeast cells and activated carbon. The decolorized oligosaccharide syrup is then passed through anion and cation exchange resin to remove the anions and cations.

[0061] The present invention also provides a method for preparing the hydrogel, comprising the following steps: preparing chitosan solution, gelatin solution and linear maltodextrin solution respectively; mixing chitosan solution, gelatin solution and linear maltodextrin solution in proportion and stirring evenly to obtain a mixed solution; and allowing the mixed solution to stand to prepare the hydrogel.

[0062] In this invention, the chitosan solution is prepared by dissolving chitosan in an aqueous acetic acid solution; the concentration of chitosan in the chitosan solution is 1~10g / 100mL; and the volume fraction of acetic acid in the aqueous acetic acid solution is 0.5%~2%.

[0063] In this invention, the gelatin solution is obtained by dissolving gelatin in water; the concentration of gelatin in the gelatin solution is 10~30g / 100mL.

[0064] In this invention, the linear maltodextrin solution is obtained by dissolving linear maltodextrin in water; the concentration of linear maltodextrin in the linear maltodextrin solution is 20~60g / 100mL.

[0065] In this invention, chitosan solution, gelatin solution and linear maltodextrin solution are mixed in a volume ratio of 8~12:8~12:1.

[0066] In this invention, the mixed solution is placed at 20~30℃ and allowed to stand for 15~30 hours.

[0067] The present invention also provides the application of the hydrogel in the preparation of products for skin wound repair, cartilage tissue engineering, and nerve injury repair.

[0068] The technical solution of the present invention will be described in detail below with reference to specific embodiments. In the following embodiments, unless otherwise specified, the reagents, materials and equipment used can be purchased commercially, prepared by conventional methods, or commonly used in the industry.

[0069] Example 1: Cell migration experiments of linear maltotetrasaccharide and linear maltpentose HaCaT cell suspension was prepared at a ratio of 5 × 10 5 Cells were seeded at a density of / well into 12-well plates. When cells reached confluence of over 90%, scratches were created using a 200 μL pipette tip. Cells were then washed three times with PBS, and 1 mL of DMEM medium supplemented with linear maltodextrose or linear maltopentose (final concentration of linear maltodextrose or linear maltopentose was 0.5% (w / w)) was added. Cells were incubated at 37°C in a 5% CO2 incubator. Scratch healing was recorded by photographing at 0 h and 24 h, and the scratch healing area was calculated using ImageJ software. Cell migration rate (CM) was calculated using the following formula: CM(%)=(S0-S) / S0×100% S0 represents the original scratch area at 0 h of the experiment, and S represents the area of ​​the cell scratch at the corresponding time.

[0070] The control group was supplemented with an equal amount of DMEM medium. After 24 h, the cell migration rate was 28.38%, while the cell migration rates of linear maltotetrasaccharide and linear maltpentose were 56.48% and 71.42%, respectively.

[0071] Example 2: This embodiment provides a method for preparing a hydrogel containing linear maltotetrasaccharide, the specific steps of which are as follows: Step (1): Weigh 4 g of chitosan (degree of deacetylation ≥ 90%), dissolve it in 100 mL of 1% acetic acid aqueous solution, and stir magnetically until completely dissolved to obtain a chitosan solution with a mass concentration of 4%.

[0072] Weigh 20 g of gelatin and dissolve it in 100 mL of deionized water. Heat the solution in a 60°C water bath and stir until completely dissolved to obtain a gelatin solution with a mass concentration of 20%.

[0073] Weigh 4 g of linear maltotetrasaccharide and dissolve it in 10 mL of deionized water to obtain a linear maltotetrasaccharide solution.

[0074] Step (2): Mix the above chitosan solution, gelatin solution and linear maltotetrasaccharide solution at a volume ratio of 1:1:0.1 and stir until homogeneous to obtain a mixed solution.

[0075] Step (3): Pour the mixed solution into the mold and place it at room temperature (25°C) for 24 h to form a physically cross-linked hydrogel.

[0076] Example 3: This embodiment provides a method for preparing a hydrogel containing linear maltopentose, the specific steps of which are as follows: Step (1): Weigh 4 g of chitosan (degree of deacetylation ≥ 90%), dissolve it in 100 mL of 1% acetic acid aqueous solution, and stir magnetically until completely dissolved to obtain a chitosan solution with a mass concentration of 4%.

