Surface-modified bacillus cereus preparation as well as preparation method and application thereof

Abstract

The invention discloses a surface-modified bacillus cereus preparation as well as a preparation method and application thereof, and relates to the technical field of viable bacteria medicines. According to the invention, dopamine and chitosan are modified on the surface of bacillus cereus to form a two-component coating bacillus cereus preparation by utilizing the spontaneous polymerization principle of dopamine and chitosan, and the two-component coating bacillus cereus preparation is used for preparing the medicine for preventing and treating enteritis. Research finds that the bacillus cereus preparation with the polydopamine and chitosan double-component coating can efficiently remove active oxygen to relieve enteritis. The preparation can remain in intestinal tracts of mice for a longer time and has a remarkable curative effect on radiation enteritis caused by 12 Gy gamma ray abdominal irradiation, and compared with an original bacterial liquid group, the weight, intestinal length, histopathology, local active oxygen removal, local inflammation, mucous membrane permeability and the like of the mice given with the preparation are obviously improved in the aspects of weight, intestinal length, histopathology, local active oxygen removal, local inflammation, mucous membrane permeability and the like.

Description

Technical Field

[0001] This invention relates to the field of live bacterial pharmaceutical technology, and more specifically to a surface-modified Bacillus cereus preparation, its preparation method, and its application. Background Technology

[0002] Radiation enteritis (RE) is a common complication following radiotherapy for tumors in the abdomen, retroperitoneum, or pelvis. Its main symptoms include nausea, vomiting, abdominal pain, diarrhea, mucosal shedding, and rectal bleeding, all caused by radiation damage to the small intestine, colon, and rectum. Reactive oxygen species (ROS) play a crucial role in the development of radiation enteritis; therefore, antioxidant therapy has become one of the main strategies for treating it. Due to the long treatment period for radiation enteritis, there is an urgent need for highly effective and safe antioxidants. Amifostine (Ami) is a free radical scavenger and is currently the only clinically available drug for preventing radiation enteritis during radiotherapy; however, its therapeutic window is narrow, and it causes significant hypotension, limiting its clinical application. Given the short half-life and significant side effects of existing antioxidants, there is an urgent need to develop more effective, longer-lasting, and safer alternatives.

[0003] The potential of probiotic therapy in the prevention and treatment of major diseases, such as inflammatory bowel disease, metabolic disorders, and malignancies, is increasingly recognized. While some wild-type and engineered probiotics have shown some efficacy against radiation-induced enteritis, no radiation-induced enteritis-specific strains based on reactive oxygen species (ROS) scavenging have yet been identified. Bacillus cereus (… Bacillus cereus Bacillus cereus (BC) is a Gram-positive, sporulating, facultative anaerobic bacterium. While it is generally believed that Bacillus probiotics can eliminate oxygen in the gut, their ability to combat oxidative stress by scavenging reactive oxygen species (ROS) has not been fully explored. Given the crucial role of ROS in the development of radiation enteritis, and the fact that live bacteria can proliferate in the gut and continuously act as living antioxidants, probiotics with highly efficient ROS scavenging capabilities may demonstrate significant therapeutic efficacy in treating radiation enteritis.

[0004] Two key factors limiting the efficacy of bacterial therapies are oral delivery efficiency and intestinal retention time. The former affects the number of viable bacteria reaching the disease site, while the latter determines the therapeutic window of the live bacteria; both significantly impact the therapeutic effect. A novel solution is to modify bacteria with safe biomaterials that can block the inhibitory effects of gastric acid, bile salts, and other factors on bacterial viability without affecting bacterial survival. Polydopamine (PDA) and chitosan are two commonly used biosafe bacterial coating materials, but the modification methods for Bacillus cereus and their effects on intestinal retention time and gastrointestinal tolerance remain unclear.

[0005] Therefore, providing a polydopamine and chitosan-modified Bacillus cereus preparation and developing it into a live bacterial drug for enteritis caused by specific reasons is a problem that urgently needs to be solved by those skilled in the art. Summary of the Invention

[0006] In view of this, the present invention is hereby proposed.

[0007] One of the objectives of this invention is to provide a Bacillus cereus preparation.

[0008] The second objective of this invention is to provide a method for preparing a surface-modified Bacillus cereus preparation.

