Bacillus velezensis and application thereof in preventing and treating inflammatory bowel disease

Abstract

The invention belongs to the technical field of biological medicines, and particularly relates to bacillus velezensis and application thereof in preventing and treating inflammatory bowel diseases. The bacillus velezensis GFZF-23 provided by the invention is preserved in the China Center for Type Culture Collection, and the preservation number of the bacillus velezensis GFZF-23 is CCTCC (China Center for Type Culture Collection) NO: M 20251699. The bacillus velezensis GFZF-23 can be used for effectively treating the inflammatory bowel disease by reducing the pathological injury of intestinal tissues, reducing the expression of inflammatory factors and immune receptor genes, enhancing the expression of related genes of intestinal mucosal barriers and remodeling the balance of intestinal flora. The invention provides a new treatment strategy for treating the inflammatory bowel disease, and has remarkable clinical application value and wide market prospect.

Description

Technical Field

[0001] This invention belongs to the field of biomedical technology, specifically relating to a type of Bacillus belye and its application in the prevention and treatment of inflammatory bowel disease. Background Technology

[0002] Inflammatory bowel disease (IBD) is a chronic, relapsing, nonspecific inflammatory bowel disease, primarily including ulcerative colitis (UC) and Crohn's disease (CD). Depending on its pathological features, IBD manifests as chronic inflammation of the intestinal mucosa, ulceration, and disruption of the intestinal barrier function. Currently, clinical treatment for IBD mainly relies on aminosalicylic acid derivatives, glucocorticoids, immunosuppressants, and biologics. While these drugs are effective in controlling inflammation, long-term use often results in serious adverse reactions, such as osteoporosis, increased susceptibility to infection, and liver and kidney damage. Furthermore, treatment costs are high, and patient compliance is poor. In recent years, the incidence of IBD has been rapidly increasing globally, becoming one of the most significant diseases affecting human health. Therefore, exploring safe, effective, and cost-effective new strategies for treating IBD has significant clinical and social value.

[0003] The human gut is home to tens of thousands of microbial species, forming a complex and sophisticated micro-ecosystem that plays an irreplaceable role in maintaining host health. Numerous clinical and basic studies have shown that gut microbiota dysbiosis is one of the core factors in the pathogenesis of IBD. IBD patients commonly exhibit abnormal gut microbiota structure, characterized by a sharp decline in the number of beneficial bacteria, excessive proliferation of opportunistic pathogens, and a significant reduction in gut microbiota diversity. This microecological imbalance not only disrupts gut immune homeostasis but also impairs intestinal barrier function, creating a vicious cycle of inflammation. With the growing popularity of microecological regulation-based treatments for IBD, the use of probiotics for intervention has become an important research direction.

[0004] Currently, some technologies have reported specific probiotic strains with therapeutic effects on IBD. For example, patent CN120290364A discloses a *Lactobacillus plantarum* strain PL-H1 derived from healthy individuals in cold regions. This strain can significantly alleviate disease indices, intestinal pathological changes, and inflammatory responses in inflammatory bowel disease (IBD) model mice. Compared to the standard strain WCSF1, this strain exhibits high tryptophan metabolism and regulates tryptophan metabolism in the IBD host gut, resulting in more significant efficacy in preventing and treating IBD. Another example is patent CN120384028A, which discloses a *Lactobacillus kefiranofaciens* 2503 strain. This *Lactobacillus kefiranofaciens* exhibits strong acid and bile salt resistance, antioxidant and free radical scavenging functions, and can colonize the gut, increasing the abundance of beneficial bacteria and reducing the abundance of pathogenic bacteria, thus showing good preventive and therapeutic effects against inflammatory bowel disease. However, overall, research on probiotic interventions for IBD is still in its early stages. Therefore, further exploration of more probiotics with therapeutic potential for IBD and systematic evaluation of their safety and efficacy are of great scientific and clinical significance.

[0005] Bacillus velezensis, belonging to the phylum Firmicutes, class Bacillus, order Bacillusales, family Bacillusaceae, and genus Bacillus, is a Gram-positive, aerobic, rod-shaped, motile bacterium capable of forming highly resistant endospores. In recent years, research on Bacillus velezensis has increased both domestically and internationally, demonstrating significant application value in biocontrol, drug development, and animal health maintenance. Studies have shown that Bacillus velezensis possesses significant probiotic properties, producing various bioactive metabolites, including lipopeptide antimicrobial compounds such as surfactants and iturobrinein, as well as functional enzymes such as proteases and cellulases. These metabolites exhibit good biological activity in antagonizing pathogens, regulating host immune function, and promoting intestinal health. In animal disease control research, *Bacillus belyceae* strains isolated from the intestines of aquatic animals such as carp, tilapia, and grouper have demonstrated excellent resistance to pathogens. They effectively antagonize common pathogens such as *Aeromonas hydrophila*, *Streptococcus agalactiae*, and *Vibrio parahaemolyticus*, and exert protective effects through multiple mechanisms, including enhancing host non-specific immunity, regulating intestinal microbiota structure, and improving oxidative stress resistance. Furthermore, these strains exhibit good biocompatibility and environmental adaptability, enabling them to survive and function physiologically in the complex intestinal environment.

