Bifidobacterium longum for significantly improving 5-ht4r repair of intestinal nerves and application thereof

Abstract

The application discloses a bifidobacterium longum capable of significantly improving 5-HT4R repair of intestinal nerves and an application thereof, and belongs to the field of microorganisms. The bifidobacterium longum CCFM1391 provided by the application can significantly increase the number of intestinal nerve glial cells and intestinal neurons, especially can increase the content of 5-HT, promote the expression of 5-HT4R, promote the secretion of neurotrophic factors, and repair the damaged intestinal nervous system of mice. At the same time, the inflammation reaction of intestinal tissues is relieved, the intestinal immune balance is regulated, and the water reabsorption capacity of the intestinal tract is improved. Therefore, a more personalized treatment scheme can be adopted for gastrointestinal diseases caused by abnormal intestinal nervous system and intestinal motility, and the application prospect is considerable.

Description

TECHNICAL FIELD

[0001] The present application relates to a bifidobacterium that significantly improves 5-HT4R repair of intestinal nerves and its application, belonging to the field of microorganisms. BACKGROUND

[0002] The gastrointestinal tract plays an important role in maintaining the normal physiological activities of the host, including but not limited to food and waste transport, digestion and absorption of nutrients, secretion of signal molecules and antibacterial substances, maintenance of intestinal barrier integrity, regulation of intestinal flora balance, and prevention of pathogen invasion. The regulation of these key physiological processes mainly depends on the enteric nervous system (ENS). The ENS is a complex neural network located in the wall of the gastrointestinal tract, responsible for regulating the digestive, absorptive and defensive functions of the gastrointestinal tract, and is therefore considered the core of the physiological regulation of the gastrointestinal tract. The activity of the ENS is not only affected by internal and external environmental factors, but also interacts with the microbial community in the intestine, together maintaining the homeostasis and health of the intestinal function.

[0003] The enteric nervous system is a complex and delicate network structure, which is composed of various neurons, nerve plexus and enteric glial cells, etc., which together maintain the normal function of the intestinal tract. The intestinal tract is colonized by tens of thousands of intestinal microorganisms, and there is a close relationship between the enteric nervous system (ENS) and intestinal microorganisms. Studies have found that intestinal microorganisms can regulate the development and function of the enteric nervous system through their metabolites and interactions with host cells. A diverse intestinal microbiota plays an important role in the maturation and function of the enteric nervous system. Intestinal microorganisms directly affect the activity and signal transduction of enteric neurons by producing neurotransmitters, hormones and metabolites, etc., thereby regulating the peristalsis, secretion and sensory function of the intestinal tract. In this regulation process, 5-HT plays an important role in the intestine. In the peripheral system, more than 90% of circulating 5-HT is synthesized by EC cells distributed in the gastrointestinal tract, which is a long-term regulator of gastrointestinal function. When the gastrointestinal tract is stimulated, 5-HT is released from the intestinal mucosa to respond to the contraction of the gastrointestinal tract, and then regulates the frequency of contraction by interacting with the nerve endings of the intermuscular nerve plexus. This regulation mechanism helps to maintain the normal movement and digestive function of the intestinal tract. In addition, drugs that act on 5-HT receptors can relieve intestinal smooth muscle spasm, reduce visceral sensitivity, adjust intestinal motility, and improve abdominal pain and intestinal function in patients with irritable bowel syndrome (IBS). Notably, activation of 5-HT4R has neurogenesis and neuroprotective effects, which can promote the proliferation of enteric neuronal cells and glial cells, thereby repairing the enteric nervous system and improving intestinal motility. Studies have shown that the maturation of the ENS induced by microbial colonization in germ-free mice depends on 5-HT4R signals, and canceling endogenous 5-HT or blocking 5-HT4R can prevent this pairing process. In addition, treatment of germ-free mice with 5-HT4R agonists can promote the differentiation and maturation of enteric nerve cells, and treatment with the 5-HT4R agonist prucalopride can protect intestinal neurons from oxidative stress. In addition, activation of 5-HT4R in the epithelium can reduce the inflammatory response in colitis mice. For example, changes in the intestinal microbiota caused by infection, stress or antibiotic use are believed to cause irritable bowel syndrome.

[0004] Intestinal motility and enteric nerve damage caused by antibiotics is mainly achieved through mechanisms such as imbalance of microbiota, direct neurotoxicity, changes in intestinal wall structure, and immune system response. These mechanisms interact with each other and collectively cause disorders of intestinal function. For such a situation, it is usually necessary to relieve symptoms and promote recovery of the intestinal tract by adjusting the use of antibiotics, supplementing probiotics, and taking other supportive therapies. However, no probiotic preparation has been found to effectively treat intestinal motility damage caused by antibiotics. Therefore, in-depth understanding of the mutual influence and action of intestinal microorganisms and the enteric nervous system, and revealing the relationship between the intestinal microorganisms affecting the function of the enteric nervous system and the 5-HT / 5-HT4R pathway, are expected to achieve the prevention and treatment of intestinal health, provide new ideas for customizing personalized probiotic treatment plans, by adjusting the intestinal microbiota, affecting the 5-HT system, and promoting the repair of the enteric nervous system. SUMMARY

[0005] TECHNICAL PROBLEM

[0006] The purpose of the present application is to provide a Bifidobacterium bifidum that can alleviate intestinal motility and enteric nerve damage caused by antibiotics, and to provide applications of the strain.

[0007] TECHNICAL SCHEME

[0008] In order to solve the above technical problems, the present application provides a Bifidobacterium bifidum that can effectively repair the enteric nervous system. Compared with the CCFM1163 strain, the expression of neuronal markers (PGP9.5) and enteric glial cell markers (GFAP and S100β) of the strain is up-regulated, and the inflammatory response is significantly improved, indicating that the strain can repair the enteric nervous system and alleviate various adverse effects caused by damaged enteric nerves, thereby helping patients with abnormal enteric nervous system and impaired intestinal motility due to taking antibiotics.

