A postbiotic hydrogel for relieving alcoholic gastric ulcer and a preparation method and application thereof

Abstract

This invention belongs to the field of biomedical technology, specifically relating to a post-biotic hydrogel for relieving alcoholic gastric ulcers, its preparation method, and its application. The post-biotic hydrogel for relieving alcoholic gastric ulcers provided by this invention is mainly prepared from post-biotics of *Lactobacillus rhamnosus* and modified lactoferrin; *Lactobacillus rhamnosus* is *Lactobacillus rhamnosus* JM023. Experiments have confirmed that the post-biotic hydrogel of this invention, when applied to a rat model of alcoholic gastric ulcers, can inhibit the occurrence of gastric ulcers by suppressing oxidative stress and inflammatory responses and increasing the secretion of protective factors. It also alleviates alcoholic gastric ulcers by regulating the expression of metabolic pathways related to oxidation, inflammation, apoptosis, and tissue repair, and by regulating the gastrointestinal flora. In other words, the post-biotic hydrogel of this invention has an effective effect in relieving the symptoms of alcohol-induced gastric ulcers and is suitable for development and application as a drug or health product for relieving alcoholic gastric ulcers.

Description

Technical Field

[0001] This invention belongs to the field of biomedical technology, specifically relating to a post-genetic hydrogel for relieving alcoholic gastric ulcers, its preparation method, and its application. Background Technology

[0002] With the development of science and technology and changes in modern lifestyles, there is still a significant demand for alcoholic products. Excessive alcohol intake places a heavy burden on the maintenance of normal physiological functions in the stomach. As the main component of alcoholic beverages, ethanol has a strong reducing effect, which can severely damage the gastric mucosa and disrupt its normal physiological environment, making it a direct cause of alcoholic gastric ulcers. Ethanol accumulated in the stomach can lead to ulcers by gradually eroding the gastric mucosal barrier, damaging gastric mucosal epithelial cells, and destroying epithelial capillaries.

[0003] Gastric ulcers, a common peptic ulcer disease, are characterized by mucosal ruptures greater than 3-5 mm, with visible depths reaching the submucosa. They primarily damage gastric tissue and threaten the health and homeostasis of the gastrointestinal tract. If left untreated, they can gradually develop into serious upper gastrointestinal diseases such as gastroesophageal reflux, gastric perforation, or even gastric cancer, seriously affecting human health.

[0004] Currently, there are many types of drugs used to treat gastric ulcers, including omeprazole (OMG), rabeprazole, ranitidine, sucralfate, and teprenone. Although these drugs exhibit strong and effective anti-ulcer capabilities, long-term use can have adverse effects on organs such as the liver and kidneys, hindering long-term remission treatment. Furthermore, the complexity of the digestive tract environment may reduce the bioavailability of these drugs, leading to weakened therapeutic effects. Due to the mucus secretion, digestion by various enzymes, and gastric emptying within the gastric mucosal barrier, drugs and other components often cannot fully contact the target site, failing to reach their maximum therapeutic potential. This can result in incomplete drug release, reduced bioavailability, increased drug dosage, and prolonged treatment time, reducing efficacy and causing unnecessary waste. Therefore, researching, discovering, and designing functional active substances and materials that can be used to alleviate and treat gastric ulcers represents a new approach to relieving or resolving gastric ulcers.

[0005] Metabiotics are mixtures produced by artificially inactivating microorganisms through processes such as heat treatment, ultrasonic treatment, high pressure, and ultraviolet irradiation. They include intact cells, cell components, cell wall components, cell secretions, and metabolites of inactivated bacteria, while retaining some of the original microbial efficacy. Compared to live probiotics, metabiotics do not require consideration of environmental influences on biological activity and can avoid issues such as the transfer of drug-resistant genes, the emergence of virulence factors, the development of different colonization patterns, and opportunistic infections caused by bacteria, exhibiting better stability and safety. However, research on the preparation and efficacy of metabiotics in the treatment of gastric ulcers remains limited.

[0006] Hydrogels, as safe and practical biomaterials, are three-dimensional networks formed by polymer cross-linking. They possess unique water absorption and structural maintenance capabilities, and have been proven to play a positive role in the treatment of gastrointestinal diseases, which is beneficial for developing more effective and long-lasting methods for the prevention and treatment of these diseases. However, current research on hydrogel drugs or health products for relieving alcoholic gastric ulcers is limited, and related drugs suffer from drawbacks such as limited variety and potential side effects.

[0007] Therefore, developing a post-biotic hydrogel that can effectively relieve alcoholic gastric ulcers, so that it can be better used as a treatment drug or natural health product for alcoholic gastric ulcers, will generate significant social benefits and application prospects. Summary of the Invention

[0008] To overcome the shortcomings of the prior art, the present invention aims to provide a post-biotic hydrogel that relieves alcoholic gastric ulcers, which has a good effect on relieving alcoholic gastric ulcers and is suitable for development as a drug or health product for relieving alcoholic gastric ulcers.

[0009] Furthermore, the present invention aims to provide a method for preparing the above-mentioned post-biotic hydrogel for relieving alcoholic gastric ulcers.

[0010] Furthermore, the present invention aims to provide the application of the above-mentioned post-biotic hydrogel for relieving alcoholic gastric ulcers.

[0011] To achieve the above objectives, the technical solution adopted by the present invention is as follows:

[0012] An epigenetic hydrogel for relieving alcoholic gastric ulcers is prepared mainly from epigenetics of Rhamnosus bacillus and modified lactoferrin.

[0013] The rhamnosus bacillus mentioned is Lacticaseibacillus rhamnosus JM023, which is deposited at the Guangdong Provincial Center for Microbial Culture Collection, with accession number GDMCC NO: 65215.

[0014] This invention isolated and screened a bacterial strain from a fecal sample of a four-month-old healthy infant. The strain's genome was extracted, and 16S rDNA amplification and sequencing were performed. Combined with morphological observation, the strain was identified as *Lactobacillus rhamnosus*, named *Lactobacillus rhamnosus* JM023. This strain is deposited at the Guangdong Provincial Center for Microbial Culture Collection (GDMCC NO: 65215), dated September 27, 2024, at the Institute of Microbiology, Guangdong Academy of Sciences, 5th Floor, Building 59, No. 100 Xianlie Middle Road, Guangzhou.

[0015] As a preferred embodiment, the modified lactoferrin is amino-modified lactoferrin.

[0016] The preparation method of the above-mentioned post-biotic hydrogel for relieving alcoholic gastric ulcers includes the following steps:

[0017] (1) Inoculate *Rhamnosus rhamnosus* into a sterile culture medium and culture until the colony count reaches 10⁻⁶. 7 ~10 9 After CFU / mL, heat inactivation was performed at 60–90 °C, and then the inactivated product was freeze-dried to obtain the metagene of Rhamnosus bacillus;

[0018] Lactoferrin, 1-hydroxybenzotriazole monohydrate, and N-(3-dimethylaminopropyl)-N'-ethylcarbodiimide hydrochloride were mixed in water, and then nano-calcium carbonate was added. After mixing thoroughly, the mixture was centrifuged, and the resulting supernatant was a solution containing modified lactoferrin.

[0019] (2) Take sodium alginate solution and mix it with the metagener of Rhamnosus bacillus prepared in step (1) and a solution containing modified lactoferrin to obtain the metagener hydrogel for relieving alcoholic gastric ulcer.

[0020] As a preferred embodiment, in step (1), the sterile culture medium is sterile MRS liquid culture medium or sterile skim milk culture medium; the inoculation volume ratio of Rhamnosus rhamnosus in the sterile culture medium is 4% to 8%. More preferably, the inoculation volume ratio of Rhamnosus rhamnosus in the sterile culture medium is 5%.

