Bifidobacterium bifidum for significantly increasing expression of brain-derived neurotrophic factor in host and application thereof

Abstract

The application discloses a bifidobacterium that can significantly improve the expression of BDNF of a host and an application thereof, and belongs to the technical field of microorganisms. The bifidobacterium CCFM1359 provided by the application can effectively improve the levels of BDNF and its specific receptor TrkB in the colon of a mouse, can also increase the number of enteric glial cells, improve the content of excitatory neurotransmitter Ach in the colon, down-regulate the content of inhibitory neurotransmitter NO, and repair the enteric nervous system. Moreover, the bacteria can down-regulate the gene expression levels of TLR2 and TNF-alpha, up-regulate the gene expression level of IL-10, reduce the inflammation level in the colon, and regulate the intestinal motility. Therefore, in the future, a more personalized treatment scheme can be adopted for patients with damaged enteric nerves (including but not limited to intestinal motility disorders), and the application prospect is very considerable.

Description

TECHNICAL FIELD

[0001] The present application relates to a Bifidobacterium bifidum strain that significantly increases the expression of BDNF in a host and an application thereof, and belongs to the field of microbial technology. BACKGROUND

[0002] The enteric nervous system (ENS) is often referred to as the "brain of the gastrointestinal tract", and the stimulation of the sensory neurons therein promotes the release of various neurotransmitters by excitatory and inhibitory neurons, activates the corresponding receptors to produce different motor signals, and ultimately the smooth muscle completes contraction or relaxation. When the enteric nervous system and various neurons are disordered, it will lead to intestinal motility disorders, and then affect the absorption of nutrients and the excretion of metabolic waste. For example, diarrhea caused by rapid intestinal motility will cause rapid loss of water and electrolytes in the human body, while slow intestinal motility will cause food residues to be unable to be discharged in time, and microorganisms will over-ferment to produce various harmful substances affecting human health. Therefore, the enteric nervous system plays a key role in the regulation of intestinal motility disorders.

[0003] Intestinal motility disorders, especially those accompanied by neurological diseases, are often found to have damage to the enteric nervous system, and brain-derived neurotrophic factor (BDNF) as a protein with neurotrophic effects has been widely recognized for its pleiotropic effects in the central nervous system and its impact on various brain diseases. However, BDNF is not only expressed in the nervous system but also in various layers of the intestine, including the enteric nervous system, intestinal mucosal epithelium, and intestinal muscle layer, and studies have shown that its expression in the intestine even exceeds that in the brain. Multiple in vivo and in vitro experiments have also shown that after BDNF specifically binds to tyrosine kinase receptor B (TrkB), it activates the downstream signaling pathway to inhibit the expression of inflammatory factors, inhibit intestinal epithelial cell apoptosis, promote colonic smooth muscle contraction, and other pathways to improve the state of colonic disease. Therefore, the role of BDNF in intestinal motility from the perspective of mediating intestinal nerve repair should be taken seriously. However, due to the great limitations of the short half-life and biological non-availability of BDNF in clinical applications, plant-derived 7,8-dihydroxyflavone (7,8-DHF) is widely studied as a small molecule mimic of BDNF.

[0004] Probiotics are "live microorganisms that, when administered in adequate amounts, confer health benefits to the host". So far, more and more animal and human experiments have proved that probiotics have significant alleviating effect on intestinal motility disorders, and also provide a model for future microecological treatment of other diseases. On the one hand, probiotics can regulate intestinal motility by regulating the level of intestinal inflammatory factors. On the other hand, probiotics can regulate the enteric nervous system by regulating the synthesis of neuroactive substances such as neurotransmitters, neurotrophic factors, etc., thereby promoting intestinal motility, and the most typical one is BDNF. SUMMARY

[0005] A number of studies have proved that probiotics can increase the BDNF content of the host to maintain the health of the host. Among them, bifidobacterium is proved to be able to effectively repair the damaged enteric nervous system of patients with intestinal motility disorders, and can also reduce the psychological pressure of patients and improve the quality of life of patients. Whether it is made into a bacterial agent or added to food to make functional food, it has very great application prospect. Through the research on the regulation of BDNF level by bifidobacterium bifidum, it will have a great impact on food science, microbiology and preventive medicine, etc. Therefore, it is necessary to explore the regulation of bifidobacterium bifidum to BDNF and the application of bifidobacterium bifidum.