[0009] The present application provides a Bifidobacterium bifidum CCFM1391, which was deposited in the Guangdong Provincial Academy of Microbiology on June 14, 2024, with the accession number GDMCC No: 64757, and the deposit address is the 5th floor of Building 59, Guangdong Provincial Microorganism Institute, 100 Middle Martyrs Road, Guangzhou.

[0010] The Bifidobacterium bifidum is from a stool sample of an adult in Wuxi, Jiangsu Province. The strain is sequenced and analyzed. The sequenced sequence is subjected to nucleic acid sequence alignment in NCBI Standard Nucleotide BLAST. The result shows that the nucleic acid sequence similarity with the Bifidobacterium bifidum is 100%. The result shows that the strain is Bifidobacterium bifidum, which is named Bifidobacterium bifidum (Bifidobacterium bifidum) CCFM1391.

[0011] The Bifidobacterium bifidum CCFM1391 has the following biological characteristics:

[0012] 1) Bacterial characteristics: Gram-positive non-spore-forming bacilli, the bacterial body is about 0.5-1.3 μm x 1.5-8 μm, and the polymorphism is obvious.

[0013] 2) Colony characteristics: After streak culture on MRS medium containing 0.1% L-cysteine hydrochloride for 48 h, obvious colonies are formed, the diameter is between 0.2-2.5 mm, round, convex or lenticular, slightly white, opaque, with smooth to mucoid soft surface, and no mycelium is formed.

[0014] 3) Growth characteristics: The optimal growth temperature of the strain is 36-38℃, and it grows well at 32-38℃, but can grow at 45℃ with high survival rate. The optimal initial pH is 6-7, and the growth is less at pH 5.5 or below. It grows well in anaerobic culture medium containing glucose, and enters the late logarithmic phase or early stationary phase after 20 h of culture, the liquid tube is turbid, and the final pH is 4.0-4.8.

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

[0016] 5) It has adhesion, and can better adhere to colon cancer cells HT-29.

[0017] 6) It significantly increases the number of enteric neurons and enteric glial cells in colon tissue, promotes 5-HT secretion and increases 5-HT4R expression, promotes the secretion of neurotrophic factors, repairs the damaged enteric nervous system of mice. At the same time, it relieves the inflammatory response of intestinal tissue, regulates the immune balance of intestinal tract, improves the water reabsorption capacity of intestinal tract, and relieves the intestinal motility disorder.

[0018] The application also provides a microbial preparation containing the Bifidobacterium bifidum CCFM1391.

[0019] The present application also provides a food product containing the above-mentioned microbial preparation of Bifidobacterium bifidum CCFM1391.

[0020] In one embodiment of the present application, the food product is a fermented food product produced by fermentation using Bifidobacterium bifidum CCFM1391, and includes a solid food product, a liquid food product, or a semi-solid food product.

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

[0022] In one embodiment of the present application, the fermented food product includes a dairy product, a soy product, or a fruit / vegetable product.

[0023] In one embodiment of the present application, the dairy product is a fermented dairy product, including fermented milk, a fermented milk beverage, butter, cheese, or milk powder; the soy product includes soy milk, a soy milk beverage, or soy milk powder; and the fruit / vegetable product includes a fermented fruit / vegetable beverage or food product fermented using Chinese cabbage, white radish, cucumber, sugar beet, yellow peach, or waxberry as a raw material.

[0024] The present application also provides a pharmaceutical product containing the above-mentioned Bifidobacterium bifidum CCFM1391 or the above-mentioned microbial preparation.

[0025] In one embodiment of the present application, the pharmaceutical product contains Bifidobacterium bifidum CCFM1391 and a pharmaceutically acceptable carrier.

[0026] In one embodiment of the present application, the carrier includes one or more of a filler, a binder, a wetting agent, a disintegrating agent, a lubricant, or a flavoring agent commonly used in medicine.

[0027] In one embodiment of the present application, the pharmaceutical product is in the form of a granule, a capsule, a tablet, a pill, or an oral solution.

[0028] In one embodiment of the present application, the Bifidobacterium bifidum CCFM1391 is added to the pharmaceutical product in an amount of at least 1 x 10 8 CFU / mL or 1 x 10 8 CFU / g.

[0029] The application also provides application of the above-mentioned Bifidobacterium bifidum CCFM1391 or the above-mentioned microbial agent in preparation of health products for helping to relieve diarrhea.

[0030] The application also provides application of the above-mentioned Bifidobacterium bifidum CCFM1391 or the above-mentioned microbial agent in preparation of drugs for relieving intestinal nerve damage, characterized in that the relieving intestinal damage comprises at least one of the following:

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

[0032] (b) increasing the gene expression of TPH2 and 5-HT4R in the intestinal tract and decreasing the gene expression of SERT;

[0033] (c) increasing the content of neurotransmitter 5-HT in the intestinal nerve damaged colon tissue;

[0034] (d) increasing the expression of neurotrophic factors GDNF and NGF in the intestinal nerve damaged colon tissue

[0035] (e) increasing the content of anti-inflammatory factors TGF-β / IL-10 and decreasing the content of pro-inflammatory factors CXCL-1 / IL-17 in the intestinal nerve damaged colon tissue;

[0036] (f) increasing the expression of AQP4 in the intestinal nerve damaged colon tissue;

[0037] (g) increasing the content of acetic acid and propionic acid in feces;

[0038] (h) relieving hypokinetic intestinal motility disorder and increasing the water reabsorption capacity of the intestinal tract.