[0021] As a preferred embodiment, in step (1), the culture temperature is 36-38°C; and the heat inactivation time is 20-40 min.

[0022] As a preferred embodiment, in step (1), the ratio of lactoferrin, 1-hydroxybenzotriazole monohydrate, N-(3-dimethylaminopropyl)-N'-ethylcarbodiimide hydrochloride, water, and nano-calcium carbonate is (0.15–0.25) g : (0.08–0.12) g : (0.08–0.12) g : (80–120) mL : (4–6) g. More preferably, the ratio of lactoferrin, 1-hydroxybenzotriazole monohydrate, N-(3-dimethylaminopropyl)-N'-ethylcarbodiimide hydrochloride, water, and nano-calcium carbonate is 0.2 g : 0.1 g : 0.1 g : 100 mL : 5 g.

[0023] As a preferred option, in step (1), the centrifugation process parameters are: a temperature of 3-5℃ and a speed of 8000-12000r / min, and centrifugation for 10-30min.

[0024] As a preferred embodiment, in step (2), the mass concentration of the sodium alginate solution is 1% to 4%. More preferably, the mass concentration of the sodium alginate solution is 2%.

[0025] As a preferred embodiment, in step (2), the ratio of sodium alginate solution, metagenic agent of Rhamnosus rhamnosus, and solution containing modified lactoferrin is (0.8–1.2) mL : (0.8–1.2) g : (0.8–1.2) mL. More preferably, the ratio of sodium alginate solution, metagenic agent of Rhamnosus rhamnosus, and solution containing modified lactoferrin is 1 mL : 1 g : 1 mL.

[0026] The above-mentioned application of post-biotic hydrogel for relieving alcoholic gastric ulcers is in the preparation of drugs or health products for relieving alcoholic gastric ulcers.

[0027] Compared with the prior art, the beneficial effects of the present invention are as follows:

[0028] The post-biotic hydrogel for alleviating alcoholic gastric ulcers provided by this invention is mainly prepared from post-biotics of Rhamnosus bacillus and modified lactoferrin. Experiments have confirmed that the post-biotic hydrogel of this invention can effectively inhibit the occurrence of gastric ulcers by inhibiting oxidative stress and inflammatory responses and increasing the secretion of protective factors. It also effectively alleviates alcoholic gastric ulcers by regulating the expression of metabolic pathways related to oxidation, inflammation, apoptosis, and tissue repair, and by modulating the gastrointestinal flora.

[0029] The method for preparing post-biotic hydrogel provided by this invention uses post-biotic derived from *Lactobacillus rhamnosus* JM023. During preparation, modified lactoferrin adsorbs nano-calcium carbonate, which is then mixed with sodium alginate containing post-biotic powder to prepare a hydrogel containing the post-biotic. Experiments have demonstrated that this post-biotic hydrogel, when applied to a rat model of alcoholic gastric ulcers, can effectively alleviate symptoms of alcohol-induced gastric ulcers, showing significant application potential in the preparation of drugs and health products for alleviating alcoholic gastric ulcers. Attached Figure Description

[0030] Figure 1 This is a graph showing the cytotoxicity results of the Lactobacillus rhamnosus JM023 postbiotic hydrogel in Experiment Example 1 of this invention;

[0031] Figure 2 This is a graph showing the effect of Lactobacillus rhamnosus JM023 postbiotic hydrogel on erythrocyte hemolysis in Experiment Example 1 of this invention;

[0032] Figure 3 This is a diagram showing the morphological changes of the stomach tissue of rats in each group in Experiment Example 3 of this invention;

[0033] Figure 4 This is a graph showing the percentage of gastric ulcer area and ulcer score of each group of rats in Experiment Example 3 of this invention;

[0034] Figure 5 The images show the TUNEL staining results of the stomach tissues of rats in each group in Experiment Example 3 of this invention.

[0035] Figure 6 The graph shows the results of SOD, CAT, GSH-Px and MDA levels in the gastric tissue of rats in each group in Experiment Example 3 of this invention;

[0036] Figure 7 The graph shows the levels of IL-6, IL-1β, TNF-α, MPO and IL-10 in the gastric tissue of rats in each group in Experiment Example 3 of this invention.

[0037] Figure 8 This is a graph showing the PGE2 and NO levels in the gastric tissues of rats in each group in Experiment Example 3 of this invention;

[0038] Figure 9 The images show the Keap1 immunohistochemical staining results of the stomach tissues of rats in each group in Experiment Example 3 of this invention.

[0039] Figure 10A This is a graph showing the expression levels of Occludin, ZO-1, Bcl-2, and Bax mRNA in the gastric tissue of rats in each group in Experiment Example 3 of this invention.

[0040] Figure 10BThis is a graph showing the expression levels of Keap1, Nrf2, and HO-1 mRNA in the gastric tissue of rats in each group in Experiment Example 3 of this invention.

[0041] Figure 11A This is a graph showing the Alpha diversity analysis results of the gastric flora of rats in each group in Experiment Example 3 of this invention;

[0042] Figure 11B This is a graph showing the differences in gastric flora at the phylum level among the rats in each group in Experiment Example 3 of this invention;

[0043] Figure 11C This is a graph showing the differences in the family level of gastric flora among the rats in Experiment Example 3 of this invention;

[0044] Figure 11D This is a graph showing the differences in the genus level of gastric flora among rats in each group in Experiment Example 3 of this invention;

[0045] Figure 12A This is a graph showing the Alpha diversity analysis results of the gut microbiota of rats in each group in Experiment Example 3 of this invention;

[0046] Figure 12B This is a graph showing the differences in gut microbiota at the phylum level among the rats in each group in Experiment Example 3 of this invention;

[0047] Figure 12C This is a graph showing the differences in gut microbiota family levels among the rat groups in Experiment Example 3 of this invention;

[0048] Figure 12D This is a graph showing the differences in gut microbiota genera among the rat groups in Experiment Example 3 of this invention. Detailed Implementation

[0049] The technical solutions and effects of the present invention will be clearly and completely described below with reference to specific embodiments and test examples. However, those skilled in the art should understand that the embodiments are only used to illustrate the technical solutions of the present invention and should not be regarded as limiting the scope of protection of the present invention. Unless otherwise specified, the test methods used in the following embodiments are conventional methods; unless otherwise specified, the raw materials used are items commonly used in the art, publicly available, or commercially obtainable.

[0050] In the following examples, the lactoferrin used was from Shanghai Yuanye Biotechnology Co., Ltd.; the sterile MRS liquid culture medium, sterile MRS solid culture medium, and sterile skim milk culture medium were all from Qingdao Haibo Biotechnology Co., Ltd.; and the nano calcium carbonate was from Shanghai Maclean Biochemical Technology Co., Ltd.

[0051] Example 1

[0052] This embodiment provides a metagenic hydrogel for relieving alcoholic gastric ulcers, mainly prepared from metagenic Lactobacillus rhamnosus and modified lactoferrin; the modified lactoferrin is amino-modified lactoferrin. The Lactobacillus rhamnosus is Lactobacillus rhamnosus JM023, which is deposited at the Guangdong Provincial Center for Microbial Culture Collection (GDMCC NO: 65215), with a deposit date of September 27, 2024, at the Institute of Microbiology, Guangdong Academy of Sciences, 5th Floor, Building 59, No. 100 Xianlie Middle Road, Guangzhou.

[0053] The preparation method of the above-mentioned post-biotic hydrogel for relieving alcoholic gastric ulcers includes the following steps:

[0054] (I) Preparation of metagenes of Lactobacillus rhamnosus: Lactobacillus rhamnosus JM023 was inoculated into sterile skim milk medium at a volume fraction of 5% and cultured under anaerobic conditions at 37°C until the total number of colonies reached 10. 7 CFU / mL, 10 8 CFU / mL, 10 9 After obtaining CFU / mL, the product was heat-inactivated at 80℃ for 30 min, and then freeze-dried to obtain low, medium, and high doses of Rhamnosus synergist powder, which was stored at -20℃ for later use.