[0006] The present application provides a bifidobacterium bifidum capable of repairing the enteric nervous system by increasing the expression level of BDNF, and provides the application of the strain. Bifidobacterium bifidum The present application provides a bifidobacterium bifidum capable of significantly increasing the protein level (61.31%) of brain-derived neurotrophic factor (BDNF) in the colon and the expression (26.51%) of its receptor TrkB gene. In addition, the strain can also significantly increase the number of enteric glial cells (S100β, 73.71%), up-regulate the excitatory neurotransmitter (Ach, 53.45%), down-regulate the inhibitory neurotransmitter (NO, 38.49%), repair the enteric nervous system, down-regulate the gene expression level of TLR2 (32.14%) and TNF-α (39.55%), up-regulate the gene expression level of IL-10 (85.18%), reduce the inflammation level in the colon, significantly shorten the intestinal transit time of the host (18.26%), increase the fecal particle number within 5 hours (200%), increase the small intestinal propulsion rate (31.9%), and promote intestinal motility. The corresponding probiotic preparation, fermented food and functional food are provided, so as to realize the regulation of the BDNF level of the body, or make the patients with damaged enteric nervous system gradually get rid of the side effects and limitations of drug treatment.

[0007] The present application provides a bifidobacterium bifidum capable of repairing the enteric nervous system by increasing the expression level of BDNF. Bifidobacterium bifidum) Bifidobacterium bifidum CCFM1359, which was preserved in Guangdong Microbial Culture Collection Center on November 9, 2023, at 5th floor, No. 59 Building, Guangzhou Martyrs' Courtyard, 100, Xianlie Road, Guangzhou, with a preservation number of GDMCC No: 63992.

[0008] The Bifidobacterium bifidum ( Bifidobacterium bifidum ) is from a fecal sample of a pregnant woman in Guangzhou. After sequencing analysis, the sequencing sequence is subjected to nucleic acid sequence alignment in NCBI Standard Nucleotide BLAST, and the results show that the strain is Bifidobacterium bifidum, which is named Bifidobacterium bifidum ( Bifidobacterium bifidum ) CCFM1359.

[0009] The Bifidobacterium bifidum CCFM1359 has the following biological characteristics:

[0010] (1) Bacterial characteristics: Gram-positive, non-spore-forming, non-motile bacteria.

[0011] (2) Colony characteristics: round, convex, smooth, and regular edge.

[0012] (3) Growth characteristics: about 54 h in MRS medium at 37 DEG C constant temperature to reach the late logarithmic phase.

[0013] (4) Strong tolerance to simulated gastrointestinal fluid.

[0014] (5) Significantly improves the relative expression of BDNF and its specific receptor TrkB in colon tissue, reduces the level of intestinal inflammation, and promotes the reconstruction of the intestinal nervous system.

[0015] The application also provides a microbial preparation containing the above-mentioned Bifidobacterium bifidum ( Bifidobacterium bifidum ) CCFM1359 or a fermentation liquor, a freeze-dried powder, a lysate, a culture or an extract thereof.

[0016] In an embodiment of the application, the amount of the Bifidobacterium bifidum CCFM1359 added in the microbial agent is at least 10 8 CFU / mL or 10 8 CFU / g.

[0017] The application also provides a medicine containing the above-mentioned Bifidobacterium bifidum ( Bifidobacterium bifidum ) CCFM1359 or the above-mentioned microbial agent.

[0018] In one embodiment of the invention, the drug comprises Bifidobacterium bifidum CCFM1359 and a pharmaceutically permissible carrier.

[0019] 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.

[0020] In one embodiment of the present invention, the dosage form of the medicine includes granules, capsules, tablets, pills, or oral liquid.

[0021] In one embodiment of the present invention, the Bifidobacterium bifidum ( Bifidobacterium bifidum The minimum addition level of CCFM1359 to a pharmaceutical product is 10%. 8 CFU / mL or 10 8 CFU / g.

