Bifidobacterium adolescentis BAS05 and application thereof in preparation of medicine for preventing or treating intestinal inflammation
By screening and identifying Bifidobacterium adolescentis BAS05, a bacterial suspension was prepared to inhibit DSS-induced neutrophil aggregation, solving the problem of the lack of domestically developed probiotic strains in my country and achieving effective treatment of intestinal inflammation.
Patent Information
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- GUANGDONG LONGSEE BIOMEDICAL CO LTD
- Filing Date
- 2023-04-10
- Publication Date
- 2026-04-17
AI Technical Summary
my country lacks functional probiotic strains with independent intellectual property rights, which affects the promotion of probiotic products. Currently, there is a lack of scientific research evidence regarding the function of probiotics in treating intestinal inflammation.
Bifidobacterium adolescentis (BAS05) was screened and isolated from the feces of centenarians in Jiaoling County, Meizhou City, Guangdong Province. It was identified as Bifidobacterium adolescentis and prepared into a bacterial suspension. This suspension was used to inhibit DSS-induced neutrophil aggregation in the zebrafish intestine and to develop drugs for the prevention or treatment of intestinal inflammation.
Bifidobacterium adolescentis BAS05 significantly inhibited DSS-induced neutrophil aggregation in the zebrafish intestine, demonstrating a good potential to alleviate intestinal inflammation, which is stronger than the existing strain 15703, providing a theoretical basis and practical guidance for the prevention or treatment of intestinal inflammation.
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Figure CN121874019A_ABST
Abstract
Description
Technical Field
[0001] This invention relates to the field of microbial technology, and more specifically to Bifidobacterium adolescentis BAS05 and its use in the preparation of drugs for the prevention or treatment of intestinal inflammation. Background Technology
[0002] Enteritis is generally caused by bacteria, viruses, fungi, or parasites. Clinically, it can be divided into proctitis and colitis depending on the location of the infection, and into acute and chronic enteritis depending on the speed of onset. Diarrhea is the most typical symptom of enteritis. In addition, patients are also prone to abdominal pain and constipation, which greatly affects their normal life. Therefore, enteritis must be treated actively.
[0003] Traditional treatments for enteritis can be divided into modern medical treatments and traditional Chinese medicine treatments. Probiotics, as an endogenous and immune defense barrier in the gut, can antagonize pathogenic bacteria and are characterized by safety, controllability, effectiveness, and minimal side effects, making them an ideal method for treating enteritis. Currently, international probiotic patent applications are concentrated in traditional research powerhouses like the US, Japan, and Russia, while my country lacks functional strains with independent intellectual property rights. The lack of strong scientific research evidence regarding the function of probiotics seriously hinders the promotion of probiotics and their products. Therefore, in-depth exploration of the functions of bacterial strains and the screening of novel probiotic strains with independent intellectual property rights, specific functional properties, and suitability for the physiological characteristics of the Chinese population are crucial for improving the core competitiveness of Chinese probiotic manufacturers and promoting the development of probiotic products in my country.
[0004] Therefore, providing Bifidobacterium adolescentis BAS05 and its application in the preparation of drugs for the prevention or treatment of intestinal inflammation is a problem that urgently needs to be solved by those skilled in the art. Summary of the Invention
[0005] In view of this, the present invention provides Bifidobacterium adolescentis BAS05 and its use in the preparation of drugs for the prevention or treatment of intestinal inflammation.
[0006] Inflammation is an immune response to tissue damage and infection, characterized by the aggregation of white blood cells (granulocytes and macrophages) around infected tissue. The zebrafish immune system is very similar to that of mammals; when trauma occurs, neutrophils and macrophages respond to traumatic inflammation almost simultaneously. Neutrophils migrate rapidly, recruiting to the site of injury first, followed by macrophages. Several hours later, the inflammation begins to subside, and macrophages and neutrophils leave the site of injury. Inducing intestinal inflammation in zebrafish with sodium dextran sulfate (DSS) stimulates an immune response in zebrafish neutrophils.
[0007] Using transgenic fluorescent neutrophil fish (green in color), the number of neutrophils in the intestine of DSS-induced zebrafish was significantly increased compared to that of normal zebrafish under a fluorescence microscope.