[0006] Although Bacillus belycera has shown broad application prospects in agricultural production, food industry, and animal health maintenance, research on its application in treating digestive system diseases such as inflammatory bowel disease in humans remains very limited. Therefore, it is necessary to further explore the therapeutic mechanisms and clinical application value of Bacillus belycera. Summary of the Invention

[0007] In view of this, one of the objectives of the present invention is to provide a strain for treating inflammatory bowel disease.

[0008] To achieve the above objectives, the present invention adopts the following technical solution:

[0009] The strain used to treat inflammatory bowel disease is Bacillus velezensis GFZF-23, which was deposited at the China Center for Type Culture Collection on July 28, 2025, with accession number CCTCC NO: M 20251699.

[0010] The Bacillus velezensis GFZF-23 can effectively treat inflammatory bowel disease by reducing pathological damage to intestinal tissues, downregulating the expression of inflammatory factors and immune receptor genes, enhancing the expression of intestinal mucosal barrier-related genes, and reshaping the balance of intestinal flora.

[0011] A second objective of this invention is to provide a method for culturing the aforementioned strain used to treat inflammatory bowel disease.

[0012] To achieve the above objectives, the present invention adopts the following technical solution:

[0013] The method for culturing strains used to treat inflammatory bowel disease includes the following steps:

[0014] S1: Inoculate Bacillus velezensis GFZF-23 strain onto TSA solid medium and incubate at 35-40℃ for 18-24 hours. After the colonies have grown well, pick a single colony.

[0015] S2: Using aseptic techniques, the single colony obtained in S1 was inoculated into TSB liquid medium and cultured with shaking at 35-40℃ and 180-250rpm to obtain Bacillus velezensis GFZF-23 in the logarithmic growth phase.

[0016] Preferably, the TSA solid culture medium comprises the following components: 15.0–20.0 g / L tryptone, 2.0–4.0 g / L soybean peptone, 4.0–6.0 g / L sodium chloride, 1.5–3.5 g / L dipotassium hydrogen phosphate, 1.5–3.5 g / L glucose, and 10.0–20.0 g / L agar, with a pH of 7.3 ± 0.5.

[0017] As a more preferred embodiment, the TSA solid culture medium comprises the following components: 17.0 g / L tryptone, 3.0 g / L soybean peptone, 5.0 g / L sodium chloride, 2.5 g / L dipotassium hydrogen phosphate, 2.5 g / L glucose, and 15.0 g / L agar, with a pH of 7.3 ± 0.2.

[0018] Preferably, the TSB liquid culture medium comprises the following components: 15.0–20.0 g / L tryptone, 2.0–4.0 g / L soybean peptone, 4.0–6.0 g / L sodium chloride, 1.5–3.5 g / L dipotassium hydrogen phosphate, and 1.5–3.5 g / L glucose, with a pH of 7.3 ± 0.5.

[0019] More preferably, the TSB liquid culture medium comprises the following components: 17.0 g / L tryptone, 3.0 g / L soybean peptone, 5.0 g / L sodium chloride, 2.5 g / L dipotassium hydrogen phosphate and 2.5 g / L glucose, with a pH of 7.3 ± 0.2.

[0020] Preferably, in S1, the culture temperature is 37°C.

[0021] As a preferred option, in S2, the cells are cultured under shaking conditions at 37°C and 200 rpm.

[0022] Preferably, in S2, the culture is carried out until the bacterial solution becomes turbid and reaches the logarithmic growth phase.

[0023] A third objective of this invention is to provide a bacterial agent for treating inflammatory bowel disease.

[0024] To achieve the above objectives, the present invention adopts the following technical solution:

[0025] A bacterial agent for treating inflammatory bowel disease, said bacterial agent containing the aforementioned bacterial strain or its metabolites and a pharmaceutically acceptable carrier or excipient.

[0026] Preferably, the dosage of Bacillus velezensis GFZF-23 in the bacterial agent is 4 × 10⁻⁶. 5 ~6×10 5 CFU / mL.

