Postbiotic hydrogel for relieving alcoholic gastric ulcer as well as preparation method and application thereof

Abstract

The invention belongs to the technical field of biological medicines, and particularly relates to a postbiotic hydrogel for relieving alcoholic gastric ulcer as well as a preparation method and application thereof. The metagen hydrogel for relieving the alcoholic gastric ulcer, provided by the invention, is mainly prepared from metagen of rhamnobacter and modified lactoferrin; the lactobacillus rhamnosus is lactobacillus rhamnosus JM023. Tests prove that when the postbiotic hydrogel is applied to an alcoholic gastric ulcer rat model, the occurrence of gastric ulcer can be inhibited by inhibiting oxidative stress and inflammatory response and improving protection factor secretion; and the alcoholic gastric ulcer is relieved by regulating and controlling the expression of oxidation, inflammation, cell apoptosis and tissue repair related metabolic pathways and regulating gastrointestinal flora. The metagen hydrogel disclosed by the invention has the effect of effectively relieving gastric ulcer symptoms caused by alcohol, and is suitable for being developed and applied as a medicine or a health-care product for relieving alcoholic gastric ulcer.

Description

Technical Field

[0001] The present invention belongs to the field of biomedicine technology, and specifically relates to a postbiotic hydrogel for relieving alcoholic gastric ulcer, and a preparation method and application thereof. Background Art

[0002] With the development of science and technology and the changes in modern lifestyles, people still have a large demand for the consumption of alcoholic products. Large amounts of alcohol intake have brought a huge burden on the maintenance of normal physiological activities in the stomach. As the main component of wine and alcoholic beverages, ethanol has a strong reducing effect, which can cause strong damage to the mucosal tissue on the surface of the stomach and destroy the normal physiological environment of the gastric mucosa. It is a direct pathogenic factor causing alcoholic gastric ulcers. Ethanol accumulated in the stomach can cause ulcers by gradually eroding the gastric mucosal barrier, damaging gastric mucosal epithelial cells, and destroying epithelial capillaries.

[0003] Gastric ulcer is a common peptic ulcer disease, characterized by mucosal rupture and damage greater than 3-5mm, with the depth reaching the submucosal layer. It mainly causes damage to the gastric tissue and threatens the healthy homeostasis of the gastrointestinal tract. If not treated in time, it can gradually develop into serious upper gastrointestinal diseases such as gastroesophageal reflux, gastric perforation and even gastric cancer, which seriously affect human health.

[0004] There are many types of drugs currently used to treat gastric ulcers, including omeprazole (OMG), rabeprazole, ranitidine, sucralfate, teprenone, etc. Although these drugs show strong and effective anti-ulcer ability, long-term use has adverse effects on organs such as the liver and kidneys, which is not conducive to long-term relief treatment. In addition, the complexity of the digestive tract environment in the body may lead to a decrease in the bioavailability of the above-mentioned therapeutic drugs, thereby weakening the therapeutic effect. Due to the mucus secretion of the gastric mucosal barrier, the digestion of multiple enzymes, and the emptying of the stomach, drugs and other ingredients often cannot fully contact the site where the drug is needed, making it difficult to maximize the therapeutic potential, and will lead to incomplete drug release, reduced bioavailability, increased drug dosage, prolonged treatment time, etc., which will reduce the efficacy and cause unnecessary waste. Therefore, researching, exploring and designing functional active substances and materials that can be used to relieve and treat gastric ulcers is a new idea for relieving or solving gastric ulcers.

[0005] Postbiotics are mixtures produced by artificial treatments such as heat treatment, ultrasonic treatment, high pressure, and ultraviolet irradiation to inactivate microorganisms. They include intact cells, cell components, cell wall components, cell secretions, and metabolites of inactivated bacteria, and retain certain original microbial efficacy. Compared with live probiotics, postbiotics do not need to consider the impact of the environment on biological activity, and can avoid the transfer of drug-resistant genes produced by bacteria, the emergence of virulence factors, the generation of different colonization patterns, and the generation of opportunistic infections, and have better stability and safety. However, in the treatment of gastric ulcers, there is still little research on the preparation and efficacy of postbiotics.

[0006] As a safe and practical biomaterial, hydrogel is a three-dimensional network formed by cross-linking polymers. It has unique water absorption and structural maintenance capabilities. It has been proven to play a good role in the treatment of gastrointestinal diseases and is conducive to the development of more effective and lasting methods for the prevention and treatment of gastrointestinal diseases. However, there are few studies on hydrogel drugs or health products for relieving alcoholic gastric ulcers, and the related drugs have the defects of being single in type and having side effects.

[0007] Therefore, developing a postbiotic hydrogel that can effectively relieve alcoholic gastric ulcers so as to better use it as a therapeutic drug or natural health product for alcoholic gastric ulcers will have greater social benefits and application prospects. Summary of the invention

[0008] In order to overcome the deficiencies in the prior art, the purpose of the present invention is to provide a postbiotic hydrogel for relieving alcoholic gastric ulcer, which has a good effect of relieving alcoholic gastric ulcer and is suitable for development as a medicine or health product for relieving alcoholic gastric ulcer.

[0009] Furthermore, the present invention also aims to provide a method for preparing the above-mentioned postbiotic hydrogel for relieving alcoholic gastric ulcer.

[0010] Furthermore, the present invention also aims to provide the application of the above-mentioned postbiotic hydrogel for relieving alcoholic gastric ulcer.

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

[0012] A postbiotic hydrogel for relieving alcoholic gastric ulcer, mainly prepared from postbiotics of rhamnosus bacillus and modified lactoferrin;

[0013] The rhamnosus bacillus is Lactobacillus rhamnosus JM023, which is deposited in Guangdong Microbiological Culture Collection Center with a deposit number of GDMCC NO: 65215.

[0014] The present invention isolates and screens a strain from a fecal sample of a healthy four-month-old infant. By extracting the genome of the strain and performing 16S rDNA amplification and sequencing, combined with morphological observation results, the strain is identified as Lactobacillus rhamnosus, named Lactobacillus rhamnosus JM023, and the Latin name is Lacticaseibacillus rhamnosusJM023. The strain is deposited in the Guangdong Microbial Culture Collection Center, with a deposit number of GDMCC NO: 65215, a deposit date of September 27, 2024, and a deposit address of 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 postbiotic hydrogel for relieving alcoholic gastric ulcer comprises the following steps:

[0017] (1) Inoculate rhamnosus bacteria in sterile culture medium and culture until the number of colonies reaches 10 7 ~10 9 CFU / mL, heat inactivation was performed at 60-90°C, and then the inactivated product was freeze-dried to obtain the postbiotics of Bacillus rhamnosus;

[0018] Lactoferrin, 1-hydroxybenzotriazole monohydrate, and N-(3-dimethylaminopropyl)-N'-ethylcarbodiimide hydrochloride are mixed in water, and then nano calcium carbonate is added, and the mixture is fully mixed and centrifuged to obtain a supernatant solution containing modified lactoferrin;

[0019] (2) Mixing the sodium alginate solution with the postbiotics of Bacillus rhamnosus prepared in step (1) and the solution containing modified lactoferrin to obtain the postbiotic hydrogel for relieving alcoholic gastric ulcer.