[0008] To achieve the above objectives, the present invention adopts the following technical solution:
[0009] A strain of Bifidobacterium adolescentis BAS05, with accession number CGMCC No. 22515, has been deposited at the China General Microbiological Culture Collection Center (CGMCC), located at No. 3, Courtyard 1, Beichen West Road, Chaoyang District, Beijing, Institute of Microbiology, Chinese Academy of Sciences, on May 13, 2021. It is classified and named Bifidobacterium adolescentis.
[0010] Furthermore, the application of the aforementioned Bifidobacterium adolescentis BAS05 in the preparation of drugs for the prevention or treatment of intestinal inflammation.
[0011] Furthermore, the application of the aforementioned Bifidobacterium adolescentis BAS05 in the preparation of a drug to inhibit DSS-induced neutrophil aggregation in the zebrafish intestine.
[0012] Furthermore, the Bifidobacterium adolescentis BAS05 is a bacterial suspension.
[0013] As can be seen from the above technical solution, compared with the prior art, the present invention discloses Bifidobacterium adolescentis BAS05 and its application in the preparation of drugs for the prevention or treatment of intestinal inflammation. Bifidobacterium adolescentis BAS05 was isolated and screened from the feces of long-lived elderly people in Jiaoling County, Meizhou City, Guangdong Province. Bifidobacterium adolescentis BAS05 can inhibit DSS-induced neutrophil aggregation in the intestine of zebrafish. This provides a theoretical reference and guiding basis for the development of drugs for the prevention or treatment of intestinal inflammation using Bifidobacterium adolescentis BAS05. Attached Figure Description
[0014] 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.
[0015] Figure 1 The attached figure shows the colony morphology of Bifidobacterium adolescentis BSA05 of the present invention on BS agar plates;
[0016] Figure 2The attached figure is a visual representation of how Bifidobacterium adolescentis BSA05 of the present invention inhibits DSS-induced neutrophil aggregation in the zebrafish intestine;
[0017] Among them, A: normal group; B: model group; C: positive control group; D: Bifidobacterium adolescentis 15703; E: Bifidobacterium adolescentis BSA05;
[0018] Figure 3 The attached figure is a statistical graph showing the inhibition of DSS-induced neutrophil aggregation in the zebrafish intestine by Bifidobacterium adolescentis BSA05 according to the present invention. Detailed Implementation
[0019] 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.
[0020] The zebrafish were Tg(mpx:EGFP) centriole cell green fluorescent zebrafish and AB line zebrafish.
[0021] Sodium dextran sulfate (DSS) (MedChemExpress); mesalazine (MedChemExpress); and Bifidobacterium adolescentis 15703 (ATCC 15703) were purchased from Beijing Bio-Bio Biotechnology Co., Ltd.
[0022] Example 1: Isolation, identification and preservation of Bifidobacterium adolescentis BAS05
[0023] (1) Separation:
[0024] 1) Dissolve the feces of the long-lived elderly (about 0.1g) in a 1.5mL centrifuge tube containing 1mL of sterile physiological saline, and mix thoroughly by pipetting with a 1mL sterile pipette tip.
[0025] 2) Add 900 μL of sterile physiological saline to each of six sterile 1.5 mL centrifuge tubes.
[0026] 3) Starting from the first one containing 10 -1 In the centrifuge tube containing the sample dilution, add 100 μL of the liquid to the second centrifuge tube (10 μL). -2 Dilute to 10 -2 ;
[0027] 4) From the second one containing 10 -2 In the centrifuge tube containing the sample dilution solution, add 100 μL of the liquid to the third centrifuge tube (10 μL). -3 Dilute to 10 -3;
[0028] 5) Repeat the previous step until the solution is diluted to 10. -4 10 -5 10 -6 10 -7 .
[0029] 6) From the container containing 10 -4 Take 100 μL of the sample dilution from the centrifuge tube and inoculate it onto MRS solid medium and BS solid medium, respectively. Spread the 100 μL of bacterial culture evenly and dry it. Be careful to spread it gently and quickly, and this must be done near an alcohol lamp flame. After spreading, mark the side of the petri dish with information including name, sample number, medium name, incubation time, dilution gradient, and incubation conditions (anaerobic / aerobic).
[0030] 7) Repeat the previous step to complete 10. -5 10 -6 10 -7 Dilution gradient dilution coating.