[0039] The application also provides a microbial agent containing the Bifidobacterium bifidum CCFM1391.

[0040] In an embodiment of the application, the number of viable Bifidobacterium bifidum CCFM1391 in the microbial agent is ≥1×10 8 CFU / g or 1×10 8 CFU / mL.

[0041] In an embodiment of the application, the microbial agent is obtained by drying a microbial liquid containing Bifidobacterium bifidum CCFM1391, and the number of viable Bifidobacterium bifidum CCFM1391 is ≥1×10 8 CFU / g or 1×10 8Powder for inhalation

[0042] In an embodiment of the present application, the drying refers to vacuum freeze-drying.

[0043] Beneficial effects

[0044] (1) The Bifidobacterium bifidum CCFM1391 of the present application has good activity, can significantly increase the number of enteric glial cells and neurons in the colon tissue, increase the content of 5-HT and the expression of 5-HT4R gene in the colon tissue (increased by 107.17% and 60.84% compared with the model, respectively), promote the secretion of neurotrophic factors GDNF and NGF, improve the inflammatory response, increase the content of acetic acid and propionic acid in the feces (increased by 91.05% and 99.87% compared with the model, respectively), increase the expression of water channel protein AQP4 in the colon (increased by 178.80% compared with the model), and regulate intestinal movement. Importantly, the Bifidobacterium bifidum CCFM1391 has a more obvious effect on the above-mentioned indicators than CCFM1163, and the expression amounts of S100β and GFAP are increased by 21.87% and 54.45% compared with the CCFM1163 group, respectively, and the expression amount of PGP9.5 is increased by 58.39%. Therefore, this strain has a more obvious effect on the repair of intestinal nerves.

[0045] (2) The present application can be considered as a drug for relieving or treating intestinal movement disorders and damaged intestinal nerves caused by the use of antibiotics, and can also be applied to drugs or some fermented foods and functional foods, thereby widely exerting its effect and having a very valuable application prospect.

[0046] Preservation of biological materials

[0047] A Bifidobacterium bifidum CCFM1391, which is taxonomically named as Bifidobacterium bifidum, was preserved in the Guangdong Microbial Culture Collection Center on June 14, 2024, with a preservation number of GDMCC No: 64757 and a preservation address of 5th Floor, Building 59, Guangdong Microbial Institute, 100 Middle Martyrs Road, Guangzhou. BRIEF DESCRIPTION OF DRAWINGS

[0048] Figure 1 : Schematic diagram of the change in the mRNA expression level of neuron and glial cell marker proteins in the colon tissue of an ABX-induced intestinal nerve damaged mouse after intervention of the Bifidobacterium bifidum CCFM1391 strain.

[0049] Figure 2Figure 6: The expression of 5-HT4R, SERT, TPH2 and the content of 5-HT in the colon tissue of ABX-induced enteric nervous system impaired mice after the intervention of Bifidobacterium bifidum CCFM1391 strain.

[0050] Figure 3 Figure 7: The expression of GDNF and NGF in the colon tissue of ABX-induced enteric nervous system impaired mice after the intervention of Bifidobacterium bifidum CCFM1391 strain.

[0051] Figure 4 Figure 8: The content of TGF-β / IL-10 and CXCL1 / IL-17 in the colon tissue of ABX-induced enteric nervous system impaired mice after the intervention of Bifidobacterium bifidum CCFM1391 strain.

[0052] Figure 5 Figure 9: The expression of AQP4 in the colon tissue of ABX-induced enteric nervous system impaired mice after the intervention of Bifidobacterium bifidum CCFM1391 strain.

[0053] Figure 6 Figure 10: The content of acetic acid and propionic acid in the feces of ABX-induced enteric nervous system impaired mice after the intervention of Bifidobacterium bifidum CCFM1391 strain.

[0054] Figure 7 Figure 11: The related indicators (the time of the first black feces, the water content of feces, and the small intestine propulsion rate) of the symptoms alleviated by Bifidobacterium bifidum CCFM1391 strain in the mice with intestinal motility disorder caused by enteric nervous system impairment. DETAILED DESCRIPTION

[0055] The male C57BL / 6J mice involved in the following examples were purchased from Zhejiang Vantong Lifetechnology Co., Ltd.

[0056] The strain information involved in the following examples is as follows:

[0057] A Bifidobacterium bifidum CCFM1391, which is classified as Bifidobacterium bifidum, and has a preservation number of GDMCC No: 64757.

[0058] A Bifidobacterium bifidum CCFM1167, which is classified as Bifidobacterium bifidum, has been disclosed in patent CN112940980A, and has a preservation number of GDMCC No: 61482.

[0059] A Bifidobacterium bifidum CCFM1163, which is classified as Bifidobacterium bifidum, has been disclosed in patent CN113025530A, and has a preservation number of GDMCC No: 61478.

[0060] The culture medium involved in the following examples is as follows:

[0061] MRS liquid medium: beef extract 10 g; tryptone 10 g; yeast powder 5 g; glucose 20 g; anhydrous sodium acetate 5 g; MgSO4·7H2O 0.1 g; MnSO4·H2O 0.05 g; diammonium hydrogen citrate 2 g; K2HPO4·3H2O 2.6 g; Tween 80 1 mL; cysteine hydrochloride 1 g. Adjust pH to 6.8±0.2; make up to 1 L. Autoclave at 115℃ for 20 min.

[0062] MRS solid medium: add 2% agar powder to the MRS liquid medium.

[0063] Preparation of Bifidobacterium bifidum bacterial suspension involved in the following examples:

[0064] Bifidobacterium bifidum CCFM1163, Bifidobacterium bifidum CCFM1167 and Bifidobacterium bifidum CCFM1391 were inoculated into MRS solid medium respectively, and cultured at 37℃ under anaerobic conditions for 48h to obtain single colonies. The prepared single colonies were inoculated into MRS liquid medium respectively, and activated at 37℃ under anaerobic conditions for 24h.