[0055] (II) Modification of lactoferrin: Dissolve 0.2g of lactoferrin in 100mL of deionized water, add 0.1g of 1-hydroxybenzotriazole monohydrate (HOBt·H2O) and 0.1g of N-(3-dimethylaminopropyl)-N′-ethylcarbodiimide hydrochloride (EDC·HCl) and stir until homogeneous. Add 5g of nano-calcium carbonate (CaCO3) at a ratio of 5g / 100mL and stir continuously until fully mixed. Centrifuge at 4℃ and 10000r / min for 20min. The supernatant obtained is the solution containing modified lactoferrin and is ready for use.

[0056] (III) Preparation of epigenetic hydrogel: A 2% (w / w) alginate solution was prepared using sodium alginate. Then, 1.0 g of the *Rhamnosus rhamnosus* epigenetic powder prepared in step (I) was added to the sodium alginate solution (1.0 mL) and stirred to disperse it evenly. This was further mixed in an equal proportion with the modified lactoferrin solution (1.0 mL) from step (II). When the system encounters acid, it will release Ca2+. 2+ Crosslinking with sodium alginate produces hydrogen bonds to form hydrogels, thus yielding low, medium, and high doses of epigenetic hydrogels.

[0057] The screening, identification, and preservation process of Lactobacillus rhamnosus JM023 involved in this embodiment is as follows:

[0058] 1. Screening

[0059] One g of fecal matter from a healthy four-month-old infant in Harbin, Heilongjiang Province, was serially diluted and spread onto sterile MRS solid medium. The medium was incubated anaerobicly at 37°C for 48 hours, and colony morphology was observed and recorded. Moist, raised, white colonies were streaked onto MRS solid medium and purified under anaerobic conditions at 37°C. This process was repeated three times to obtain purified single colonies. Single colonies were then streaked onto MRS solid medium and incubated anaerobicly at 37°C for 48 hours to obtain the selected strain. The colonies of this strain on MRS medium were raised, smooth, round, moist, white, opaque, and 0.8–1.6 mm in diameter.

[0060] 2. Identification

[0061] The genome of the *Lactobacillus rhamnosus* strain JM023 obtained from the above screening was extracted. The 16S rDNA of the strain was amplified and sequenced (the nucleotide sequence of the amplified *Lactobacillus rhamnosus* JM023 16S rDNA is shown in SEQ ID NO.1). The obtained sequence was compared with the nucleic acid sequence in NCBI-Blast. The results showed that the strain had 100% homology with *Lactobacillus rhamnosus* strain LR-B1. Based on the morphological observation results, the strain was identified as *Lactobacillus rhamnosus* and named *Lactobacillus rhamnosus* JM023.

[0062] The primers used for 16S rDNA amplification are as follows:

[0063] 27F upstream primer: 5'-AGTCTCTGATCATGCCTCAG-3' (as shown in SEQ ID NO.2);

[0064] 1492R downstream primer: 5'-AAGGAGGTGCTCCAGCC-3' (as shown in SEQ ID NO.3);

[0065] The 16S rDNA amplification procedure is as follows:

[0066] 95℃ for 5 min; 35 cycles (95℃ for 30 s; 55℃ for 30 s; 72℃ for 2 min); 72℃ for 10 min.

[0067] The nucleotide sequence of the 16S rDNA of Lactobacillus rhamnosus JM023 is as follows (SEQ ID NO.1):

[0068]

[0069] 3. Save

[0070] Single colonies of *Lactobacillus rhamnosus* JM023 were inoculated into sterile MRS liquid medium and cultured anaerobically at 37°C for 17 h to obtain a bacterial suspension. Sterilized 80% (v / v) glycerol aqueous solution was added to the obtained bacterial suspension, mixed well, and stored in glycerol tubes at -80°C. Simultaneously, the obtained *Lactobacillus rhamnosus* was biopreserved at the Guangdong Provincial Microbial Culture Collection Center, accession number GDMCC NO: 65215, deposit date September 27, 2024, address: Institute of Microbiology, Guangdong Academy of Sciences, 5th Floor, Building 59, No. 100 Xianlie Middle Road, Guangzhou.

[0071] To verify the technical effects achieved by the present invention, the toxicity and safety evaluation, adhesion evaluation, and animal experiments were conducted on the post-genetic hydrogel prepared according to the present invention. The experimental results were analyzed using SPSS one-way ANOVA with multiple sample means. Pairwise comparisons were performed using the LSD test and SNK test. All experiments were repeated at least three times. Results are expressed as mean ± standard deviation, with p < 0.05 indicating statistical significance. GraphPad Prism and Origin were used to plot the experimental data. The specific experimental process and results are as follows.

[0072] Example 1: Toxicity and Safety Evaluation of Postgenetic Hydrogels

[0073] 1.1 Cytotoxicity Assay: GES-1 cells (human gastric mucosal cells) were cultured in DMEM medium supplemented with 10% fetal bovine serum and 1% penicillin-dextrose antibody, and then 100 μL of high-dose post-biotic hydrogel (10 μg / mL) prepared in Example 1 containing different concentrations (0, 50, 100, 200, 400 μg / mL) was added. 9 GES-1 cells were cultured using a medium containing CFU / mL instead of the above medium. After 1, 2, 3, 4, and 5 days of co-culture, 10 μL of CCK-8 solution was added to the above solution, and cytotoxicity was detected using the CCK-8 assay.

[0074] 1.2 Hemolysis Test: Fresh rat blood was centrifuged at 1500 r / min for 5 min. The precipitate was washed with sterile physiological saline, centrifuged again, and the precipitate was collected. 1.2 mL of high-dose (10 μL / mL) hemolysis test solution containing different concentrations (0, 50, 100, 200, 400 μL / mL) prepared in Example 1 was used to treat the hemolysis. 9Red blood cells were dispersed in physiological saline of a metagenic hydrogel (CFU / mL) and mixed thoroughly. Simultaneously, equal volumes of sterile physiological saline and deionized water were used to disperse the red blood cell precipitate, serving as negative and positive controls, respectively. After incubation at 37°C for 2 hours, the absorbance of the supernatant at 540 nm was measured using a microplate reader.

[0075] The results of the cytotoxicity assay of post-genetic gel using GES-1 cells are as follows: Figure 1 As shown. In the experimental procedure, a group of normally cultured cells was selected daily as a control. (From...) Figure 1 It was found that the cell viability of GES-1 cells treated with 0 μL / mL epigenetic hydrogel for 1-5 days was close to 100%, indicating normal cell viability. Furthermore, the cells were evenly distributed across the groups, with no significant differences in quantity or activity (p > 0.05). After 5 days of co-culturing with 0-200 μL / mL epigenetic hydrogel, the cell viability of GES-1 cells was 100.00±0.07%, 98.41±1.70%, 96.61±2.27%, and 95.93±3.04%, respectively, maintaining high viability. After 5 days of culture with 400 μL / mL epigenetic hydrogel, the cell viability was 94.39±3.01%, showing no significant difference from the normal level, indicating that the epigenetic hydrogel had no significant toxic effect on GES-1 cells. The main components of the hydrogel in this invention are sodium alginate, lactoferrin, and various active ingredients contained in post-genetic agents. It has no toxic side effects and can also provide nutrition, showing no significant inhibitory effect on the proliferation of GES-1 cells.