[0031] 8) After coating, place the petri dishes in anaerobic conditions at 37°C and incubate for 48 hours. Observation and recording can be carried out after 48 hours.
[0032] 9) Use an inoculation loop to pick up a single colony from the plate and streak it onto BS solid medium. Incubate anaerobically at 37°C for 48 hours to obtain pure colonies.
[0033] 10) Inoculate the pure colonies on the plate into BS liquid medium, anaerobic culture at 37°C for 12-16 h, add 20% glycerol, and store in a -80°C refrigerator.
[0034] (2) Molecular biological identification of the strain: Genomic DNA was extracted from the obtained strain, and the full-length 16S rDNA fragment was amplified by PCR using the universal 16S rDNA primers 27F and 1492R. Then, sequencing was performed to identify the species of the strain.
[0035] The primer sequences for universal primers 27F and 1492R are as follows:
[0036] 27F: 5'-AGAGTTTGATCCTGGCTCAG-3'; SEQ ID NO.1;
[0037] 1492R: 5'-GGTTACCTTGTTACGACTT-3'; SEQ ID NO. 2.
[0038] Experimental results: The strains screened from the feces of centenarians in Jiaoling County, Meizhou City, Guangdong Province were identified as Bifidobacterium adolescentis by morphological observation and 16S rDNA identification. The 16S rDNA sequence of strain BAS05 is shown in SEQ ID NO.3.
[0039]
[0040] Single colonies of strain BAS05 were inoculated onto BS solid medium and grew well under aerobic conditions at 37°C. The colonies were milky white, with regular edges, spherical shape, and smooth surface. Figure 1 The strain BAS05 has been deposited at the China General Microbiological Culture Collection Center (CGMCC), located at No. 3, Courtyard 1, Beichen West Road, Chaoyang District, Beijing, Institute of Microbiology, Chinese Academy of Sciences. The deposit date was May 13, 2021. The classification name is Bifidobacterium adolescentis, and the accession number is CGMCC No. 22515.
[0041] Example 2: Preparation of Bifidobacterium adolescentis BSA05 bacterial suspension (cells)
[0042] After activation and culture, *Bifidobacterium adolescentis* BSA05 was inoculated into BS liquid medium and cultured at 37°C for 24 h. The culture was then centrifuged at 4°C, 6000 rpm for 10 min to obtain a bacterial pellet. The bacterial pellet was washed twice with PBS, and the cells were resuspended in PBS to adjust the cell concentration to 1 × 10⁻⁶. 6 A bacterial suspension (bacterial cells) was obtained at CFU / mL.
[0043] Example 3: Preparation of Bifidobacterium adolescentis 15703 bacterial suspension (cells)
[0044] After activation and culture, *Bifidobacterium adolescentis* 15703 was inoculated into BS liquid medium and cultured at 37°C for 24 h. The culture was then centrifuged at 4°C and 6000 rpm for 10 min to obtain a bacterial pellet. The bacterial pellet was washed twice with PBS, and the cells were resuspended in PBS to adjust the cell concentration to 1 × 10⁻⁶. 6 A bacterial suspension (bacterial cells) was obtained at CFU / mL.
[0045] Example 4: Effect of Bifidobacterium adolescentis BSA05 on DSS-induced neutrophil aggregation in the zebrafish intestine.
[0046] Healthy Tg(mpx:EGFP) zebrafish (3dpf) were selected and placed in 6-well cell culture plates, one well per group, with 15 fish per well (15 fish per well, neutrophil count in the intestine of each fish). 5 mL of PBS was added to the normal control group, and 1 × 10⁻⁶ cells were added to the model group, positive control group (mesalazine), and Bifidobacterium adolescentis 15703 intervention group (1 × 10⁻⁶ cells). 6 CFU / mL), Bifidobacterium adolescentis BSA05 intervention group (1×10 6All zebrafish (CFU / mL) were added to 5 mL of 0.5% DSS solution (w / w) and incubated at 28°C for 72 h in a biochemical incubator. Each group of zebrafish was then washed three times with PBS. The normal and model groups were then added to 5 mL of PBS; the positive control group was added to 5 mL of 50 μg / mL mesalazine solution; and the Bifidobacterium adolescentis 15703 intervention group was added to 1×10⁻⁶ CFU / mL solution. 6 CFU / mL Bifidobacterium adolescentis 15703, 5 mL per well; Bifidobacterium adolescentis BSA05 intervention group added 1×10 6 CFU / mL Bifidobacterium adolescentis BSA05, 5 mL per well, was incubated in a biochemical incubator at 28℃ for 48 h. The neutrophil aggregation in the zebrafish intestine was observed under a fluorescence microscope and photographed.
