Bacillus velezensis tk1926 and its use in gastric mucosa repair

Abstract

The application belongs to the technical field of microbial medicine, and specifically discloses a bacillus velezensis TK1926 and application thereof in repair of gastric mucosa. Through systematic evaluation of multiple models and multiple indexes, it is confirmed that TK1926 fermentation treatment can comprehensively enhance the gastric mucosa protection and treatment effect of radix codonopsis. Compared with unfermented radix codonopsis, fermented radix codonopsis shows improved performance in anti-inflammatory, antioxidant, barrier repair and pathogen inhibition, which is the result of multi-dimensional synergistic effect such as anti-inflammatory-antioxidant-barrier repair-regulation of gastric environment. The fermentation process improves the bioavailability and efficacy of the original active ingredients of radix codonopsis through biological transformation, and may produce new beneficial metabolites, thereby exerting a synergistic enhancement effect. The fermentation product provided by the application can be used for preparing medicines to repair gastric mucosa damage, and provides a new method for intervention of gastric mucosa damage related diseases, and has a good application prospect.

Description

Technical Field

[0001] This invention relates to the field of microbial medicine technology, and in particular to Bacillus vesiculosus TK1926 and its application in gastric mucosal repair. Background Technology

[0002] Gastric mucosal injury is a crucial area of ​​research in digestive system diseases, with complex causes closely related to social environment and lifestyle. The gastric mucosa plays a fundamental barrier role in maintaining the integrity and normal function of the stomach. This membranous structure prevents direct damage from factors such as gastric acid and pepsin. However, when damaging factors outweigh protective factors, gastric mucosal injury occurs. Persistent damage can lead to deeper gastric lesions such as gastritis, gastric ulcers, and gastric cancer. While current clinical treatments (such as proton pump inhibitors and bismuth preparations) can alleviate symptoms, they suffer from side effects and high recurrence rates. Codonopsis pilosula, a traditional Chinese medicine often used for "tonifying the middle energizer, replenishing qi, strengthening the spleen and benefiting the lungs," has a long history and some modern pharmacological evidence supporting its effectiveness in improving gastrointestinal function and repairing gastric mucosal damage. However, traditional Codonopsis pilosula slices or extracts may have low bioavailability and relatively slow onset of action due to their high-molecular-weight polysaccharides and saponins, thus requiring further improvement in their efficacy.

[0003] In recent years, microbial fermentation technology has provided new insights for the modernization of traditional Chinese medicine research. This technology, through the enzymatic action of microorganisms, may transform macromolecules in Codonopsis pilosula into more easily absorbed small molecules, while simultaneously generating new active metabolites and removing some impurities. This could potentially achieve "enhanced efficacy and reduced toxicity," thereby strengthening its pharmacological activity and reducing potential side effects. Bacillus belyceae is a recognized safe strain capable of producing various enzymes and active metabolites. However, currently, there is a lack of publicly reported technical solutions for using Bacillus belyceae to ferment Codonopsis pilosula for the repair of gastric mucosal damage. Existing technologies have not revealed the effects and pathways by which specific Bacillus belyceae fermenting Codonopsis pilosula promotes gastric mucosal structural repair, reduces inflammatory responses, or enhances mucosal barrier function.

[0004] Therefore, developing a method for fermenting Codonopsis pilosula based on Bacillus to obtain fermentation products with significant gastric mucosal damage repair effects is of great scientific significance and application value for enriching the fermentation technology system of traditional Chinese medicine and expanding the means of intervention for gastric mucosal damage. Summary of the Invention

[0005] The purpose of this invention is to address the above-mentioned shortcomings of the prior art by providing a Bacillus vesiculus TK1926 and its application in gastric mucosal repair. By fermenting Codonopsis pilosula with Bacillus vesiculus TK1926, a fermented product of Codonopsis pilosula is obtained, which shows a significant synergistic effect in gastric mucosal repair compared with unfermented Codonopsis pilosula.

[0006] To achieve the above objectives, the present invention adopts the following technical solution:

[0007] The first aspect of the present invention is to provide a Bacillus velezensis TK1926, which was deposited on December 10, 2025, at the China General Microbiological Culture Collection Center (CGMCC), located at No. 3, Courtyard 1, Beichen West Road, Chaoyang District, Beijing. The classification name is Bacillus velezensis, and the accession number is CGMCC No:36999.

[0008] A second aspect of the invention is to provide the use of Bacillus vesiculosus TK1926 in the preparation of a medicament for repairing alcoholic gastric mucosal damage or Helicobacter pylori-associated gastric mucosal damage.

[0009] A third aspect of the present invention is to provide a fermentation product with gastric mucosa repair function, wherein the fermentation product is obtained by fermenting Codonopsis pilosula raw material with Bacillus berberis TK1926 as described above.

[0010] Furthermore, the fermentation products include fermentation broth, fermentation supernatant, fermentation extract, or freeze-dried powder thereof.

[0011] A fourth aspect of the invention is to provide a composition comprising the above-described fermentation product.

[0012] A fifth aspect of the present invention is to provide a method for preparing a fermentation product with gastric mucosa repair function, comprising the following steps:

[0013] Codonopsis pilosula raw materials are provided, crushed and sieved to obtain Codonopsis pilosula powder;

[0014] Sterile water is added to the Codonopsis pilosula powder according to a preset ratio, and after sterilization, Codonopsis pilosula matrix is ​​obtained.

[0015] The above-mentioned Bacillus berreatus TK1926 was inoculated into the Codonopsis pilosula substrate for fermentation to obtain fermentation broth;

[0016] The fermentation broth was freeze-dried to obtain the fermentation product.

[0017] Further, 2% to 5% by volume of secondary seed culture of Bacillus belye TK1926 was inoculated into the Codonopsis pilosula matrix.

[0018] Furthermore, the fermentation conditions are as follows: shake culture at 30~37℃ and 150~200 rpm for 24~48h.

[0019] Furthermore, the freeze-drying process is as follows: pre-freezing at a depth below -40°C for 2~4 hours; then, under a vacuum of 10~30 Pa, first raising the temperature to -20°C to -10°C and maintaining it for 20~40 hours, and then raising it to 25~35°C and maintaining it for 6~12 hours.

[0020] A sixth aspect of the present invention is to provide the use of the above-described fermentation product and composition in the preparation of a medicament for repairing gastric mucosal damage.

[0021] Furthermore, the gastric mucosal injury includes at least one of the following: alcoholic gastric mucosal injury, nonsteroidal anti-inflammatory drug-induced gastric mucosal injury, stress-induced gastric mucosal injury, or Helicobacter pylori-associated gastric mucosal injury.

[0022] Compared with the prior art, the beneficial effects of the present invention are:

[0023] (1) This invention utilizes the fermentation products obtained from the fermentation of Codonopsis pilosula and Bacillus vesiculosus TK1926 to demonstrate a comprehensive enhancement effect in multidimensional gastric mucosal repair. Whether used as a preventive protective group or as a treatment group after injury, the Bacillus vesiculosus fermented Codonopsis pilosula group showed superior effects compared to the corresponding unfermented Codonopsis pilosula group in improving gastric mucosal histopathology, regulating inflammatory balance, alleviating oxidative stress, repairing barrier function, and regulating the gastric environment. This indicates that fermentation treatment not only enhances the preventive and protective capabilities of Codonopsis pilosula but also strengthens its therapeutic and repair effects.

[0024] (2) Fermented Codonopsis promotes faster and more complete structural recovery: The degree of gastric mucosal damage was directly assessed by histopathological scoring and section observation of gastric mucosal tissue, and inflammation was graded. In the Helicobacter pylori infection model, Codonopsis fermented with Bacillus belyceae showed better improvement on gastric mucosal pathological damage induced by Helicobacter pylori than unfermented Codonopsis, with the treatment group showing better results than the protection group; in the acute ethanol injury model, fermented Codonopsis had a better protective effect against gastric mucosal pathological damage induced by ethanol. Section observation showed that the typical mucosal epithelial cell shedding, congestion and edema, and dense infiltration of inflammatory cells in the model group were significantly improved after intervention; in the Hp model, the gastric mucosal congestion and bleeding in the fermented Codonopsis treatment group was milder, the damage site was shallower, and the local necrosis was significantly reduced; in the acute ethanol injury model, the gastric mucosal epithelial cell loosening and inflammatory cell infiltration in the fermented Codonopsis protection group were milder. This indicates that fermented Codonopsis can more effectively promote the structural and functional recovery of gastric mucosal tissue.