[0076] Biomaterials used in vivo must also possess good blood compatibility. The effects of different gel concentrations on erythrocyte hemolysis are as follows: Figure 2 As shown. By Figure 2 It was observed that the red blood cells in the positive control group (PC group) ruptured under the osmotic pressure of deionized water, turning the solution red. The hemolysis rates of the epigenetic hydrogel at concentrations of 50, 100, 200, and 400 μL / mL were 1.18±0.28%, 1.32±0.29%, 1.66±0.27%, and 3.10±0.32%, respectively, meeting the safety requirement of a hemolysis rate of less than 5%. Therefore, the epigenetic hydrogel of this invention demonstrates good biocompatibility and meets the safety requirements for in vivo use.

[0077] Experimental Example 2: Evaluation of the in vitro adhesiveness of post-genetic hydrogels

[0078] GES-1 cells were co-cultured with gastric mucosain and transferrin to simulate the microenvironment of normal and inflamed gastric tissue in vivo. The co-culture microenvironment was divided into three groups: PBS, MUC, and TRP. PBS served as the control group, MUC as the normal microenvironment group, and TRP as the inflamed microenvironment group. After GES-1 cells adhered to the culture dish, they were cultured in high-glucose medium containing 5% gastric mucosain and 100 μg / mL transferrin for 12 h. Then, they were cultured in DiD fluorescently labeled metagenic hydrogel for 2 h, washed three times slowly with PBS, and imaged using laser confocal scanning microscopy.

[0079] This experiment evaluated targeting by constructing microenvironments of normal and inflamed tissues in vitro and measuring fluorescence residue. The results showed that, compared to the PBS group, the MUC group showed only a small amount of red DiD fluorescence, indicating virtually no post-inflammatory hydrogel residue, while the TRP group showed abundant red fluorescence, indicating that post-inflammatory hydrogel was still adhered to the culture dish. This demonstrates that, compared to the normal environment, the post-inflammatory hydrogel of this invention can specifically adhere to inflamed tissue sites.

[0080] Experimental Example 3: Animal Experiment Evaluation of the Effects of Postbiotic Hydrogels

[0081] Animal model establishment: Eighty healthy SPF-grade male SD rats (4-5 weeks old, weighing 200-250g) were selected and housed at a temperature of 22±2℃ and a relative humidity of 60±5%, maintaining a 12-hour light / dark cycle. All rats were administered the model via gavage once daily. After one week of acclimatization feeding with normal diet and free access to water, the rats were randomly divided into 8 groups (n=10 per group): blank control group (NC), ulcer group (GU), post-regenerative group (PLR), hydrogel group (GEL), and low-dose (10g) of the drug from Example 1. 7 CFU / mL) post-biotic hydrogel group (LPG), medium dose (10) of Example 1 8 CFU / mL) post-biotic hydrogel group (MPG), high dose (10) of Example 1 9 Postbiotic hydrogel (CFU / mL) and positive control group (OME).

[0082] Subsequently, the control group was administered 5 mL / kg·d of sterile PBS solution by gavage daily, while the other groups were administered 5 mL / kg·d of 60% ethanol aqueous solution by gavage daily to establish the model. One hour later, the control group and the model group were administered 5 mL / kg·d of sterile PBS solution by gavage, while the other groups were administered the corresponding test substance by gavage at a dose of 5 mL / kg·d. During the feeding period, the alcohol dosage was adjusted according to the rat's body weight and mortality rate. The survival curve and weekly body weight changes of the rats were recorded, and the feeding was continued for 6 weeks.

[0083] The animal administration methods were designed according to the groupings in Table 1, and the animals were administered via gavage. The low, medium, and high doses of metagenic hydrogels (LPG, MPG, and HPG) were the metagenic hydrogels prepared in Example 1 of this invention. The preparation process of the hydrogel group (GEL) involved was as follows: a 2% (w / w) alginate solution was prepared using sodium alginate, and then mixed in an equal proportion with the modified lactoferrin solution (1.0 mL) from step (II) of Example 1 to form a hydrogel. The preparation method of the metagenic solution involved in the metagenic group (PLR) was as follows: 1.0 g of *Rhamnosus rhamnosus* metagenic powder prepared in step (I) of Example 1 was dissolved in PBS solution to obtain the metagenic solution.

[0084] Table 1. Grouping and Gavage Procedures in Animal Experiments

[0085]

[0086] Sample Collection and Processing: After the final gavage, rats were fasted for 12 hours but given free access to water. Phenobarbital was injected into the groin, and blood was collected. Serum was collected after centrifugation and stored at -20°C. Rats were euthanized by cervical dislocation. After disinfection, the rats were dissected, and gastric tissue, gastric contents, and cecal contents were collected. After observation and photography, a portion of the gastric tissue was used for ROS staining and Western blotting analysis. Another portion was fixed with 4% paraformaldehyde and stored at 4°C for pathological staining. The remaining portion was flash-frozen in liquid nitrogen and stored at -80°C for subsequent biochemical assays.

[0087] 3.1. Gastric histopathological staining

[0088] Gastric tissue was fixed in 4% formaldehyde solution and then routinely embedded in paraffin and sectioned. The sections were then immersed sequentially in xylene and ethanol of different concentrations at room temperature, and washed with double-distilled water. The washed sections were stained with Hematoxylin and eosin (H&E) according to the instructions. After staining, the pathological morphology was observed and photographed under a 200x optical microscope. The morphological observation of rat gastric tissue is as follows: Figure 3 As shown. The percentage of ulcer area and ulcer score in rat gastric tissue are as follows. Figure 4 As shown. Figure 3 In the table, (a) represents the normal group (NC group), (b) represents the model group (GU group), (c) represents the post-natal group (PLR group), (d) represents the hydrogel group (GEL group), (e) represents the low-dose post-natal hydrogel group (LPG group), (f) represents the medium-dose post-natal hydrogel group (MPG group), (g) represents the high-dose post-natal hydrogel group (HPG group), and (h) represents the allomeprazole group (OME group).

[0089] Depend on Figure 3It was found that the gastric tissue of the NC group rats was intact, with normal morphology and a smooth gastric mucosa surface, showing no damage. The GU group exhibited more severe damage, with bleeding, ulceration, and other ulceration symptoms on the surface, and edema in some damaged areas. After gavage with post-biotic and hydrogel, the area of ​​damaged gastric tissue decreased, but some bleeding symptoms still existed, and the effect was slightly weaker than that of the OME group. The post-biotic hydrogel group of this invention showed better results than the post-biotic, hydrogel, and OME groups. This indicates that the use of post-biotic hydrogel in this invention further improved the bleeding situation and effectively reduced the damaged area.

[0090] Figure 4 middle, Figure 4 'a' represents the percentage of ulcer area in the rat's gastric tissue. Figure 4 b represents the rat's gastric ulcer score. Figure 4 It was found that the gastric mucosal damage area in the GU group reached 42.73±4.22%, and the pathological score was concentrated in the range of multiple small ulcers and a few large ulcers (4-5 points), which was different from the NC group. The damage area and score of the GEL and PLR groups were significantly smaller than those of the GU group, while the ulcer proportions of the LPG, MPG, and HPG groups decreased to 11.73±3.71%, 9.7±2.87%, and 9.67±3.54%, respectively, and the ulcer scores were concentrated in the range of congestion and a few bleeding points (0.5-2 points). In addition, the results of the post-biotic gel group of the present invention were close to those of the normal group, showing similar or even better effects than those of the OME group. This indicates that the post-biotic and hydrogel alone have a relieving effect on ethanol-induced gastric ulcers, but the post-biotic hydrogel of the present invention shows a stronger therapeutic effect, with an effect close to or better than omeprazole, which can significantly reduce the degree of surface damage and is expected to restore the gastric tissue to a near-normal level.

