Bifidobacterium longum for repairing enteric nerve and improving intestinal motility and application thereof

Abstract

The application discloses a bifidobacterium longum for repairing intestinal nerves and improving intestinal motility and an application thereof, and belongs to the field of microorganisms. The bifidobacterium longum CCFM1389 provided by the application can significantly increase the number of intestinal nerve glial cells and intestinal neurons, promote the release of excitatory neurotransmitters ACh and SP, repair the damaged intestinal nervous system of mice, improve the expression of TJ protein, improve the intestinal permeability, improve the pathological damage of colon tissue, and relieve the intestinal motility disorder. Therefore, a more personalized treatment scheme can be adopted for gastrointestinal diseases caused by abnormal intestinal nervous system, and the application prospect is considerable.

Description

Technical Field

[0001] This invention relates to a strain of Bifidobacterium bifidum that repairs intestinal nerves and improves intestinal motility, and its applications, belonging to the field of microbiology. Background Technology

[0002] Currently, antibiotics are still widely used in human medicine to treat various bacterial infections. A 2021 article published in Nature investigated the effects of 144 antibiotics on human gut commensal bacteria, showing that approximately half of the tested gut bacteria strains failed to survive after treatment with these antibiotics. When the balance of the gut microbiota is disrupted, it can lead to the overgrowth of harmful bacteria, resulting in damage to intestinal wall cells or the intestinal mucosa. This damage can affect the lining of the enteric nervous system, thereby affecting intestinal motility and the intestine's ability to absorb water, with diarrhea becoming one of the most common symptoms.

[0003] Animal studies on probiotics' ability to improve impaired intestinal motility have largely focused on Bifidobacteria and Lactobacillus. Research indicates that Bifidobacteria exhibit interspecies differences in alleviating intestinal motility. Specifically, *Bifidobacterium bifidum*, *Bifidobacterium longum*, and *Bifidobacterium infantis* showed the best effects, *Bifidobacterium adolescentis* and *Bifidobacterium breve* were partially effective, while *Bifidobacterium animalis* was ineffective. *Bifidobacterium bifidum* not only improves intestinal motility but also repairs enteric nerve damage. Studies on germ-free mice have found that under germ-free conditions, the number, distribution, and function of intestinal neurons may be affected, leading to abnormalities in intestinal motility and sensory function. Furthermore, germ-free mice may exhibit abnormalities in the intestinal immune system, further affecting the normal function of the nervous system. By comparing the enteric nervous systems of germ-bearing and germ-free mice, researchers have found that gut microbiota can regulate intestinal neuronal survival and promote neurogenesis, playing a crucial role in the development and maturation of the enteric nervous system.

[0004] To some extent, antibiotic-treated mice may exhibit phenotypes similar to germ-free mice, depending on factors such as the type of antibiotic, dosage, and duration of treatment. Currently, no method has been found that can completely and effectively alleviate antibiotic-induced intestinal problems. In managing this issue, appropriate antibiotics can be selected based on the specific circumstances, or a rational drug use strategy can be adopted. Furthermore, adjusting the diet to increase the intake of foods rich in probiotics and prebiotics may also have some effect on reducing antibiotic-induced intestinal problems, but the effect is limited. Additionally, during or after antibiotic use, supplementing with probiotics as advised by a doctor can help restore the balance of the intestinal microecology and maintain intestinal health. However, no probiotic preparations have yet been found that can effectively treat antibiotic-induced intestinal motility damage. Therefore, screening for strains effective in treating antibiotic-associated diarrhea will help in developing effective probiotic preparations to repair intestinal nerve function and regulate intestinal motility. Summary of the Invention

[0005] [Technical Issues]

[0006] The purpose of this invention is to provide a strain of Bifidobacterium bifidum that can alleviate antibiotic-induced damage to intestinal motility and enteric nerves, and to provide the application of this strain.

[0007] [Technical Solution]

[0008] To address the aforementioned technical problems, this invention provides a strain of *Bifidobacterium bifidum* that can effectively repair the enteric nervous system. Compared with the normalized CCFM1163 strain, this strain shows upregulated expression of neuronal markers (PGP9.5) and enteric glial cell markers (GFAP and S100β), and significantly improved colonic pathological damage. This indicates that the strain can repair the enteric nervous system and alleviate various adverse effects caused by enteric nerve damage, thus helping patients with enteric nervous system abnormalities and impaired intestinal motility due to antibiotic use.

[0009] This invention provides a strain of Bifidobacterium bifidum CCFM1389, which was deposited on July 11, 2024 at the Guangdong Provincial Center for Microbial Culture Collection, with accession number GDMCC No: 64859, located at the Institute of Microbiology, Guangdong Academy of Sciences, 5th Floor, Building 59, No. 100 Xianlie Middle Road, Guangzhou.