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

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

[0022] As a preferred solution, 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. Further 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 solution, in step (1), the process parameters of the centrifugation are: temperature 3-5°C, 8000-12000 r / min, and centrifugation for 10-30 min.

[0024] As a preferred solution, 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 solution, in step (2), the ratio of the amount of sodium alginate solution, the postbiotics of rhamnosus Bacillus, and the solution containing modified lactoferrin is (0.8-1.2) mL: (0.8-1.2) g: (0.8-1.2) mL. Further preferably, the ratio of the amount of sodium alginate solution, the postbiotics of rhamnosus Bacillus, and the solution containing modified lactoferrin is 1 mL: 1 g: 1 mL.

[0026] The application of the postbiotic hydrogel for relieving alcoholic gastric ulcer is used in the preparation of medicines or health products for relieving alcoholic gastric ulcer.

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

[0028] The postbiotic hydrogel for relieving alcoholic gastric ulcer provided by the present invention is mainly prepared from the postbiotics of rhamnosus bacillus and modified lactoferrin. Experiments have confirmed that the postbiotic hydrogel of the present invention can effectively inhibit the occurrence of gastric ulcers by effectively inhibiting oxidative stress and inflammatory response and increasing the secretion of protective factors, and can effectively relieve alcoholic gastric ulcers by regulating the expression of metabolic pathways related to oxidation, inflammation, apoptosis, and tissue repair and regulating gastrointestinal flora.

[0029] The preparation method of the postbiotic hydrogel provided by the present invention uses a postbiotic derived from Lactobacillus rhamnosus JM023. During the preparation, modified lactoferrin is used to adsorb nano-calcium carbonate, which is mixed with sodium alginate introduced with postbiotic powder to prepare a postbiotic-containing hydrogel. Experiments have confirmed that the postbiotic hydrogel is applied to an alcoholic gastric ulcer rat model, and can effectively relieve the symptoms of gastric ulcer caused by alcohol, and has outstanding application prospects in the field of medicines and health products for relieving alcoholic gastric ulcers. BRIEF DESCRIPTION OF THE DRAWINGS

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

[0031] Figure 2 This is a result diagram of the effect of the Lactobacillus rhamnosus JM023 postbiotic hydrogel on red blood cell hemolysis in Experimental Example 1 of the present invention;

[0032] Figure 3 The morphological changes of gastric tissues of rats in each group in Experimental Example 3 of the present invention are shown in FIG.

[0033] Figure 4 The ulcer area percentage and ulcer score of each group of rats in Experimental Example 3 of the present invention are shown in FIG.

[0034] Figure 5 The results of TUNEL staining of gastric tissues of rats in each group in Experimental Example 3 of the present invention are shown in FIG.

[0035] Figure 6 The results of the SOD, CAT, GSH-Px and MDA levels in the gastric tissues of rats in each group in Experimental Example 3 of the present invention are shown in FIG.

[0036] Figure 7 The results of the IL-6, IL-1β, TNF-α, MPO and IL-10 levels in the gastric tissues of rats in each group in Experimental Example 3 of the present invention are shown in FIG.

[0037] Figure 8 The results of PGE2 and NO levels in gastric tissue of rats in each group in Experimental Example 3 of the present invention are shown in FIG.

[0038] Fig. 9 The Keap1 immunohistochemical staining results of the gastric tissues of rats in each group in Experimental Example 3 of the present invention are shown in FIG.

[0039] Fig. 10A The result diagram of the expression level of mRNA of Occludin, ZO-1, Bcl-2 and Bax in the gastric tissue of each group of rats in Experimental Example 3 of the present invention;

[0040] Fig. 10BThe graph is a result of the expression level of Keap1, Nrf2 and HO-1 mRNA in the gastric tissue of each group of rats in Experimental Example 3 of the present invention;

[0041] Fig.11A This is a graph showing the Alpha diversity analysis results of the gastric flora of rats in each group in Experimental Example 3 of the present invention;

[0042] Fig. 11B This is a graph showing the differences in the bacterial flora level of the gastric flora of the rats in each group in Experimental Example 3 of the present invention;

[0043] Fig. 11C This is a graph showing the differences in the bacterial family level of the gastric flora of rats in each group in Experimental Example 3 of the present invention;

[0044] Fig.11D This is a graph showing the differences in the gastric flora of rats in each group at the genus level in Experimental Example 3 of the present invention;

[0045] Fig. 12A This is a graph showing the Alpha diversity analysis results of the intestinal flora of each group of rats in Experimental Example 3 of the present invention;

[0046] Fig. 12B This is a graph showing the differences in the bacterial flora level of the intestinal flora of each group of rats in Experimental Example 3 of the present invention;

[0047] Fig. 12C This is a graph showing the differences in the bacterial family level of the intestinal flora of rats in each group in Experimental Example 3 of the present invention;

[0048] Fig.12D This is a graph showing the differences in the intestinal flora at the genus level between the rats in each group in Experimental Example 3 of the present invention. DETAILED DESCRIPTION

[0049] The technical scheme and technical effects of the present invention will be clearly and completely described below in conjunction with specific embodiments and test examples. However, it should be understood by those skilled in the art that the embodiments are only used to illustrate the technical scheme of the present invention and should not be regarded as limiting the scope of protection of the present invention. The test methods used in the following examples are conventional methods unless otherwise specified; the raw materials used are all commonly used in the art, available to the public or commercially available, unless otherwise specified.

[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 nano-calcium carbonate was from Shanghai McLean Biochemical Technology Co., Ltd.

[0051] Example 1

[0052] This embodiment provides a postbiotic hydrogel for relieving alcoholic gastric ulcer, which is mainly prepared from postbiotics of rhamnosus bacteria and modified lactoferrin; the modified lactoferrin is amino-modified lactoferrin. Rhamnosus bacteria is Lactobacillus rhamnosus JM023, Latin name Lacticaseibacillus rhamnosus JM023, which is deposited in Guangdong Microbiological Culture Collection Center, with a deposit number of GDMCC NO: 65215, a deposit date of September 27, 2024, and a deposit address of Guangdong Academy of Sciences Institute of Microbiology, 5th Floor, Building 59, No. 100 Xianlie Middle Road, Guangzhou.

[0053] The preparation method of the above-mentioned postbiotic hydrogel for relieving alcoholic gastric ulcer comprises the following steps:

[0054] (I) Preparation of postbiotics 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 was 10 7 CFU / mL, 10 8 CFU / mL, 10 9 CFU / mL, and then heat-inactivated at 80°C for 30 min. The inactivated product was freeze-dried to obtain low, medium and high doses of rhamnosus postbiotic powder, respectively, and stored at -20°C for use.

[0055] (ii) Modification of lactoferrin: Dissolve 0.2 g of lactoferrin in 100 mL of deionized water, add 0.1 g of 1-hydroxybenzotriazole monohydrate (HOBt·H2O) and 0.1 g of N-(3-dimethylaminopropyl)-N′-ethylcarbodiimide hydrochloride (EDC·HCl) respectively and stir evenly, add 5 g of nano calcium carbonate (CaCO3) at a ratio of 5 g / 100 mL and continue stirring. After fully mixing, centrifuge at 4°C and 10,000 r / min for 20 min. The supernatant obtained is a solution containing modified lactoferrin for standby use.