[0065] The activated 3rd generation bacterial solution was inoculated into 1L MRS liquid medium at an inoculation amount of 2%, and after shaking and mixing, it was cultured in a constant temperature anaerobic incubator at 37°C for 18h. Centrifugation was performed at 8000g / min and 4°C for 10min, the supernatant was removed, and the bacterial body was resuspended in 10% sterilized skimmed milk solution to prepare two Bifidobacterium bifidum CCFM1163 bacterial suspensions, two Bifidobacterium bifidum CCFM1167 bacterial suspensions, and two Bifidobacterium bifidum CCFM1391 bacterial suspensions, each with a final concentration of 5x10 9 CFU / mL, and the resulting bacterial solution was stored in a -80°C refrigerator for one week.

[0066] Before the animal experiment, the bacterial solution stored in the refrigerator was taken out, and the number of viable bacteria was determined by plate coating method. The order of magnitude of the initial and one-week-stored viable bacteria did not change, indicating that the storage of the bacterial solution did not affect the experiment and could be used for animal experiments.

[0067] The detection method of the expression amount of S100β / GFAP / PGP9.5 gene, GDNF / NGF gene, AQP4 gene, and TPH2 / SERT / 5-HT4R gene in the following examples is as follows:

[0068] Real-time fluorescent quantitative polymerase chain reaction (qRT-PCR) was used to determine the expression amount of S100β / GFAP / PGP9.5 gene, GDNF / NGF gene, AQP4 gene, and TPH2 / SERT / 5-HT4R gene. First, RNA was extracted from fresh tissue, and the specific method is as follows:

[0069] 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.

[0070] Primers for the mouse S100β / GFAP / PGP9.5 gene, GDNF / NGF gene, AQP4 gene, TPH2 / SERT / 5-HT4R gene, and internal reference gene GAPDH gene are shown in Table 1.

[0071] Table 1 Primer sequences

[0072]

[0073]

[0074] qRT-PCR reaction system and conditions:

[0075] 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:

[0076] Table 2 qRT-PCR reaction system

[0077]

[0078] The gene qRT-PCR reaction conditions are as follows:

[0079] 95℃ 30s; 95℃ 10s, 60℃ 30s, 40 cycles in total. GAPDH gene was used as internal reference gene, and the results were analyzed by CFX96 Manager software.

[0080] The detection method of the contents of anti-inflammatory factor TGF-β / IL-10, pro-inflammatory factor CXCL1 / IL-17 and 5-HT in the colon tissue involved in the following examples is as follows:

[0081] The expression amounts of anti-inflammatory factor TGF-β / IL-10, pro-inflammatory factor CXCL1 / IL-17 and 5-HT in the colon tissue were quantified by ELISA method. The specific method is as follows: the colon tissue was rinsed with pre-cooled PBS to remove residual blood, and the surrounding adipose tissue was removed. After weighing, the colon tissue was cut into small pieces. The cut tissue was crushed with PBS solution at a weight to volume ratio of 1:9 on a high-throughput tissue crusher. Finally, the homogenate was centrifuged at 5000 x g for 5-10 minutes, and the supernatant was detected. The experiment was carried out according to the corresponding kit instruction. The anti-inflammatory factor TGF-β / IL-10, pro-inflammatory factor CXCL1 / IL-17 and 5-HT in the tissue were calculated according to the standard curve.

[0082] The detection method of the contents of acetic acid and propionic acid in the feces of mice involved in the following examples is as follows:

[0083] The specific method is as follows: 20 mg of freeze-dried feces was weighed, resuspended with 500 μL of saturated NaCl solution, and 20 μL of 10% H2SO4 solution was added. 1000 μL of anhydrous ether was added, shaken uniformly, and fatty acids were extracted, and then centrifuged at 12000 rpm at 4℃ for 15 min. The upper ether phase was taken, and 0.25 g of anhydrous Na2SO4 was added for drying. After standing for 30 min, the upper ether phase was taken by centrifugation at 12000 rpm at 4℃ for 5 min. The content of short-chain fatty acids in the freeze-dried feces of mice was determined by GC-MS. Rtx-Wax column (column length 30 m, inner diameter 25 μm) was used; the carrier gas was He, the flow rate was 2 mL / min; the injection volume was 1 μL, the temperature was raised to 140℃ at 7.5℃ / min, then raised to 200℃ at 60℃ / min and maintained for 3 min, and the ionization temperature was 20℃; the analysis was carried out in full scan mode, and the standard curve was obtained by external standard method, so as to calculate the concentrations of acetic acid and propionic acid.

[0084] Example 1: Obtaining Bifidobacterium bifidum

[0085] 1. Isolation and screening of Bifidobacterium bifidum:

[0086] (l) Collect the fecal samples of adults in Wuxi, Jiangsu Province using disposable sterile sampling device, spread the fecal samples on MRS medium with 0.08% (mass percentage) cysteine, and enrich in an anaerobic incubator (N2:CO2:H2=80:10:10) for 12 h;

[0087] (2) Dilute the fecal samples with sterile normal saline, spread on solid plates of MRS with 0.08% (mass percentage) L-cysteine hydrochloride and 100 μg / mL sterile mupirocin and 50 U / mL sterile nystatin, and incubate for 24-48 h;

[0088] (3) Select single colonies with the basic morphology of Bifidobacterium for plate streaking and purification, and isolate the selected strains;

[0089] (4) Gram stain the single colonies cultured in liquid MRS with 0.08% (mass percentage) cysteine for 24 h, and select Gram-positive bacteria for subsequent tests.