[0010] The *Bifidobacterium bifidum* strain was obtained from a fecal sample of an adult in Wuxi City, Jiangsu Province. Sequencing analysis of the strain and alignment of the sequenced data with NCBI Standard Nucleotide BLAST showed a 100% similarity to other *Bifidobacterium* strains. Therefore, the strain was identified as *Bifidobacterium bifidum* and named *Bifidobacterium bifidum* CCFM1389.

[0011] The Bifidobacterium bifidum CCFM1389 has the following biological characteristics:

[0012] 1) Bacterial characteristics: It is a Gram-positive non-spore-forming bacillus with a cell size of approximately 0.5-1.3 μm × 1.5-8 μm and obvious pleomorphism.

[0013] 2) Colony characteristics: After streaking on MRS medium containing 0.1% L-cysteine ​​hydrochloride for 48 hours, obvious colonies are formed, with a diameter between 0.2-2.5 mm. They are round, convex or lenticular, slightly white, opaque, and have a smooth to mucous soft surface. They do not form mycelia.

[0014] 3) Growth characteristics: The optimal growth temperature for this strain is 36-38℃, with good growth at 32-38℃. However, it can also grow at 45℃ with a high survival rate. The optimal initial pH is 6-7, with reduced growth at pH 5.5 or below. It grows well in anaerobic culture medium containing glucose, entering the late logarithmic phase or early stationary phase after 20 hours of culture, with turbidity in the liquid tube, and the final pH reaching 4.0-4.8.

[0015] 4) It has good tolerance to simulated gastrointestinal fluid.

[0016] 5) It has adhesive properties and can adhere well to HT-29 colon cancer cells.

[0017] 6) Significantly increases the number of enteric neurons and glial cells in the colon tissue, promotes the release of enteric neurotransmitters, repairs the intestinal mechanical barrier and improves the pathological damage of colon tissue, and alleviates intestinal motility disorders.

[0018] The present invention also provides a microbial preparation containing the above-mentioned Bifidobacterium bifidum CCFM1389.

[0019] The present invention also provides a food product containing a microbial preparation of the aforementioned Bifidobacterium bifidum CCFM1389.

[0020] In one embodiment of the present invention, the food is a fermented food, which is produced by fermentation using Bifidobacterium bifidum CCFM1389. The fermented food includes solid food, liquid food, and semi-solid food.

[0021] In one embodiment of the present invention, the amount of Bifidobacterium bifidum added to the food is at least 1× 8 CFU / mL or 1×10 8 CFU / g.

[0022] In one embodiment of the present invention, the fermented food includes dairy products, soy products, or fruit and vegetable products.

[0023] In one embodiment of the present invention, the dairy product is a fermented dairy product, including fermented milk, fermented milk beverage, cream, cheese or milk powder; the soy product includes soy milk, soy milk beverage, soy milk powder; the fruit and vegetable product includes fermented fruit and vegetable beverages or foods made from cabbage, white radish, cucumber, beet, yellow peach or bayberry.

[0024] The present invention also provides a medicine containing the above-mentioned Bifidobacterium bifidum CCFM1389 or the above-mentioned microbial agent.

[0025] In one embodiment of the invention, the drug comprises Bifidobacterium bifidum CCFM1389 and a pharmaceutically permissible carrier.

[0026] In one embodiment of the present invention, the carrier includes one or more of the following commonly used in medicine: fillers, adhesives, wetting agents, disintegrants, lubricants, and flavoring agents.

[0027] In one embodiment of the present invention, the dosage form of the medicine includes granules, capsules, tablets, pills, or oral liquid.

[0028] In one embodiment of the present invention, the amount of Bifidobacterium bifidum CCFM1389 added to the pharmaceutical product is at least 1×10⁻⁶. 8 CFU / mL or 1×10 8 CFU / g.

[0029] The present invention also provides the application of the above-mentioned Bifidobacterium bifidum CCFM1389 or the above-mentioned microbial agent in the preparation of health products that help relieve diarrhea.

[0030] This invention also provides the application of the above-mentioned Bifidobacterium bifidum CCFM1389 or the above-mentioned microbial agent in the preparation of a medicine for relieving intestinal nerve damage, characterized in that the relief of intestinal damage includes at least one of the following:

[0031] a) Increase the number of enteric glial cells and enteric neurons;

[0032] b) Increase the levels of neurotransmitters ACh and SP in colon tissue damaged by enteric nerves;

[0033] c) Increase the gene expression of tight junction proteins Occludin, Claudin-3 and ZO-1 in the gut;

[0034] d) Improves the pathological damage of colon tissue with impaired enteric nerves.

[0035] e) Relieves intestinal motility disorders caused by weakened motility and improves the intestine's ability to reabsorb water.

[0036] The present invention also provides a bacterial agent containing the aforementioned Bifidobacterium bifidum CCFM1389.