[0056] (III) Preparation of postbiotic hydrogel: A 2% alginate solution was prepared using sodium alginate, and then the postbiotic powder (1.0 g) of Bacillus rhamnosus prepared in step (I) was added to the sodium alginate solution (1.0 mL) and stirred to make it evenly dispersed, and then mixed with the solution containing modified lactoferrin (1.0 mL) in step (II) in equal proportions. When the system encounters acid, Ca 2+ Cross-linking with sodium alginate produces hydrogen bonds to form hydrogels, thus obtaining low, medium and high dose postbiotic hydrogels.

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

[0058] 1. Screening

[0059] Take 1g of feces from a healthy four-month-old baby in Harbin, Heilongjiang Province, spread it on sterile MRS solid medium after gradient dilution, and culture it in an anaerobic environment at 37℃ for 48h, observe and record the colony morphology; pick white colonies with moist surface, protrusions, and streak them on MRS solid medium, and purify and culture them under anaerobic conditions at 37℃, repeat this operation 3 times to obtain purified single colonies; pick single colonies and streak them on MRS solid medium, and culture them anaerobically at 37℃ for 48h to obtain the screened strain. The colonies of this strain on MRS medium are protruding, with smooth, round, moist, white and opaque surfaces, and a diameter of 0.8-1.6mm.

[0060] 2. Identification

[0061] The genome of the Lactobacillus rhamnosus JM023 strain obtained by the above screening was extracted, and the 16S rDNA of the strain was amplified and sequenced (the nucleotide sequence of the 16S rDNA of the amplified Lactobacillus rhamnosus JM023 is shown in SEQ ID NO.1), and the obtained sequence was compared with the nucleic acid sequence in NCBI-Blast. The results showed that the homology between the strain and the Lacticaseibacillus rhamnosus strain LR-B1 was 100%. Combined with the results of morphological observation, 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°C for 5 min; 35 cycles (95°C for 30 s; 55°C for 30 s; 72°C for 2 min); 72°C for 10 min.

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

[0068]

[0069] 3. Save

[0070] A single colony of Lactobacillus rhamnosus JM023 was picked and inoculated into a sterile MRS liquid culture medium, and cultured at 37°C for 17 hours under anaerobic conditions to obtain a bacterial solution; a sterilized 80% (v / v) glycerol aqueous solution was added to the obtained bacterial solution and mixed well, and then stored in a glycerol tube at -80°C. At the same time, the above-obtained Lactobacillus rhamnosus was biologically preserved, and it was preserved in the Guangdong Provincial Microbiological Culture Collection Center, with a preservation number of GDMCC NO: 65215, a preservation date of September 27, 2024, and a preservation address of the Institute of Microbiology, Guangdong Academy of Sciences, 5th Floor, Building 59, No. 100 Xianlie Middle Road, Guangzhou.

[0071] In order to confirm the technical effect that can be achieved by the technical solution of the present invention, the toxicity safety evaluation, adhesion evaluation and animal test of the postbiotic hydrogel prepared by the present invention are carried out as follows. The test results were analyzed by SPSS one-way analysis of variance (ANOVA) for multiple groups of sample means, and the LSD test and SNK test were used for pairwise comparison. All tests were repeated at least three times, and the results were expressed as mean ± standard deviation, indicating that p < 0.05 indicated that the results were significantly different. GraphPad Prism and Origin were used to plot the test data. The test process and test results are as follows.

[0072] Experimental Example 1: Toxicity and Safety Evaluation of Postbiotic Hydrogel

[0073] 1.1 Cytotoxicity test: GES-1 cells (human gastric mucosal cells) were cultured in DMEM culture medium supplemented with 10% fetal bovine serum and 1% double antibody, and then 100 μL of high-dose postbiotic hydrogel (10 μg / mL) prepared in Example 1 containing different concentrations (0, 50, 100, 200, 400 μg / mL) was used. 9 CFU / mL) culture medium was used to replace the above culture medium for GES-1 cell culture. After culturing for 1d, 2d, 3d, 4d, and 5d, 10μL CCK-8 solution was added to the above solution, and the cytotoxicity was detected by CCK-8 method.

[0074] 1.2. Red blood cell hemolysis test: Fresh rat blood was centrifuged at 1500 r / min for 5 min, the precipitate was washed with sterile saline, and the precipitate was collected after centrifugation. 1.2 mL of high-dose (10 9CFU / mL) of postbiotic hydrogel was dispersed with saline and mixed. At the same time, equal volumes of sterile saline and deionized water were used to disperse the red blood cell precipitate as negative and positive controls. After 2 h in a 37°C water bath, the absorbance of the supernatant at 540 nm was measured using an ELISA reader.

[0075] The results of the cytotoxicity test of postbiotic gel using GES-1 cells are shown in Figure 1 As shown. During the experiment, a group of normal cultured cells were selected as controls every day. Figure 1 It can be seen that the cell activity was close to 100% under the treatment of 0μL / mL postbiotic hydrogel for 1-5 days, indicating that the GES-1 cell activity was normal, and the cells were evenly distributed among the groups, with no significant difference in number and activity (p>0.05). After GES-1 was co-cultured with 0-200μL / mL postbiotic hydrogel for 5 days, the cell viability was 100.00±0.07%, 98.41±1.70%, 96.61±2.27%, and 95.93±3.04%, respectively, and it still maintained a high activity. After culturing with 400μL / mL postbiotic hydrogel for 5 days, the cell viability was 94.39±3.01%, which showed no significant difference from the normal level, indicating that postbiotic hydrogel had no obvious toxic effect on GES-1 cells. The main components of the hydrogel of the present invention are sodium alginate, lactoferrin and various active ingredients contained in postbiotics, which have no toxic side effects and can also provide nutrition, and show no obvious inhibitory effect on the proliferation of GES-1 cells.

[0076] Biomaterials used in vivo must also have good blood compatibility. The effects of different gel concentrations on red blood cell hemolysis are shown in Figure 2. Figure 2 As shown. Figure 2 It can be seen that the red blood cells in the positive control group (PC group) burst under the osmotic pressure of deionized water, and the solution appeared red. The hemolysis rates of the postbiotic 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, it is confirmed that the postbiotic hydrogel of the present invention has good biocompatibility and meets the safety requirements for in vivo use.

[0077] Experimental Example 2: Evaluation of in vitro adhesion of postbiotic hydrogels

[0078] GES-1 cells were co-cultured with gastric mucosa and transferrin to simulate the normal and inflammatory gastric tissue microenvironment in vivo. During co-culture, the microenvironment was divided into three groups: PBS, MUC and TRP. PBS was the control group, MUC was the normal microenvironment group, and TRP was the inflammatory microenvironment group. After GES-1 cells were cultured in a culture dish until they adhered to the wall, high-glucose culture medium containing 5% gastric mucosa and 100μg / mL transferrin was cultured in the culture dish for 12h, and then the postbiotic hydrogel labeled with DiD fluorescent dye was cultured in the culture dish for 2h, slowly washed with PBS 3 times, and imaged with a laser confocal scanning microscope.