[0022] The present invention also provides the above-mentioned Bifidobacterium bifidum ( Bifidobacterium bifidum The use of CCFM1359 or the above-mentioned microbial agents in the preparation of drugs that can accelerate intestinal peristalsis, repair damaged intestinal nerves in patients with intestinal motility disorders, or promote intestinal motility.

[0023] In one embodiment of the present invention, the dosage form of the medicine includes granules, capsules, tablets, pills, or oral liquid.

[0024] In one embodiment of the present invention, the amount of Bifidobacterium bifidum added to the drug is at least 10. 8 CFU / mL or 10 8 CFU / g.

[0025] In one embodiment of the invention, the pharmaceutical product comprises Bifidobacterium bifidum CCFM1359 and a carrier permitted in the pharmaceutical field.

[0026] The present invention also provides the above-mentioned Bifidobacterium bifidum ( Bifidobacterium bifidum Application of CCFM1359 or the above-mentioned microbial agents in the preparation of functional products having at least one of the following functions:

[0027] (a) Significantly increased the protein level of brain-derived neurotrophic factor (BDNF) in the colon (61.31%) and the expression of its receptor TrkB gene (26.51%).

[0028] (b) Significantly increased the number of enteric glial cells (S100β, 73.71%), upregulated excitatory neurotransmitters (Ach, 53.45%), downregulated inhibitory neurotransmitters (NO, 38.49%), and repaired the enteric nervous system;

[0029] (c) Downregulates the gene expression levels of TLR2 (32.14%) and TNF-α (39.55%), upregulates the gene expression level of IL-10 (85.18%), and reduces the level of inflammation in the colon;

[0030] (d) Significantly shortened the host intestinal transit time (18.26%), increased the number of fecal particles within 5 hours (200%), improved the small intestinal propulsion rate (31.9%), and promoted intestinal motility;

[0031] The present invention also provides a product containing the aforementioned Bifidobacterium bifidum ( Bifidobacterium bifidum CCFM1359 microbial agent.

[0032] In one embodiment of the present invention, the bacterial agent contains Bifidobacterium bifidum (Bifidobacterium bifidum). Bifidobacterium bifidum The number of live bacteria in CCFM1359 is ≥10. 8 cfu / g or 10 8 cfu / mL.

[0033] In one embodiment of the present invention, the bacterial agent is a solution containing Bifidobacterium bifidum (Bifidobacterium bifidum). Bifidobacterium bifidum The number of viable bacteria obtained by drying the CCFM1359 bacterial solution was ≥10. 8 cfu / g or 10 8 Powder with cfu / mL.

[0034] In one embodiment of the present invention, the drying refers to vacuum freeze drying.

[0035] In one embodiment of the present invention, the product is a pharmaceutical product.

[0036] Beneficial effects

[0037] (1) The Bifidobacterium bifidum CCFM1359 of the present invention exhibits good activity, certain acid and alkali resistance, and adhesiveness. It can significantly increase the levels of BDNF and its specific receptor TrkB in the colon of mice, increase the number of enteroglial cells, increase the content of the excitatory neurotransmitter Ach in the colon, downregulate the content of the inhibitory neurotransmitter NO, and repair the damaged enteronergic system. Furthermore, this bacterium can downregulate the gene expression levels of TLR2 and TNF-α, upregulate the gene expression level of IL-10, and reduce the level of inflammation in the colon. This includes, but is not limited to, accelerating intestinal transit, increasing the number of fecal particles in five hours, and improving the small intestinal propulsion rate, thereby promoting intestinal peristalsis.

[0038] (2) This invention can be regarded as a drug for repairing intestinal nerve damage, while reducing the level of inflammation in the colon. It can also be applied to pharmaceuticals, thus playing a wide role and having a very valuable application prospect.

[0039] (3) The application also provides application of the Bifidobacterium bifidum CCFM1359 in preparation of a drug for relieving a laxative colon. BRIEF DESCRIPTION OF DRAWINGS

[0040] Figure 1 : The Bifidobacterium bifidum (Bifidobacterium bifidum) Bifidobacterium bifidum CCFM1359 strain intervention, the expression level of BDNF gene and protein in the colon tissue of the intestinal movement disorder mouse is changed.