[0025] (3) Fermented Codonopsis pilosula more effectively reshapes inflammatory balance: In both models, the levels of pro-inflammatory factors (TNF-α, IL-8, IL-6) were significantly increased in the model groups. After treatment with fermented Codonopsis pilosula, the levels of these pro-inflammatory factors decreased significantly, while the level of the anti-inflammatory factor IL-10 increased significantly. This indicates that one of the core mechanisms of action of fermented Codonopsis pilosula is to effectively curb the inflammatory cascade of the gastric mucosa by regulating immune balance, inhibiting the excessive release of pro-inflammatory mediators, and promoting the expression of anti-inflammatory factors.

[0026] Fermented Codonopsis pilosula is more effective because the fermentation process may: Firstly, enhance immunomodulatory activity: fermentation may degrade the large polysaccharides in Codonopsis pilosula into smaller, more easily absorbed polysaccharides or oligosaccharides, which can more effectively bind to receptors on the surface of immune cells. Secondly, produce new anti-inflammatory metabolites: Bacillus belye may produce or transform Codonopsis pilosula components during fermentation to generate small molecules with anti-inflammatory activity (such as short-chain fatty acids), creating a synergistic effect with unfermented Codonopsis pilosula.

[0027] (4) Fermented Codonopsis provides stronger antioxidant defense: In the model group, the MDA content, representing the degree of oxidative damage, was significantly increased, while the SOD activity, representing antioxidant capacity, was significantly decreased. Fermented Codonopsis treatment reversed this trend, significantly increasing SOD activity and decreasing MDA content. The results indicate that fermented Codonopsis has antioxidant activity, which can enhance the body's ability to scavenge oxygen free radicals, reduce the damage of lipid peroxidation to gastric mucosal cells, and create a favorable microenvironment for mucosal repair.

[0028] The enhancing effect of fermented Codonopsis pilosula stems from its ability to improve the body's endogenous antioxidant capacity and directly reduce oxidative damage. The antioxidants originally present in Codonopsis pilosula (such as phenols and flavonoids) may be converted into more bioactive or easily absorbed forms through fermentation, improving the utilization rate of antioxidant components. Extracts from fermented Codonopsis pilosula may more effectively activate intracellular antioxidant signaling pathways such as Nrf2, prompting cells to produce more SOD and other antioxidant enzymes, thereby fundamentally enhancing the ability of gastric mucosal cells to resist oxidative stress.

[0029] (5) Fermented Codonopsis pilosula effectively repairs the gastric mucosal barrier: ELISA was used to detect the expression of tight junction protein (ZO-1), assess the damage of *Helicobacter pylori* to the mucosal barrier, verify the repair effect on the gastric barrier, and evaluate the integrity of the mucosal barrier. In the model group, the expression of tight junction protein ZO-1 was significantly downregulated, indicating severe damage to the mucosal barrier. Fermented Codonopsis pilosula treatment significantly upregulated the expression of ZO-1. This indicates that fermented Codonopsis pilosula can repair and enhance the mechanical barrier of the gastric mucosa, reduce the reverse permeation of gastric acid, pepsin, and harmful substances, which is a key link in preventing further damage and promoting healing.

[0030] Fermented Codonopsis pilosula can more effectively repair and maintain the physical barrier of the gastric mucosa. The active ingredients produced by fermentation may more effectively promote the synthesis of tight junction proteins such as ZO-1 by epithelial cells; its strong anti-inflammatory and antioxidant effects create a favorable microenvironment for barrier repair, reducing the damage of inflammatory factors and oxygen free radicals to tight junction structures.

[0031] (6) Fermented Codonopsis pilosula helps restore gastric homeostasis: Gastric acid secretion and pepsin activity are affected by Hp infection, which can inhibit gastric acid secretion. Gastric function damage can be assessed by detecting gastric juice pH and pepsinogen I / II ratio. High gastric acid environment exacerbates the corrosion of the mucosa by ethanol, and inhibiting gastric acid secretion is one of the repair mechanisms. In the model group, the gastric environment deteriorated, and the fermented Codonopsis pilosula treatment group showed an increase in gastric juice pH (decreased acidity), while the pepsinogen I / II ratio significantly rebounded. Pepsinogen I is mainly secreted by the chief cells of the gastric fundic glands, and a decrease in its ratio to pepsinogen II is a marker of gastric mucosal atrophy. The rebound in the ratio suggests that fermented Codonopsis pilosula may help maintain the normalization of gastric mucosal function and reduce the erosion of aggressive factors.

[0032] (7) It showed a significant repair effect in a gastric mucosal injury model caused by Helicobacter pylori infection. The experimental results showed that the urease activity in the model group was extremely high, while the urease activity in the fermented Codonopsis pilosula treatment group was significantly reduced, indicating that it can more effectively inhibit the colonization and activity of Helicobacter pylori. In addition to being related to the improved gastric environment, fermentation may have directly produced certain bacteriocins or antibacterial metabolites with inhibitory effects on Helicobacter pylori.

[0033] (8) In the ethanol-induced gastric mucosal injury model, the gastric ulcer index of the Bacillus vesiculosus-fermented Codonopsis pilosula group was significantly lower, the ulcer area was smaller, and the healing was faster. This directly demonstrates the protective effect of fermented Codonopsis pilosula against acute chemical injury and its ability to promote ulcer healing, comprehensively reflecting its overall advantages in anti-inflammatory, antioxidant, and cell protection aspects.

[0034] (9) Validation results obtained using different injury mechanism models showed that the Bacillus vesiculosus fermented Codonopsis pilosula product exhibited stable and consistent repair effects in both infectious and chemical gastric mucosal injury models. This indicates that the fermented product does not act on a single injury pathway, but rather promotes the recovery of gastric mucosal structure and function through multiple synergistic mechanisms.

[0035] (10) Compared with unfermented Codonopsis pilosula, the present invention significantly improves the bioavailability and biological effects of active ingredients through Bacillus belye fermentation, thereby exhibiting superior efficacy in repairing gastric mucosal damage. Furthermore, compared with current gastric mucosal protection methods that primarily rely on a single mechanism of action, the fermentation product described in this invention has advantages such as milder action, higher safety, and wider applicability, making it more suitable for long-term use or as an adjunct to other therapies.

[0036] (11) Based on the above experimental results, the fermentation product of Bacillus vesiculosus fermented Codonopsis pilosula provided by the present invention can be used to prepare drugs to repair gastric mucosal damage, providing a new method for the intervention of gastric mucosal damage-related diseases, and has good application prospects. Attached Figure Description

[0037] Figure 1 Pathological section of gastric mucosa tissue from mouse H&E;

[0038] Figure 2 A histogram showing the pathological scores of mouse gastric mucosal tissue;

[0039] Figure 3 The graphs are bar charts of inflammatory factors in mouse cells, where a is the IL-6 bar chart, b is the IL-8 bar chart, c is the TNF-α bar chart, and d is the IL-10 bar chart.

[0040] Figure 4 The graphs are bar charts representing oxidative stress in mice, where a is the SOD bar chart and b is the MDA bar chart.

[0041] Figure 5 A bar graph of tight junction proteins in mouse gastric tissue;

[0042] Figure 6 The graph shows the relationship between gastric acid secretion and pepsin activity. In the graph, a is a bar chart of the pH value of mouse gastric juice; b is a bar chart of mouse pepsinogen I / II.