[0047] Data were statistically processed using SPSS 19.0 software. Experimental data are expressed as mean ± SEM values. T-tests were used for analysis, comparing the results with the normal group: #### p<0.001, compared with the model group: ****P<0.001.
[0048] The experimental results of Bifidobacterium adolescentis BSA05 inhibiting DSS-induced neutrophil aggregation in the zebrafish intestine are as follows: Figure 2 and Figure 3 As shown.
[0049] Depend on Figure 2 and Figure 3 It was found that a small number of neutrophils (12.67±1.71) were aggregated in the intestine of zebrafish in the normal group, while a large number of neutrophils were aggregated in the intestine of zebrafish in the model group. Furthermore, the number of neutrophils in the intestine of zebrafish in the model group was 32.47±2.46, which was significantly different from that in the normal group (12.67±1.71) (P<0.001), indicating that the DSS-induced zebrafish intestinal inflammation model was successfully established.
[0050] Depend on Figure 2 and Figure 3 It was found that only a small number of neutrophils were aggregated in the intestines of zebrafish in the positive control group (methalazine). Furthermore, the number of neutrophils in the intestines of zebrafish in the positive control group (methalazine) was 14.20±1.57, which was significantly different from that in the model group (32.47±2.46) (P<0.001). Therefore, methalazine has an effect in relieving intestinal inflammation, consistent with clinical results, indicating that this trial evaluating its efficacy in relieving intestinal inflammation is effective. The Bifidobacterium adolescentis 15703 intervention group (1×10⁻⁶) 6 The zebrafish gut containing CFU / mL showed a large accumulation of neutrophils, similar to the model group; meanwhile, the Bifidobacterium adolescentis 15703 intervention group (1×10⁻⁶ CFU / mL) showed similar activity. 6The number of neutrophils in the zebrafish intestine (CFU / mL) was 29.40±2.79, which was not significantly different from that in the model group (32.47±2.46) (P>0.05). Furthermore, only a small number of neutrophils were aggregated in the zebrafish intestine of the Bifidobacterium adolescentis BSA05 group, similar to the positive control group (mesalazine); Meanwhile, the number of neutrophils in the Bifidobacterium adolescentis BSA05 intervention group (1×10⁻⁶ CFU / mL) was also significantly different from that in the model group (1×10⁻⁶ CFU / mL). 6 The number of neutrophils in the zebrafish intestine (CFU / mL) was 16.40±2.05, which was significantly different from that in the model group (32.47±2.46) (P<0.001). Therefore, the above results indicate that at the same concentration, *Bifidobacterium adolescentis* BSA05 has a stronger inhibitory effect on DSS-induced neutrophil aggregation in the zebrafish intestine than *Bifidobacterium adolescentis* 15703, demonstrating a good potential to alleviate intestinal inflammation.
[0051] 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 strain of Bifidobacterium adolescentis BAS05, characterized in that, Its accession number is CGMCCNo.22515.
2. The use of Bifidobacterium adolescentis BAS05 as described in claim 1 in the preparation of drugs for the prevention or treatment of intestinal inflammation.
3. Use of Bifidobacterium adolescentis BAS05 according to claim 2 for the preparation of a medicament for the prevention or treatment of intestinal inflammation, characterized in that, The Bifidobacterium adolescentis BAS05 is a bacterial suspension.
4. The use of Bifidobacterium adolescentis BAS05 as described in claim 1 in the preparation of a drug to inhibit DSS-induced neutrophil aggregation in the zebrafish intestine.
5. The application of Bifidobacterium adolescentis BAS05 according to claim 4 in the preparation of a drug for inhibiting DSS-induced neutrophil aggregation in the zebrafish intestine, characterized in that, The Bifidobacterium adolescentis BAS05 is a bacterial suspension.