[0027] More preferably, the dosage of Bacillus velezensis GFZF-23 in the bacterial agent is 5 × 10⁻⁶. 5 CFU / mL.

[0028] Preferably, the dosage form of the microbial agent is any one of liquid, solid, and semi-solid formulations.

[0029] The fourth objective of this invention is to provide the use of the aforementioned strain and / or the aforementioned bacterial agent in the preparation of a medicament for the prevention, relief and / or treatment of inflammatory bowel disease.

[0030] The fifth objective of this invention is to provide an application of the aforementioned strain and / or the aforementioned microbial agent in the preparation of a drug having any one or more of the functions described in 1) to 4):

[0031] 1) Inhibit the expression of IL-1β, IL-6, MYD88 and / or TLR4 immune receptor genes;

[0032] 2) Promotes the expression of claudin11b and / or MUC2.1;

[0033] 3) Regulates the structure of the intestinal flora and improves flora imbalance;

[0034] 4) Reduces pathological damage to intestinal tissues and intestinal inflammation, and improves intestinal morphology.

[0035] Preferably, the reduction of intestinal tissue pathological damage includes reducing the degree of crypt damage, reducing inflammatory cell infiltration, and / or reducing the degree of fibrosis.

[0036] The sixth objective of this invention is to provide an application of the aforementioned strain and / or the aforementioned bacterial agent in the preparation of a medicament for reducing the abundance of harmful bacteria in the intestine, wherein the harmful bacteria include Flavobacterium.

[0037] The beneficial effects of this invention are as follows:

[0038] The Bacillus velezensis GFZF-23 provided by this invention has a good therapeutic effect on inflammatory bowel disease (IBD). Studies have shown that Bacillus velezensis GFZF-23 can effectively treat IBD by significantly improving host survival, improving overall morphology, reducing pathological damage to intestinal tissues, downregulating the expression of inflammatory factors and immune receptor genes, enhancing the expression of intestinal mucosal barrier-related genes, and reshaping the balance of intestinal flora. This invention provides a new microecological treatment option for IBD, which has significant clinical implications and broad market application prospects. Attached Figure Description

[0039] Figure 1 The figure shows the results of detecting the effect of different concentrations of sodium dextran sulfate (DSS) on the survival rate of zebrafish juveniles;

[0040] Figure 2 The figure shows the results of detecting the effect of Bacillus velezensis on the survival rate of juvenile zebrafish with DSS-induced colitis.

[0041] Figure 3 The figure shows the results of detecting the effect of Bacillus velezensis on the survival curve of juvenile zebrafish with DSS-induced colitis.

[0042] Figure 4 Figure 1 shows the results of detecting the effect of Bacillus velezensis on the overall morphology of juvenile zebrafish with DSS-induced colitis.

[0043] Figure 5 Representative images of hematoxylin and eosin (H&E) staining for the effects of Bacillus velezensis on the intestinal histopathology of DSS-induced colitis in juvenile zebrafish;

[0044] Figure 6 Figure showing the results of detecting the effect of Bacillus velezensis on the expression of the inflammation-related gene IL-1β in DSS-induced colitis in zebrafish juveniles;

[0045] Figure 7 Figure showing the results of detecting the effect of Bacillus velezensis on the expression of the inflammation-related gene IL-6 in DSS-induced colitis in zebrafish juveniles;

[0046] Figure 8 Figure showing the results of detecting the effect of Bacillus velezensis on the expression of the inflammation-related gene Myd88 in DSS-induced colitis in zebrafish juveniles;

[0047] Figure 9 Figure 1 shows the results of detecting the effect of Bacillus velezensis on the expression of the immune receptor gene TLR4 in DSS-induced colitis in zebrafish juveniles.

[0048] Figure 10 Figure 1 shows the results of detecting the effect of Bacillus velezensis on the expression of the intestinal barrier gene muc2.1 in DSS-induced colitis in zebrafish juveniles.

[0049] Figure 11 Figure 1 shows the results of detecting the effect of Bacillus velezensis on the expression of claudin11b, a tight junction protein gene, in juvenile zebrafish with DSS-induced colitis.

[0050] Figure 12 The figure shows the results of detecting the effect of Bacillus velezensis on the gut microbiota composition at the genus level in juvenile zebrafish with DSS-induced colitis. Detailed Implementation

[0051] The technical solution of the present invention will be described more clearly and completely below with reference to specific embodiments. Obviously, the described embodiments are only a part of the embodiments of the present invention, and not all of them. Therefore, based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the protection scope of the present invention.