[0009] A third objective of this invention is to provide applications of the above-mentioned formulation.

[0010] To achieve the above objectives, the present invention adopts the following technical solution: A surface-modified Bacillus cereus preparation, wherein the surface of Bacillus cereus is modified with a two-component coating of dopamine and chitosan.

[0011] Preferably, the surface-modified Bacillus cereus preparation can be further prepared into live bacteria freeze-dried powder, live bacteria tablets, or live bacteria capsules.

[0012] A second aspect of the present invention provides a method for preparing a surface-modified Bacillus cereus preparation.

[0013] A method for preparing a surface-modified Bacillus cereus preparation, the preparation process including: 1) Prepare Tris-HCl buffer solution with pH 8.3~8.7. Dissolve dopamine and chitosan separately in Tris-HCl buffer solution to prepare dopamine solution and chitosan solution. 2) The Bacillus cereus liquid was shaken and cultured, the bacterial cells were collected by centrifugation and resuspended in the dopamine solution obtained in step 1) to obtain a bacterial suspension; 3) Stir the bacterial suspension obtained in step 2) at room temperature, and then add the chitosan solution obtained in step 1) to the stirred bacterial suspension in multiple batches, stirring after each addition. After completion, centrifuge and discard the supernatant to collect the bacterial cells, thus obtaining the surface-modified Bacillus cereus preparation.

[0014] Preferably, the Tris-HCl buffer concentration is 1-50 mM, the dopamine solution concentration is 0.1-1.0 mg / mL, the chitosan concentration is 1-10 mg / mL, and the Bacillus cereus liquid is cultured with shaking until OD. 600 The concentration was 0.2–1.0, and the cell density after resuspending was 1–9 × 10⁻⁶. 8 CFU / mL.

[0015] Preferably, in step 3, chitosan solution is added to the bacterial suspension every 20-40 minutes until the third addition of chitosan solution, and then stirring is continued for another 20-40 minutes. The volume ratio of bacterial suspension to chitosan solution added each time is 100-300:1.

[0016] The third aspect of this invention provides the application of surface-modified Bacillus cereus preparations in the preparation of drugs for the prevention and treatment of enteritis.

[0017] Preferably, the enteritis includes radiation enteritis, ulcerative colitis, Crohn's disease, and other enteritis with diarrhea symptoms.

[0018] As can be seen from the above technical solution, compared with the prior art, the technical effects achieved by the present invention are as follows: (1) The reactive oxygen species scavenging efficiency of the Bacillus cereus preparation with two components of polydopamine and chitosan was significantly improved compared with that of the original Bacillus cereus.

[0019] (2) The oral live bacteria delivery efficiency and intestinal retention time of the surface-modified Bacillus cereus preparation in this invention are significantly improved.

[0020] (3) The surface-modified Bacillus cereus preparation of the present invention can significantly alleviate the symptoms of enteritis caused by specific reasons. Attached Figure Description

[0021] To more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the drawings used in the description of the embodiments or the prior art will be briefly introduced below. Obviously, the drawings described below are only embodiments of the present invention. For those skilled in the art, other drawings can be obtained based on the provided drawings without creative effort.

[0022] Figure 1The attached figure shows the appearance of Bacillus cereus before and after surface modification with polydopamine and chitosan; BC represents unmodified Bacillus cereus, and BC@PCS represents Bacillus cereus with surface modification with polydopamine and chitosan.

[0023] Figure 2 The attached image shows transmission electron microscope (TEM) images of BC and BC@PCS.

[0024] Figure 3 The attached figures show the average particle size and potential of BC and BC@PCS; A: Average particle size of both; B: Zeta potential of both. n = 3, *** P <0.001.

[0025] Figure 4 The attached figure shows the growth curves of BC and BC@PCS; n = 3.

[0026] Figure 5 The attached figure shows the bacterial survival rate in BC and BC@PCS after freeze-drying; n = 2, ** P <0.01.

[0027] Figure 6 The attached figures show the tolerance of BC and BC@PCS to simulated gastric juice; A: Plate colony photographs of both BC and BC@PCS after co-incubation with simulated gastric juice for 0.5h, 1.0h, 1.5h, and 2.0h; B: Bacterial survival rate of both BC and BC@PCS after co-incubation with simulated gastric juice for 0.5h, 1.0h, 1.5h, and 2.0h. n = 3, *** P <0.001.