[0037] In one embodiment of the present invention, the viable count of Bifidobacterium bifidum CCFM1389 in the bacterial agent is ≥1×10⁻⁶. 8 CFU / g or 1×10 8 CFU / mL.

[0038] In one embodiment of the present invention, the bacterial agent is obtained by drying a bacterial solution containing Bifidobacterium bifidum CCFM1389, resulting in a viable bacterial count ≥1×10⁻⁶. 8 CFU / g or 1×10 8 Powder with CFU / mL.

[0039] In one embodiment of the present invention, the drying refers to vacuum freeze drying.

[0040] Beneficial effects

[0041] (1) The *Bifidobacterium bifidum* CCFM1389 strain of this invention exhibits superior activity, significantly increasing the number of enteroglial cells and neurons in colonic tissue, increasing the gene expression levels of tight junction proteins Occludin, Claudin-3, and ZO-1 in colonic tissue, enhancing the expression of excitatory neurotransmitters, improving pathological damage in colonic tissue, alleviating intestinal motility disorders caused by weakened motility, and improving the intestinal reabsorption capacity. Importantly, *Bifidobacterium bifidum* CCFM1389 shows significantly better effects than CCFM1163 in terms of the number of enteroglial cells and neurons, ACh and SP content, and tight junction protein gene expression levels. Compared with normalized CCFM1163, the expression levels of S100β and GFAP increased by 10.61% and 44.28%, respectively, and the expression level of PGP9.5 increased by 20.80%. Therefore, this strain has a more significant effect on the repair of enteroneurin.

[0042] (2) This invention can be regarded as a drug to relieve or treat intestinal motility disorders and enteric nerve damage caused by antibiotic use. It can also be applied to pharmaceuticals or some fermented foods and functional foods, thereby playing a wide role and having a very valuable application prospect.

[0043] Preservation of biological materials

[0044] A strain of Bifidobacterium bifidum (CCFM1389), taxonomically named Bifidobacterium bifidum, was deposited on July 11, 2024, at the Guangdong Provincial Microbial Culture Collection Center (GDMCC No.: 64859), located at the Guangdong Institute of Microbiology, 5th Floor, Building 59, No. 100 Xianlie Middle Road, Guangzhou. Attached Figure Description

[0045] Figure 1 Schematic diagram showing changes in the expression levels of neuronal and glial cell marker proteins mRNA in the colon tissue of mice with ABX-induced enteric nerve damage after intervention with Bifidobacterium bifidum strain CCFM1389.

[0046] Figure 2 Schematic diagram showing changes in the levels of neurotransmitters ACh and SP in the colonic tissue of ABX-induced enteric nerve damage mice after intervention with Bifidobacterium bifidum CCFM1389 strain. Schematic diagram showing changes in the transcriptional levels of three TJ proteins in ABX-induced enteric nerve damage mice after intervention with Bifidobacterium bifidum CCFM1389 strain.

[0047] Figure 3 Schematic diagram of the changes in transcriptional levels of three TJ proteins in ABX mice with enteric nerve damage after intervention with Bifidobacterium bifidum strain CCFM1389.

[0048] Figure 4 Schematic diagram and histopathological scoring table of H&E staining results of colon tissue in mice with ABX-induced enteric nerve damage after intervention with Bifidobacterium bifidum CCFM1389 strain.

[0049] Figure 5 Schematic diagram of the relevant indicators (time to first black stool, fecal water content, and small intestinal propulsion rate) of Bifidobacterium bifidum strain CCFM1389 in alleviating symptoms of intestinal motility disorders caused by enteric nerve damage. Detailed Implementation

[0050] The male C57BL / 6J mice used in the following examples were purchased from Zhejiang Vital River Laboratory Animal Technology Co., Ltd.

[0051] The strain information involved in the following examples is as follows:

[0052] A strain of Bifidobacterium bifidum (CCFM1389) was classified as Bifidobacterium bifidum and its accession number is GDMCC No: 64859.

[0053] A strain of Bifidobacterium bifidum, CCFM1163, has been taxonomically named Bifidobacterium bifidum. Its patent has been published as CN113025530A, and its accession number is GDMCC No: 61478.

[0054] The culture media involved in the following examples are as follows:

[0055] MRS liquid culture medium: 10g beef extract; 10g tryptone; 5g yeast extract; 20g glucose; 5g anhydrous sodium acetate; 0.1g MgSO4·7H2O; 0.05g MnSO4·H2O; 2g diammonium citrate; 2.6g K2HPO4·3H2O; 1mL Tween 80; 1g cysteine ​​hydrochloride. Adjust pH to 6.8±0.2; bring volume to 1L. Autoclave at 115℃ for 20min.

[0056] MRS solid medium: 2% agar powder is added to the MRS liquid medium.