[0052] In this invention, it should be understood that the terminology used is merely for describing particular embodiments and is not intended to limit the invention. Furthermore, regarding numerical ranges in this invention, it should be understood that each intermediate value between the upper and lower limits of the range is also specifically disclosed. Every smaller range between any stated value or intermediate value within a stated range, and any other stated value or intermediate value within said range, is also included in this invention. The upper and lower limits of these smaller ranges may be independently included or excluded from the range.

[0053] Unless otherwise stated, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art. While only preferred methods and materials have been described herein, any methods and materials similar or equivalent to those described herein may be used in the implementation or testing of this invention. All references to this specification are incorporated by way of citation to disclose and describe methods and / or materials associated with those references. In the event of any conflict with any incorporated reference, the content of this specification shall prevail.

[0054] In the embodiments of this invention, the materials, reagents, animals, or instruments involved, unless otherwise specified, can all be purchased commercially. The related experimental methods, unless otherwise specified, are conventional techniques in the art. Non-essential improvements and adjustments made to the implementation schemes by those skilled in the art based on the above-described invention are still within the scope of protection of this invention.

[0055] In this invention, Bacillus velezensis GFZF-23 is deposited at the China Center for Type Culture Collection (CCTCC), named Bacillus velezensis GFZF-23, classified as Bacillus velezensis, with accession number CCTCC NO: M 20251699, deposit date July 28, 2025, and depositor is Pei Desheng.

[0056] Example 1. Culture of Bacillus velezensis GFZF-23

[0057] (a) Subculture and preservation of Bacillus velezensis GFZF-23

[0058] Bacillus velezensis strain GFZF-23 was inoculated onto TSA solid medium (17.0 g / L tryptone, 3.0 g / L soybean peptone, 5.0 g / L sodium chloride, 2.5 g / L dipotassium hydrogen phosphate, 2.5 g / L glucose, 15.0 g / L agar, pH 7.3±0.2) and incubated at 37℃ for 18-24 hours. After good colony growth, single colonies were picked and aseptically inoculated into TSB liquid medium (17.0 g / L tryptone, 3.0 g / L soybean peptone, 5.0 g / L sodium chloride, 2.5 g / L dipotassium hydrogen phosphate, 2.5 g / L glucose, pH 7.3±0.2). The medium was then shaken at 37℃ and 200 rpm until the culture became turbid and reached the logarithmic growth phase.

[0059] To maintain the viability of the strain and establish a working strain library, subculturing was performed every 7-10 days. An appropriate amount of bacterial culture was taken from the liquid culture and inoculated into fresh TSB liquid medium at a 1:50 ratio, and cultured under the same conditions. Simultaneously, well-grown bacterial cultures were streaked onto TSA solid medium, and single colonies were obtained and stored at 4°C as working strains for short-term preservation.

[0060] For long-term preservation of bacterial strains, fresh bacterial suspension in the logarithmic growth phase is mixed with sterile glycerol at a 1:1 ratio to prepare a glycerol-bacterial suspension with a final concentration of 20%. The suspension is aliquoted into sterile cryovials (1.6 mL per tube), labeled with the strain name, storage date, and other information, and then stored at -80°C for long-term preservation. The preserved strains can be retrieved and directly inoculated onto TSA solid medium for resuscitation when needed.

[0061] (b) Culture and amplification of Bacillus velezensis GFZF-23

[0062] Remove cryovials from glycerol cultures stored at -80℃, thaw rapidly at room temperature, and streak an appropriate amount of bacterial culture onto TSA solid medium using a sterile inoculation loop. Incubate at 37℃ for 18-24 hours until single colonies appear. Select morphologically typical and uniformly sized single colonies and inoculate them into 5 mL of TSB liquid medium. Incubate at 37℃ with shaking at 200 rpm until the growth phase reaches logarithmic growth, using this as the seed culture. Transfer the seed culture in the logarithmic growth phase to fresh TSB liquid medium at a 1:50 inoculation ratio. Incubate at 37℃ with shaking at 200 rpm. Depending on the experimental scale, the bacterial culture can be expanded to 50 mL, 100 mL, or larger volumes of medium. Maintain aseptic technique during incubation, ensuring a stable temperature and appropriate shaking speed.

[0063] (c) Determination of the logarithmic growth phase of Bacillus velezensis GFZF-23

[0064] A single activated bacterial strain was picked from TSA solid medium and inoculated into TSB liquid medium, and cultured overnight at 37°C and 200 rpm with shaking. The next day, 6 μL of bacterial culture and 300 μL of TSB liquid medium were added to the well plate at a ratio of 1:50. The experiment was repeated three times, with three parallel controls each time.