[0043] Figure 7 Comparison of urease activity detection in rat gastric juice;

[0044] Figure 8 Pathological section of gastric mucosa tissue from mouse H&E;

[0045] Figure 9 A histogram showing the pathological scores of mouse gastric mucosal tissue;

[0046] Figure 10 The graphs are bar charts of inflammatory factors in mouse cells, where a is the IL-6 bar chart, b is the IL-8 bar chart, c is the TNF-α bar chart, and d is the IL-10 bar chart.

[0047] Figure 11 The charts show bar charts for oxidative stress in mice, with a for SOD stress and b for MDA stress.

[0048] Figure 12 A bar graph of tight junction proteins in mouse gastric tissue;

[0049] Figure 13The graph shows the relationship between gastric acid secretion and pepsin activity. In the graph, a is a bar chart of the pH value of mouse gastric juice; b is a bar chart of mouse pepsinogen I / II.

[0050] Figure 14 A histogram of the gastric mucosal ulceration index in mice;

[0051] Figure 15 The phylogenetic tree analysis results for Bacillus belyssus TK1926 are shown in the figure. Detailed Implementation

[0052] To make the objectives, technical solutions, and advantages of this invention clearer, the specific embodiments and accompanying drawings are described in further detail below. Where specific techniques or conditions are not specified in the embodiments, they are performed according to the techniques or conditions described in the literature in this field or according to the product instructions. Reagents or instruments whose manufacturers are not specified are all commercially available conventional products.

[0053] To facilitate understanding of the technical solutions of this invention, unless otherwise stated, the following terms used in this specification have the following meanings. Those skilled in the art should understand that the following definitions are for illustrative purposes only and should not be construed as limiting the scope of protection of this invention.

[0054] Terminology Explanation:

[0055] In this invention, "fermented Codonopsis pilosula" or "Bacillus velezensis fermented Codonopsis pilosula" both refer to the fermentation system and its products obtained by using Codonopsis pilosula as a fermentation substrate and acting on it with Bacillus velezensis. The fermentation process may be accompanied by the biotransformation, degradation or metabolite generation of components in Codonopsis pilosula. The preferred Bacillus velezensis is Bacillus velezensis TK1926, which was deposited on December 10, 2025, at the China General Microbiological Culture Collection Center (CGMCC). The address of the depository is No. 3, Courtyard 1, Beichen West Road, Chaoyang District, Beijing. The classification name is Bacillus velezensis, and the accession number is CGMCC No:36999.

[0056] It should be noted that fermented Codonopsis pilosula does not limit the specific fermentation method, inoculation method, or fermentation container form.

[0057] In this invention, "fermentation product" refers to the substance obtained by fermenting Codonopsis pilosula with Bacillus belye TK1926, including but not limited to fermentation broth, fermentation supernatant, fermentation extract, and its freeze-dried or concentrated form. Of course, the fermentation product can contain bacterial components or be a processed product without live bacteria.

[0058] In this invention, "gastric mucosal injury" specifically refers to a state of damage to the structure or function of the gastric mucosa caused by infectious or non-infectious factors, including but not limited to mucosal congestion, edema, erosion, bleeding, inflammatory infiltration, or impaired barrier function. It can be caused by Helicobacter pylori infection, chemical irritation (such as ethanol), drug irritation, or stress factors.

[0059] In this invention, "gastric mucosal repair" refers to the repair of damaged gastric mucosa. This process improves the condition of the damaged gastric mucosa by promoting the recovery of its tissue structure, reducing inflammation, enhancing mucosal barrier function, or improving pathological morphology. Repair is not limited to a complete return to normal; any improvement shown in pathological or functional indicators is considered sufficient to repair gastric mucosal damage.

[0060] In this invention, "Helicobacter pylori-associated gastric mucosal injury" refers to a state of gastric mucosal inflammation and tissue damage caused or involved by Helicobacter pylori infection, characterized by elevated levels of inflammatory factors, destruction of gastric mucosal structure, or decreased barrier function.

[0061] In this invention, "chemically irritated gastric mucosal injury" refers to the state of injury caused by chemical irritants acting on the gastric mucosa, and the chemical irritants are not limited to ethanol.

[0062] In this invention, "composition" refers to a formulation system comprising the fermentation product described in this invention and pharmaceutically acceptable excipients, and may be a pharmaceutical composition. "Pharmaceutically acceptable excipients" refers to carriers, excipients, diluents, or stabilizers that are safe for human use within a reasonable dosage range and do not affect the gastric mucosal repair function of the fermentation product.

[0063] To obtain the optimal fermentation substrate, this invention first conducted parallel fermentation screening on 33 common Chinese medicinal herbs (including: Bupleurum chinense, Atractylodes macrocephala, Paeonia suffruticosa, Poria cocos, Alisma plantago-aquatica, Angelica sinensis, Citrus aurantium, Epimedium brevicornu, Coix lacryma-jobi, Magnolia officinalis, Cannabis sativa, Cinnamomum cassia, Dioscorea opposita, Lycium barbarum, Lilium brownii, Nelumbo nucifera, Polygonatum sibiricum, Citrus reticulata peel, Crataegus pinnatifida, Euryale ferox, Dolichos lablab, Polyporus umbellatus, Amomum villosum, Alpinia oxyphylla, Polygonatum odoratum, Pueraria lobata, Cassia tora, Cornus officinalis, Codonopsis pilosula, Coptis chinensis, Gardenia jasminoides, Taraxacum mongolicum powder, and raw malt powder), including the following steps:

[0064] Each medicinal material was prepared into a powder suspension of the same concentration (12% w / v) to serve as the fermentation substrate;

[0065] Inoculate with the same amount of Bacillus vesiculus seed culture and carry out liquid fermentation under completely identical fermentation conditions (temperature, time, pH, stirring rate, etc.);

[0066] After fermentation, the supernatant of each fermentation was collected, and the diameter of the inhibition zone against Escherichia coli (a representative Gram-negative bacterium) was determined using the Oxford cup method. Preliminary antibacterial activity can indirectly reflect the amount or conversion efficiency of antibacterial and potential anti-inflammatory components in the fermentation product.

[0067] In this systematic screening, the supernatant of the *Bacillus belye*-fermented *Codonopsis pilosula* group showed the most significant inhibition zone diameter, suggesting that fermentation may have produced richer or stronger bioactive metabolites. Based on this, *Codonopsis pilosula* was selected as the optimal fermentation substrate for further in-depth development and efficacy verification.

[0068] The first aspect of this invention is to provide an application of Bacillus velezensis TK1926 in the preparation of a gastric mucosa repair drug. Bacillus velezensis TK1926 was deposited on December 10, 2025, at the China General Microbiological Culture Collection Center (CGMCC), located at No. 3, Courtyard 1, Beichen West Road, Chaoyang District, Beijing, with accession number CGMCC No:36999.

[0069] A second aspect of this invention provides a fermentation product obtained by fermenting Codonopsis pilosula with Bacillus veitchii TK1926, which has the effect of repairing gastric mucosal damage. By selecting Bacillus veitchii TK1926 to ferment Codonopsis pilosula raw material, the metabolic transformation ability of Bacillus veitchii is utilized to cause some components in Codonopsis pilosula to undergo biotransformation, thereby obtaining a fermentation product with enhanced biological activity in repairing gastric mucosal damage.

[0070] In this invention, the Codonopsis pilosula raw material can be the medicinal root of Codonopsis pilosula, or it can exist in sliced ​​or powdered form. The Codonopsis pilosula raw material can be washed, pulverized, extracted, or sterilized before fermentation, but this invention does not limit the specific processing methods; the purpose of pretreatment is only to improve the stability and repeatability of the fermentation process. The Codonopsis pilosula raw material can be used directly as a fermentation substrate, or it can be mixed with water or a buffer solution to form a fermentation system.

[0071] In some implementations, the fermentation process includes:

[0072] The processed Codonopsis pilosula is crushed and sieved to obtain Codonopsis pilosula powder; it is added to a fermentation container and mixed with water to form a fermentation system, which is then inoculated with Bacillus belye and fermented under suitable conditions.