[0057] The preparation of Bifidobacterium bifidum suspensions involved in the following examples:

[0058] Bifidobacterium bifidum CCFM1163 and Bifidobacterium bifidum CCFM1389 were inoculated into MRS solid medium and cultured at 37°C under anaerobic conditions for 48 h to obtain single colonies. The obtained single colonies were then inoculated into MRS liquid medium and cultured at 37°C under anaerobic conditions for 24 h to activate them.

[0059] After activation for three generations, the bacterial culture was inoculated at a rate of 2% into 1 L of MRS liquid medium. After shaking to mix, the culture was incubated at 37°C for 18 h in a constant-temperature anaerobic incubator. The culture was then centrifuged at 8000 g / min at 4°C for 10 min. After discarding the supernatant, the cells were washed twice with sterile physiological saline, and centrifuged again under the same conditions. After discarding the supernatant, the cells were resuspended in 10% sterile defatted emulsion to prepare a final concentration of 5 × 10⁻⁶ for each culture. 9 CFU / mL Bifidobacterium bifidum CCFM1163 and CCFM1389 bacterial suspensions were obtained to prepare bacterial solutions for gavage. These solutions were then frozen at -80°C for one week.

[0060] Before conducting animal experiments, the frozen bacterial culture was removed from the freezer, and the number of viable bacteria was determined using the plate spread method at the initial stage and after one week of freezing. The order of magnitude of the initial and one-week viable bacterial counts did not change, indicating that freezing the bacterial culture did not affect the experiment and it could be used for animal experiments.

[0061] The following examples illustrate the methods for detecting the expression levels of the S100β / GFAP / PGP9.5 and Occludin / ZO-1 / Claudin-3 genes:

[0062] The expression levels of the S100β / GFAP / PGP9.5 and Occludin / ZO-1 / Claudin-3 genes were determined using real-time quantitative polymerase chain reaction (qRT-PCR). First, RNA was extracted from fresh tissue using the following method:

[0063] 0.2g of fresh colon tissue obtained from mouse dissection was repeatedly ground in a mortar (180℃, 4h high-temperature enzyme inactivation) with liquid nitrogen. Then, 1mL of TrizoL reagent was added to the mortar, and grinding continued until the liquid was basically clear. The mixture was then collected into a 1.5mL enzyme-free centrifuge tube and allowed to stand at room temperature for 15min. 200μL of chloroform solution was added to the centrifuge tube, and the mixture was gently shaken for 15s. The mixture was allowed to stand at room temperature for 10min, and then centrifuged at 4℃ and 12000r / min for 15min. 600μL of the colorless upper aqueous phase was transferred to another enzyme-free centrifuge tube, and 500μL of isopropanol was added. Invert the tube to mix thoroughly, let stand at room temperature for 10 min. After standing, centrifuge at 12000 rpm for 10 min at 4 °C, discard the supernatant, leaving the white precipitate formed at the bottom of the centrifuge tube containing RNA. Add 1 mL of 75% ethanol solution prepared with DEPC water, vortex to resuspend, centrifuge at 7500 rpm for 5 min at 4 °C, discard the supernatant, and allow to evaporate and dry at room temperature. Add 30 μL of RNase-free water to the dried RNA. After the RNA dissolves, determine the RNA concentration and purity using Nanodrop, and assess the RNA quality by agarose gel electrophoresis. Using the extracted total RNA as a template, reverse transcribe cDNA according to the instructions of the Comvita HiFiScript gDNARemovaL RT MasterMix reverse transcription kit and store at -20 °C.

[0064] The primers for the mouse S100β / GFAP / PGP9.5 gene, Occludin / ZO-1 / Claudin-3 gene, and internal reference gene GAPDH are shown in Table 1.

[0065] Table 1 Primer sequences

[0066]

[0067] qRT-PCR reaction system and conditions:

[0068] use PCR amplification was performed using the CFX96TM real-time quantitative PCR instrument, and fluorescence signals were read. The qRT-PCR reaction system was as follows:

[0069] Table 2 qRT-PCR reaction system

[0070]

[0071] The gene qRT-PCR reaction conditions are as follows:

[0072] The temperature was set at 95℃ for 30 seconds, then at 95℃ for 10 seconds, followed by 60℃ for 30 seconds, for a total of 40 cycles. The GAPDH gene was used as an internal reference gene, and the results were analyzed using CFX96Manager software.

[0073] Example 1: Obtaining Bifidobacterium bifidum

[0074] 1. Isolation and screening of Bifidobacterium bifidum:

[0075] (l) Feces were collected from adults in Wuxi, Jiangsu Province using a disposable sterile fecal collection device. The fecal samples were spread on MRS medium containing 0.08% cysteine ​​and enriched in an anaerobic incubator (N2:CO2:H2 = 80:10:10) for 12 hours.

[0076] (2) After serially diluting the fecal samples with sterile physiological saline, spread them on solid plates containing sterile 100 μg / mL mupirocin and 50 U / mL nystatin MRS+ 0.08% L-cysteine ​​hydrochloride and incubate for 24-48 h.