[0028] Figure 7 The attached figure shows the effects of BC and BC@PCS on superoxide anions (•O2). - The removal efficiency; n = 3, *** P <0.001.

[0029] Figure 8 The attached figures show the retention of BC and BC@PCS in the mouse intestine; A: In vivo imaging of the mouse intestine at 4h, 6h, 8h, and 12h after gavage administration of BC and BC@PCS, with both BC and BC@PCS labeled with fluorescence; B: Number of Bacillus cereus in the mouse intestine at 4h, 6h, 8h, and 12h after gavage administration of BC and BC@PCS. n = 3, * P <0.05, *** P <0.001.

[0030] Figure 9 The attached figures show the therapeutic effects of BC and BC@PCS on mice with radiation enteritis; A: Schematic diagram of the experimental procedure; B: Appearance of the small intestine and colon of mice in different groups on day 3.5 after irradiation; C: H&E pathological sections of small intestine tissue in different groups of mice on days 3.5 and 7 after irradiation; D: Ki67 immunohistochemical sections of small intestine tissue in different groups of mice on day 3.5 after irradiation; E: Statistical analysis of small intestine length in different groups of mice on day 3.5 after irradiation; F: Immunofluorescence sections of reactive oxygen species (ROS) in small intestine tissue of different groups of mice on day 3.5 after irradiation, with ROS labeled as red fluorescence and cell nuclei labeled as blue fluorescence; except for the small intestine length statistics (… Figure 9 E, n = 4), the remaining experiments n = 3, *** P <0.001.

[0031] Figure 10 The attached figures show the effects of BC and BC@PCS on tight junction proteins, inflammatory factors, and intestinal permeability in the mouse intestine; A: Immunofluorescent sections of Occludin from small intestinal tissue of different groups of mice, with Occludin labeled as yellow fluorescence and cell nuclei labeled as blue fluorescence; B: Immunofluorescent sections of ZO-1 from small intestinal tissue of different groups of mice, with ZO-1 labeled as green fluorescence and cell nuclei labeled as blue fluorescence; C: TNF-α content in small intestinal tissue of different groups of mice; D: IL-6 content in small intestinal tissue of different groups of mice; E: IL-1β content in small intestinal tissue of different groups of mice; F: Diamine oxidase (DAO) content in small intestinal tissue of different groups of mice; F: Serum fluorescence intensity of different groups of mice. n = 3, ns represents no statistically significant difference, *** P <0.001. Detailed Implementation

[0032] The technical solutions of the embodiments of the present invention will be clearly and completely described below with reference to the accompanying drawings. Obviously, the described embodiments are only some embodiments of the present invention, and not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention. Example 1: A surface-modified Bacillus cereus preparation

[0033] Bacillus cereus ( Bacillus cereusBacillus cereus lyophilized powder (Bacillus cereus strain number: CMCC63303) was purchased from Beijing Bio-Biobio Biotechnology Co., Ltd. The lyophilized powder was aseptically dispersed in enzyme-free water in a clean bench, then added to NB liquid medium (containing 3.0 g beef meal, 0.5 g sodium chloride, and 1.0 g peptone per 100 mL). The medium was incubated at 30°C and 220 rpm in a shaking incubator until OD reached its limit. 600 The value was 0.6. Take 6 μL of bacterial solution and count the bacteria using a bacterial counter (Countstar Mira FL Pro, Shanghai Ruiyu Biotechnology Co., Ltd.).

[0034] Dopamine hydrochloride (batch number: KCDGP59), low-viscosity chitosan (batch number: KCDIC23), and Tris-HCl (batch number: KYGK010) were purchased from Beijing Innocare Technology Co., Ltd. A 10 mM Tris-HCl buffer solution was prepared, and the pH was adjusted to 8.5 with 1N sodium hydroxide. Then, a 0.5 mg / mL dopamine solution and a 5 mg / mL chitosan solution were prepared using this buffer. The bacterial culture was centrifuged at 3500 rpm for 5 minutes, and the culture medium was discarded to collect the bacterial cells. The bacterial cells were resuspended in the 0.5 mg / mL dopamine solution to achieve a cell density of 2 × 10⁻⁶ cells / mL. 8 CFU / mL. Take 1 mL of the above bacterial culture and place it at 25°C with magnetic stirring at 200 rpm for 30 minutes. Then add 5 μL of the above chitosan solution. During the reaction, add 5 μL of chitosan solution to the mixture every 30 minutes until the third addition. After reacting for 30 minutes, centrifuge at 3500 rpm for 5 minutes, discard the supernatant, and resuspend the bacteria in 15 mL of 10 mM Tris-HCl buffer to obtain the polydopamine and chitosan two-component surface-modified Bacillus cereus preparation (abbreviated as BC@PCS). Example 2: A surface-modified Bacillus cereus preparation