[0090] 2. Preliminary identification of Bifidobacterium: fructose-6-phosphate phosphoketolase assay

[0091] (1) Culture the lactic acid bacteria selected in step 1 in liquid MRS with 0.08% (mass percentage) cysteine for 24 h, and then centrifuge 1 mL of the culture at 8000 rpm for 2 min;

[0092] (2) Wash twice with 0.05M KH2PO4 solution containing 0.08% (mass percentage) cysteine and having pH 6.5;

[0093] (3) Resuspend in 200 μL of the above phosphate buffer with 0.25% (mass percentage) Triton X-100 added;

[0094] (4) Add 50 μL of a mixture of sodium fluoride with a concentration of 6 mg / mL and sodium iodoacetate with a concentration of 10 mg / mL, and 50 μL of fructose-6-phosphate with a concentration of 80 mg / mL, and incubate at 37°C for 1 h;

[0095] (5) Add 300 μL of light amine hydrochloride with a concentration of 0.139 g / mL and pH 6.5, and stand at room temperature for 10 min;

[0096] (6) Add 200 μL of 15% (mass percentage) trichloroacetic acid and 4M HCl, respectively;

[0097] (7) Add 200 μL of 0.1M HCl containing 5% (mass percentage) ferric trichloride. If the system turns red rapidly, it is F6PPK positive, and it can be preliminarily determined to be Bifidobacterium.

[0098] 3. Molecular biological identification of Bifidobacterium

[0099] (1) Single bacterial genome extraction: 1 mL of bacteria (cultured for 12-48 h) screened in step 2 and activated for 3 generations was centrifuged at 10,000 rpm for 2 min, and the supernatant was discarded to obtain the bacteria; after the bacteria were washed with 1 mL of sterile water and centrifuged at 10,000 rpm for 2 min, the supernatant was discarded to obtain the bacteria; 200 μL of SDS lysis solution was added, and the bacteria were incubated at 80°C for 30 min; 200 μL of phenol-chloroform solution was added to the bacterial lysis solution, wherein the composition and volume ratio of the phenol-chloroform solution were Tris-saturated phenol: chloroform: isopropyl alcohol = 25:24:1, and after being mixed well, the mixture was centrifuged at 12,000 rpm for 5-10 min, and 200 μL of the supernatant was taken; 400 μL of ice ethanol or ice isopropyl alcohol was added to 200 μL of the supernatant, which was placed at -20°C for 1 h, and then centrifuged at 12,000 rpm for 5-10 min, and the supernatant was discarded; 500 μL of 70% (volume percentage) ice ethanol was added to resuspend the precipitate, and then the mixture was centrifuged at 12,000 rpm for 1-3 min, and the supernatant was discarded; the precipitate was dried in a 60°C oven or naturally dried; and 50 μL of ddH2O was added to dissolve the precipitate for PCR;

[0100] (2) 16S rDNA PCR:

[0101] A. 50 μL of bacterial 16S rDNA PCR reaction system: 10×Taq buffer, 5 μL; dNTP, 5 μL; 27F, 0.5 μL; 1492R, 0.5 μL; Taq enzyme, 0.5 μL; template, 0.5 μL; ddH2O, 38 μL.

[0102] B. PCR conditions: 95°C for 5 min; 95°C for 10 s; 55°C for 30 s; 72°C for 30 s; step 2-4 30×; 72°C for 5 min; 12°C for 2 min;

[0103] C. Prepare 1% agarose gel, then mix the PCR product with 10,000×loading buffer, load 2 μL, run at 120 V for 30 min, and then perform gel imaging;

[0104] D. The obtained PCR product was sent to a professional sequencing company, and the obtained sequencing results were searched and similarity aligned in GeneBank using BLAST, and were identified as Bifidobacterium bifidum, named Bifidobacterium bifidum CCFM1391, and preserved at -80°C for standby use.

[0105] Example 2: Bifidobacterium bifidum CCFM1391 increases the number of enteric glial cells and enteric neurons in the colon tissue of enteric nervous system damaged mice

[0106] The specific steps are as follows:

[0107] (1) Preparation of Bifidobacterium bifidum CCFM1391 bacterial suspension

[0108] After the Bifidobacterium bifidum CCFM1389 strain was taken out from the-80℃ refrigerator, it was streaked on MRS solid culture medium and cultured at 37℃ for 48h. A single colony was picked on MRS liquid culture medium and cultured anaerobically at 37℃ for 20h to prepare a seed liquid.

[0109] The prepared seed liquid was inoculated into new MRS liquid culture medium at an inoculation amount of 2%(v / v) and cultured anaerobically at 37℃ for 16h. The same process was repeated for one generation to prepare Bifidobacterium bifidum CCFM1391 fermentation broth.

[0110] Then, the prepared Bifidobacterium bifidum CCFM1389 fermentation broth was centrifuged at 8000r / min and 4℃ for 10min, and then resuspended with 10% skim milk to prepare a bacterial suspension for animal experiments.

[0111] (2) 40 healthy male C57BL / 6J mice aged 6 weeks were adaptively placed in the environment for 1 week and randomly divided into 5 groups: a control group, a model group, a Bifidobacterium bifidum CCFM1163 group (positive bacteria group) (disclosed in patent CN113025530A), a Bifidobacterium bifidum CCFM1167 group (positive bacteria group) (disclosed in patent CN112940980A), and a Bifidobacterium bifidum CCFM1391 group, each containing 8 mice. The dose of the bacterial suspension for gavage was 5×10 9 CFU / mL, and gavage was started at 9am every day, with 0.2mL each time.