[0091] 3.2 TUNEL staining of gastric tissue

[0092] At room temperature, slides were sequentially immersed in dewaxing solution and anhydrous ethanol, and then washed with double-distilled water. After spin-drying, proteinase K working solution (stock solution:PBS = 1:9) was added to cover the tissue, and the slides were incubated at 37°C for 20 min. After incubation, the slides were washed in PBS. After spin-drying, permeabilization working solution was added to the tissue, and the slides were incubated at room temperature for 20 min, followed by washing with PBS. Buffer was added to the inner circle to cover the tissue, and the slides were incubated at room temperature for 20 min. Then, the TUNEL kit was followed according to the instructions. After processing, the slides were mounted with anti-fluorescence quenching mounting medium and observed and images were acquired under a fluorescence microscope. The TUNEL staining results for detecting apoptosis in rat gastric tissue cells are shown below. Figure 5 As shown. Figure 5In the table, (a) represents the normal group (NC group), (b) represents the model group (GU group), (c) represents the post-natal group (PLR group), (d) represents the hydrogel group (GEL group), (e) represents the low-dose post-natal hydrogel group (LPG group), (f) represents the medium-dose post-natal hydrogel group (MPG group), (g) represents the high-dose post-natal hydrogel group (HPG group), and (h) represents the allomeprazole group (OME group).

[0093] Depend on Figure 5 Normal cells exhibit blue fluorescence, while apoptotic cells fluoresce red. The NC group showed a lower number of red fluorescent cells, indicating a low apoptosis rate, which can be attributed to normal cellular life processes. In contrast, the GU group showed an abnormally increased number of red fluorescent cells, indicating irritative damage leading to mass cell death. After intervention treatment in each group, the number of apoptotic cells decreased to varying degrees, with apoptotic cells mainly distributed at the mucosal margin and sloughed-off portions. The apoptosis status in the HPG group was similar to that in the normal group. This indicates that PLR, GEL, and OME can all protect cells from ethanol damage to some extent. The introduction of PLR enhanced the protective effect of GEL, and the epigenetic hydrogel HPG group of this invention showed the strongest alleviating effect, restoring apoptosis levels to normal.

[0094] 3.3 Levels of inflammatory and protective factors in gastric tissue

[0095] This experiment measured the levels of biomolecules SOD, CAT, GSH-Px, MDA, cytokines IL-6, IL-1β, TNF-α, MPO, IL-10, and protective factors PGE2 and NO in gastric tissue according to the kit instructions.

[0096] Ethanol generates free radicals in gastric tissue, attacking lipids and other biomolecules, causing lipid peroxidation. This leads to a decrease in SOD, CAT, and GSH-Px, and an increase in MDA, thereby exacerbating mucosal damage and ulcer formation. The levels of related enzymes in rat gastric tissue, such as SOD (…),… Figure 6 a) CAT Figure 6 b) GSH-Px Figure 6 c) and MDA Figure 6 The measurement results of d) are as follows Figure 6 As shown. Figure 6 In this context, the meanings of each experimental group are the same as in Section 3.1. Figure 6It was found that, compared with the NC group, ethanol gavage significantly reduced the activities of three enzymes and the level of MDA in the GU group, indicating that ethanol had caused oxidative damage to gastric tissue. Compared with the GU group, all other groups showed varying degrees of inhibition of this damage. After gavage with post-biotics, the oxidative stress level of gastric tissue could be significantly improved, and the post-biotic hydrogel could significantly enhance this improvement. Among them, LPG showed a similar effect to omeprazole, and the enzyme activities of the MPG and HPG groups were no significantly different from those of the NC group, and the MDA level was close to the normal level. This indicates that the post-biotic hydrogel can effectively alleviate the excessive oxidation of tissue caused by ethanol and showed a synergistic effect different from the effect of single components. This may be attributed to the fact that the hydrogel, when attached to the damaged site, can prolong the action time of the post-biotic active substances and protect the tissue from ethanol damage, thus preventing ethanol-induced gastric damage.

[0097] When the gastric mucosa is attacked and damaged, and MDA is produced in large quantities, an inflammatory response often occurs. To investigate the effect of post-biotic hydrogels on ethanol-induced gastric tissue inflammation, IL-6 (…) was measured. Figure 7 a) IL-1β Figure 7 b) TNF-α Figure 7 c) and MPO Figure 7 d) IL-10 Figure 7 e) Expression levels of marker inflammatory factors, results as follows Figure 7 As shown. Figure 7 In this context, the meanings of each experimental group are the same as in Section 3.1. Figure 7 It was found that, compared with the NC group, the GU group showed a significant increase in the levels of pro-inflammatory cytokines, indicating that ethanol had induced a significant inflammatory response in the stomach. Compared with the GU group, all treatment groups reduced the levels of pro-inflammatory cytokines and increased the levels of anti-inflammatory cytokines to varying degrees. Among them, the LPR group showed an anti-inflammatory effect similar to that of omeprazole (OME group), and slightly better than the GEL group. Post-inflammatory hydrogel showed a stronger anti-inflammatory effect, with medium and high doses of post-inflammatory hydrogel showing better effects than the low-dose group. It significantly reduced the levels of pro-inflammatory factors IL-6, IL-1β, TNF-α, and MPO, bringing their levels close to those of the NC group, while significantly increasing the production of the anti-inflammatory factor IL-10, exhibiting the best anti-inflammatory effect. This indicates that medium and high doses of post-inflammatory hydrogel can effectively inhibit the occurrence of inflammatory responses, which is one of the mechanisms by which it alleviates gastric mucosal damage.

[0098] PGE2 and NO are two protective factors related to the gastric mucosal barrier defense mechanism. PGE2 (… Figure 8 a) and NO( Figure 8 b) The measurement results are as follows Figure 8 As shown. Figure 8In this context, the meanings of each experimental group are the same as in Section 3.1. Figure 8 It was found that, compared to the NC group, ethanol significantly reduced the PGE2 and NO levels in the GU group. Both post-biotic and hydrogel administration significantly increased the levels of protective factors. Specifically, the levels of both after the post-biotic and hydrogel mixture were not significantly different from those in the OME group and were close to those in the NC group, indicating that the post-biotic hydrogel may also exert an anti-ulcer effect by upregulating the levels of gastric mucosal protective factors and promoting the repair of the gastric mucosal barrier.

[0099] 3.4 Immunohistochemical analysis

[0100] Gastric tissue was fixed in 4% neutral formalin at room temperature for 2 days, embedded in paraffin, and then 4μm tissue sections were prepared. The sections were then treated sequentially with xylene and ethanol of different concentrations until dewaxed and hydrated. The sections were placed in a repair chamber filled with EDTA (pH 9.0) and heated in a microwave oven, maintaining boiling for 20 minutes, while preventing excessive evaporation of the buffer solution and drying. After repair, the sections were allowed to cool naturally, and then washed three times with PBS for 5 minutes each time. After incubation with 3% H2O2 at room temperature in the dark for 25 minutes, the sections were washed three more times with PBS. The sections were placed in a humidified chamber in the dark, and 3% BSA was evenly added to the tissue for blocking at room temperature for 30 minutes. The BSA was then removed, and the prepared primary antibody was added and incubated overnight at 4°C. The chamber was then removed and allowed to return to room temperature. The sections were washed three times with PBS and incubated with secondary antibody at room temperature for 1 hour. The sections were then washed three more times with PBS, slightly dried, and DAB chromogenic solution was added. The chromogenic time was controlled under a microscope. The sections were rinsed with tap water before terminating the chromogenic process. After hematoxylin nuclear counterstaining and dehydration mounting, the results were interpreted under a white light microscope, and cross-sectional images were acquired under a scanner for immunoassay scoring.