[0073] The fermentation temperature can be 30~37℃, and the culture can be shaken at 150-200 rpm. The fermentation time can be 24~48h. After fermentation, the fermentation broth or fermentation supernatant can be obtained by centrifugation, filtration, etc., and can be further concentrated, freeze-dried or extracted to obtain fermentation products in different forms.

[0074] More specifically, the preparation method of Codonopsis pilosula fermented with Bacillus belysinensis includes the following steps:

[0075] The preserved Bacillus belye was streaked onto a nutrient broth solid medium and incubated at 35-37℃ for 18-24 hours.

[0076] Pick a single colony and inoculate it into primary seed liquid medium, and incubate at 35-37℃ and 150-200 rpm for 20-24 hours with shaking.

[0077] Transfer the primary seed culture to the secondary seed culture medium at a volume ratio of 2%-5%, and culture under the same conditions for 20-24 hours to obtain a high-density bacterial suspension, which is used as the inoculum.

[0078] The dried Codonopsis pilosula slices are pulverized and passed through a 300-500 mesh sieve to obtain Codonopsis pilosula powder. The powder is dissolved in sterile water at a mass ratio of 1:10-18, stirred until homogeneous, packaged, sterilized at 121℃ for 30 minutes, and then cooled for later use.

[0079] Under aseptic conditions, the secondary seed culture obtained in the above steps is inoculated into the Codonopsis pilosula matrix prepared in the above steps at an inoculation rate of 2%-5% (v / v), and cultured with shaking at 30-37℃ and 150-200 rpm for 24-48 hours.

[0080] Furthermore, the post-processing and vacuum freeze-drying of the Bacillus vesicular fermented Codonopsis pilosula specifically includes the following steps:

[0081] After fermentation, the fermentation broth was directly subjected to vacuum freeze-drying: first, it was pre-frozen at a depth below -40℃ for 2-4 hours; then, under a vacuum of 10-30 Pa, it was sublimated and desorbed by staged heating (maintaining -20℃ to -10℃ for 20-40 hours, then raising it to 25-35℃ and maintaining it for 6-12 hours) to obtain a loose and porous freeze-dried powder. Meanwhile, the unfermented Codonopsis pilosula reference standard used in the experiment required the same freeze-drying process.

[0082] The fermentation products obtained according to the present invention can be in liquid, semi-solid, or solid form, particularly fermentation broth, fermentation supernatant, fermentation extract, or lyophilized powder. These products can be used alone or in combination with pharmaceutical or food excipients to prepare compositions. The fermentation products obtained by the above methods maintain their stability, thereby facilitating the development and application of subsequent formulations.

[0083] The fermentation product provided by this invention exhibits excellent repair effects on the gastric mucosa, demonstrating good repair efficacy in both Helicobacter pylori-induced gastric mucosal damage models and chemically induced gastric mucosal damage models (e.g., ethanol-induced damage). In the Helicobacter pylori-induced damage model, the fermentation product can alleviate the inflammatory response of the gastric mucosa caused by infection and promote the repair of damaged gastric mucosal structures. In the chemically induced damage model, the fermentation product can reduce pathological changes such as congestion, edema, and erosion of the gastric mucosa and limit the extent of damage. These results indicate that the fermentation product of this invention is suitable for repairing gastric mucosal damage caused by multiple damage mechanisms, and is not limited to a single model.

[0084] In view of the above-mentioned technical effects, the fermentation product or its composition described in this invention can be used to prepare a medicine for repairing gastric mucosal damage. The route of administration can be oral, and the dosage form can be a liquid preparation, a solid preparation, or any other preparation suitable for gastric action.

[0085] The invention has now been generally described, and will be more readily understood by referring to the following embodiments, which are provided by way of example and not by way of limitation.

[0086] The embodiments of this invention employ two different gastric mucosal injury models, each with a clearly defined scoring system, such as... Figure 2 (Corresponding to the pyloric infection model) and Figure 9 (As shown in the ethanol-induced model), the histopathological scores of both models follow the conventional semi-quantitative scoring methods in this field.

[0087] The scoring criteria and data processing methods for the Helicobacter pylori infection model are as follows:

[0088] (1) The gastric mucosal histopathological scoring of the pyloric infection model includes the following four evaluation dimensions:

[0089] Inflammatory cell infiltration: Assess the degree of infiltration of inflammatory cells (such as neutrophils and lymphocytes) in the gastric mucosa and submucosa;

[0090] Glandular atrophy: Assess the degree of reduction, structural damage, or disappearance of the gastric intrinsic glands;

[0091] Intestinal metaplasia: assessing the replacement of gastric mucosal epithelium with intestinal-type epithelium;

[0092] Atypical hyperplasia: assessing the degree of structural and cellular atypia of gastric mucosal glands.

[0093] (2) A semi-quantitative scoring method of 0-3 points is used for all four dimensions. The specific standards are as follows:

[0094] 0 points: No obvious lesions were observed, and the tissue structure was normal;

[0095] 1 point: Mild lesion (mild inflammatory cell infiltration, slight atrophy of individual glands, focal intestinal metaplasia or mild dysplasia);

[0096] 2 points: Moderate lesion (moderate inflammatory cell infiltration, partial glandular atrophy, multifocal intestinal metaplasia or moderate dysplasia);

[0097] 3 points: Severe lesion (diffuse inflammatory cell infiltration, widespread atrophy and disappearance of glands, widespread intestinal metaplasia or severe dysplasia).

[0098] (3) Calculation method of total score

[0099] Gastric mucosal histopathological score = inflammatory cell infiltration score + glandular atrophy score + intestinal metaplasia score + dysplasia score.

[0100] The scoring criteria and data processing methods for the ethanol-induced model are as follows:

[0101] (1) Scoring Dimensions

[0102] The histopathological scoring of gastric mucosa in the ethanol-induced model includes the following three evaluation dimensions: inflammatory cell infiltration, glandular atrophy, and dysplasia;

[0103] It should be noted that the acute ethanol injury model usually does not involve intestinal metaplasia, a chronic lesion indicator. Therefore, the scoring dimensions differ from those of the pyloric infection model, which is consistent with the pathological characteristics of different injury models.

[0104] (2) The 0-3 point semi-quantitative scoring method was adopted, with the same standard as the scoring of the pyloric infection model.

[0105] (3) Calculation method of total score

[0106] Gastric mucosal histopathological score = inflammatory cell infiltration score + glandular atrophy score + dysplasia score.

[0107] Example 1

[0108] 1. Isolation and screening of Bacillus belyssus TK1926:

[0109] Using fermented soybean paste as the isolation source, 5g of the sample to be isolated was placed in a test tube, and an appropriate amount of sterile physiological saline was added to dilute it. After shaking and mixing, the diluted sample was spread onto a nutrient broth solid medium. The spread plates were incubated at 37℃ for 24h, and the colony morphology was observed and recorded. Single colonies were picked and cultured in shake flasks at 220rpm and 37℃ for 48h. The target microorganisms of the single colonies were then screened. 600μL of the bacterial solution was placed in a sterile centrifuge tube, and 200μL of 30% glycerol was added. The solution was then stored at -80℃ to obtain the bacterial strain.

[0110] 2. Strain identification:

[0111] The isolated strain was amplified using universal 16S rDNA primers: 27F 5'-AGAGTTTGATCCTGGCTCAG-3' and 1492R 5'-GGTTACCTTGTTACGACTT-3'. The amplified products were sequenced to obtain the specific gene sequences, which were then uploaded and compared with the NCBI database. The results showed 100% similarity to *Bacillus belyssii*. Phylogenetic tree analysis was also performed, and the constructed results are shown below. Figure 15 As shown.

[0112] The sequencing results were compared with the nucleic acid sequences in NCBI, and a phylogenetic tree was constructed. Finally, Bacillus belysium was obtained and named Bacillus belysium TK1926.