[0112] The experimental animal grouping and treatment method are shown in Table 3:

[0113] Table 3 Experimental animal grouping

[0114]

[0115]

[0116] After the experiment, the gene expression levels of S100β and GFAP, which are characteristic proteins of enteric glial cells in the colon of mice, were used to characterize the number of glial cells in the enteric nerve, and PGP9.5 was used to evaluate the overall neuronal density.

[0117] The enteric nervous system is composed of enteric neurons and enteric glial cells. Enteric neurons are nerve cells within the intestinal tract that make up the bulk of the enteric nervous system (ENS), responsible for controlling functions such as movement, secretion, and sensation in the gut. Enteric glial cells are another type of cell that play a supportive and regulatory role in the function of neurons in the gut, including providing nutritional support, maintaining microenvironment stability between neurons, and participating in signal transduction, among others. These two types of cells together make up the enteric nervous system, which plays an important role in maintaining gut function and balancing the internal environment of the body. The number of glial cells can well reflect the health status of the enteric nervous system, and S100β and GFAP are characteristic proteins of glial cells, and the amount of S100β and GFAP can reflect the number of glial cells. PGP9.5 is a pan-neuron marker used to assess overall neuron density, and the selective afferent fiber marker calretinin is used to assess changes in nerve fiber number and density. By Figure 1 It can be seen that the expression levels of 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) compared with the control group, and the expression level of pan-neuron marker PGP9.5 was reduced by 52.15% (p<0.01) compared with the control group. After intragastric administration of CCFM1391, the expression levels of marker protein genes S100β and GFAP were increased by 150.70% and 130.51% (p<0.01) compared with the model group, and PGP9.5 was increased by 156.38%; the expression levels of S100β and GFAP were increased by 16.50% and 14.38% compared with CCFM1167 group, and the expression level of PGP9.5 was increased by 34.18%; the expression levels of S100β and GFAP were increased by 21.87% and 54.45% compared with CCFM1163 group, and the expression level of PGP9.5 was increased by 58.40%. This shows that Bifidobacterium CCFM1391 is involved in the reconstruction of the enteric nervous system, can restore the intestinal nerve health of mice with damaged intestinal nerves, and the effect is better than that of positive control strains CCFM1163 and CCFM1167.

[0118] Example 3: Bifidobacterium CCFM1391 increases the expression of TPH2 and 5-HT4R and reduces the expression of SERT in the colon tissue of mice with damaged enteric nervous system, and increases the content of 5-HT

[0119] C57BL / 6J mice were divided into groups, modeled and treated as in Example 2. The expression level of 5-HT in the colon tissue was quantified by ELISA method, and the expression levels of TPH2 and 5-HT genes were determined by real-time fluorescent quantitative polymerase chain reaction (qRT-PCR).

[0120] 5-Hydroxytryptamine (5-HT), also known as serotonin, is an important neurotransmitter that plays a crucial role in regulating various physiological processes in the enteric nervous system. These processes include regulating intestinal peristalsis and neuronal activity, stimulating intestinal mucosal cells to secrete mucus and water, and directly and indirectly regulating intestinal smooth muscle activity, thus maintaining intestinal function and health. 5-HT4R, as a specific 5-HT receptor, plays a unique and important role in regulating intestinal function through its specific signal transduction pathway and expression in specific tissues. For example, activation of 5-HT4R in the enteric nervous system can exert neurogenic and neuroprotective effects. Results include... Figure 2 As shown, the 5-HT content in mice with enteric nerve damage was significantly reduced by 32.22% compared to the control group (p<0.05), and the 5-HT4R gene expression level was significantly reduced by 49.83% compared to the control group (p<0.05). After gavage administration of CCFM1391, the 5-HT content and 5-HT4R gene expression level increased by 107.17% and 71.65% respectively compared to the model group (p<0.05), by 7.31% and 80.87% respectively compared to the CCFM1167 group, and by 42.42% and 80.07% respectively compared to the CCFM1163 group.

[0121] TPH2 is often referred to as a neuronal rate-limiting isoform because it primarily functions in neurons. TPH2 catalyzes the conversion of tryptophan to 5-hydroxytryptophan in neurons and is one of the key enzymes in serotonin synthesis. SERT, short for serotonin transporter, plays a crucial role in the regulation of the neurotransmitter serotonin (5-HT). Its main function is to recycle 5-HT from the synaptic cleft back into the neuron for reuse or degradation. As shown in the figure, the expression level of the TPH2 gene in the model group mice was reduced by 55.87% compared to the control group (p<0.01), while the expression level of the SERT gene was increased by 31.27% compared to the control group. After gavage administration of CCFM1391, the expression level of TPH2 gene increased by 219.75% compared with the model group (p<0.0001), 13.85% compared with the CCFM1167 group, and 104.25% compared with the CCFM1163 group; the expression level of SERT gene decreased by 38.92% compared with the model group and tended to be similar to that of the normal group.

[0122] Therefore, the results show that Bifidobacterium bifidum CCFM1391 can increase the expression of TPH2 gene and decrease the expression of SERT gene, thereby increasing the content of 5-HT and activating the expression of 5-HT4R, thus achieving the purpose of repairing the enteric nervous system.

[0123] Example 4: Bifidobacterium CCFM1391 improves the expression of glial cell-derived neurotrophic factor (GDNF) and nerve growth factor (NGF) in the colon tissue of mice with damaged enteric nervous system

[0124] C57BL / 6J mice were grouped, modeled and treated as in Example 2. Real-time fluorescent quantitative polymerase chain reaction (qRT-PCR) was used to determine the expression levels of GDNF and NGF genes.