[0065] The growth curves of the strains were determined using a BIOSCREEN fully automated growth curve analyzer. The change in optical density at 600 nm (OD600) was measured every 0.5 hours for 24–48 hours, with blank TSB liquid medium used as a control. A constant temperature of 37°C was maintained throughout the experiment to ensure environmental stability.

[0066] Based on the measurement results, a time-OD600 growth curve of the strain was plotted. By analyzing different stages of the curve, the time points of the lag phase, logarithmic growth phase, stationary phase, and death phase of the strain were determined. The period when the curve shows a linear increase and the specific growth rate is the logarithmic growth phase, providing a reference for the optimal timing of cell culture and collection in subsequent experiments.

[0067] (d) Collection and resuspension preparation of Bacillus velezensis GFZF-23 cells

[0068] The *Bacillus velezensis* GFZF-23 bacterial culture, cultured to the logarithmic growth phase, was collected and resuspended. The culture was transferred to a sterile centrifuge tube and centrifuged at 4000 rpm for 10 minutes at 4°C. The supernatant was discarded, and the bacterial pellet was collected. The bacterial cells were resuspended in sterile zebrafish culture water and washed again under the same conditions. This washing step was repeated 2-3 times to remove residual components from the culture medium.

[0069] After the final centrifugation, discard the supernatant and resuspend the bacteria in sterile zebrafish culture water. Gently mix until the bacteria are completely dispersed, avoiding clumping. The prepared bacterial suspension should be stored at 4°C and used within 2 hours to ensure bacterial activity.

[0070] (e) Determination of the correlation between optical density values ​​and viable cell concentration of Bacillus velezensis GFZF-23 bacterial suspension

[0071] The prepared bacterial suspension was analyzed using a spectrophotometer to determine its optical density (OD600) at 600 nm, and a standard curve was established comparing the optical density with the viable cell concentration. A series of dilutions were prepared from the bacterial suspension using a 10-fold serial dilution method, with a dilution gradient of 10... -1 10 -2 10 -3 10 -4 10 -5 10 -6 The OD600 values ​​of each dilution were measured.

[0072] The viable cell concentration of bacterial suspensions at each dilution was determined using the plate count method. 100 μL of bacterial suspension at each dilution was evenly spread onto TSA solid medium plates, with three replicates for each dilution. After incubation at 37°C for 18–24 hours, colony counting was performed. Plates with colony counts ranging from 30 to 300 were selected for statistical analysis, and the viable cell concentration (CFU / mL) corresponding to each dilution was calculated.

[0073] Calculation formula: C=(N÷V)×M

[0074] Where C: bacterial concentration; N: average number of colonies grown on the plate at the dilution; V: volume (mL) of diluent used when plating; M: dilution factor.

[0075] Standard curve fitting analysis determined that when the OD600 value of the bacterial suspension was in the range of 0.03-0.06, the corresponding bacterial concentration was 10. 6 CFU / mL. Based on this standard relationship, in subsequent water inoculation experiments, this concentration of bacterial suspension was diluted at a ratio of 1:10 and added to achieve a final concentration of Bacillus velezensis GFZF-23 of 5 × 10⁻⁶ CFU / mL in the water. 5 CFU / mL.

[0076] Example 2. Establishment of a juvenile zebrafish model of colitis

[0077] (a) Zebrafish husbandry and laboratory animal preparation

[0078] Wild-type AB zebrafish were obtained from the fish model organism experimental platform of the School of Public Health, Chongqing Medical University. Adult fish were raised under standard conditions, with the culture system maintained at a constant temperature of 28±0.5℃, pH 7.0-7.5, and a 14 / 10 hour light / dark cycle. Adult fish were fed brine shrimp twice daily. Embryos were collected using a natural mating method, with sexually mature males and females placed in a 1:1 ratio in a mating tank overnight. The partition was removed the following morning to initiate mating. Fresh fertilized embryos were collected within one hour of mating and placed in culture dishes containing embryo culture medium for development in a 28.5℃ constant temperature incubator. During development, the culture medium was changed daily, and dead or abnormally developing embryos were promptly removed.

[0079] Three days after fertilization (3 days post-fertilization), healthy juveniles that showed normal development, good vitality, and no deformities were selected. The selected juveniles were randomly divided into three biological replicates per group, with each replicate containing 30 juveniles. Each replicate was placed in a six-well cell culture plate, with 8 mL of zebrafish culture water added to each well. Constant environmental conditions were maintained throughout the experiment to ensure the stability of the juveniles' physiological state.