[0101] Immunohistochemical analysis (IHC) was used to detect the effects of the gel on the expression of Keap1, Nrf2, HO-1, p-NF-κB, Occludin, and ZO-1 proteins. The aim was to explore the mechanism of action of post-genetic hydrogel in antioxidation, anti-inflammation, inhibition of apoptosis, and promotion of tight junction protein expression. The staining results are shown below. Figure 9 As shown. The meaning of each experimental group is the same as in Section 3.1. From Figure 9 It was found that the GU group had an increased number of Keap1-positive cells and decreased Nrf2 and HO-1 expression levels compared to the NC group, with a significant change in histochemical score (p < 0.05). This indicates that ethanol induces oxidative stress by activating the Keap1 pathway and reduces the levels of downstream antioxidant enzymes such as HO-1 and SOD by inhibiting Nrf2 expression. After intervention, the levels of Nrf2 and HO-1 were significantly increased (p < 0.05), with the HPG group showing the most significant effect. This suggests that PLR and GEL mainly promote the expression of antioxidant enzymes through the Keap1 and Nrf2 signaling pathways, thereby inhibiting oxidative stress.

[0102] 3.5 mRNA expression

[0103] Total RNA was extracted using an RNA extraction kit. Approximately 1g of rat stomach tissue was rapidly frozen in liquid nitrogen, then quickly ground into powder and transferred to a 1.5mL enzyme-free centrifuge tube. The laminar flow hood was wiped with chloroform before extraction of total RNA from the rat stomach tissue according to the Simply P kit instructions, protected from light. All equipment was treated with DEPC water, and the test areas were wiped with chloroform. The extracted total RNA was collected in enzyme-free centrifuge tubes, and RNA integrity was assessed by agarose gel electrophoresis. RNA purity and concentration were determined using a nucleic acid protein analyzer. An OD260 / OD280 value within the range of 1.8–2.1 indicated good RNA purity, suitable for subsequent experiments.

[0104] The expression of related genes (Occludin, ZO-1, Bcl-2, Bax) and (Keap1, Nrf2, HO-1) was determined by RT-PCR to explore the protective effect of postbiotic hydrogel on gastric tissue. Figure 10A The results show the mRNA expression levels of the related genes Occludin, ZO-1, Bcl-2, and Bax. Figure 10B This represents the results of mRNA expression level measurements for the related genes Keap1, Nrf2, and HO-1. Figure 10A , Figure 10B Compared with the NC group, the GU group showed significantly decreased expression of genes related to tight junction proteins (Occludin, ZO-1), genes related to apoptosis inhibition (Bcl-2), and genes related to antioxidant enzymes (Nrf2) (p < 0.05), while significantly increased expression of genes related to apoptosis (Bax) and oxidation (Keap1) (p < 0.05), and increased expression of the oxidation-related HO-1 gene. This indicates that ethanol induces the expression of oxidation-related and apoptosis-related genes and inhibits the expression of tight junction proteins, thereby causing oxidative damage to tissues. Compared with the GU group, the expression levels of Occludin, ZO-1, Bcl-2, Nrf2, and HO-1 in the gastric tissue of rats in each intervention group were increased, while the expression levels of Bax and Keap1 were decreased. Specifically, after gavage administration of different doses of post-biotic hydrogel, the expression levels of each gene showed significant differences compared with the GU group. This indicates that both post-genetic agents and hydrogels can significantly inhibit oxidative stress and apoptosis by altering the expression levels of related mRNAs, while hydrogels loaded with post-genetic agents exhibit better effects.

[0105] 3.6 Determination of the abundance of gastrointestinal flora

[0106] DNA was extracted from rat gastric and cecal contents using the TIANamp DNA extraction kit. DNA quality and concentration were assessed by agarose gel electrophoresis and a Nanodrop spectrophotometer. The V3-V4 hypervariable region of the 16S rRNA gene was amplified using upstream primer (5′-ACTCCTACGGGAGGCAGCA-3′) and downstream primer (5′-GGACTACHVGGGTWTCTAAT-3′). The PCR amplification program was as follows: initial denaturation: 98℃ for 2 min; cycles: 98℃ for 15 s, 55℃ for 30 s, 72℃ for 30 s, 25 cycles; hold: 72℃ for 5 min.

[0107] The PCR amplification products and target fragments were processed sequentially as follows: electrophoresis detection, gel extraction and recovery, quantitative PCR, equimolar sample mixing, sequencing library preparation, and 2×300bp paired-end sequencing. The sequencing platform was Illumina-MiSeq. QIIME software was used to call VSEARCH to examine and extract chimeric sequences, which were grouped according to 97% similarity. The Alpha diversity index was calculated, and linear discriminant analysis (LEfSe) was used to assess the composition at each taxonomic level and perform statistical analysis.

[0108] Alpha diversity is one of the indicators for evaluating the abundance and diversity of gut microbiota. It includes the Chao1, Simpson, and Observed Species indices for evaluating microbiota abundance, and the Shannon and Faith pd indices for evaluating microbiota diversity. The results of alpha diversity characterization of rat gastric microbiota are as follows: Figure 11A As shown. By Figure 11A It was found that, compared with the NC group, all indices in the GU group increased, indicating that ulceration led to the enrichment of bacteria on the mucosal surface and an increase in the abundance and diversity of gastric flora. After gavage with either metabiotic or hydrogel alone, the abundance and diversity indices in the rat stomach decreased. The combination of the two also showed an effect of reducing the Chao1, Simpson, and Observed species indices in ulcerated rats, with the HPG group showing Chao1 and Observed species indices closer to those of the NC group. This indicates that metabiotics and hydrogel can reduce the abundance and diversity of gastric flora in ulcerated rats, and the recovery effect of high-dose metabiotics combined with hydrogel is closer to the normal level.

[0109] By comparing the taxonomic differences in the composition of gastric flora in different groups of rats, the effects of ethanol and post-biotic hydrogel on the gastric flora of rats were analyzed. The results are as follows: Figure 11B-11D As shown.

[0110] The results of the phylum-level compositional analysis of rat gastric microbiota are as follows: Figure 11B As shown, the rat stomach was mainly composed of Firmicutes, Bacteroidetes, Proteobacteria, Actinobacteria, Spirochaetes, TM7, Tenericulates, Deferribacteres, Verrucomicrobia, and Cyanobacteria. Among them, Firmicutes, Bacteroidetes, Proteobacteria, and Actinobacteria had the highest abundance, accounting for more than 90% of the total. In the NC group, the proportions of the four phyla were 79.00%, 9.21%, 7.68%, and 3.43%, respectively. In the GU group, the abundance of Firmicutes and Actinobacteria decreased to 69.24% and 0.32%, respectively, while the abundance of Bacteroidetes and Proteobacteria increased to 16.09% and 13.91%, respectively, with the total relative abundance of both increasing. This outcome could be reversed after PLR and GEL intervention. Gavage with postbiotic hydrogel reduced the proportions of Bacteroidetes and Proteobacteria while increasing the abundance of Firmicutes and Actinobacteria. Bacteroidetes and Proteobacteria contain various pathogenic bacteria; their increased abundance can disrupt the homeostasis of the gastric microenvironment and induce disease. This reflects an altered gastric microenvironment in rats with alcoholic ulcers, and the damaged mucosal wounds are more susceptible to infection and other complications. After treatment with postbiotic hydrogel, the gastric flora of rats improved at the phylum level and gradually returned to balance.

[0111] The results of the analysis of rat gastric flora at the family level are as follows: Figure 11CAs shown, the gut microbiota of each group of rats mainly consisted of Peptostreptococcaceae, Lactobacillaceae, Turicibacteraceae, Erysipelotrichaceae, Clostridiaceae, S24-7, Pseudomonadaceae, Ruminococcaceae, Lachnospiraceae, and Enterobacteriaceae at the family level. Compared with the NC group, the gastric microbiota composition at the family level in the GU group showed significant changes, with a decrease in Lactobacillaceae and an increase in the abundance of Erysipelotrichaceae and Pseudomonadaceae. After treatment with different intervention groups, the abundance of Erysipelotrichaceae and Pseudomonadaceae decreased, while the abundance of Lactobacillaceae increased to varying degrees. The microbial composition of the MPG and HPG groups was closest to that of the NC group. Furthermore, the PLR, GEL, PG, and OME groups showed certain differences in their microbial composition at the family level. This may be due to differences in the component content and mode of action of each intervention agent, leading to varying effects on the gastric microbiota of rats and consequently, differences in the microbial composition after intervention. This indicates that some pathogenic bacteria overgrow on the gastric mucosa surface of rats with gastric ulcers, while the growth of beneficial bacteria is inhibited. Treatment with various intervention agents can reverse this situation, with the HPG group showing the most significant effect, bringing the microbial abundance closest to the normal group.