[0035] Images were taken of BC and BC@PCS suspensions and the centrifuged bacterial cells. The modified Bacillus cereus preparation appeared black, while the unmodified Bacillus cereus appeared pale yellow. Figure 1 Take 5 μL of the diluted BC and BC@PCS suspension and place it on a copper grid for 1 minute. Allow it to air dry naturally. Add 5 μL of 2% phosphotungstic acid staining solution (batch number: A20211126, purchased from Wuhan Saiweier Biotechnology Co., Ltd.). After standing for 1 minute, absorb the excess phosphotungstic acid staining solution with filter paper. Observe the bacterial surface morphology using a transmission electron microscope. Figure 2BC showed visible pili, while BC@PCS did not. This is due to the pili covering the surface after polydopamine and chitosan modification. A suitable amount of diluted BC and BC@PCS suspensions were placed in a particle size potential detection cup, and particle size and zeta potential were measured using a laser particle size analyzer (Zetasizer Nano ZS, Malvern Instruments). The particle size of BC@PCS was larger than that of BC (…). Figure 3 In A), both have negative potentials, and BC@PCS > BC ( Figure 3 (B in the text). Take 10 μL of the bacteria, with a bacterial density of 1×10⁻⁶. 8 The two types of live BC and BC@PCS bacteria, each at CFU / mL, were inoculated into 1 mL of NB medium and cultured at 30°C with shaking at 220 rpm for 48 hours. The OD of the bacterial culture was measured at 0, 3, 6, 12, 24, and 48 hours. 600 They found no difference between the two. Figure 4 This indicates that surface modification does not affect the viability of Bacillus cereus.

[0036] The density of fresh bacteria prepared according to the above method was 1×10⁻⁶. 8 Two bacterial suspensions, BC and BC@PCS, at CFU / mL, were tested for bacterial viability using a bacterial fluorescent dye (batch number: B24011, Shanghai Ruiyu Biotechnology Co., Ltd.) according to the reagent supplier's instructions. After drying the bacterial suspensions using a freeze dryer (LGJ-30, Beijing Songyuan Huaxing Technology Development Co., Ltd.), they were resuspended in an equal volume of Tris-HCl buffer. The same method was used to test bacterial viability again. The results showed that the viability rate of BC@PCS was 92%, and that of BC was 76%, indicating that the two-component surface modification with polydopamine and chitosan can better protect bacteria and improve viability. The above results are attached. Figure 5 Presented in the middle.

[0037] Prepare simulated gastric fluid (SGF) as follows: Take 80 mL of purified water, add 1.64 mL of dilute hydrochloric acid (batch number: 20220617, purchased from Sinopharm Chemical Reagent Co., Ltd.) and 1 g of pepsin (batch number: 628F031, purchased from Beijing Solarbio Science & Technology Co., Ltd.), shake well, and dilute with water to 100 mL. The dilute hydrochloric acid is obtained by diluting 23.4 mL of concentrated hydrochloric acid with water to 100 mL. Prepare mannitol polymyxin agar plates as follows: Weigh 9.8g of mannitol egg yolk polymyxin agar base (batch number: 422H031, purchased from Beijing Solarbio Science & Technology Co., Ltd.), dissolve it in 200mL of distilled water by heating, autoclave at 121℃ for 15 minutes, and when cooled to 60℃, add polymyxin B (batch number: P2091068, purchased from Shanghai Adamas Reagent Co., Ltd.) to a final concentration of 1μg / mL, shake well, pour into a pre-prepared bacterial culture plate, and allow it to cool and solidify before use. Take 1mL of a 1×10⁻⁶ agar plate. 8 CFU / mL of BC and BC@PCS bacterial suspensions were added to 1 mL of artificial gastric fluid and incubated at 37°C. At 30 min, 1 h, 1.5 h, and 2 h, 50 μL of each suspension was centrifuged at 3500 rpm for 5 minutes and resuspended in an equal volume of sterile PBS. 50 μL of each suspension was then spread onto mannitol-polymyxin agar plates and incubated overnight at 37°C. The next day, the growth of different groups on the agar plates was observed, and viable cell counts were performed. The survival rates of BC and BC@PCS in artificial gastric acid at different time points were calculated. Figure 6 (A and B in the sample) It was found that the bacterial survival rate of BC@PCS was significantly higher than that of BC.