[0079] This experiment constructs the microenvironment of normal and inflammatory tissues in vitro, and measures the residual fluorescence to evaluate the targeting. The results show that compared with the PBS group, only a small amount of red DiD fluorescence appeared in the MUC group, indicating that there was basically no residual postbiotic hydrogel, while a large amount of red fluorescence was present in the TRP group, indicating that it still retained the postbiotic hydrogel adhered to the culture dish. This shows that compared with the normal environment, the postbiotic hydrogel of the present invention can specifically adhere to the site of inflammatory tissue.

[0080] Experimental Example 3: Animal Experiment to Evaluate the Effect of Postbiotic Hydrogel

[0081] Establishment of animal model: 80 healthy SPF male SD rats (4-5 weeks old) weighing 200-250g were selected and kept at a temperature of 22±2°C and a relative humidity of 60±5%, maintaining a 12-hour light / dark cycle. All rats were gavaged regularly once a day. After one week of adaptive feeding with normal feed and free drinking water, the rats were randomly divided into 8 groups (10 in each group): blank control group (NC), ulcer group (GU), postbiotic group (PLR), hydrogel group (GEL), low dose (10 7 CFU / mL) postbiotic hydrogel group (LPG), the medium dose (10 8 CFU / mL) postbiotic hydrogel group (MPG), high dose (10 9 CFU / mL) postbiotic hydrogel (HPG) and positive control group (OME).

[0082] After that, the normal group was gavaged with 5 mL / kg·d sterile PBS solution daily, and the other groups were gavaged with 5 mL / kg·d 60% ethanol aqueous solution daily to establish the model. One hour later, the normal group and the model group were gavaged with 5 mL / kg·d sterile PBS solution, and the other groups were gavaged with the corresponding test substances at a dose of 5 mL / kg·d. During the feeding period, the amount of alcohol was adjusted according to the weight and mortality of the rats, and the survival curve and weekly weight changes of the rats were recorded. The feeding was continued for 6 weeks.

[0083] Among them, the gavage method of animals is designed in groups according to Table 1, and gavage treatment is performed. Among them, low, medium and high doses of postbiotic hydrogels (LPG, MPG and HPG) are the postbiotic hydrogels prepared in Example 1 of the present invention. The preparation process of the hydrogel involved in the hydrogel group (GEL) is: using sodium alginate to prepare an alginate solution with a mass fraction of 2%, and further mixing it with the solution containing modified lactoferrin (1.0mL) in step (ii) of Example 1 in equal proportions to prepare a hydrogel. The preparation method of the postbiotic solution involved in the postbiotic group (PLR) is: take the postbiotic powder (1.0g) of Bacillus rhamnosus prepared in step (i) of Example 1 and dissolve it in PBS solution to obtain a postbiotic solution.

[0084] Table 1. Grouping and gavage of animal experiments

[0085]

[0086] Sample collection and processing: After the last oral gavage, the rats were fasted for 12 hours and allowed to drink water freely. Phenobarbital was injected into the groin, blood was collected, and serum was collected after centrifugation and stored at -20°C. The rats were killed by dislocation of the neck, and the rats were disinfected and dissected. Gastric tissue, gastric contents and cecal contents were collected. After observation and photography, a part of the gastric tissue was taken for ROS staining and WB analysis, a part was fixed with 4% paraformaldehyde and stored at 4°C for pathological staining, and the rest was quickly frozen with liquid nitrogen and stored at -80°C for subsequent biochemical index determination of rats.

[0087] 3.1. Gastric Histopathological Staining

[0088] The stomach tissue was fixed with 4% formaldehyde solution for routine paraffin embedding and sectioning. The sections were sequentially immersed in xylene and different concentrations of ethanol at room temperature and washed with double distilled water. The washed sections were stained with H&E according to the instructions. After staining, the pathological morphology was observed and photographed under a 200x optical microscope. Figure 3 The percentage of gastric ulcer area and ulcer score of rats are shown in Figure 4 shown. Figure 3 In the figure, (a) represents the normal group (NC group), (b) represents the model group (GU group), (c) represents the postbiotic group (PLR group), (d) represents the hydrogel group (GEL group), (e) represents the low-dose postbiotic hydrogel group (LPG group), (f) represents the medium-dose postbiotic hydrogel group (MPG group), (g) represents the high-dose postbiotic hydrogel group (HPG group), and (h) represents the allomeprazole group (OME group).

[0089] Depend on Figure 3It can be seen that the gastric tissue of rats in the NC group is intact, with normal morphology, a smooth gastric mucosal surface, and no damage. The GU group showed more serious damage, with ulcer symptoms such as bleeding and ulceration on the surface, and edema in some damaged areas. After intragastric administration of postbiotics and hydrogels, the area of ​​damaged gastric tissue was reduced, but there were still some bleeding symptoms, and the effect was slightly weaker than that of the OME group. The postbiotic hydrogel group of the present invention has better effects than the postbiotic, hydrogel, and OME groups. It shows that the use of postbiotic hydrogel in the present invention further improves the bleeding situation and effectively reduces the area of ​​damage.

[0090] Figure 4 middle, Figure 4 a represents the percentage of gastric ulcer area in rats, Figure 4 b represents the score of gastric tissue ulcer in rats. Figure 4 It can be seen that the proportion of gastric mucosal injury area in the GU group reached 42.73±4.22%, and the pathological scores were concentrated in the range of multiple small ulcers and a small number of large ulcers (4-5 points), which was different from the NC group. The proportion of injury area in the GEL group and the PLR ​​group was significantly reduced compared with the GU group, while the proportion of ulcers in the LPG, MPG, and HPG groups was reduced to 11.73±3.71%, 9.7±2.87%, and 9.67±3.54%, respectively. The ulcer scores were concentrated in the range of congestion and a small number of bleeding points (0.5-2 points). In addition, the results of the postbiotic gel group of the present invention were close to those of the normal group, showing an effect similar to or even better than that of the OME group. This shows that when postbiotics and hydrogels act alone, they have a relieving effect on ethanol-induced gastric ulcers, but the postbiotic hydrogel of the present invention shows a stronger therapeutic effect, and the effect is close to or better than omeprazole, which can greatly reduce the degree of surface damage and is expected to restore gastric tissue to a level close to normal.

[0091] 3.2. TUNEL staining of gastric tissue

[0092] At room temperature, immerse the slices in dewaxing solution and anhydrous ethanol in turn, and wash with double distilled water. After the slices are dried, add proteinase K working solution (stock solution: PBS = 1:9) to cover the tissue and incubate in a 37°C incubator for 20 minutes. After incubation, place the slides in PBS for washing. After drying, add membrane permeabilization working solution to the tissue and incubate at room temperature for 20 minutes, and wash with PBS. Add buffer in the circle to cover the tissue and incubate at room temperature for 20 minutes. Then follow the instructions of the TUNEL kit. After the treatment is completed, seal the slide with anti-fluorescence quenching sealing agent, observe under a fluorescence microscope and collect images. The TUNEL staining results for the detection of apoptosis in rat gastric tissue cells are shown in Figure 2. Figure 5 shown. Figure 5In the figure, (a) represents the normal group (NC group), (b) represents the model group (GU group), (c) represents the postbiotic group (PLR group), (d) represents the hydrogel group (GEL group), (e) represents the low-dose postbiotic hydrogel group (LPG group), (f) represents the medium-dose postbiotic hydrogel group (MPG group), (g) represents the high-dose postbiotic hydrogel group (HPG group), and (h) represents the allomeprazole group (OME group).