[0125] GDNF (glial cell-derived neurotrophic factor) maintains the health of the intestinal tract mainly by supporting the survival and function of neurons, can promote the regeneration and repair of damaged neurons, and also has a protective effect on intestinal mucosal cells. NGF is a neurotrophic factor, which is an important protein that plays a role in promoting the growth, development and maintenance of neurons, and plays an important role in the growth and regeneration of neurons. By Figure 3 It can be seen that the GDNF and NGF levels of the model mice were reduced by 51.73% and 48.36% (p<0.01) compared with the control group, indicating that there was damage to the enteric nervous system. The GDNF and NGF levels of the CCFM1167 group were increased by 75.19% and 34.91% compared with the model group, and the GDNF and NGF levels of the CCFM1163 group were increased by 58.66% and 48.54% compared with the model group; after gavage with CCFM1391, the GDNF and NGF levels were significantly increased by 188.45% and 137.35% (p<0.01%) compared with the model group, and the effect was more significant than that of the positive control group.

[0126] Bifidobacterium CCFM1391 can nourish the enteric nervous system by increasing the expression of GDNF and NGF, and restore normal enteric nervous function.

[0127] Example 5: Bifidobacterium CCFM1391 increases the content of anti-inflammatory factors TGF-β / IL-10 and reduces the content of pro-inflammatory factors CXCL1 / IL-17 in the colon tissue of mice with damaged enteric nervous system

[0128] C57BL / 6J mice were grouped, modeled and treated as in Example 2. ELISA was used to quantify the content of anti-inflammatory factors TGF-β / IL-10 and pro-inflammatory factors CXCL1 / IL-17 in the colon tissue.

[0129] There is a close interaction between the enteric nervous system and the immune system. When the enteric nervous system is damaged or dysfunctional, this interaction can be disrupted, leading to increased inflammatory response of the immune system to the intestinal tissue. For example, increased inflammatory response caused by the release of inflammatory mediators, activation of immune cells, etc.

[0130] TGF-β (transforming growth factor-beta) and IL-10 (interleukin-10) are two important immune modulatory factors that work together in the gut to regulate the balance of intestinal immunity and inhibit the occurrence and progression of inflammatory reactions. CXCL1 (also known as GRO-α) and IL-17 are two cytokines that play important roles in the gut and are involved in regulating immune responses and inflammatory reactions, but their directions of action are different: CXCL1 mainly promotes inflammatory reactions and attracts granulocytes and other inflammatory cells, while IL-17 is a pro-inflammatory factor that can enhance the intensity and duration of inflammatory reactions. The balance and regulation of these factors in the gut are crucial for maintaining the immune balance and health of the gut. As shown in the results Figure 4 As shown in the results, compared with the control group, the model group significantly reduced two anti-inflammatory factors and significantly increased two pro-inflammatory factors (p<0.05). After gavage with CCFM1391, the contents of TGF-β and IL-10 were significantly increased by 201.01% and 27.33% (p<0.05) compared with the model group; the contents of CXCL and IL-17 were significantly reduced by 51.44% and 54.69% (p<0.01%) compared with the model group, and the effects were better than those of the positive control groups CCFM1167 and CCFM1163.

[0131] Bifidobacterium CCFM1391 plays a role in relieving intestinal inflammation by increasing the production of inhibitory inflammatory factors and reducing the production of pro-inflammatory factors.

[0132] Example 6: Bifidobacterium CCFM1391 increases the expression of AQP4 in the colon tissue of mice with damaged enteric nervous system

[0133] C57BL / 6J mice were grouped, modeled, and treated as in Example 2. Real-time fluorescent quantitative polymerase chain reaction (qRT-PCR) was used to determine the expression level of AQP4 gene.

[0134] Antibiotics can affect the water balance mechanism of intestinal cells, leading to decreased water reabsorption capacity and increased permeability of water through the intestinal mucosal barrier due to damage to the mucosal barrier. Aquaporin-4 (AQP4) is a water channel protein that plays an important role in the gut and regulates water absorption and secretion. Its dysfunction can lead to water balance disorders and affect intestinal function and health. From the results Figure 5It can be seen that the AQP4 level of the intestinal nerve damaged mice was reduced by 59.80% (p<0.001) compared with the control group, indicating that there was water balance disorder in the intestinal tract. The AQP4 gene expression of the CCFM1167 group was increased by 131.88% (p<0.0001) compared with the model group; the AQP4 gene expression of the CCFM1163 group was increased by 100.92% compared with the model group, and the AQP4 gene expression after the CCFM1391 was increased by 178.80% (p<0.0001) compared with the model group, which was more significant than the positive control group.

[0135] It is speculated that CCFM1391 can improve the abnormal water reabsorption capacity of the intestinal tract caused by antibiotics by increasing the expression of AQP4

[0136] Example 7: Bifidobacterium CCFM1391 increases the content of acetic acid and propionic acid in the feces of mice with damaged enteric nervous system

[0137] The C57BL / 6J mice were grouped, modeled and treated as in Example 2. The content of acetic acid and propionic acid in the feces was detected by gas chromatography-mass spectrometry (GC-MS) targeting.

[0138] Short-chain fatty acids, especially acetic acid and propionic acid, are believed to be able to promote intestinal peristalsis. They affect the contraction and relaxation of intestinal smooth muscle through various mechanisms, thereby regulating the frequency and intensity of intestinal peristalsis. Acetic acid and propionic acid can stimulate neurons in the intestinal tract, promote nerve conduction, and increase the frequency and amplitude of intestinal peristalsis. This effect may be through stimulating neurons in the nervous system of the gastrointestinal tract (enteric nervous system), enhancing gastrointestinal motility. As can be seen from the figure, the content levels of acetic acid and propionic acid in the model mice were reduced by 50.95% and 46.78% (p<0.01%) compared with the control group. After the CCFM1391 was administered, the content of acetic acid and propionic acid was significantly increased by 91.05% and 99.87% (p<0.05) compared with the model group, and the content of acetic acid and propionic acid was increased by 15.77% and 76.33% respectively compared with the CCFM1163 group. The ability to increase the content of short-chain fatty acids is superior to the positive control strains CCFM1163 and CCFM1167.