[0077] (3) Select single colonies that conform to the basic morphology of Bifidobacterium for streak plate purification, and screen and isolate the selected strains;

[0078] (4) After culturing the above single colonies in liquid MRS + 0.08% cysteine ​​culture medium for 24 hours, Gram staining was performed, and Gram-positive bacteria were selected for subsequent experiments.

[0079] 2. Preliminary identification of Bifidobacteria: Fructose-6-phosphate phosphoketonease assay

[0080] (1) The lactic acid bacteria screened in step 1 were cultured in liquid MRS + 0.08% cysteine ​​culture medium for 24 h, and then 1 mL of culture was centrifuged at 8000 rpm for 2 min.

[0081] (2) Wash twice with a 0.05M KH2PO4 solution containing 0.08% (mass percentage) cysteine ​​at pH 6.5;

[0082] (3) Resuspend in 200 μL of the above phosphate buffer with 0.25% (w / w) Triton X-100 added;

[0083] (4) Add 50 μL of a mixture of sodium fluoride at a concentration of 6 mg / mL and sodium iodoacetate at a concentration of 10 mg / mL, and 50 μL of fructose-6-phosphate at a concentration of 80 mg / mL, and incubate at 37°C for 1 h;

[0084] (5) Add 300 μL of hydroxylamine hydrochloride with a concentration of 0.139 g / mL and pH 6.5, and let it stand at room temperature for 10 min;

[0085] (6) Add 200 μL of 15% (mass percentage) trichloroacetic acid and 4M HCl respectively;

[0086] (7) Add 200 μL of 0.1 M HCl containing 5% (mass percentage) ferric chloride. If the system quickly turns red, it is F6PPK positive, and it can be preliminarily identified as Bifidobacterium.

[0087] 3. Molecular biological identification of Bifidobacteria

[0088] (1) Single-cell genome extraction: Take 1 mL of the bacterial cells selected and activated for 3 generations in step (II) (cultured for 12-48 h) into a 1.5 mL centrifuge tube, centrifuge at 10000 rpm for 2 min, discard the supernatant to obtain the bacterial cells; wash the bacterial cells with 1 mL of sterile water, centrifuge at 10000 rpm for 2 min, discard the supernatant to obtain the bacterial cells; add 200 μL Incubate SDS lysis buffer at 80°C for 30 min. Add 200 μL of phenol-chloroform solution to the bacterial lysis buffer, where the composition and volume ratio of the phenol-chloroform solution is Tris-saturated phenol:chloroform:isoamyl alcohol = 25:24:1. Mix thoroughly by inversion, centrifuge at 12000 rpm for 5–10 min, and collect 200 μL of the supernatant. Add 400 μL of ice-cold ethanol or ice-cold isopropanol to 200 μL of the supernatant, incubate at -20°C for 1 h, centrifuge at 12000 rpm for 5–10 min, and discard the supernatant. Resuspend the precipitate in 500 μL of 70% (v / v) ice-cold ethanol, centrifuge at 12000 rpm for 1–3 min, and discard the supernatant. Dry in a 60°C oven or air dry. Redissolve the precipitate in 50 μL of ddH2O for PCR.

[0089] (2) 16S rDNA PCR:

[0090] A. Bacterial 16S rDNA 50μL PCR reaction system: 10×Taq buffer, 5μL; dNTP, 5μL; 27F, 0.5μL; 1492R, 0.5μL; Taq enzyme, 0.5μL; template, 0.5μL; ddH2O, 38μL.

[0091] B. PCR conditions: 95℃ for 5 min; 95℃ for 10 s; 55℃ for 30 s; 72℃ for 30 s; step 2-4 30×; 72℃ for 5 min; 12℃ for 2 min;

[0092] C. Prepare a 1% agarose gel, then mix the PCR product with 10000× loading buffer, load 2 μL, run at 120V for 30 min, and then perform gel imaging;

[0093] D. The obtained PCR product was sent to a professional sequencing company. The sequencing results were compared with those obtained by searching and similarity in GeneBank using BLAST. It was identified as Bifidobacterium bifidum and named Bifidobacterium bifidum CCFM1389. It was stored at -80℃ for later use.

[0094] Example 2: Bifidobacterium bifidum CCFM1389 increases the number of enteric glial cells and enteric neurons in the colon tissue of mice with impaired enteric nervous system.

[0095] The specific steps are as follows:

[0096] (1) Preparation of Bifidobacterium bifidum (CCFM1389) bacterial suspension

[0097] After the Bifidobacterium bifidum CCFM1389 strain was taken out of the -80℃ freezer, it was streaked on MRS solid medium and cultured at 37℃ for 48h. Single colonies were picked and cultured in MRS liquid medium and anaerobic cultured at 37℃ for 20h to prepare seed culture.