[0093] Depend on Figure 5 It can be seen that normal cells show blue fluorescence and apoptotic cells show red fluorescence. The number of red fluorescence in the NC group is small, indicating that the cell apoptosis rate is low, which can be attributed to the normal cell life process. In contrast, the number of red fluorescence in the GU group increased abnormally, indicating that the cells were damaged by irritation, resulting in a large number of deaths. After intervention treatment in each group, the number of apoptotic cells decreased to varying degrees, and the apoptotic cells were mainly distributed at the edge of the mucosa and the detached part. Among them, the apoptosis in the HPG group was similar to that in the normal group. This shows that PLR, GEL and OME can protect cells from ethanol damage to a certain extent. The introduction of PLR enhances the protective effect of GEL, and the postbiotic hydrogel HPG group of the present invention shows the strongest relief effect, which can restore the level of cell apoptosis to normal.

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

[0095] This test 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 produces free radicals in gastric tissue that attack lipids and other biomolecules, causing lipid peroxidation, leading to a decrease in SOD, CAT, and GSH-Px and an increase in MDA, which in turn exacerbates the destruction of the mucosal layer and the formation of ulcers. Figure 6 a) CAT( Figure 6 b) GSH-Px( Figure 6 c) and MDA( Figure 6 d) The results of the determination are as follows Figure 6 shown. Figure 6 In this section, the meanings of the test groups are the same as those in Section 3.1. Figure 6It can be seen that compared with the NC group, ethanol gavage significantly reduced the activities of the three enzymes and the level of MDA in the GU group, indicating that ethanol has caused oxidative damage to gastric tissue, while compared with the GU group, the other groups showed a trend of inhibiting this damage to varying degrees. After gavage with postbiotics, the oxidative stress level of gastric tissue can be well improved, and gavage with postbiotic hydrogel can significantly enhance this improvement effect. Among them, LPG showed an effect similar to omeprazole, and the enzyme activity of the MPG and HPG groups had no significant difference from the NC group, and the MDA level was also close to the normal level, indicating that the postbiotic hydrogel can effectively alleviate the excessive oxidation of tissues caused by ethanol, and showed a synergistic effect different from the action of a single component. This may be attributed to the fact that the hydrogel attached to the damaged site can prolong the action time of the postbiotic active substances and protect the tissue from ethanol damage, etc., to prevent ethanol-induced gastric damage.

[0097] When the gastric mucosal layer is attacked and destroyed, and MDA is produced in large quantities, it is often accompanied by an inflammatory response. In order to explore the effect of postbiotic hydrogel on gastric tissue inflammation caused by ethanol, IL-6 ( 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 signature inflammatory factors. Figure 7 shown. Figure 7 In this section, the meanings of the test groups are the same as those in Section 3.1. Figure 7 It can be seen that compared with the NC group, the levels of proinflammatory cytokines in the GU group increased significantly, indicating that ethanol has caused a significant inflammatory response in the stomach. Compared with the GU group, each treatment group can reduce the level of proinflammatory cytokines and increase the level 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 was slightly better than the GEL group. Postbiotic hydrogel showed a stronger anti-inflammatory effect, among which the medium and high doses of postbiotic hydrogel were better than the low dose group, which can significantly reduce the levels of proinflammatory factors IL-6, IL-1β, TNF-α and MPO, making their content close to that of the NC group, and significantly increase the production of anti-inflammatory factor IL-10, showing the best anti-inflammatory effect. It shows that medium and high doses of postbiotic hydrogel can effectively inhibit the occurrence of inflammatory response, which is also one of its ways to relieve gastric mucosal damage.

[0098] PGE2 and NO are two protective factors related to the gastric mucosal barrier defense. Figure 8 a) and NO( Figure 8 b) The measurement results are as follows Figure 8 shown. Figure 8In this section, the meanings of the test groups are the same as those in Section 3.1. Figure 8 It can be seen that compared with the NC group, ethanol can cause a significant decrease in the PGE2 and NO content in the GU group, and the level of protective factors can be significantly increased after oral administration of postbiotics and hydrogels alone. Among them, the levels of the two after the postbiotics and hydrogels were mixed were not significantly different from those of the OME group, and were close to those of the NC group, indicating that the postbiotic hydrogel may also play an anti-ulcer role by upregulating the level of gastric mucosal protective factors and promoting the repair of the gastric mucosal barrier.

[0099] 3.4 Immunohistochemical analysis

[0100] Gastric tissue was fixed with 4% neutral formalin at room temperature for 2 days, embedded in paraffin, and then 4 μm tissue sections were prepared. Then, xylene and different concentrations of ethanol were used to dewax the sections and put them into water. The tissue sections were placed in a repair box filled with EDTA (pH 9.0) and heated in a microwave oven to maintain boiling for 20 minutes, and excessive evaporation of the buffer was prevented to cause dry sections. After the repair was completed, it was cooled naturally and the slides were washed 3 times in PBS, each time for 5 minutes. After adding 3% H2O2 and incubating at room temperature and away from light for 25 minutes, they were washed 3 times with PBS. The sections were placed in a light-proof humidified box, 3% BSA was evenly dripped on the tissue, and after blocking at room temperature for 30 minutes, the BSA on the sections was shaken off, and the prepared primary antibody was added and incubated overnight at 4°C. After that, the humidified box was taken out and returned to room temperature, washed 3 times with PBS and incubated with the secondary antibody at room temperature for 1 hour, and then the slides were washed 3 times with PBS, and after being slightly dried, DAB color developing solution was added. The color development time was controlled under a microscope, and the color development was terminated after the sections were rinsed with tap water. The nuclear sections were counterstained with hematoxylin and dehydrated and sealed, and the results were interpreted under a white light microscope. The cross-sectional images were obtained under a scanner for immune scoring.

[0101] Immunohistochemistry (IHC) was used to detect the effect of the gel on the expression of Keap1, Nrf2, HO-1, p-NF-κB, Occludin and ZO-1 proteins, aiming to explore the mechanism of action of postbiotic hydrogel in anti-oxidation, anti-inflammation, inhibition of cell apoptosis and promotion of tight junction protein expression. The staining results are shown in Fig. 9 The meaning of each test group is the same as that in Section 3.1. Fig. 9 It can be seen that the number of Keap1-positive cells in the GU group increased compared with the NC group, the expression of Nrf2 and HO-1 decreased, and the histochemical score changed significantly (p < 0.05), indicating that ethanol induced oxidative stress by activating the Keap1 pathway and reduced 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 increased significantly (p < 0.05), and the effect of the HPG group was the most significant, indicating that PLR and GEL mainly promoted the expression of antioxidant enzymes through the Keap1 and Nrf2 signaling pathways, thereby inhibiting oxidative stress.