[0113] The 16S rDNA sequencing sequence of the bacterial strain is shown in SEQ ID NO:1.

[0114] Example 2

[0115] Screening of fermented Chinese medicinal materials with antibacterial activity.

[0116] 1. Materials

[0117] Bacterial strain: Bacillus velezensis CGMCC No. 36999.

[0118] Chinese medicinal herbs: Bupleurum, Atractylodes macrocephala, Moutan bark, Poria cocos, Alisma plantago-aquatica, Angelica sinensis, Citrus aurantium, Epimedium, Coix seed, Magnolia officinalis, Cannabis sativa seed, Cinnamon twig, Dioscorea opposita, Lycium barbarum, Lilium brownii, Nelumbo nucifera seed, Polygonatum sibiricum, Citrus reticulata peel, Crataegus pinnatifida, Euryale ferox seed, Dolichos lablab, Polyporus umbellatus, Amomum villosum, Alpinia oxyphylla, Polygonatum odoratum, Pueraria lobata, Cassia tora seed, Cornus officinalis, Codonopsis pilosula, Coptis chinensis, Gardenia jasminoides, Taraxacum mongolicum, and Hordeum vul

[0119] Indicator bacterium: Escherichia coli.

[0120] Culture media: LB medium, nutrient broth medium.

[0121] 2. Methods

[0122] 2.1 Activation and scale-up culture of bacterial strains

[0123] (1) Purification of bacterial strains

[0124] Remove the glycerol cryovial of Bacillus velezensis TK1926 (CGMCC No. 36999) stored at -80℃ and thaw it on ice. Using an inoculation loop, take a small amount of the bacterial suspension and streak it onto a nutrient broth solid medium plate (10 g / L peptone, 5 g / L beef extract, 5 g / L sodium chloride, 15 g / L agar powder, pH 7.2±0.2). Incubate upside down in a 37℃ incubator for 24 hours until single colonies are fully formed.

[0125] (2) Large-scale culture of microbial strains

[0126] Preparation of primary seed culture: Pick a plump single colony from the above plate and inoculate it into a 250 mL Erlenmeyer flask containing 50 mL of nutrient broth liquid seed culture medium. Incubate at 37℃ and 220 rpm for 20-24 h to obtain primary seed culture.

[0127] Preparation of secondary seed culture (fermentation inoculum): Transfer the primary seed culture to a secondary liquid seed culture medium Erlenmeyer flask at an inoculation rate of 2%-5% (v / v). Continue culturing under the same conditions (37℃, 220 rpm) for 20-24 hours until the cell growth enters the late logarithmic phase. This secondary seed culture is then used for inoculation.

[0128] 2.2 Preparation of Fermentation Substrate for Traditional Chinese Medicine

[0129] Weigh 6 g of each herbal powder and place them separately in 250 ml Erlenmeyer flasks. Add 50 ml of purified water, dilute, and stir to dissolve. Sterilize at 121°C for 20 minutes and then cool.

[0130] 2.3 Inoculation and Fermentation

[0131] Under aseptic conditions, the secondary seed liquid prepared in step 2.1 was inoculated into the traditional Chinese medicine fermentation substrate prepared in step 2.2 at an inoculation rate of 2% (v / v).

[0132] The culture medium after inoculation was placed in a shaker (220 rpm) and cultured at 37°C with ventilation for 24 hours.

[0133] 2.4 Fermentation product treatment

[0134] After fermentation, the fermentation broth was centrifuged at 4℃ and 12000 rpm for 10 min, and the supernatant was collected to obtain fermentation supernatant samples of each Chinese herbal medicine. These samples were stored at 4℃ for later use.

[0135] 2.5 Determination of antibacterial activity (Oxford cup method)

[0136] a. After activating the Escherichia coli indicator bacteria, adjust the bacterial concentration to 1×10⁻⁶ with sterile physiological saline. 8 CFU / mL.

[0137] b. Spread 100 μL of bacterial suspension evenly onto an LB agar plate.

[0138] c. Place sterile Oxford cups at equal intervals on a plate, and add 200 μL of the corresponding herbal fermentation supernatant to each Oxford cup. Each group should have 3 replicates.

[0139] d. After diffusion at 4℃ for 4 h, transfer the plate to a 37℃ incubator for 18-24 h.

[0140] e. Measure the diameter of the inhibition zone (including the outer diameter of the Oxford cup) using vernier calipers and take the average value.

[0141] 3. Results

[0142] The inhibition zone assay results showed that among the Bacillus vesiculosus fermentation supernatants of 33 kinds of Chinese medicinal materials, the fermented Codonopsis pilosula supernatant produced the largest inhibition zone diameter, (20.5 ± 1.8) mm, significantly larger than that of the other fermentation groups. No inhibition zone was observed in the unfermented Codonopsis pilosula aqueous extract. These results indicate that Bacillus vesiculosus fermentation significantly enhances the antibacterial activity of Codonopsis pilosula, suggesting that it may have produced new or higher concentrations of antibacterial and anti-inflammatory active ingredients through biotransformation. Therefore, Codonopsis pilosula is the preferred fermentation substrate for further in-depth research and development.

[0143] Example 3

[0144] Preparation of Codonopsis pilosula samples fermented by Bacillus belyssus TK1926.

[0145] 1. Activation and scale-up culture of microbial strains

[0146] Same as step 2.1 in Example 1.

[0147] 2. Preparation of Codonopsis pilosula fermentation substrate

[0148] Weigh out the Codonopsis pilosula slices, break down their cell walls and grind them into a fine powder with a particle size of about 400 mesh. Add 8-16 times (w / v) of purified water, dilute and stir to dissolve. Based on this Codonopsis pilosula solution, you can selectively add 0.5%-3% glucose or jujube powder as an auxiliary carbon source, and 0.1%-1% soybean powder or yeast extract as a nitrogen source. Adjust the pH to 6.5-7.5, dispense into fermentation Erlenmeyer flasks, sterilize at 121℃ for 20 minutes, cool, and you will get the liquid fermentation culture medium.

[0149] 3. Inoculation and Fermentation

[0150] Under aseptic conditions, the secondary seed culture prepared in step 1 was inoculated into the Codonopsis pilosula liquid fermentation medium prepared in step 2 at an inoculation rate of 2%-5% (v / v).

[0151] Place the inoculated culture medium in a shaker (180-220 rpm) and incubate at 30-37℃ with ventilation for 20-36 hours.

[0152] 4. Pretreatment and vacuum freeze drying

[0153] After fermentation, the fermentation product (fermentation broth) is transferred to the tray of a vacuum freeze dryer and spread into a uniform thin layer with a thickness of no more than 10 mm.

[0154] The pretreated fermentation product was placed in a vacuum freeze dryer and processed according to the following procedure:

[0155] Pre-freezing stage: The material temperature is rapidly reduced to below -40℃ and maintained for 2-4 hours to completely freeze the material.

[0156] First drying stage (sublimation drying): Start the vacuum system and reduce the pressure inside the drying chamber to 10-30 Pa. Under this vacuum condition, slowly raise the shelf temperature to -20℃ to -10℃ and maintain it for 20-40 hours, causing most of the free ice crystals in the material to sublimate.

[0157] Analysis and drying (secondary drying) stage: The shelf temperature is further slowly raised to 25℃-35℃ and maintained for 6-12 hours to remove the remaining adsorbed water in the material.

[0158] Vacuum breaking and discharge: After drying, sterile nitrogen or dry air is introduced to atmospheric pressure, and the dried product is removed. A freeze-dried Codonopsis pilosula powder with uniform color and a loose, porous, sponge-like texture is obtained.

[0159] Comparative Example 1

[0160] Preparation of unfermented Codonopsis pilosula samples

[0161] Parallel processing was performed on the unfermented Codonopsis pilosula control sample:

[0162] The unfermented Codonopsis pilosula reference standard (Codonopsis pilosula powder solution of the same concentration) used for the experimental comparative example was subjected to the same sterilization treatment and then to the same vacuum freeze-drying process as described above.