[0098] The prepared seed culture was inoculated into a new MRS liquid medium at an inoculation rate of 2% (v / v) and cultured anaerobically at 37°C for 16 h. The culture was repeated for one generation in the same manner to prepare the fermentation broth of Bifidobacterium bifidum CCFM1389.

[0099] The prepared Bifidobacterium bifidum CCFM1389 fermentation broth was then centrifuged at 8000 r / min and 4℃ for 10 min, and then resuspended in 10% skim milk to obtain a bacterial suspension for animal experiments.

[0100] (2) Thirty-two healthy male C57BL / 6J mice aged 6 weeks were acclimatized to the environment for 1 week and randomly divided into 4 groups: control group, model group, Bifidobacterium bifidum CCFM1163 group (positive bacteria group) (disclosed in patent CN113025530A), and Bifidobacterium bifidum CCFM1389 group. Each group contained 8 mice, and the dose of bacterial suspension administered by gavage was 5 × 10⁻⁶. 9 CFU / mL, administered via gavage starting at 9 AM every morning, 0.2 mL each time.

[0101] The grouping and treatment methods for experimental animals are shown in Table 3:

[0102] Table 3 Grouping of experimental animals

[0103]

[0104] After the experiment, the gene expression levels of S100β and GFAP, characteristic proteins of enteric glial cells in the mouse colon, were used to characterize the number of glial cells in the enteric nerve, and PGP9.5 was used to assess the overall neuronal density.

[0105] The enteric nervous system is composed of enteric neurons and enteric glial cells. Enteric neurons are nerve cells within the intestine, forming the main body of the enterovagus nerve system (ENS), responsible for controlling intestinal motility, secretion, and sensation. Enteric glial cells are another type of cell that supports and regulates neuronal function in the intestine, including providing nutritional support, maintaining the stability of the interneuronal microenvironment, and participating in signal transduction. These two types of cells together constitute the enteric nervous system, playing a crucial role in maintaining intestinal function and balancing the body's internal environment. The number of glial cells is a good indicator of the health of the enteric nervous system. S100β and GFAP are characteristic proteins of glial cells, and their levels reflect the number of glial cells. PGP9.5 is a pan-neuronal biomarker used to assess overall neuronal density, and the selective afferent fiber biomarker calreticulin assesses changes in the number and density of nerve fibers. Figure 1The results showed that the expression levels of the enteric glial cell markers S100β and GFAP in the model group mice were reduced by 56.89% (p<0.01) and 44.76% (p<0.05), respectively, compared with the control group. The expression level of the panneuronal marker PGP9.5 was reduced by 52.15% (p<0.01) compared with the control group, indicating enteric nerve damage in the model mice. After gavage administration of CCFM1389, the expression levels of the marker protein genes S100β and GFAP increased by 127.56% and 115.33%, respectively, compared with the model group (p<0.0001), and PGP9.5 increased by 95.54%. The expression levels of S100β and GFAP increased by 10.61% and 44.28% respectively compared with the CCFM1163 group, and the expression level of PGP9.5 increased by 20.80%, indicating that the Bifidobacterium bifidum group CCFM1389 participated in the reconstruction of the enteric nervous system, could restore the enteric nerve health of mice with enteric nerve damage, and the effect was better than that of CCFM1163.

[0106] Bifidobacterium bifidum CCFM1389 can repair enteric nervous system damage caused by antibiotics, thereby promoting colonic peristalsis and improving colonic motility.

[0107] Example 3: Bifidobacterium bifidum CCFM1389 can increase the levels of neurotransmitters ACh and SP in the colon tissue of mice with enteric nerve damage.

[0108] The grouping, modeling, and treatment methods for C57BL / 6J mice are the same as in Example 2.

[0109] ELISA was used to quantify ACh and SP in colon tissue. The specific method is as follows: Colon tissue was rinsed with pre-cooled PBS to remove residual blood and surrounding adipose tissue. The tissue was weighed and then minced. The minced tissue was mixed with PBS solution at a weight-to-volume ratio of 1:9 and homogenized using a high-throughput tissue homogenizer. The homogenate was then centrifuged at 5000×g for 5-10 minutes, and the supernatant was collected for analysis. The experiment was conducted according to the instructions of the corresponding kits, and the contents of ACh and SP in the tissue were calculated based on the standard curve.