[0102] 3.5 mRNA expression

[0103] The total RNA was extracted using an RNA extraction kit. About 1 g of rat stomach tissue was taken, quickly frozen with liquid nitrogen, and then quickly ground into powder and transferred to a 1.5 mL enzyme-free centrifuge tube. The clean bench was wiped with chloroform in advance, and the total RNA of rat stomach tissue was extracted in the dark according to the instructions of the Simply P kit. All supplies must be treated with DEPC water, and the test area must be wiped with chloroform. The extracted total RNA was collected into an enzyme-free centrifuge tube, and the integrity of the RNA was detected by agarose gel electrophoresis, and the purity and concentration of the RNA were determined by a nucleic acid protein concentration meter. When the OD260 / OD280 value was in the range of 1.8 to 2.1, it indicated that the extracted RNA had good purity and could be used for subsequent experiments.

[0104] RT-PCR was used to determine the expression of related genes (Occludin, ZO-1, Bcl-2, Bax) and (Keap1, Nrf2, HO-1) to explore the protective effect of postbiotic hydrogel on gastric tissue. Fig. 10A The results of the determination of mRNA expression of related genes Occludin, ZO-1, Bcl-2, and Bax. Fig. 10B The results are for the determination of mRNA expression levels of related genes Keap1, Nrf2, and HO-1. Fig. 10A , Fig. 10B It can be seen that compared with the NC group, the expression of Occludin and ZO-1 genes related to tight junction protein expression, Bcl-2 genes related to inhibition of cell apoptosis, and Nrf2 genes related to antioxidant enzymes in the GU group were significantly reduced (p < 0.05), the expression of Bax genes related to cell apoptosis and Keap1 genes related to oxidation were significantly increased (p < 0.05), and the expression of HO-1 gene related to oxidation increased. This shows that ethanol can induce the expression of oxidation-related genes and cell apoptosis genes, inhibit the expression of tight junction proteins, and thus cause 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, and the expression levels of Bax and Keap1 were decreased. Among them, after intragastric administration of different doses of postbiotic hydrogel, the expression levels of each gene showed significant differences from the GU group. This indicates that both postbiotics and hydrogels can significantly inhibit oxidative stress and cell apoptosis by changing the expression levels of related mRNAs, and the hydrogels exhibit better effects after being loaded with postbiotics.

[0105] 3.6 Determination of gastrointestinal flora abundance

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

[0107] The PCR amplification products and target fragments were processed in the following steps: electrophoresis detection, gel recovery, fluorescence quantification, equimolar mixing of samples, preparation of sequencing libraries, and 2×300bp double-end sequencing. The sequencing platform was the Illumina-MiSeq platform. The QIIME software called VSEARCH to check and propose chimeric sequences, merged and divided according to 97% similarity, and calculated the Alpha diversity index. The linear discriminant analysis effect (LEfSe) was used to evaluate the composition at each classification level and perform statistical analysis.

[0108] Alpha diversity is one of the indicators for evaluating the abundance and diversity of the microbiota, including Chao1, Simpson, and Observed species indices for evaluating the richness of the microbiota and Shannon and Faith pd indices for evaluating the diversity of the microbiota. Fig.11A As shown. Fig.11A It can be seen that compared with the NC group, various indexes of the GU group increased, indicating that the occurrence of ulcers led to the enrichment of bacterial species on the mucosal surface, and the abundance and diversity of gastric flora increased. After the relief of postbiotics and hydrogels alone, the richness and diversity indexes in the rat stomach decreased. After the combination of the two, it also showed the effect of reducing the Chao1, Simpson, and Observed species indices of ulcer rats, among which the HPG group showed Chao1 and Observed species indices closer to the NC group. It shows that postbiotics and hydrogels can reduce the richness and diversity of gastric flora in ulcer rats, and the recovery effect after high-dose postbiotics and hydrogels are combined is closer to normal levels.

[0109] By comparing the taxonomic differences in the composition of the gastric flora in each group of rats, the effects of ethanol and postbiotic hydrogel on the gastric flora of rats were analyzed. Figures 11B-11D shown.

[0110] The results of the composition analysis of rat gastric flora at the phylum level are as follows Fig. 11B As shown in the figure, the stomach of rats is mainly composed of Firmicutes, Bacteroidetes, Proteobacteria, Actinobacteria, Spirochaetes, TM7, Tenericutes, Deferribacteres, Verrucomicrobia, Cyanobacteria, etc. Among them, Firmicutes, Bacteroidetes, Proteobacteria and Actinobacteria have the highest abundance, accounting for more than 90% in total. The proportions of the four bacterial phyla in the NC group 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, and the abundance of Bacteroidetes and Proteobacteria increased to 16.09% and 13.91%, respectively. The sum of their relative abundance increased, and this result could be changed after intervention with PLR and GEL. Intragastric administration of postbiotic hydrogels can reduce the proportion of Bacteroidetes and Proteobacteria, while increasing the abundance of Firmicutes and Actinobacteria. Among them, Bacteroidetes and Proteobacteria contain a variety of pathogenic bacteria, and their increased abundance can destroy the microenvironment homeostasis maintained in the stomach and induce the occurrence of diseases, reflecting that the gastric microenvironment of rats with alcoholic ulcers has changed, and the wounds on the mucosal surface are more susceptible to infection and other complications. After treatment with postbiotic hydrogels, the gastric flora of rats can be improved at the phylum level and gradually restored to balance.

[0111] The results of the analysis of rat gastric flora at the family level are as follows Fig. 11CAs shown. The microbiota of rats in each group was mainly concentrated in Peptostreptococcaceae (Peptostreptococcus family), Lactobacillaceae (Lactobacillus family), Turicibacteraceae (Zurichella family), Erysipelotrichaceae (Erysipelotrichaceae), Clostridiaceae (Clostriaceae), S24-7, Pseudomonadaceae (Pseudomonadaceae), Ruminococcaceae (Verrucomicrobia family), Lachnospiraceae (Lachnospiraceae), Enterobacteriaceae, etc. Compared with the NC group, the composition of the gastric microbiota in the GU group at the family level changed significantly, with a decrease in Lactobacillaceae, etc., and an increase in the abundance of Erysipelotrichaceae and Pseudomonadaceae. After treatment with different intervention groups, the abundance of Erysipelotrichaceae and Pseudomonadaceae decreased, and the abundance of Lactobacillaceae increased to varying degrees. The microbial composition of the MPG and HPG groups was close to that of the NC group. In addition, the PLR, GEL, PG and OME groups showed certain differences in the microbial composition at the family level. This may be due to the different content and mode of action of each intervention agent, which has different effects on the gastric microbial flora of rats, leading to differences in the microbial composition after intervention. This indicates that some pathogenic bacteria overgrow on the surface of the gastric mucosa of rats with gastric ulcer, and the growth of probiotics is inhibited. This situation can be changed after treatment with various intervention agents. Among them, the effect of the HPG group makes the microbial abundance closest to that of the normal group.