[0077] Weigh 20 g of gelatin and dissolve it in 100 mL of deionized water. Heat the solution in a 60°C water bath and stir until completely dissolved to obtain a gelatin solution with a mass concentration of 20%.

[0078] Weigh 4 g of linear maltopentose and dissolve it in 10 mL of deionized water to obtain a linear maltopentose solution.

[0079] Step (2): Mix the above chitosan solution, gelatin solution and linear maltopentose solution in a volume ratio of 1:1:0.1 and stir until homogeneous to obtain a mixed solution.

[0080] Step (3): Pour the mixed solution into the mold and place it at room temperature (25°C) for 24 h to form a physically cross-linked hydrogel.

[0081] Scanning electron microscopy (SEM) analysis of the hydrogel prepared in Example 3 is as follows: Figure 1 As shown, the material exhibits a rich cross-linked porous structure, attributed to the electrostatic interaction between the cationic groups on the chitosan molecular chain and the anionic groups on the gelatin molecular chain. Simultaneously, the numerous hydroxyl groups on the linear malt oligosaccharide molecular chain form hydrogen bonds with the chitosan and gelatin molecular chains, synergistically reinforcing each other to form a matrix material with an interpenetrating network structure. Its dense and regular honeycomb structure promotes the transport of nutrients, achieving the goals of moisturizing, providing nutrition, and promoting wound healing.

[0082] Comparative Example 1: Step (1): Weigh 4 g of chitosan (degree of deacetylation ≥ 90%), dissolve it in 100 mL of 1% acetic acid aqueous solution, and stir magnetically until completely dissolved to obtain a chitosan solution with a mass concentration of 4%.

[0083] Weigh 20 g of gelatin and dissolve it in 100 mL of deionized water. Heat the solution in a 60°C water bath and stir until completely dissolved to obtain a gelatin solution with a mass concentration of 20%.

[0084] Step (2): Mix the above chitosan solution and gelatin solution at a volume ratio of 1:1 and stir until homogeneous to obtain a mixed solution.

[0085] Step (3): Pour the mixed solution into the mold and place it at room temperature (25°C) for 24 h to form a physically cross-linked hydrogel.

[0086] Comparative Example 2: Step (1): Weigh 10 g of gelatin, dissolve it in 100 mL of deionized water, heat and stir in a 60℃ water bath until completely dissolved, and obtain a gelatin solution with a mass concentration of 10%.

[0087] Step (2): Pour the solution into the mold and place it at room temperature (25°C) for 24 h to form a physically cross-linked hydrogel.

[0088] Test Example 1: Antibacterial test The antibacterial properties of the hydrogel were evaluated using *Escherichia coli* and *Staphylococcus aureus*. Both bacteria were inoculated separately into LB agar and incubated at 37°C for 24 h to obtain bacterial suspensions of a specific concentration. The suspensions were then diluted with sterile water to achieve a colony concentration of approximately 1 × 10⁻⁶. 6 CFU / mL was used to obtain the bacterial suspension for the experiment. The experimental groups were: a) blank control; b) comparative example 1; c) example 3. 0.2 g of sterile hydrogel was placed in a 24-well plate, and 10 μL of the bacterial suspension was added on top of the hydrogel. The plates were incubated at 37°C for 6 h. Wells without hydrogel served as blank controls. After incubation, 990 μL of PBS was added to each well, and bacteria were collected by repeated pipetting. The bacterial suspension was then diluted a certain factor, and 100 μL of the bacterial suspension was spread onto LB agar plates. The plates were incubated at 37°C for 12 h. After incubation, the colonies on the plates were observed, photographed, and counted. The inhibition rate was calculated using the following formula: Antibacterial rate (%) = (C Sample C Blank ) / C Blank *100% Among them, C Sample and C Blank These represent the number of colonies in the hydrogel sample group and the blank control group, respectively.

[0089] like Figure 2As shown, Example 3 exhibited inhibition rates greater than 99.99% against both *Escherichia coli* and *Staphylococcus aureus*, while Comparative Example 1 showed inhibition rates of 99.68% and 96.66% against *Escherichia coli* and *Staphylococcus aureus*, respectively. The addition of linear maltpentose synergistically enhances the positive charge density with the chitosan / gelatin hydrogel, irreversibly disrupting bacterial cell membrane integrity and inducing intracellular reactive oxygen species bursts, thereby improving the antibacterial effect. In summary, the hydrogel obtained by this invention possesses superior antibacterial properties and can effectively inhibit the growth of common bacteria such as *Escherichia coli* and *Staphylococcus aureus*.