[0110] The enteric nervous system (ENS) is composed of numerous neurons and glial cells, capable of independently regulating and controlling gastrointestinal function. It is considered the body's second brain, also known as the gut-brain complex. The ENS is rich in excitatory and inhibitory neurotransmitters, which can directly act on gastrointestinal smooth muscle cells to regulate normal intestinal motility. ACh is one of the most important neurotransmitters for stimulating gastrointestinal motility. Released by cholinergic neurons, it specifically binds to M receptors on gastrointestinal smooth muscle cells, causing a decrease in intracellular cyclic adenosine monophosphate (cAMP), cell membrane depolarization, and leading to the release of glycoproteins (GAP). 2+Channel opening, intracellular Ga 2+ Increased concentration regulates gastrointestinal motility and promotes the contraction of gastrointestinal smooth muscle. Substance P (SP) is an excitatory neurotransmitter composed of 11 amino acids, widely distributed throughout the gastrointestinal tract. It can directly bind to corresponding ion channels on intestinal smooth muscle cells, causing intestinal contraction. Figure 2 It was found that after enteric nerve damage, the levels of the excitatory neurotransmitters ACh and SP in the mouse intestine decreased by 35.57% and 42.03% respectively compared with the control group (p<0.05). After gavage administration of CCFM1389, the levels of ACh and SP in the small educational model group increased significantly by 56.80% and 50.49% respectively (p<0.05), and the intervention effect was more significant than that of the positive control group CCFM1163.

[0111] Bifidobacterium bifidum CCFM1389 can regulate intestinal peristalsis by repairing the enteric nervous system, promoting the release of excitatory neurotransmitters ACh and SP.

[0112] Example 4: Bifidobacterium bifidum CCFM1389 can increase the expression levels of Occludin / Claudin-3 / ZO-1 genes in colonic tissue of mice after damage to the colonic mechanical barrier.

[0113] The grouping, modeling, and treatment methods for C57BL / 6J mice were the same as in Example 2. The expression levels of Occludin, Claudin-3, and ZO-1 genes were determined using real-time quantitative polymerase chain reaction (qRT-PCR).

[0114] Occludin, Claudin-3, and ZO-1 are all tight junction proteins in the gut, playing crucial roles in maintaining the integrity and function of the intestinal epithelial barrier. Occludin, a transmembrane protein located on the cell membrane, interacts with neighboring cells' Occludin via its extracellular structure, forming tight junctions between intestinal epithelial cells. These junctions act as "pads" between intestinal epithelial cells, playing a key role in preventing fluids and solutes from entering the body through the intercellular spaces. Claudin-3, also a transmembrane protein, participates in the formation of tight junctions between intestinal epithelial cells, similar to Occludin. Claudin-3 plays an important role in these junctions, helping to regulate intercellular permeability and selectivity. ZO-1 is a binding protein located intracellularly, connecting the tight junction proteins Occludin and Claudin to the cytoskeleton of intestinal epithelial cells. By binding to the cytoskeleton, ZO-1 stabilizes the structure of tight junctions and also regulates their formation and stability. In summary, these tight junction proteins play a synergistic role in the connections between intestinal epithelial cells, maintaining the integrity and function of the intestinal epithelial barrier, preventing the penetration of harmful substances, and simultaneously maintaining selective permeability to nutrients. Figure 3 It was found that after enteric nerve injury, the expression of tight junction proteins Occludin, Claudin-3 and ZO-1 in mouse colon tissue was significantly reduced (p<0.01). After gavage administration of CCFM1389, the gene expression levels of Occludin, Claudin-3 and ZO-1 were significantly increased compared with the model group, and increased by 21.18%, 17.81% and 12.59% respectively compared with CCFM1163 group.

[0115] In summary, intervention with Bifidobacterium bifidum CCFM1389 increased the expression of TJ proteins (Occludin, Claudin-3, ZO-1) after enteric nerve damage and reduced intestinal permeability.

[0116] Example 5: Bifidobacterium bifidum CCFM1389 reduces pathological damage to colon tissue with impaired enteric nerves in mice.

[0117] The grouping, modeling, and treatment methods for C57BL / 6J mice are the same as in Example 2.

[0118] H&E staining of mouse colon tissue sections was used to characterize pathological damage in mouse colon tissue. After dissection, fresh tissue was collected and soaked in paraformaldehyde fixative, then rinsed overnight. The samples were dehydrated sequentially with 70%, 80%, and 90% ethanol solutions for 30 min each, followed by two 20-min immersions in 95% and 100% ethanol solutions. The samples were then immersed in a mixture of equal parts pure ethanol and xylene for 15 min, followed by 15 min of xylene pre-wash and 15 min of xylene post-wash until clear. Finally, the samples were immersed in a 1:1 mixture of xylene and paraffin for 15 min, followed by 50-60 min of paraffin I and paraffin II permeation to remove the clearing agent. The treated samples were then embedded, sectioned, spread, baked, stained with H&E, and mounted. Colonic pathological damage was quantified using a pathological scoring scale.

[0119] Table 4 Pathology Scoring Table

[0120]

[0121] Observe the pathological sections of mouse colon tissue, such as Figure 4 As shown, compared with the control group, the colonic tissue of mice in the model group showed a thinner mucus layer, shorter crypt structures, fewer goblet cells, and inflammatory infiltration. However, after oral administration of Bifidobacterium bifidum CCFM1389, the mucus layer in the colonic tissue of mice thickened, the boundaries of goblet cells became clearer, and the smooth muscle structure was more intact. According to the pathological scores, CCFM1163 reduced the level by 51.28% compared with the model group, and CCFM1389 reduced it by 64.10% compared with the model group, which was superior to the positive control strain.