[0112] The analysis results of rat gastric flora at the genus level are as follows Fig.11DAs shown. The bacterial flora of each rat at the genus level was mainly concentrated in Turicibacter, Lactobacillus, Allobaculum, Pseudomonas, Staphylococcus, Streptococcus, Oscillospira, Prevotella, Adlercreutzia, Corynebacterium, etc. Compared with the NC group, the abundance of Allobaculum and Pseudomonas in the GU group increased significantly, and the abundance of Lactobacillus decreased, which was alleviated to varying degrees after treatment with different interventions. After PLR gavage intervention, the abundance of Lactobacillus in rats increased significantly, while the abundance of Pseudomonas decreased, indicating that postbiotic gavage caused a significant change in the gastric flora of rats and other dominant strains appeared. The changes in the abundance of the GEL group also showed that it promoted the proliferation of Lactobacillus and other bacterial genera. LPG, MPG and HPG treatments also increased the abundance of beneficial bacterial genera such as Lactobacillus, and the intestinal flora disorder was alleviated to varying degrees. Among them, the recovery effect of the HPG group was the most obvious. Compared with the GU group, the relative abundance of Lactobacillus increased in rats under its influence, and the total abundance of Pseudomonas and other bacterial genera was lower. The concentration at the genus level was closest to 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 shows that when gastric ulcers occur, the composition of the surface flora will also change, and different doses of postbiotic hydrogels can improve the gastric flora disorder in rats with gastric ulcers by regulating the gastric flora.

[0113] The results of Alpha diversity characterization of rat intestinal flora are as follows Fig. 12A As shown. Fig. 12AIt can be seen that compared with the NC group, all indexes of the GU group were reduced, indicating that the homeostasis of the intestinal flora in rats may be affected, and the abundance and diversity of the flora were reduced. After the relief of postbiotic gavage, the richness and diversity index of the flora in the rat intestine can be improved. After compounding with hydrogel, it can also show that the Chao1, Shannon, Faith pd and Observed species indexes of GU rats are significantly improved. Among them, the HPG group showed the highest index level in Chao1, Shannon and Observed species, and the results of its five indexes were close to normal levels. It shows that postbiotics can improve the richness and diversity of the intestinal flora of ulcer rats, and the recovery effect after high-dose postbiotic hydrogel gavage intervention is closer to the normal level. After the intervention of hydrogel alone, only the Faith pd index result showed an upward trend, indicating that the hydrogel had no significant alleviating effect on the changes in the intestinal flora of ulcer rats. In addition, the effect of omeprazole in the treatment of gastric ulcer has been confirmed, but the therapeutic effect has not shown a significant connection with the restoration of the homeostasis of intestinal flora.

[0114] By comparing the taxonomic differences in the composition of the cecal flora in each group of rats, the effects of gastric ulcer and postbiotic hydrogel intervention on the intestinal flora of rats were analyzed. Figures 12B-12D shown.

[0115] The results of the analysis of rat intestinal flora at the phylum level are as follows Fig. 12BAs shown. The main bacterial phyla in the rat intestine are Firmicutes, Bacteroidetes, Proteobacteria, Actinobacteria, Spirochaetes, TM7, Verrucomicrobia, Tenericutes, Deferribacteres, Elusimicrobia, etc. Among them, Firmicutes, Bacteroidetes, Proteobacteria and Actinobacteria are the four most abundant phyla, with a total proportion of more than 90%. The proportions of the four phyla in the NC group were 50.97%, 34.79%, 7.08% and 3.65%, respectively. In the GU group, the abundance of Bacteroidetes decreased to 20.54%, and the abundance of Firmicutes and Verrucomicrobia increased to 64.23% and 2.95%. After intervention with PLR and GEL, the abundance of these two phyla was found to be reduced, while the abundance of other phyla such as Bacteroidetes increased. Oral administration of postbiotic hydrogel can also reduce the abundance of Firmicutes and Verrucomicrobia in the intestine, while increasing the abundance of Bacteroidetes and Actinobacteria. The abundance of Firmicutes and Bacteroidetes in the MPG group recovered to 51.91% and 34.50%, which is closest to the level of the NC group (50.97% and 34.79%). Similar to the gastric flora, the changes in the abundance of Firmicutes and Bacteroidetes may reflect the disruption of intestinal homeostasis, leading to the proliferation of multiple pathogens, which can increase the host's chance of infection by producing harmful substances and reduce the body's resistance, thereby affecting the healing of ulcers and increasing the risk of other diseases. After different treatments, the intestinal flora of rats can be improved at the phylum level and show a trend of gradually restoring balance. The effect is more obvious in the MPG and HPG groups. This may be because some components of postbiotics can also act as prebiotics, promote the proliferation of probiotics, and change the composition of the flora. OME also showed a certain restorative effect on the intestinal flora in this process and can also increase the abundance of Proteobacteria.

[0116] Then, the effect of postbiotic hydrogel on intestinal flora was further analyzed by characterizing the composition of the flora at the family level. Fig. 12CAs shown. The microbiota of rats in each group was mainly concentrated in S24-7, Ruminococcaceae, Helicobacteraceae, Erysipelotrichaceae, Peptostreptococcaceae, Lachnospiraceae, Lactobacillaceae, Paraprevotellaceae, Coriobacteriaceae, Desulfovibrionaceae, etc. at the family level. Compared with the NC group, the composition of the intestinal flora of rats in the GU group 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 homeostasis of intestinal flora. The treatment of different intervention groups can significantly reduce the abundance of Helicobacteraceae in rats with ulcers. After oral administration of postbiotics alone, the abundance of Peptostreptococcaceae, Lactobacillaceae, Coriobacteriaceae, etc. in rats increased. These bacterial families contain many microorganisms that are beneficial to the host and have been found to inhibit and alleviate the occurrence of various chronic diseases of the host. This reflects that postbiotics contain a variety of active ingredients that can promote the proliferation of probiotics and help the recovery of intestinal flora. GEL oral administration showed a significant inhibitory effect on Helicobacteraceae, which may be due to the good antibacterial effect of lactoferrin contained in it. After hydrogel loading, LPG, MPG and HPG groups can further reduce the increase in the abundance of Erysipelotrichaceae under the action of postbiotics and promote the recovery of the flora to normal levels. The abundance of beneficial bacteria under HPG intervention can reach the level closest to that of the NC group. The OME group showed that pathogenic bacteria such as Helicobacteraceae dominated, confirming that its effective anti-ulcer effect did not affect the flora. Similar to the results of gastric flora, the intervention groups also showed certain differences in the family level composition of intestinal flora, but all played a positive role in increasing the abundance of beneficial bacteria.