[0080] (b) Optimization and determination of sodium dextran sulfate (DSS) concentration for constructing inflammatory bowel disease

[0081] To establish a stable and reproducible intestinal inflammation model, a concentration gradient experiment was used to determine the optimal DSS concentration. Control group (CON) and treatment groups with 0.35%, 0.30%, 0.25%, and 0.20% DSS were established. The DSS (quality standard: MW: 36000-50000) was prepared fresh using standard zebrafish culture water, ensuring the solution pH was within the physiological range.

[0082] DSS exposure treatment began at 3 days post-exposure (dpf) and continued until the 7-dpf experimental endpoint. The DSS solution was replaced daily during treatment to prevent drug degradation and bacterial contamination. Figure 1 As shown, DSS concentration exhibited a clear dose-response relationship with the survival rate of zebrafish juveniles. With increasing DSS concentration, the survival rate of juveniles decreased significantly, reflecting the concentration-dependent nature of DSS-induced intestinal inflammation. The survival rates for each concentration group were: 0.35% DSS group 28.8%, 0.30% DSS group 41.1%, 0.25% DSS group 64.4%, and 0.20% DSS group 83.3%. Based on the survival rate assessment results, 0.20% DSS was ultimately determined to be the optimal concentration for modeling. The 0.20% DSS group achieved a survival rate of 83.3%, maintaining good animal welfare while inducing a significant inflammatory response, providing a stable and reliable disease model basis for subsequent Bacillus velezensis GFZF-23 treatment experiments.

[0083] (c) Experimental group design and sample collection and analysis

[0084] Based on previous results, four experimental groups were set up: control group (CON), Bacillus belysinus group (B), 0.20% DSS model group (DSS), and DSS combined with Bacillus belysinus treatment group (DB). Treatment started at 3 days post-exposure (dpf) and continued until the experimental endpoint at 7 days post-exposure (dpf).

[0085] All groups had their treatment solutions changed daily at set times. The CON group received fresh zebrafish culture water, the DSS group received 0.20% DSS solution, and the bacterial suspension groups received freshly prepared bacterial suspension to ensure stable bacterial activity and concentration. The Bacillus velezensis GFZF-23 dosage was determined based on the previously established OD600-CFU standard curve, and the bacterial suspension was prepared to achieve a final concentration of 5 × 10⁻⁶ CFU in the water. 5 CFU / mL.

[0086] During the experiment, the survival status of each group of juvenile fish was observed and recorded at a fixed time every morning. At the end of the 7-day post-flood (dpf) experiment, the overall morphological characteristics of the juvenile fish were observed and photographed under a stereomicroscope. Sample collection consisted of three parts: morphological analysis samples were fixed with 4% paraformaldehyde at 4°C for subsequent paraffin sectioning and hematoxylin-eosin (H&E) staining; gut microbiome analysis samples were collected whole fish under sterile conditions, immediately placed in sterile cryovials, flash-frozen in liquid nitrogen, and stored at -80°C for 16S rRNA gene sequencing analysis of gut microbiota structure changes; molecular biology detection samples were also flash-frozen in liquid nitrogen and stored at -80°C for qPCR detection of gene expression levels.

[0087] Example 3. Analysis of the effects of Bacillus velezensis GFZF-23 on the survival rate and survival curve of zebrafish juveniles.

[0088] During the experiment, the survival status of each group of juvenile fish was observed and recorded at a fixed time every morning, continuously monitored from 0 dpf to 7 dpf, and the number of surviving individuals was counted and the survival rate was calculated daily. GraphPad Prism 10.0 software was used for data processing and statistical analysis. A survival rate bar chart was created to visually display the final survival rate of each group, and Kaplan-Meier survival curves were plotted to assess the significance of survival differences between groups.

[0089] Survival analysis results as follows Figure 2 As shown, the final survival rates of each group at the end of the experiment were: CON group: 100%, B group: 100%, DSS group: 88.3%, and DB group: 94.4%. The survival curve analysis results are as follows: Figure 3As shown, during the 7-day observation period (0-7 dpf), the DSS group exhibited significant mortality starting at 4 dpf, with a continuously declining survival rate, reflecting the significant impact of DSS-induced intestinal inflammation on juvenile fish survival. In contrast, although the DB group experienced mortality after 3 dpf, the decline was more gradual, and the survival rate was significantly higher than that of the DSS group. The CON and B groups maintained a 100% survival rate throughout the entire observation period, indicating that Bacillus velezensis GFZF-23 itself has no toxic side effects on zebrafish juveniles. Therefore, Bacillus velezensis GFZF-23 can effectively improve the survival of DSS-induced colitis in zebrafish juveniles, demonstrating a good protective effect.

[0090] Example 4. Analysis of the effects of Bacillus velezensis GFZF-23 on morphological and histological changes in zebrafish juveniles.