[0122] Example 6: The effect of Bifidobacterium bifidum CCFM1389 on the relief of symptoms related to intestinal motility disorders in mice caused by enteric nerve damage.

[0123] The grouping, modeling, and treatment methods for C57BL / 6J mice are the same as in Example 2. Specific methods are as follows:

[0124] After gavage in week 5, mice were placed individually in cages lined with absorbent paper, and their feces were collected and weighed to obtain the wet weight. After freeze-drying, the wet weight was obtained. The water content of the feces was calculated using the following formula.

[0125] Fecal moisture content = (fecal wet weight - fecal dry weight) / fecal wet weight × 100%

[0126] Each mouse was given 0.2 mL of ink by gavage, and the time when each mouse excreted its first black stool was recorded from the start of the gavage.

[0127] Before sacrifice, each mouse was given 0.2 mL of ink by gavage. After 30 minutes, the mice were sacrificed and dissected. The upper part of the small intestine from the lower end of the pylorus to the cecum was cut off. The total length of the small intestine was measured as the "total length of the small intestine", and the length from the pylorus to the front of the ink was measured as the "ink propulsion length". The propulsion rate of the small intestine was calculated according to the following formula.

[0128] Small intestine propulsion rate = (Ink propulsion length (cm)) / (Total small intestine length (cm)) × 100%

[0129] Results of fecal water content, time to first black stool, and small intestinal propulsion rate are as follows: Figure 5 As shown in the figure, compared with the control group, the fecal water content of mice after modeling increased to 1.2815 times that of the blank control group, the time to first black stool was prolonged to 1.7416 times that of the blank control group, and the small intestinal propulsion rate was shortened to 83.15% of the normal group, indicating that the model mice have intestinal motility disorders caused by enteric nerve damage. Compared with the model group, gavage treatment with Bifidobacterium bifidum CCFM1389 can reduce the fecal water content of mice with enteric nerve damage to 84.45% of the model group and shorten the time to first black stool to 72.12% of the model group. However, after intervention with the positive control strain CCFM1163, there was no significant difference in the time to first black stool and fecal water content compared with the model group. CCFM1389 intervention reduced the time to first black stool by 27.88% and the fecal water content by 15.71% compared with the model group, showing better results than the positive strain CCFM1163 group. This indicates that gavage treatment with Bifidobacterium bifidum CCFM1389 can alleviate the symptoms of intestinal motility disorders in mice caused by enteric nerve damage.

[0130] Therefore, the results show that Bifidobacterium bifidum CCFM1389 can effectively repair the damaged enteric nervous system, improve intestinal inflammation and colonic pathological damage caused by enteric nerve damage, and alleviate intestinal motility disorders caused by enteric nerve damage.

[0131] Although the present invention has been disclosed above with reference to preferred embodiments, it is not intended to limit the present invention. Anyone skilled in the art can make various modifications and alterations without departing from the spirit and scope of the present invention. Therefore, the scope of protection of the present invention should be determined by the claims.

Claims

1. A strain of Bifidobacterium bifidum ( Bifidobacterium bifidum CCFM1389 was deposited on July 11, 2024, at the Institute of Microbiology, Guangdong Academy of Sciences, with accession number GDMCC No: 64859. The deposit address is 5th Floor, Building 59, No. 100 Xianlie Middle Road, Guangzhou, Guangdong Academy of Microbiology.

2. A microbial inoculum containing the Bifidobacterium bifidum CCFM1389 as described in claim 1.

3. Food containing Bifidobacterium bifidum CCFM1389 as described in claim 1 or the microbial agent as described in claim 2.

4. The food product as described in claim 3, characterized in that, The food products include fermented foods.

5. The food product as described in claim 3 or 4, characterized in that, The amount of Bifidobacterium added in the food is at least 1 x 10 8 CFU / mL or 1 x 10 8 CFU / g.

6. A pharmaceutical product containing the Bifidobacterium bifidum CCFM1389 of claim 1 or the microbial agent of claim 2.

7. The pharmaceutical product as described in claim 6, characterized in that, The dosage forms of the medicine include granules, capsules, tablets, pills, or oral liquids.

8. The pharmaceutical product as described in claim 6 or 7, characterized in that, The Bifidobacterium is added in the pharmaceutical product in an amount of at least 1 x 10 8 CFU / mL or 1 x 10 8 CFU / g.

9. The use of Bifidobacterium bifidum CCFM1389 as described in claim 1 or the microbial agent as described in claim 2 in the preparation of health products that help with bowel movements.

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