[0112] Analysis results of rat gastric flora at the genus level are as follows: Figure 11DAs shown, the gut microbiota of the rats at the genus level mainly consisted of *Turicibacter*, *Lactobacillus*, *Allobaculum*, *Pseudomonas*, *Staphylococcus*, *Streptococcus*, *Oscillospira*, *Prevotella*, *Adlercreutzia*, and *Corynebacterium*. Compared with the NC group, the abundance of *Allobaculum* and *Pseudomonas* in the GU group was significantly increased, while the abundance of *Lactobacillus* decreased, with varying degrees of mitigation after treatment with different interventions. After PLR gavage intervention, the abundance of *Lactobacillus* in the rats significantly increased, while the abundance of *Pseudomonas* decreased, indicating that postbiotic gavage significantly altered the gastric microbiota of the rats and introduced other dominant strains. The changes in the abundance of bacteria in the GEL group also showed that it promoted the proliferation of bacteria such as Lactobacillus. Treatment with LPG, MPG, and HPG also increased the abundance of beneficial bacteria such as Lactobacillus, and alleviated intestinal flora imbalance to varying degrees. The HPG group showed the most significant recovery effect, and compared with the GU group, the rats under its influence had a relatively increased abundance of Lactobacillus and a lower total abundance of bacteria such as Pseudomonas. The concentration at the genus level was closest to that of the NC group, indicating that it can regulate the gastric flora by promoting the growth of probiotics and inhibiting the growth of pathogenic bacteria. This suggests that when gastric ulcers occur, the composition of the surface flora also changes, and different doses of postbiotic hydrogel can improve the gastric flora imbalance in rats with gastric ulcers by regulating the gastric flora.

[0113] Alpha diversity results of rat gut microbiota characterization, as follows Figure 12A As shown. By Figure 12AIt was found that compared with the NC group, all indices in the GU group decreased, indicating that the intestinal flora homeostasis of rats may be affected, with reduced abundance and diversity of the flora. After the relief was achieved by gavage administration of postbiotics, the abundance and diversity indices of the intestinal flora in rats increased. When combined with hydrogel, it also significantly increased the Chao1, Shannon, Faith pd, and Observed species indices in GU rats. The HPG group showed the highest index levels in Chao1, Shannon, and Observed species, and all five index results were close to normal levels. This indicates that postbiotics can improve the abundance and diversity of the intestinal flora in ulcer rats, and the recovery effect after high-dose postbiotic hydrogel gavage intervention is closer to normal levels. After hydrogel intervention alone, only the Faith pd index showed an increasing trend, indicating that hydrogel had no significant alleviating effect on the changes in the intestinal flora of ulcer rats. Furthermore, the efficacy of omeprazole in treating gastric ulcers has been confirmed, but the therapeutic effect did not show a significant correlation with the restoration of intestinal flora homeostasis.

[0114] By comparing the taxonomic differences in the cecal microbiota composition of rats from different groups, the effects of gastric ulcer and post-genetic hydrogel intervention on the rat gut microbiota were analyzed. The results are as follows: Figure 12B-12D As shown.

[0115] The results of the analysis of rat gut microbiota at the phylum level are as follows: Figure 12BAs shown, the main phyla in the rat intestine are Firmicutes, Bacteroidetes, Proteobacteria, Actinobacteria, Spirochaetes, TM7, Verrucomicrobia, Tenericulates, Deferribacteres, and Elusimicrobia. Firmicutes, Bacteroidetes, Proteobacteria, and Actinobacteria are the four most abundant phyla, accounting for over 90% of the total. In the NC group, the proportions of these four phyla were 50.97%, 34.79%, 7.08%, and 3.65%, respectively. In the GU group, the abundance of Bacteroidetes decreased to 20.54%, while the abundances of Firmicutes and Verrucomicrobia increased to 64.23% and 2.95%, respectively. Interventions using PLR and GEL revealed a decrease in the abundance of Firmicutes and Verrucomicrobia, while increasing the abundance of other phyla such as Bacteroidetes. Gavage administration of biogenic hydrogel also reduced the abundance of Firmicutes and Verrucomicrobia in the gut, while increasing the abundance of Bacteroidetes and Actinobacteria. In the MPG group, the abundance of Firmicutes and Bacteroidetes recovered to 51.91% and 34.50%, respectively, most similar to the levels in the NC group (50.97% and 34.79%). Similar to the gastric flora, changes in the abundance of Firmicutes and Bacteroidetes may reflect a disruption of intestinal homeostasis, leading to the proliferation of various pathogenic bacteria. These pathogens can increase the host's chances of infection by producing harmful substances, leading to decreased immunity and consequently affecting ulcer healing and increasing the risk of other diseases. After treatment in different groups, the gut microbiota of rats improved at the phylum level and showed a gradual trend of restoration to balance. The MPG and HPG groups showed more significant effects, which may be because some components of the postbiotic can also act as prebiotics, promoting the proliferation of probiotics and altering the composition of the gut microbiota. OME also showed a certain restorative effect on the gut microbiota during this process and could also increase the abundance of proteobacteria.

[0116] Subsequently, the influence of postbiotic hydrogels on the gut microbiota was further analyzed by characterizing the composition of the gut microbiota at the family level. The results of the family-level analysis of rat gut microbiota are as follows: Figure 12CAs shown, the gut microbiota of each group of rats mainly consisted of S24-7, Ruminococcaceae, Helicobacteraceae, Erysipelotrichaceae, Peptostreptococcaceae, Lachnospiraceae, Lactobacillaceae, Pararaprevotellaceae, Coriobacteriaceae, and Desulfovibrionaceae at the family level. Compared with the NC group, the gut microbiota composition of the GU group rats also changed significantly at the family level, with a decrease in the abundance of Peptostreptococcaceae and Lactobacillaceae, and an increase in the abundance of Ruminococcaceae, Helicobacteraceae, and Erysipelotrichaceae, indicating that the occurrence of ulcers had an adverse effect on the maintenance of gut microbiota homeostasis. All treatments in different intervention groups significantly reduced the abundance of Helicobacteraceae in ulcerated rats. After gavage administration of metabiotics alone, the abundance of Peptostreptococcaceae, Lactobacillaceae, and Coriobacteriaceae increased. These bacterial families contain many beneficial microorganisms that have been found to inhibit and alleviate various chronic diseases in the host, reflecting that metabiotics contain various active ingredients that can promote the proliferation of probiotics and help restore the intestinal flora. GEL gavage showed a significant inhibitory effect on Helicobacteraceae, possibly due to the good antibacterial effect of lactoferrin contained therein. After loading with hydrogel, the LPG, MPG, and HPG groups further reduced the increase in Erysipelotrichaceae abundance under metabiotic treatment and promoted the restoration of the flora to normal levels. The abundance of beneficial bacteria under HPG intervention reached levels closest to the NC group. The OME group showed a dominant presence of pathogenic bacteria such as Helicobacteraceae, confirming that its effective anti-ulcer effect does not affect the gut microbiota. Similar to the gastric microbiota results, the intervention groups also showed some differences in the family composition of the gut microbiota, but all played a positive role in increasing the abundance of beneficial bacteria.