[0163] Example 4

[0164] Study on the protective and therapeutic effects of Bacillus vesiculosus TK1926 fermented Codonopsis pilosula preparation on gastric mucosal damage induced by Helicobacter pylori infection.

[0165] 1. Animal selection: Strain: SPF-grade C57BL / 6 mice (male, 6-8 weeks old, weighing 18-22g); acclimatization for 7 days; Number: 6 mice per group. Drug: Lyophilized powder prepared in Example 2 and Comparative Example 2.

[0166] 2. Grouping

[0167] Blank control group (normal diet + saline) N=6; Hp model group (Hp infection + saline) N=6; Hp positive drug group (quadruple therapy (omeprazole + amoxicillin + clarithromycin + bismuth potassium citrate), administered post-infection) N=6; Hp protection group - unfermented Codonopsis pilosula (administered pre-infection + Hp infection) - sample N=6; Hp protection group - fermented Codonopsis pilosula (administered pre-infection + Hp infection) - sample N=6; Hp treatment group - unfermented Codonopsis pilosula (administered post-infection + Hp infection) - sample N=6; Hp treatment group - fermented Codonopsis pilosula (administered post-infection + Hp infection) - sample N=6

[0168] 3. Establishment of Hp infection model

[0169] First, antibiotics were used to eliminate stomach bacteria: Mice were first administered a mixed antibiotic solution (azithromycin 10 mg / ml, ampicillin 10 mg / ml, gentamicin 1.2 mg / ml) by gavage for one week, 0.2 ml daily for 7 consecutive days, followed by pyloric transplantation seven days later. Mice were fasted for 24 hours and deprived of water for 6 hours before transplantation. One hour before transplantation, 0.2 mL of 2% sodium bicarbonate solution was administered by gavage, and 1 × 10⁻⁶ liters of pyloric bacteria solution were administered daily by gavage. 9 CFU was administered via gavage on days 1, 4, 7, 11, and 15 for colonization. Once a positive urine test was confirmed, gavage treatment could begin.

[0170] 4. Dosing regimen

[0171] Hp protection group: The experimental samples were administered via gavage 4 weeks before infection, at fixed time points, and the medication was continuously administered during the infection period (administered 1 hour before gavage). The medication was discontinued after infection.

[0172] Hp treatment group: medication was started on day 1 after infection and continued for 4 weeks, in sync with the protection group.

[0173] Hp-positive drug group: Treatment began on day 1 after infection, using quadruple therapy (omeprazole 6 mg / kg, bismuth potassium citrate 75 mg / kg, clarithromycin 75 mg / kg, amoxicillin 150 mg / kg), twice daily for 14 days, followed by observation for 2 weeks after discontinuation of treatment. The control group and model group received normal saline.

[0174] Example 1 and Comparative Example 1 sample gavage dosage: The mice were designed to be gavaged twice a day, therefore, the single gavage dose was 0.81 g / kg.

[0175] 5. Detection Indicators

[0176] 5.1 H&E gastric mucosal histopathological scoring and section observation

[0177] Hematoxylin and eosin (H&E) staining of gastric tissue can observe the degree of gastric mucosal damage, quantify inflammatory cell infiltration, glandular atrophy, and intestinal metaplasia. It can clarify the type of pathological changes (such as mucosal epithelial dissolution and hemorrhagic foci formation) and the state of repair (such as new epithelial coverage), directly assess the degree of gastric mucosal damage, and provide morphological basis for inflammation grading.

[0178] 5.2 Inflammatory factors

[0179] ELISA kits detect serum cytokine concentrations. During an inflammatory response, blood cytokine levels change, including pro-inflammatory factors (TNF-α, IL-8, IL-6) and anti-inflammatory factors (IL-10). *Helicobacter pylori* (Hp) promotes IL-8 secretion by activating the NF-κB pathway, mediating gastric epithelial cell apoptosis. Elevated IL-10 levels reflect activation of anti-inflammatory repair mechanisms.

[0180] 5.3 Oxidative stress markers (superoxide dismutase SOD, malondialdehyde MDA)

[0181] MDA is an end product of lipid peroxidation, reflecting the degree of oxidative damage to cell membranes; ELISA kits use specific antibodies to detect MDA concentrations in gastric tissue or serum. Superoxide dismutase (SOD) is detected spectrophotometrically; SOD is a key antioxidant enzyme that scavenge superoxide radicals, and decreased SOD activity indicates impaired antioxidant capacity.

[0182] 5.4 Tight junction proteins

[0183] ELISA was used to detect tight junction protein (ZO-1) expression to assess the damage of *Helicobacter pylori* (Hp) to the mucosal barrier. The repair effect on the gastric barrier was validated, and the integrity of the mucosal barrier was evaluated.

[0184] 5.5 Gastric acid secretion and pepsin activity

[0185] Gastric acid secretion intensity can be assessed immediately by measuring the pH of gastric juice (normal range 1.5-3.5). Pepsin activity is highly correlated with pH (optimal pH ≈ 2.0), and activity decreases significantly when the pH deviates from this range. *Helicobacter pylori* infection can inhibit gastric acid secretion; gastric function impairment can be assessed by detecting gastric juice pH and the pepsinogen I / II ratio. A high-acid environment exacerbates mucosal corrosion, and inhibiting gastric acid secretion is one of the repair mechanisms. The PGI / PGII (pepsinogen I / pepsinogen II) ratio is an important indicator for screening gastric diseases; a decreased ratio may indicate an increased risk of diseases such as gastric mucosal atrophy.

[0186] 5.6 Urease activity in gastric juice

[0187] The colonization status of *Helicobacter pylori* (Hp) can be rapidly determined by detecting urease activity in gastric tissue homogenate or gastric juice. Urease secreted by *Hp* decomposes urea to produce ammonia and carbon dioxide, neutralizing gastric acid to promote its own survival; its activity directly reflects bacterial colonization density and infection intensity. This study validates the success of infection in animal models and evaluates the effectiveness of interventions in bacterial clearance.

[0188] 6. Results Analysis

[0189] Note: In the figure, N represents the blank group, M represents the model group, H represents the positive drug group, UF represents the unfermented Codonopsis pilosula experimental group, and F represents the fermented Codonopsis pilosula experimental group; P represents the protection group and T represents the treatment group.

[0190] 6.1 H&E gastric mucosal histopathological scoring and section observation

[0191] Pathological section observation reveals that, Figure 1 In the blank control group N mice, the mucosal epithelial cells and lamina propria structure were clear and intact, and no obvious pathological changes such as mucosal damage were observed. Figure 1 In the model group M mice, a large number of inflammatory cells infiltrated the gastric mucosa, and the lamina propria and submucosa were loose, with a pathological score significantly higher than that of the normal group (P<0.001). In the experimental groups, the fermented Codonopsis pilosula protection group and the treatment group... Figure 1 The gastric mucosa of mice showed milder congestion and bleeding, shallower lesions, and reduced local necrosis. The pathological scores of both the protection group (P<0.001) and the treatment group (P<0.001) were significantly lower than those of the model group, and the treatment group showed better results than the protection group. This indicates that Codonopsis pilosula fermented with Bacillus belycei has a better effect on improving the pathological damage to the gastric mucosa induced by Helicobacter pylori in mice than unfermented Codonopsis pilosula.

[0192] 6.2 Inflammatory Factors

[0193] Experimental results are as follows Figure 3 As shown, Figure 3In the diagram, 'a' represents IL-6. The levels of TNF-α, IL-8, and IL-6 in the model group were significantly higher (P<0.001) than in the control group, while the IL-10 level was significantly lower, indicating that the Helicobacter pylori infection model was successfully established and the mice in the model group developed an inflammatory response. The levels of TNF-α, IL-8, and IL-6 in both the protective and treatment groups were lower than in the model group, while the IL-10 level was higher. Furthermore, the levels of TNF-α, IL-8, and IL-6 in both the fermented Codonopsis pilosula protective and treatment groups were significantly lower (P<0.001) than in the model group, while the IL-10 level was significantly higher (P<0.001). Fermented Codonopsis pilosula was more effective than unfermented Codonopsis pilosula, and the overall therapeutic effect was better than the protective effect. This indicates that the experimental samples can upregulate anti-inflammatory factors and downregulate pro-inflammatory factors in Helicobacter pylori-infected gastric tissue, and simultaneously have a protective and therapeutic effect against Helicobacter pylori infection in mice.