[0091] At the end of the experiment (7 days post-exposure), representative juvenile fish were randomly selected from each group for overall morphological observation. The juvenile fish were placed under a stereomicroscope, and whole-fish morphological images were observed and photographed at appropriate magnification. By comparing the overall morphological differences among the juvenile fish in each group, the effects of DSS-induced inflammatory response on juvenile development and the intervention effect of Bacillus velezensis GFZF-23 were evaluated.

[0092] Analysis results as follows Figure 4 As shown, there were significant differences in morphological characteristics among the groups of juvenile fish. The CON group juveniles developed normally, with symmetrical body shapes, a naturally straight spine, and no obvious developmental abnormalities. The B group juveniles also exhibited normal morphological characteristics, demonstrating that Bacillus velezensis GFZF-23 had no adverse effects on juvenile development. In contrast, the DSS group juveniles showed obvious morphological abnormalities, mainly spinal deformities and lateral curvature, suggesting that DSS-induced intestinal inflammation had a significant negative impact on normal juvenile development. Notably, the DB group showed a significantly reduced degree of spinal deformity and improved morphological abnormalities compared to the DSS group, indicating that Bacillus velezensis GFZF-23 can effectively alleviate the adverse effects of DSS-induced colitis on the morphological development of zebrafish juveniles, demonstrating a good protective intervention effect.

[0093] Histological analysis of juvenile fish in each group was performed using conventional paraffin embedding and sectioning techniques. The fixed juvenile fish were embedded whole and sections were prepared. Representative images of the intestinal cross-section were observed and photographed under an optical microscope after H&E staining to analyze the differences in intestinal histopathology among groups, assess the degree of DSS-induced intestinal inflammation damage, and evaluate the intestinal tissue protective effect of Bacillus velezensis GFZF-23.

[0094] Analysis results as follows Figure 5 As shown, there were significant differences in intestinal tissue structure among the groups. In the DSS-induced colitis model group, the intestinal tissue exhibited typical inflammatory pathological changes, mainly characterized by significant thinning of the lamina propria, severe damage to the intestinal gland crypt structure, and complete loss of crypt structure in some areas. Simultaneously, abnormal thickening of the mucosal layer was observed, with localized areas showing chronic inflammatory features such as fibrosis and scar tissue formation. These pathological changes reflect the successful induction of an intestinal inflammatory injury model by DSS. In contrast, the intestinal tissue structure of the juvenile fish in group B remained well-preserved, with intact mucosal morphology, clear crypt structure, and very mild inflammatory cell infiltration, similar to the control group, further confirming the safety of Bacillus velezensis GFZF-23. In the DB group, the degree of intestinal tissue pathological damage was significantly reduced compared to the DSS group, characterized by relatively intact mucosal structure, reduced crypt damage, significantly decreased inflammatory cell infiltration, and decreased fibrosis. These results indicate that Bacillus velezensis GFZF-23 can effectively alleviate DSS-induced intestinal histopathological damage and exert a significant tissue protective effect.

[0095] Example 5. Analysis of the effects of Bacillus velezensis GFZF-23 on the expression of inflammatory factors, immune receptors and intestinal barrier-related genes in zebrafish juveniles.

[0096] At the end of the experiment (7 days post-exposure), juvenile fish were randomly selected from each group for gene expression analysis. Total RNA was extracted using TRIzol reagent, and RNA concentration and purity were detected using a NanoDrop spectrophotometer to ensure the A260 / A280 ratio was between 1.8 and 2.0. RNA was reverse transcribed into cDNA using a reverse transcription kit, followed by quantitative real-time PCR (qRT-PCR) to detect the expression levels of target genes. Target genes detected included: inflammation-related genes (IL-1β, IL-6, Myd88), immune receptor genes (TLR4), and intestinal barrier-related genes (muc2.1, claudin11b). GAPDH was used as an internal reference gene, and the relative expression levels of each gene were calculated using the 2^(-ΔΔCt) method. Statistical analysis was performed using GraphPad Prism 10.0 software. Data are expressed as mean ± standard error (Mean ± SEM) (N = 3). One-way ANOVA was used for comparisons among multiple groups, with a significance level set at P < 0.05. In the results figures and tables, the following notation system was used to indicate statistical significance: * indicates a significant difference compared to the CON group (*P < 0.05; **P < 0.01; ***P < 0.001; ****P < 0.0001); + indicates a significant difference compared to the DSS group (+P < 0.05; ++P < 0.01; +++P < 0.001; ++++P < 0.0001).