[0139] Example 8: Bifidobacterium CCFM1391 alleviates the symptoms related to intestinal motility disorder caused by damaged enteric nervous system in mice

[0140] The C57BL / 6J mice were grouped, modeled and treated as in Example 2. The specific method is as follows:

[0141] After the end of the intragastric administration in the 5th week, the mice were placed individually in a cage box padded with water-absorbing paper, and the feces were collected, weighed as wet weight, and freeze-dried as dry weight. The water content of the feces was calculated according to the following formula.

[0142] Fecal water content = (wet fecal weight - dry fecal weight) / wet fecal weight x 100%

[0143] Each mouse was given 0.2 mL of ink by gavage, and the time of the first black stool was recorded from the start of gavage.

[0144] Before the mice were sacrificed, each mouse was given 0.2 mL of ink by gavage, and 30 min later, the mice were dissected, the upper end from the lower end of the pylorus to the cecum was cut, the total length of the small intestine was measured as "total length of the small intestine", and the ink front was measured from the pylorus to the ink front as "ink propulsion length". The small intestine propulsion rate was calculated according to the following formula.

[0145] Small intestine propulsion rate = (ink propulsion length (cm)) / (total length of small intestine (cm)) x 100%

[0146] The fecal water content, the time of the first black stool, and the small intestine propulsion rate results are shown in Table 1. Figure 6 As can be seen from the figure, compared with the control group, the fecal water content of the model mice increased to 1.2816 times that of the blank control group, the time of the first black stool was prolonged to 1.7416 times that of the blank control group, and the small intestine propulsion rate was reduced to 83.15% of the control group, that is, the model mice had intestinal motility disorder caused by impaired enteric nerves. Compared with the model group, the fecal water content of the mice treated with Bifidobacterium bifidum CCFM1391 by gavage was reduced to 82.64% of the model group, the time of the first black stool was shortened to 61.54% of the model, and the small intestine propulsion rate was increased to 1.064 times that of the model group; compared with the CCFM1163 group, the fecal water content of the mice treated with Bifidobacterium bifidum CCFM1391 by gavage was reduced by 16.97%, the time of the first black stool was shortened by 27.52%, and the small intestine propulsion rate was increased by 17.21%. It is shown that Bifidobacterium bifidum CCFM1391 can alleviate the symptoms of intestinal motility disorder caused by impaired enteric nerves in mice, and the effect is better than that of the positive control strain CCFM1163.

[0147] Therefore, it can be seen from the results that Bifidobacterium bifidum CCFM1391 can effectively repair the damaged enteric nervous system, improve the symptoms of intestinal inflammation and abnormal secretion of neurotrophic factors caused by impaired enteric nerves, and can alleviate the intestinal motility disorder caused by impaired enteric nerves.

[0148] Although the present application has been disclosed with reference to the preferred embodiments, it is not intended to limit the present application, and any person skilled in the art can make various modifications and modifications without departing from the spirit and scope of the present application, and therefore the protection scope of the present application should be defined by the claims.

Claims

1. A Bifidobacterium bifidum CCFM1391, which was deposited on June 14, 2024 in the Guangdong Provincial Microbiology Institute, and the deposit number is GDMCC No: 64757, and the deposit address is the 5th floor of Building 59, Guangdong Provincial Microbiology Institute, 100, Martyrs' Avenue, Guangzhou. 2.A microbial inoculant comprising the Bifidobacterium bifidum CCFM1391 of claim 1. 3.A food comprising the Bifidobacterium bifidum CCFM1391 of claim 1 or the microbial inoculant of claim 2.

4. The food product as described in claim 3, characterized in that, The food includes fermented food.

5. The food product according to claim 3 or 4, wherein The Bifidobacterium bifidum CCFM1391 is added in the food in an amount of at least 1 x 10 8 CFU / mL or 1 x 10 8 CFU / g. 6.A medicine comprising the Bifidobacterium bifidum CCFM1391 of claim 1 or the microbial inoculant of claim 2.

7. The pharmaceutical product according to claim 6, characterized in that The dosage form of the medicine includes granules, capsules, tablets, pills, or oral liquid.

8. The pharmaceutical product according to claim 6 or 7, characterized in that The Bifidobacterium bifidum CCFM1391 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.Use of the Bifidobacterium bifidum CCFM1391 of claim 1 or the microbial inoculant of claim 2 in the preparation of a health product for helping to lubricate the intestines and defecate.

10. Use of Bifidobacterium bifidum CCFM1391 of claim 1 or the microbial inoculant of claim 2 for the manufacture of a medicament for alleviating enteric nerve damage, characterized in that, The relief of intestinal damage includes at least one of the following: a) increasing the number of enteric glial cells and enteric neurons; b) increasing the gene expression of TPH2 and 5-HT4R and decreasing the gene expression of SERT in the intestines; c) increasing the content of neurotransmitter 5-HT in the colon tissue with damaged enteric nerves; d) increasing the expression of neurotrophic factors GDNF and NGF in the colon tissue with damaged enteric nerves; e) increasing the content of anti-inflammatory factors TGF-β / IL-10 and decreasing the content of pro-inflammatory factors CXCL-1 / IL-17 in the colon tissue with damaged enteric nerves; f) increasing the expression of AQP4 in the colon tissue with damaged enteric nerves; g) increasing the content of acetic acid and propionic acid in feces; h) relieving hypokinetic intestinal motility disorders and improving the water reabsorption capacity of the intestines.

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