[0194] 6.3 Markers of Oxidative Stress

[0195] Experimental results are as follows Figure 4 As shown, by Figure 4 As can be seen from 'a', the SOD levels in both the protective and treatment groups of the experimental samples were higher than those in the model group, and the SOD level in the treatment group was higher than that in the protective group. Figure 4 Figure b shows that the MDA levels in the experimental sample protection group and treatment group were significantly lower than those in the model group (P<0.001); the improvement in SOD and MDA levels in mice after fermented Codonopsis pilosula intervention was better than that in unfermented Codonopsis pilosula, indicating that the degree of oxidative damage in mice after fermented Codonopsis pilosula intervention was lower.

[0196] 6.4 Tight junction proteins

[0197] Experimental results are as follows Figure 5 As shown, Figure 5 The ZO-1 expression level in the model group was significantly lower than that in the blank group. In the experimental group, the ZO-1 expression levels in both the fermented and unfermented Codonopsis pilosula groups were significantly higher than those in the model group (P<0.001), and the fermented Codonopsis pilosula group was higher than that in the unfermented Codonopsis pilosula group. The therapeutic effect was better than the protective effect, indicating that fermented Codonopsis pilosula has a protective and improving effect on repairing the gastric barrier.

[0198] 6.5 Gastric acid secretion and pepsin activity

[0199] Experimental results are as follows Figure 6 As shown, Figure 6 The pH value of the model group (a) was significantly higher than that of the control group (P<0.001). Figure 6The result in b shows that the PGI / PGII ratio was significantly lower than that of the control group, indicating that the Helicobacter pylori infection model was successfully established. The pH values ​​of both the fermented Codonopsis pilosula protection group and the treatment group were significantly (P<0.001) lower than those of the model group, while the PGI / PGII ratios were significantly higher. This suggests that fermented Codonopsis pilosula can regulate the gastric environment in Helicobacter pylori-infected areas and inhibit gastric acid secretion.

[0200] 6.6 Urease Activity Detection

[0201] Experimental results are as follows Figure 7 As shown, the rapid urease test results indicated that the model group was purplish-red, a positive result, indicating successful *Helicobacter pylori* colonization. The unfermented *Codonopsis pilosula* group was light pink, a weak positive result; the fermented *Codonopsis pilosula* group was orange-red, a weak positive result. This suggests that the amount of ammonia produced in the experimental groups after drug intervention was lower, and the fermented group showed better results, resulting in a less significant increase in pH, possibly due to a lower *Helicobacter pylori* count.

[0202] Example 5

[0203] Study on the protective and therapeutic effects of Bacillus vesiculosus TK1926 fermented Codonopsis pilosula on ethanol-induced acute gastric mucosal injury.

[0204] 1. Animal selection, strain: same as in Example 3.

[0205] 2. Grouping

[0206] The blank control group (normal diet + saline) was used in conjunction with normal controls of Helicobacter pylori animals; the ethanol model group (ethanol stimulation + saline); the ethanol positive drug group (sucralfate); the ethanol protection group (administered before ethanol stimulation); and the ethanol treatment group (administered after ethanol stimulation).

[0207] 3. Establishment of the ethanol stimulation model

[0208] Each group was administered the corresponding drug via gavage. The ethanol protection group began gavage administration of experimental samples 14 days prior to stimulation, continuing until 24 hours after stimulation. An additional dose was administered before ethanol gavage on the day of stimulation. The ethanol model group and the ethanol blank control group were given physiological saline (0.9%). The positive control group was administered ranitidine via gavage once daily for 14 consecutive days. On day 13, mice were fasted for 24 hours, but water was permitted. Three hours after gavage on day 14, except for the blank control group which was administered physiological saline (15 mL / kg), all other groups were administered 75% ethanol (0.5 mL / 100g body weight) via gavage to induce acute gastric mucosal injury. The ethanol treatment group was administered experimental samples immediately after stimulation, and the administration was repeated at 6 and 12 hours after stimulation (a total of 3 times).

[0209] 4. Dosing regimen

[0210] Example 2 and Comparative Example 1 sample gavage dosage: The mice were designed to be gavaged twice a day, therefore, the single gavage dose was 0.81 g / kg.

[0211] The blank control group (normal diet + saline) had N=6; the model group (saline) had N=6; the positive control group (ranitidine administered by gavage) had N=6; the protection group (unfermented Codonopsis pilosula samples) had N=6; the ethanol protection group (14 days before stimulation) had N=6; the protection group (fermented Codonopsis pilosula samples) had N=6; the treatment group (unfermented Codonopsis pilosula samples) had N=6; and the treatment group (fermented Codonopsis pilosula samples) had N=6. The ulcer index was calculated by observing the degree of gastric mucosal damage, verifying the successful modeling.

[0212] 5. Detection Indicators

[0213] The common detection indicators are the same as those in Example 2, 5.1-5.5.

[0214] 5.6 Gastric Ulcer Index

[0215] After dissection, the stomach tissue was cut along the greater curvature, and the gastric mucosal damage (such as bleeding points, linear ulcers, etc.) was observed and scored. The gastric ulcer index directly quantifies the severity of gastric mucosal damage and is a core indicator for assessing the success of model establishment and the efficacy of drugs.

[0216] 6. Results and Analysis

[0217] 6.1 H&E gastric mucosal histopathological scoring and section observation

[0218] Experimental results of the ethanol stimulation model are as follows: Figures 8-9 As shown, Figure 8 In the control group, the mucosal epithelial cells and lamina propria of the mice were clearly intact, with no obvious pathological changes such as mucosal damage. In the model group, the entire gastric mucosa of the mice showed obvious damage, including mucosal sloughing defects and vascular congestion. Figure 9 The pathological score of the group was significantly higher than that of the normal group (P<0.001); the fermented Codonopsis pilosula protection group Figure 9 The degree of loosening of gastric mucosal epithelial cells and inflammatory cell infiltration in mice was milder, and the pathological score was significantly lower than that in the model group (P<0.01); indicating that fermented Codonopsis pilosula has a good protective effect against gastric mucosal pathological damage induced by ethanol in mice.

[0219] 6.2 Inflammatory factors

[0220] Experimental results are as follows Figure 10As shown in the figure, a represents IL-6, b represents IL-8, c represents TNF-α, and d represents IL-10. The results showed that the levels of TNF-α, IL-8, and IL-6 in the model group were significantly higher than those in the control group (P<0.001), while the IL-10 level was significantly lower, indicating that the ethanol-induced model was successfully established and the mice in the model group developed an inflammatory response. The levels of IL-8, IL-6, and TNF-α in the fermented Codonopsis pilosula protective group and the treatment group were significantly lower than those in the model group (P<0.001), and all indicators were lower than those in the corresponding unfermented Codonopsis pilosula groups. The IL-10 level in the fermented Codonopsis pilosula protective group and the treatment group were significantly higher than those in the model group (P<0.001), and the protective effect was better than the treatment effect. This indicates that fermented Codonopsis pilosula has both protective and therapeutic effects on ethanol-induced gastric mucosal damage in mice.

[0221] 6.3 Markers of Oxidative Stress

[0222] Experimental results are as follows Figure 11 As shown in the figure, a represents SOD level and b represents MDA level. The results showed that the MDA level in the model group was significantly higher than that in the blank group (P<0.001), while the MDA level in the experimental group was significantly lower than that in the model group (P<0.001). Moreover, the MDA levels in the fermented Codonopsis pilosula treatment group and the protection group were lower than those in the unfermented group, and the protective effect was more significant than the treatment effect. The SOD level in the experimental group was higher than that in the model group, and the SOD levels in the fermented Codonopsis pilosula treatment group and the protection group were significantly higher than those in the model group (P<0.001). This indicates that fermented Codonopsis pilosula has a certain protective and repairing effect on ethanol-induced gastric mucosal damage in mice.