[0097] Analysis results as follows Figure 6-11 As shown, the expression levels of inflammation-related genes IL-1β, IL-6, Myd88, and the immune receptor gene TLR4 in the DSS group were significantly upregulated compared with the control group (****P<0.0001), while the expression of intestinal barrier-related genes claudin11b and MUC2.1 was significantly downregulated (***P<0.001). Compared with the DSS group, the DB group showed significantly downregulated expression of inflammation-related genes and TLR4 (++++P<0.0001), significantly upregulated expression of the intestinal barrier-related gene claudin11b (++++P<0.0001), and some recovery of MUC2.1 expression. There were no significant differences in gene expression levels between the B group and the control group. The results indicate that Bacillus velezensis GFZF-23 can effectively inhibit DSS-induced inflammatory response, regulate immune receptor expression, and promote the repair of intestinal barrier function genes.

[0098] Example 6. Analysis of the effects of Bacillus velezensis GFZF-23 on the gut microbiota structure of zebrafish juveniles.

[0099] Analysis results as follows Figure 12As shown, the gut microbiota composition of zebrafish juveniles in each group was analyzed by 16S rRNA gene sequencing. At the genus level, the gut microbiota of all four groups was mainly composed of Aeromonas and Pseudomonas. After DSS treatment, the microbiota structure was significantly altered, with an increase in the relative abundance of Flavobacterium. The microbiota composition of the DB group was somewhat regulated compared to the DSS group, with a recovery in the relative abundance of Flavobacterium, while the ratio of Shinella and Chryseobacterium was altered. The results indicate that DSS-induced intestinal inflammation alters the gut microbiota structure of zebrafish juveniles, and Bacillus velezensis GFZF-23 intervention can regulate the gut microbiota balance to some extent.

Claims

1. A bacterial strain for treating inflammatory bowel disease, characterized in that, The strain is Bacillus velezensis GFZF-23, which was deposited at the China Center for Type Culture Collection on July 28, 2025, with accession number CCTCC NO: M 20251699.

2. The method for culturing the strain according to claim 1, characterized in that, Includes the following steps: S1: Inoculate Bacillus velezensis GFZF-23 strain onto TSA solid medium and incubate at 35-40℃ for 18-24 hours. After the colonies have grown well, pick a single colony. S2: Using aseptic techniques, the single colony obtained in S1 was inoculated into TSB liquid medium and cultured with shaking at 35-40℃ and 180-250rpm to obtain Bacillus velezensis GFZF-23 in the logarithmic growth phase.

3. The cultivation method according to claim 2, characterized in that, The TSA solid culture medium comprises the following components: 15.0–20.0 g / L tryptone, 2.0–4.0 g / L soybean peptone, 4.0–6.0 g / L sodium chloride, 1.5–3.5 g / L dipotassium hydrogen phosphate, 1.5–3.5 g / L glucose, and 10.0–20.0 g / L agar, with a pH of 7.3 ± 0.

5.

4. The cultivation method according to claim 2, characterized in that, The TSB liquid culture medium comprises the following components: 15.0–20.0 g / L tryptone, 2.0–4.0 g / L soybean peptone, 4.0–6.0 g / L sodium chloride, 1.5–3.5 g / L dipotassium hydrogen phosphate, and 1.5–3.5 g / L glucose, with a pH of 7.3 ± 0.

5.

5. A bacterial agent for treating inflammatory bowel disease, characterized in that, The microbial agent contains the strain of claim 1 or its metabolites, as well as a pharmaceutically acceptable carrier or excipient.

6. The microbial agent according to claim 5, characterized in that, The dosage of Bacillus velezensis GFZF-23 in the bacterial agent is 4 × 10⁻⁶. 5 ~6×10 5 CFU / mL.

7. The microbial agent according to claim 5, characterized in that, The dosage form of the microbial agent is any one of liquid, solid, and semi-solid formulations.

8. The use of the strain of claim 1 and / or the bacterial agent of any one of claims 5 to 7 in the preparation of a medicament for the prevention, relief and / or treatment of inflammatory bowel disease.

9. The use of the strain according to claim 1 and / or the microbial agent according to any one of claims 5 to 7 in the preparation of a drug having one or more functions as described in 1) to 4): 1) Inhibit the expression of IL-1β, IL-6, MYD88 and / or TLR4 immune receptor genes; 2) Promotes the expression of claudin11b and / or MUC2.1; 3) Regulates the structure of the intestinal flora and improves flora imbalance; 4) Reduces pathological damage to intestinal tissues and intestinal inflammation, and improves intestinal morphology.

10. The use of the strain according to claim 1 and / or the bacterial agent according to any one of claims 5 to 7 in the preparation of a medicament for reducing the abundance of harmful intestinal bacteria, characterized in that, The harmful bacteria include Flavobacterium.

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