[0038] Take concentrations of 1×10 8 The free radical scavenging abilities of BC and BC@PCS bacterial cultures at CFU / mL were detected using an SOD kit (batch number 20221111, purchased from Nanjing Jiancheng Bioengineering Institute). The detection method can be found in the reagent manufacturer's instructions. It was found that BC@PCS exhibited significantly higher superoxide anion scavenging ability than BC (CFU / mL). Figure 7 ).

[0039] Animal experiments were conducted to verify the intestinal retention ability of BC@PCS. Male, SPF-grade, C57BL / 6J mice with a body weight of 21±2 g were used as the animal model. The animal quality certificate was SCXK (Beijing) 2021-0006, and they were purchased from Beijing Vital River Laboratory Animal Technology Co., Ltd. During the feeding process, the mice had free access to water and food, with 12 h of darkness / light each day. Three mice were housed in each cage. After three days of acclimation to the environment, the experiment began. All experiments were carried out in accordance with the regulations of the local animal protection association. 5 μL of a FITC solution with a concentration of 50 mg / mL (batch number: I0201, purchased from Xiamen Shengguang Biotechnology Co., Ltd.) was used to label Bacillus cereus. After centrifugation at 3500 rpm for 5 minutes, the supernatant was discarded, and the bacterial cells were washed three times with phosphate buffer. The Bacillus cereus bacterial solution was coated and modified according to the method described in Example 1. Then, 200 μL of a BC and BC@PCS solution with a concentration of 1×10 10 CFU / mL was administered to the mice by gavage. The retention of Bacillus cereus in the body was observed by small animal in vivo imaging at 4 h, 6 h, 8 h, and 12 h. At 4 hours after gavage, the fluorescence intensity of BC@PCS in the small intestine of the mice was significantly higher than that of the BC group, indicating an increase in the retention time of BC@PCS in the small intestine. At 6 to 8 hours after gavage, the fluorescence intensity in the intestine of the BC group began to decrease, probably due to the intestinal peristalsis of the mice causing its excretion. In contrast, BC@PCS still had a higher fluorescence intensity. At the 12th hour after gavage, in vivo imaging showed that a large amount of BC@PCS still remained in the intestine, while most of the BC had been excreted from the body ( Figure 8 A and B in). The above results indicate that the modification with a polydopamine / chitosan two-component coating prolongs the retention time of Bacillus cereus in the intestine, which is more conducive to its continuous action in the intestine. Example 3 Experiment on the prevention and treatment of enteritis with a surface-modified Bacillus cereus preparation

[0040] Taking radiation enteritis as an example, the effect of the surface-modified Bacillus cereus preparation in preventing and treating enteritis caused by specific reasons was verified. The model establishment and treatment were carried out according to the following procedure. The experimental animals were exactly the same as those in Example 2.

[0041] (1) Experimental grouping, model construction, and drug administration C57BL / 6J male mice were randomly divided into 7 groups, with 10 mice in each group, namely: normal group (Healthy), model group (Model), amifostine group (Ami, positive drug group), Bacillus cereus bacterial solution group (BC), polydopamine and chitosan two-component surface-modified Bacillus cereus group prepared in Example 1 (BC@PCS). The BC@PCS and BC groups were given the corresponding bacterial cells by gavage to the mice 1 day before and 1 h before irradiation and continuously for 3 days after irradiation. The dosage was 0.2 mL of a solution with a concentration of 1×10 8CFU / mL bacterial suspension was administered to mice in the Ami group via intraperitoneal injection of 0.2 mL amifostine 30 minutes prior to irradiation, at a dose of 150 g / kg. The animal model of radiation enteritis was established as follows: After anesthesia and fixation, the mice were covered except for the abdomen with lead bricks, exposing the entire abdomen. 60 The entire abdomen of the mice was irradiated with a single dose of 12 Gy at a dose rate of 71.39 cGy / min. All mice were positioned at the same distance from the radiation source. The complete experimental procedure is described below. Figure 9 A in the middle.