[0090] Test Example 2: Cytotoxicity assay The cytotoxicity of the hydrogels was studied using the CCK-8 assay. 0.5 g of hydrogel samples from Examples 2 and 3, and Comparative Examples 1 and 2 were sterilized under UV irradiation for 30 min, and then extracted with 1 mL of DMEM medium for 24 h to obtain sample extracts. Cells were seeded into 96-well plates, with 100 μL of cell suspension added to each well. After cell attachment, the culture medium was removed, and 100 μL of sample extract was added. The blank control group received an equal volume of DMEM medium. After co-culturing for 24 h, the extract or culture medium was removed, and 10 μL of CCK-8 reagent and 100 μL of DMEM medium were added to each well. The plates were incubated at 37°C for 1 h in a 5% CO2 incubator. The absorbance of the 96-well plates at 450 nm was measured using a microplate reader.

[0091] like Figure 3 As shown, the cell survival rate test results indicate that the cell survival rates of the blank control group, comparative example 1, comparative example 2, example 2 and example 3 are all close to or higher than 100%, confirming that the product of the present invention does not produce cytotoxicity and has good biocompatibility.

[0092] Test Example 3: Cell migration experiment Preparation of hydrogel extract: 0.5 g of hydrogel samples from Examples 2 and 3, and Comparative Examples 1 and 2 were sterilized under ultraviolet irradiation for 30 min, and 5 mL of DMEM medium was added to each sample for extraction at 37℃ for 24 h to obtain hydrogel extracts for each experimental group.

[0093] HaCaT cell suspension was prepared at a ratio of 5 × 10 5Cells were seeded at a density of / wells into 12-well plates. When cells reached confluence (over 90%), scratch wounds were created by using a 200 μL pipette tip. Cells were then washed three times with PBS, and 1 mL of hydrogel extraction buffer was added to each well. The blank control group received an equal volume of DMEM medium. Cells were incubated at 37°C in a 5% CO2 incubator. Wound healing was recorded by photographing at 0 h and 48 h, and the wound healing area was calculated using ImageJ software. Cell migration rate (CM) was calculated using the following formula: CM(%)=(S0-S) / S0×100% S0 represents the original scratch area at 0 h of the experiment, and S represents the area of ​​the cell scratch at the corresponding time.

[0094] like Figure 4 As shown, the cell migration rate in Example 3 reached 64.6%, the cell migration rate in Example 2 reached 50.39%, the comparative example 1 was 43.5%, the comparative example 2 was 34.8%, and the blank control group was 29.8%. The cell migration rates in Examples 2 and 3 were significantly higher than those in other groups (p<0.01), indicating that the hydrogel of the present invention can significantly promote the migration of HaCaT cells, and the addition of linear maltodextrin has a significant synergistic effect.

[0095] The embodiments provided above are not intended to limit the scope of the invention, nor are the described steps intended to limit the order of execution. Any obvious modifications made to the invention by those skilled in the art based on existing common knowledge also fall within the scope of protection defined by the claims.

Claims

1. A hydrogel containing linear maltodextrin, characterized in that, The hydrogel comprises a gel network constructed with chitosan and gelatin as a matrix, and linear maltodextrin loaded in the gel network.

2. The hydrogel according to claim 1, characterized in that, The linear maltodextrin is one or more of linear maltotriose, linear maltotetraose, linear maltopentose, linear maltohexaose, and linear maltoheptaose.

3. The hydrogel according to claim 1 or 2, characterized in that, The amount of linear maltodextrin added is 4% to 20% of the total mass of chitosan and gelatin.

4. The hydrogel according to claim 1, characterized in that, The mass ratio of chitosan to gelatin is 1:2 to 1:

20.