[0117] The results of the analysis of rat intestinal flora at the genus level are as follows Fig.12DAs shown. At the genus level, the bacterial flora of rats in each group was mainly concentrated in Helicobacter, Allobaculum, Oscillospira, Lactobacillus, CF231, Desulfovibrio, Prevotella, Ruminococcaceae Ruminococcus, Adlercreutzia, Treponema, etc. Compared with the NC group, the abundance of Helicobacter in the GU group increased significantly, and the abundance of Lactobacillus decreased, reflecting that gastric ulcer caused the abundance of Helicobacter pylori and other bacteria in the intestine to increase and inhibit the growth of beneficial bacteria such as Lactobacillus through competition for nutrition, which promoted the dominant strains of pathogenic bacteria, caused intestinal flora disorder and thus affected the health of the body. After LPR gavage intervention, the abundance of Helicobacter in rats was significantly reduced, and the abundance of Allobaculum, Lactobacillus and Adlercreutzia increased, indicating that postbiotics may promote the proliferation of beneficial bacteria in the intestine by providing prebiotic-like effects, thereby repairing the balance of intestinal flora. The GEL group showed a certain inhibitory effect on the Helicobacter genus. The intervention of postbiotic hydrogel can also significantly reduce the abundance of Helicobacter and increase the abundance of Allobaculum and Lactobacillus, proving that it can also regulate the intestinal flora by promoting the proliferation of beneficial bacteria and inhibiting the growth of pathogenic bacteria, and regulating the abundance ratio of other bacterial genera to normal levels. In addition, compared with the GU group, OME also showed no effect on the intestinal flora at the genus level. This shows that the occurrence of gastric ulcer not only destroys the balance of gastric flora, but also affects the intestinal flora to a certain extent. PLR, LPG, MPG and HPG may also indirectly help repair ulcers by regulating the microbial composition of intestinal flora to maintain the homeostasis of the microenvironment.

[0118] Based on the above results, it can be seen that the Lactobacillus rhamnosus JM023 postbiotic hydrogel of the present invention has the following specific effects:

[0119] ① It has good biocompatibility and meets the safety requirements for in vivo use. ② The postbiotic hydrogel of the present invention can specifically adhere to the site of inflamed tissue. ③ Compared with postbiotics and hydrogels alone, the postbiotic hydrogel complex of the present invention shows a stronger therapeutic effect on alcoholic ulcers, and the effect is close to or better than omeprazole, which can greatly reduce the degree of surface damage and is expected to restore gastric tissue to a near normal level. ④ The Lactobacillus rhamnosus JM023 postbiotic hydrogel provided by the present invention can restore the level of cell apoptosis to a normal level compared with Lactobacillus rhamnosus postbiotics and hydrogels alone. ⑤ Compared with the treatment of postbiotics and hydrogels alone, the postbiotic hydrogel complex can relieve the oxidative stress of gastric tissue to a certain extent, and the postbiotic hydrogel complex can enhance the relief effect. ⑥ The Lactobacillus rhamnosus JM023 postbiotic hydrogel provided by the present invention shows a stronger anti-inflammatory effect, which can significantly reduce the levels of pro-inflammatory factors IL-6, IL-1β, TNF-α and MPO, and significantly increase the production of anti-inflammatory factors IL-10, showing the best anti-inflammatory effect. ⑦ The Lactobacillus rhamnosus JM023 postbiotic hydrogel provided by the present invention also plays an anti-ulcer role by upregulating the level of gastric mucosal protective factors and promoting the repair of the gastric mucosal barrier. ⑧ The Lactobacillus rhamnosus JM023 postbiotic hydrogel provided by the present invention significantly increased the levels of Nrf2 and HO-1 after intervention (p < 0.05), among which the HPG group had 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 postbiotic hydrogel provided by the present invention can significantly inhibit oxidative stress and cell apoptosis by changing the expression level of related mRNA. ⑩ The Lactobacillus rhamnosus JM023 postbiotic hydrogel provided by the present invention can reduce the richness and diversity of the gastrointestinal flora of ulcer rats, and the recovery effect after the high dose of postbiotics and hydrogels are combined is closer to the normal level.

[0120] In summary, the Lactobacillus rhamnosus JM023 postbiotic hydrogel provided by the present invention can effectively relieve alcoholic gastric ulcer, is suitable for development as a new drug or health product, and has good application prospects in the field of preparation of drugs or health products for relieving alcoholic gastric ulcer.

[0121] The above is only a preferred embodiment of the present invention. It should be pointed out that for ordinary technicians in this technical field, several improvements and modifications can be made without departing from the principle of the present invention. These improvements and modifications should also be regarded as the scope of protection of the present invention.

Claims

1. A postbiotic hydrogel for relieving alcoholic gastric ulcer, characterized in that: It is mainly prepared from postbiotics of Bacillus rhamnosus and modified lactoferrin; The rhamnosus bacillus is Lactobacillus rhamnosus ( Lacticaseibacillus rhamnosus ) JM023, which is deposited in Guangdong Microbiological Culture Collection Center with the deposit number GDMCC NO: 65215.

2. The postbiotic hydrogel for relieving alcoholic gastric ulcer according to claim 1, characterized in that: The modified lactoferrin is amino-modified lactoferrin.

3. A method for preparing a postbiotic hydrogel for relieving alcoholic gastric ulcer according to claim 1, characterized in that: The following steps are involved: (1) Inoculate rhamnosus into sterile culture medium and culture until the colony count reaches 10 7 ~10 9 CFU / mL, heat inactivation was performed at 60-90°C, and then the inactivated product was freeze-dried to obtain the postbiotics of Bacillus rhamnosus; Lactoferrin, 1-hydroxybenzotriazole monohydrate, and N-(3-dimethylaminopropyl)-N'-ethylcarbodiimide hydrochloride are mixed in water, and then nano calcium carbonate is added, and the mixture is fully mixed and centrifuged to obtain a supernatant solution containing modified lactoferrin; (2) Mixing the sodium alginate solution with the postbiotics of Bacillus rhamnosus prepared in step (1) and the solution containing modified lactoferrin to obtain the postbiotic hydrogel for relieving alcoholic gastric ulcer.

4. The method for preparing the postbiotic hydrogel for relieving alcoholic gastric ulcer according to claim 3, characterized in that: In step (1), the sterile culture medium is a sterile MRS liquid culture medium or a sterile skim milk culture medium; and the inoculation volume ratio of the rhamnosus Bacillus in the sterile culture medium is 4% to 8%.

5. The method for preparing the postbiotic hydrogel for relieving alcoholic gastric ulcer according to claim 3, characterized in that: In step (1), the culture temperature is 36-38°C; and the heat inactivation time is 20-40 minutes.

6. The method for preparing the postbiotic hydrogel for relieving alcoholic gastric ulcer according to claim 3, characterized in that: In step (1), the amount 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.

7. The method for preparing the postbiotic hydrogel for relieving alcoholic gastric ulcer according to claim 3, characterized in that: In step (1), the centrifugal process parameters are: temperature 3-5°C, 8000-12000 r / min, and centrifugation for 10-30 min.

8. The method for preparing the postbiotic hydrogel for relieving alcoholic gastric ulcer according to any one of claims 3 to 7, characterized in that: In step (2), the mass concentration of the sodium alginate solution is 1% to 4%.

9. The method for preparing the postbiotic hydrogel for relieving alcoholic gastric ulcer according to any one of claims 3 to 7, characterized in that: In step (2), the dosage ratio of the sodium alginate solution, the postbiotics of Bacillus rhamnosus, and the solution containing modified lactoferrin is (0.8~1.2) mL: (0.8~1.2) g: (0.8~1.2) mL.

10. A use of the postbiotic hydrogel for relieving alcoholic gastric ulcer according to claim 1 or 2 or the postbiotic hydrogel for relieving alcoholic gastric ulcer prepared according to any one of claims 3 to 9, characterized in that: Application in the preparation of medicines or health products for relieving alcoholic gastric ulcers.

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