[0042] (2) Examination of intestinal appearance and length On day 3.5 after modeling, four mice in each group were anesthetized and sacrificed. The small intestine was harvested, photographed, and its length was recorded.

[0043] (3) Histopathological examination of small intestine Mice were anesthetized and sacrificed on days 3.5 and 7 after irradiation. A 1 cm section of small intestine tissue was dissected and removed from 5 cm below the stomach. The intestinal contents were rinsed with physiological saline and then transferred to 4% paraformaldehyde solution for fixation for 1 day. Subsequently, the tissue was dehydrated, embedded in paraffin, and hematoxylin-eosin (H&E) staining was performed to observe the pathological morphology of the intestinal tissue.

[0044] (4) ROS analysis of small intestinal tissue On day 3.5 after modeling, mice were dissected and a 2 cm long small intestine tissue was taken from 6 cm from the lower end of the stomach. The intestinal contents were rinsed with physiological saline and the tissue was stained with ROS. The staining steps were as follows: circling, staining, counterstaining cell nuclei, mounting, and image acquisition.

[0045] (5) Investigation of tight junction proteins in small intestinal tissue The expression of tight junction proteins in small intestinal cells after irradiation was investigated using Occludin and ZO-1. The specific steps mainly included paraffin dehydration, antigen retrieval, serum blocking by drawing circles, addition of double antibiotics, DAPI staining of cell nuclei, mounting, etc., and observation under a fluorescence microscope.

[0046] (6) Investigation of inflammatory factors in small intestinal tissue On day 7 post-irradiation of mice, a 2 cm section of small intestine tissue was harvested from a location 6 cm below the stomach. Nine times the volume of pre-cooled phosphate-buffered saline (PBS) was added, and the tissue was ground with grinding beads for 360 s at a frequency of 70 Hz. The homogenate was then centrifuged at 5000 × g for 15 min at 4 °C. The supernatant was collected to detect the levels of IL-6, IL-1β, and TNF-α in the small intestine tissue using ELISA kits (Mouse TNF-α ELISA kit: batch number ml0020015-J, Mouse IL-6 ELISA kit: batch number ml063159-J, Mouse IL-1β ELISA kit: batch number ml301814-J, all purchased from Shanghai Enzyme-Link Biotechnology Co., Ltd.). The detection method can be found in the ELISA kit instructions.

[0047] (7) Investigation of intestinal mucosal permeability On day 7 post-irradiation, mice were tested for diamine oxidase (DAO) activity in small intestinal tissue using an ELISA kit (batch number: ml002199, purchased from Shanghai Enzyme-Link Biotechnology Co., Ltd.) in the same manner as in Example (6). On day 10 post-irradiation, three mice were taken from each group and fasted for 12 hours beforehand. 0.2 mL of FITC-dextran solution (batch number: P2537089, purchased from Shanghai Adamas Beta Reagent Co., Ltd.) was administered by gavage at a dose of 500 mg / kg. Blood was collected from the eyeballs 4 hours later. The blood samples were placed at 4°C and allowed to stand for 12 hours. Serum was then obtained by centrifugation at 3000 rpm for 20 minutes at 4°C. 100 μL of serum was collected, and the fluorescence intensity was measured using a multi-functional fluorescent microplate reader at an excitation wavelength of 495 nm and an emission wavelength of 520 nm.