5. The hydrogel according to claim 2, characterized in that, The linear maltopentose was prepared by the following method: S1. Prepare starch milk or maltodextrin milk as the reaction substrate, add linear maltopentose generating enzyme, then adjust the pH of the reaction system to 5.5~7.0, heat to liquefy, and after liquefaction, cool down to carry out saccharification reaction to obtain the enzymatic hydrolysis reaction solution. S2. After cooling the enzymatic hydrolysis reaction solution obtained in step S1, yeast is added to carry out the reaction to obtain an oligosaccharide syrup containing linear maltopentose. S3. The oligosaccharide syrup containing linear maltopentose is decolorized, filtered, and treated with anion and cation exchange resins to obtain the maltopentose.

6. The hydrogel according to claim 5, characterized in that, The concentration of the starch milk or maltodextrin milk is 10%~30% w / w; the starch is selected from one or more of tapioca starch, ordinary corn starch, waxy corn starch, potato starch, rice starch and wheat starch.

7. The hydrogel according to claim 5, characterized in that, The amino acid sequence of the linear maltopentose generating enzyme is shown in SEQ ID NO.1; the amount added is 25~100 U / g dry substrate.

8. The hydrogel according to claim 5, characterized in that, The liquefaction process is carried out at 80-100°C for 5-30 minutes.

9. The hydrogel according to claim 5, characterized in that, The saccharification reaction is carried out at a temperature of 50-70°C for 24-96 hours.

10. The hydrogel according to claim 5, characterized in that, In step S2, the temperature after cooling is 30~40°C.

11. The hydrogel according to claim 5, characterized in that, The amount of yeast added is 1% to 5% of the mass of starch milk or maltodextrin milk; the reaction time after adding yeast is 10 to 40 hours.

12. The hydrogel according to claim 5, characterized in that, The linear maltopentose is specifically prepared by the following method: (1) Prepare 10%~30% corn starch milk according to the mass ratio and keep it at 60°C for 15 min; add linear maltopentose generating enzyme according to the enzyme dosage of 25~100 U / g dry substrate, adjust the pH to 5.0~7.0, adjust the reaction temperature to 80~100°C and liquefy for 5~30 min, and then immediately cool it down to 50~70°C and carry out the enzymatic hydrolysis reaction for 24~96 h to obtain the enzymatic hydrolysis reaction solution; (2) After inactivating the enzyme in the enzymatic hydrolysis reaction solution obtained in step (1), cool it to 30~40°C, add 1%~5% active yeast powder of starch milk or maltodextrin milk and continue the reaction for 10~40 h to obtain oligosaccharide syrup containing linear maltopentose. (3) Add activated carbon to the oligosaccharide syrup in step (2), decolorize at 40~60℃ for 10~60 min, then centrifuge and filter to remove yeast cells and activated carbon. The decolorized oligosaccharide syrup is then passed through anion and cation exchange resin to remove the anions and cations.

13. A method for preparing the hydrogel according to any one of claims 1 to 12, characterized in that, The process includes the following steps: preparing chitosan solution, gelatin solution and linear maltodextrin solution respectively; mixing chitosan solution, gelatin solution and linear maltodextrin solution in proportion and stirring evenly to obtain a mixed solution; and allowing the mixed solution to stand to prepare the hydrogel.

14. The preparation method according to claim 13, characterized in that, The chitosan solution is prepared by dissolving chitosan in an aqueous acetic acid solution.

15. The preparation method according to claim 14, characterized in that, The concentration of chitosan in the chitosan solution is 1~10g / 100mL.

16. The preparation method according to claim 14, characterized in that, The volume fraction of acetic acid in the acetic acid aqueous solution is 0.5% to 2%.

17. The preparation method according to claim 13, characterized in that, The gelatin solution is obtained by dissolving gelatin in water; the concentration of gelatin in the gelatin solution is 10~30g / 100mL.

18. The preparation method according to claim 13, characterized in that, The linear maltodextrin solution is obtained by dissolving linear maltodextrin in water; the concentration of linear maltodextrin in the linear maltodextrin solution is 20~60g / 100mL.

19. The preparation method according to claim 13, characterized in that, Chitosan solution, gelatin solution and linear maltodextrin solution are mixed at a volume ratio of 8~12:8~12:

1.

20. The preparation method according to claim 13, characterized in that, The hydrogel is prepared by placing the mixed solution at 20-30℃ and letting it stand for 15-30 hours.

21. The use of the hydrogel according to any one of claims 1 to 12 in the preparation of products for skin wound repair, cartilage tissue engineering, and nerve injury repair.

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