[0117] The results of the analysis of rat gut microbiota at the genus level are as follows: Figure 12DAs shown in the figure, the gut microbiota of each group of rats mainly consisted of Helicobacter, Allobaculum, Oscillospira, Lactobacillus, CF231, Desulfovibrio, Prevotella, Ruminococcus, Adlercreutzia, and Treponema. Compared with the NC group, the abundance of Helicobacter was significantly increased and the abundance of Lactobacillus was decreased in the GU group. This reflects that gastric ulcers lead to an increase in the abundance of Helicobacter pylori and other bacteria in the intestine, which inhibits the growth of beneficial bacteria such as Lactobacillus through nutrient competition, promoting the dominance of pathogenic bacteria and causing intestinal flora imbalance, thus affecting the body's health. Following LPR gavage intervention, the abundance of *Helicobacter* in rats significantly decreased, while the abundance of genera such as *Allobaculum*, *Lactobacillus*, and *Adlercreutzia* increased. This indicates that postbiotics may promote the proliferation of beneficial bacteria in the gut by providing a prebiotic-like effect, thereby restoring the balance of the gut microbiota. The GEL group showed a certain inhibitory effect on *Helicobacter*. Postbiotic hydrogel intervention also significantly reduced the abundance of *Helicobacter* and increased the abundance of *Allobaculum* and *Lactobacillus*, demonstrating that it can also regulate the gut microbiota by promoting the proliferation of beneficial bacteria and inhibiting the growth of pathogenic bacteria, and restore the abundance ratio of other genera to normal levels. Furthermore, compared with the GU group, OME also showed no effect on the gut microbiota at the genus level. This suggests that the occurrence of gastric ulcers disrupts the balance of gastric flora, and the intestinal flora is also affected to some extent. PLR, LPG, MPG, and HPG may indirectly help ulcer repair by regulating the composition of intestinal flora and maintaining the homeostasis of the microenvironment.

[0118] Based on the above results, the Lactobacillus rhamnosus JM023 post-biotic hydrogel of the present invention has the following specific effects:

[0119] ① It exhibits good biocompatibility, meeting the safety requirements for in vivo use. ② The post-biotic hydrogel of this invention can specifically adhere to inflamed tissue sites. ③ Compared to post-biotics and hydrogels alone, the post-biotic hydrogel complex of this invention shows a stronger therapeutic effect on alcoholic ulcers, with an effect close to or better than omeprazole, significantly reducing the degree of surface damage and potentially restoring gastric tissue to near-normal levels. ④ The *Lactobacillus rhamnosus* JM023 post-biotic hydrogel provided by this invention, compared to *Lactobacillus rhamnosus* post-biotics and hydrogels alone, can restore cell apoptosis levels to normal levels. ⑤ Compared to treatment with post-biotics and hydrogels alone, which can alleviate oxidative stress in gastric tissue to a certain extent, the post-biotic hydrogel complex can further enhance the alleviating effect. ⑥ The *Lactobacillus rhamnosus* JM023 post-biotic hydrogel provided by this invention exhibits a stronger anti-inflammatory effect, significantly reducing the levels of pro-inflammatory factors IL-6, IL-1β, TNF-α, and MPO, while significantly increasing the production of the anti-inflammatory factor IL-10, demonstrating the best anti-inflammatory effect. ⑦ The *Lactobacillus rhamnosus* JM023 post-biotic hydrogel provided by this invention also exerts an anti-ulcer effect by upregulating the level of gastric mucosal protective factors and promoting the repair of the gastric mucosal barrier. ⑧ The *Lactobacillus rhamnosus* JM023 post-biotic hydrogel provided by this invention significantly increased the levels of Nrf2 and HO-1 after intervention (p<0.05), with the HPG group showing the most significant effect, indicating that PLR and GEL mainly promote the expression of antioxidant enzymes through the Keap1 and Nrf2 signaling pathways, thereby inhibiting oxidative stress. ⑨ The *Lactobacillus rhamnosus* JM023 post-biotic hydrogel provided by this invention can significantly inhibit oxidative stress and apoptosis by changing the expression level of related mRNAs. ⑩ The *Lactobacillus rhamnosus* JM023 post-biotic hydrogel provided by this invention can reduce the richness and diversity of the gastrointestinal flora in ulcer rats, while the recovery effect after high-dose post-biotic hydrogel combination is closer to the normal level.

[0120] In summary, the Lactobacillus rhamnosus JM023 post-biotic hydrogel provided by this invention can effectively relieve alcoholic gastric ulcers and is suitable for development as a novel drug or health product. It has good application prospects in the field of drug or health product preparation for relieving alcoholic gastric ulcers.

[0121] The above description is only a preferred embodiment of the present invention. It should be noted that for those skilled in the art, several improvements and modifications can be made without departing from the principle of the present invention, and these improvements and modifications should also be considered within the scope of protection of the present invention.

Claims

1. A method for preparing a post-natal hydrogel for relieving alcoholic gastric ulcers, characterized in that, Includes the following steps: (1) Inoculate Lactobacillus rhamnosus into a sterile culture medium and culture until the colony count reaches 10. 7 ~10 9 After CFU / mL, heat inactivation was performed at 60-90℃, and then the inactivated product was freeze-dried to obtain the metagener of Lactobacillus rhamnosus; Lactoferrin, 1-hydroxybenzotriazole monohydrate, and N-(3-dimethylaminopropyl)-N'-ethylcarbodiimide hydrochloride were mixed in water, and then nano-calcium carbonate was added. After mixing thoroughly, the mixture was centrifuged, and the resulting supernatant was a solution containing modified lactoferrin. (2) Take sodium alginate solution and mix it with the postgenetic of Lactobacillus rhamnosus prepared in step (1) and a solution containing modified lactoferrin to obtain the postgenetic hydrogel for relieving alcoholic gastric ulcers. In step (1), the Lactobacillus rhamnosus is Lactobacillus rhamnosus ( Lacticaseibacillus rhamnosus JM023 is deposited at the Guangdong Provincial Center for Microbial Culture Collection, with accession number GDMCC NO: 65215; In step (1), the ratio of lactoferrin, 1-hydroxybenzotriazole monohydrate, N-(3-dimethylaminopropyl)-N'-ethylcarbodiimide hydrochloride, water, and nano-calcium carbonate is (0.15~0.25)g∶(0.08~0.12)g∶(0.08~0.12)g∶(80~120)mL∶(4~6)g; In step (2), the ratio of sodium alginate solution, post-liferating agent of Lactobacillus rhamnosus, and solution containing modified lactoferrin is (0.8~1.2)mL∶(0.8~1.2)g∶(0.8~1.2)mL.

2. The method for preparing the post-biotic hydrogel for relieving alcoholic gastric ulcers according to claim 1, characterized in that, In step (1), the sterile culture medium is sterile MRS liquid culture medium or sterile skim milk culture medium; the inoculation volume ratio of Lactobacillus rhamnosus in the sterile culture medium is 4%~8%; in step (2), the mass concentration of the sodium alginate solution is 1%~4%.

3. The method for preparing the post-natal hydrogel for relieving alcoholic gastric ulcers according to claim 1, characterized in that, In step (1), the culture temperature is 36~38℃; the heat inactivation time is 20~40min.

4. The method for preparing the post-natal hydrogel for relieving alcoholic gastric ulcers according to claim 1, characterized in that, In step (1), the centrifugation process parameters are: 3~5℃, 8000~12000r / min, centrifugation for 10~30min.

5. A post-natal hydrogel for relieving alcoholic gastric ulcers, prepared by the method described in any one of claims 1 to 4.

6. The application of a post-natal hydrogel for relieving alcoholic gastric ulcers as described in claim 5, characterized in that, Application in the preparation of drugs for relieving alcoholic gastric ulcers.

Images