[0048] The small intestine length of the irradiated mice was significantly shorter, while the small intestine length of the BC@PCS group was significantly increased compared to the model group. Figure 9 B and E in the irradiated mice); the small intestinal tissue structure of the irradiated mice was severely damaged ( Figure 9 C and D in the formula lead to increased intestinal permeability and decreased barrier protection, while BC@PCS effectively protects the intestinal barrier function, maintaining it in a state similar to that of the healthy group. Figure 10 The levels of reactive oxygen species (ROS) in the small intestinal tissue of irradiated mice were significantly increased, while BC@PCS effectively scavenged reactive oxygen species (ROS). Figure 9 The F in the serum reduces oxidative damage and simultaneously lowers the DAO content in the serum. Figure 10 (F in the text); an examination of inflammatory factors in small intestinal tissue showed that after irradiation, the levels of pro-inflammatory factors IL-1β, TNF-α, and IL-6 increased, while BC@PCS effectively inhibited the occurrence and development of inflammation, and the levels of pro-inflammatory factors IL-1β, TNF-α, and IL-6 decreased (F in the text). Figure 10The C, D, and E values ​​indicate that BC@PCS can effectively inhibit oxidative damage and suppress the occurrence and development of radiation enteritis. Immunofluorescence examination of small intestinal tissues from mice in each group revealed low expression of all three tight junction proteins in the irradiated mice, indicating severe intestinal damage. In contrast, the BC@PCS group showed high expression of both tight junction proteins in the intestines, almost identical to the healthy group. Figure 10 (A and B in the table) indicates that BC@PCS significantly increases the expression of tight junction proteins and maintains normal intestinal function. The above data suggest that BC@PCS has a preventive and therapeutic effect on radiation enteritis.

[0049] The various embodiments in this specification are described in a progressive manner, with each embodiment focusing on the differences from other embodiments. The same or similar parts between the various embodiments can be referred to each other.

[0050] The above description of the disclosed embodiments enables those skilled in the art to make or use the invention. Various modifications to these embodiments will be readily apparent to those skilled in the art, and the general principles defined herein may be implemented in other embodiments without departing from the spirit or scope of the invention. Therefore, the invention is not to be limited to the embodiments shown herein, but is to be accorded the widest scope consistent with the principles and novel features disclosed herein.

Claims

1. A surface-modified Bacillus cereus preparation, characterized in that, The formulation contains a two-component coating of dopamine and chitosan on the surface of Bacillus cereus.

2. The surface-modified Bacillus cereus preparation according to claim 1, characterized in that, The dosage forms include live bacteria freeze-dried powder, live bacteria tablets, or live bacteria capsules.

3. The method for preparing a surface-modified Bacillus cereus preparation according to claim 1, characterized in that, The preparation process includes: 1) Prepare Tris-HCl buffer solution with pH 8.3~8.

7. Dissolve dopamine and chitosan separately in Tris-HCl buffer solution to prepare dopamine solution and chitosan solution. 2) The Bacillus cereus liquid was shaken and cultured, the bacterial cells were collected by centrifugation and resuspended in the dopamine solution obtained in step 1) to obtain a bacterial suspension; 3) Stir the bacterial suspension obtained in step 2) at room temperature, and then add the chitosan solution obtained in step 1) to the stirred bacterial suspension in multiple batches, stirring after each addition. After completion, centrifuge and discard the supernatant to collect the bacterial cells, thus obtaining the surface-modified Bacillus cereus preparation.

4. The method for preparing a surface-modified Bacillus cereus preparation according to claim 3, characterized in that, The Tris-HCl buffer concentration is 1-50 mM, the dopamine solution concentration is 0.1-1.0 mg / mL, the chitosan concentration is 1-10 mg / mL, and the Bacillus cereus liquid is cultured with shaking until OD. 600 The concentration was 0.2–1.0, and the cell density after resuspending was 1–9 × 10⁻⁶. 8 CFU / mL.

5. In the preparation method of a surface-modified Bacillus cereus preparation according to claim 3, in step 3, chitosan solution is added to the bacterial suspension every 20-40 minutes until the chitosan solution is added for the third time, and then stirring is continued for 20-40 minutes. The volume ratio of bacterial suspension to chitosan solution added each time is 100-300:

1.

6. The use of the surface-modified Bacillus cereus preparation according to claim 1 or 2, or the surface-modified Bacillus cereus preparation prepared by the preparation method according to any one of claims 3-5, in the preparation of drugs for the prevention and treatment of enteritis.

7. The application of the surface-modified Bacillus cereus preparation according to claim 6 in the preparation of drugs for the prevention and treatment of enteritis, characterized in that, The enteritis mentioned includes radiation enteritis, ulcerative colitis, and Crohn's disease.

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