[0223] 6.4 Tight junction proteins

[0224] Experimental results are as follows Figure 12 As shown, the ZO-1 expression level in the model group was significantly lower than that in the blank group (P<0.001), while the ZO-1 expression levels in the fermented Codonopsis protective group and the treatment group were significantly higher than those in the model group (P<0.001). This indicates that fermented Codonopsis has a protective and repairing effect on the gastric mucosal barrier damaged by ethanol stimulation, and the effect is better than that of unfermented Codonopsis.

[0225] 6.5 Gastric acid secretion and pepsin activity

[0226] Figure 13 The pH value of model group a was significantly higher than that of the blank group (P<0.001). Figure 13The PGI / PGII ratio in group b was significantly lower than that in the control group, indicating that the pyloric infection model was successfully established. The pH values ​​of the fermented Codonopsis pilosula treatment group, the protective group, and the unfermented Codonopsis pilosula protective group were all significantly (P<0.001) lower than those of the model group; the PGI / PGII ratios of the fermented Codonopsis pilosula treatment group and the protective group were significantly (P<0.001) higher than those of the model group; and the PGI / PGII ratio of the high-dose treatment group was significantly higher (P<0.01) than that of the model group. This indicates that fermented Codonopsis pilosula can regulate the gastric environment in response to ethanol-induced gastric mucosal damage, and its protective effect is good.

[0227] 6.6 Gastric Ulcer Index Detection

[0228] Analysis of the gastric ulcer index in mice revealed that... Figure 14 The gastric ulcer index of the model group mice was significantly higher than that of the blank group (P<0.001), while the gastric ulcer index of the fermented Codonopsis pilosula protective group was significantly lower than that of the model group (P<0.001). The results indicate that fermented Codonopsis pilosula has a protective effect against gastric mucosal damage caused by ethanol stimulation.

[0229] Results and Discussion:

[0230] Gastric mucosal injury is a common pathological condition, mainly caused by various factors such as infection (e.g., Helicobacter pylori), ethanol, and drugs. Its pathogenesis involves multiple aspects, including uncontrolled inflammatory response, exacerbated oxidative stress, disruption of the mucosal barrier integrity, and enhanced attack factors of gastric acid / protease. Helicobacter pylori infection is a major cause of chronic gastritis and peptic ulcers; its virulence factors (such as urease and vacuole toxin) can induce strong local and systemic inflammatory responses and disrupt the gastric mucosa's self-protective mechanisms. Ethanol, on the other hand, causes acute gastric mucosal injury and even ulcers by directly corroding the gastric mucosal epithelium, stimulating the release of large amounts of pro-inflammatory factors, and inducing excessive production of reactive oxygen species.

[0231] Traditional Codonopsis pilosula contains polysaccharides and other active ingredients with relatively large molecular weights, which may limit their direct absorption by the human body. However, after fermentation with Bacillus belye, this process, through biotransformation, degrades the large polysaccharides into smaller molecules, making them easier for the body to absorb and resulting in a significant synergistic effect. Related studies have shown that smaller molecules can more easily cross the intestinal barrier, thereby greatly improving bioavailability and allowing the same amount of Codonopsis pilosula to exert a greater efficacy.

[0232] This invention systematically evaluated the efficacy of Bacillus belysin fermented Codonopsis pilosula and unfermented Codonopsis pilosula in protecting and treating gastric mucosa using a Helicobacter pylori infection model and an acute ethanol injury model. Experimental results showed that Bacillus belysin fermented Codonopsis pilosula exhibited significantly better results than unfermented Codonopsis pilosula in the following indicators, confirming that fermentation treatment can significantly enhance the gastric mucosal repair capacity of Codonopsis pilosula.

[0233] This invention, through a systematic evaluation using multiple models and indicators, confirms that fermentation treatment with Bacillus vesiculosus TK1926 can comprehensively enhance the gastric mucosal protective and therapeutic effects of Codonopsis pilosula. Compared to unfermented Codonopsis pilosula, fermented Codonopsis pilosula exhibits significant improvements in anti-inflammatory, antioxidant, barrier repair, and pathogen inhibition effects, resulting from a multi-dimensional synergistic effect encompassing anti-inflammatory, antioxidant, barrier repair, and gastric environment regulation. The mechanism underlying its remarkable efficacy may lie in the fact that the fermentation process, through biotransformation, improves the bioavailability and efficacy of the original active ingredients of Codonopsis pilosula and may generate new beneficial metabolites, thereby exerting a synergistic enhancing effect.

[0234] In conclusion, Bacillus vesiculosus fermented Codonopsis pilosula shows a significant improvement in efficacy against various gastric diseases, including Helicobacter pylori-associated gastritis and alcoholic acute gastric injury, compared to traditional Codonopsis pilosula. It is a gastric mucosal protectant with great development potential, providing a solid scientific basis for its development as a high-quality candidate product for gastric health.

[0235] For any points not covered above, existing technologies shall apply.

[0236] Although specific embodiments of the present invention have been described in detail by way of examples, those skilled in the art should understand that the above examples are for illustrative purposes only and are not intended to limit the scope of the invention. Those skilled in the art can make various modifications or additions to the described specific embodiments or use similar methods to replace them, without departing from the direction of the invention or exceeding the scope defined by the appended claims. Those skilled in the art should understand that any modifications, equivalent substitutions, improvements, etc., made to the above embodiments based on the technical essence of the present invention should be included within the protection scope of the present invention.

Claims

1. A Bacillus velezensis TK1926, which was deposited on December 10, 2025, at the China General Microbiological Culture Collection Center (CGMCC), located at No. 3, Courtyard 1, Beichen West Road, Chaoyang District, Beijing. The classification name is Bacillus velezensis, and the accession number is CGMCC No:36999.

2. A fermentation product with gastric mucosa repair function, characterized in that, The fermentation product is the fermentation broth or freeze-dried powder of the fermentation broth obtained by fermenting Codonopsis pilosula raw material with Bacillus vesiculosus TK1926 as described in claim 1.

3. A composition, characterized in that, The composition comprises the fermentation product of claim 2.

4. A method for preparing a fermentation product with gastric mucosa repair function, characterized in that, Includes the following steps: Codonopsis pilosula raw materials are provided, crushed and sieved to obtain Codonopsis pilosula powder; Sterile water is added to the Codonopsis pilosula powder according to a preset ratio, and after sterilization, Codonopsis pilosula matrix is ​​obtained. The Bacillus berberis TK1926 of claim 1 was inoculated into the Codonopsis pilosula substrate for fermentation to obtain a fermentation broth; The fermentation broth was freeze-dried to obtain the fermentation product.

5. The method according to claim 4, characterized in that, At a volume ratio of 2% to 5%, secondary seed culture of Bacillus vesiculosus TK1926 was inoculated into the Codonopsis pilosula matrix.

6. The method according to claim 4, characterized in that, The fermentation conditions are: shaking culture at 30~37℃ and 150~200rpm for 24~48h.

7. The method according to claim 4, characterized in that, The freeze-drying process is as follows: pre-freeze at a depth below -40℃ for 2~4 hours; then, under a vacuum of 10~30 Pa, first raise the temperature to -20℃ to -10℃ and maintain it for 20~40 hours, then raise it to 25~35℃ and maintain it for 6~12 hours.

8. The use of the fermentation product of claim 2 and the composition of claim 3 in the preparation of a drug for repairing alcoholic gastric mucosal damage.

9. The use of the fermentation product of claim 2 and the composition of claim 3 in the preparation of a drug for repairing Helicobacter pylori-related gastric mucosal damage.

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