[0041] Figure 2 : The Bifidobacterium bifidum (Bifidobacterium bifidum) CCFM1359 strain intervention, the expression level of BDNF gene and protein in the colon tissue of the intestinal movement disorder mouse is changed.

[0042] Figure 3 : The Bifidobacterium bifidum (Bifidobacterium bifidum) Bifidobacterium bifidum CCFM1359 strain intervention, the expression level of S100β in the colon tissue of the intestinal movement disorder mouse is changed.

[0043] Figure 4 : The Bifidobacterium bifidum (Bifidobacterium bifidum) Bifidobacterium bifidum CCFM1359 strain intervention, the expression level of excitatory neurotransmitter Ach and inhibitory neurotransmitter NO in the colon tissue of the intestinal movement disorder mouse is changed.

[0044] Figure 5 : The Bifidobacterium bifidum (Bifidobacterium bifidum) Bifidobacterium bifidum CCFM1359 strain intervention, the expression level of inflammatory factors in the colon tissue of the intestinal movement disorder mouse is changed.

[0045] Figure 6 : The Bifidobacterium bifidum (Bifidobacterium bifidum) Bifidobacterium bifidum CCFM1359 strain intervention, the expression level of inflammatory factors in the colon tissue of the intestinal movement disorder mouse is changed.

[0046] Note: The symbols above the column chart represent the significance level of data, * represents p <0.05, ** represents p <0.01, *** represents p <0.001, (compared with the model group), # represents p <0.05, ## represents p <0.01, ### represents p <0.001, #### represents p <0.0001 (compared with the blank group).

[0047] Preservation of biological material

[0048] Bifidobacterium bifidum (CCFM1359) Bifidobacterium bifidum , which is taxonomically named as: Bifidobacterium bifidum , was preserved in Guangdong Microbial Culture Collection Center on November 09, 2023, at an address of 5th Floor, Building 59, Guangzhou Martyrs' Courtyard, 100, Renmin Road, and a preservation number of GDMCC No: 63992. DETAILED DESCRIPTION

[0049] Male C57BL / 6J mice involved in the following examples were purchased from Zhejiang Vintone Lihua Experimental Animal Technology Co., Ltd.

[0050] The culture medium involved in the following examples is as follows:

[0051] 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; L-cysteine hydrochloride 1 g, adjust pH to 6.8±0.2; constant volume to 1 L. Autoclave at 115°C for 20 min.

[0052] MRS solid medium: 2% agar powder is added on the basis of MRS liquid medium.

[0053] The preparation method of Bifidobacterium bifidum bacterial suspension involved in the following examples is as follows:

[0054] Bifidobacterium bifidum was inoculated into MRS solid medium and cultured at 37°C under anaerobic conditions for 48 h to obtain single colonies, and the prepared single colonies were inoculated into MRS liquid medium and cultured at 37°C under anaerobic conditions for 18 h for activation.

[0055] The bacterial liquid after 3 generations of activation was inoculated into 1 L of MRS liquid medium at an inoculation amount of 2% (v / v), and after oscillation and mixing, it was cultured at 37°C in an anaerobic environment for 12 h. Centrifugation was performed at 8000 g / min and 4°C for 10 min, the supernatant was removed, and the bacterial body was resuspended in 10% sterilized skimmed milk solution after washing twice with sterile normal saline and centrifugation under the same conditions. The final concentration of Bifidobacterium bifidum CCFM1359 bacterial suspension prepared was 5×10 9 CFU / mL, and thus the pre-gavage standby bacterial liquid was obtained, which was stored in a-80°C refrigerator for one week.

[0056] Before the animal experiment, the bacteria liquid stored in the refrigerator was taken out, and the initial and one-week after freezing active bacteria number was determined by plate coating method. The order of magnitude of the initial and one-week after freezing active bacteria number did not change, which indicated that the bacteria liquid freezing would not affect the experiment, and could be used for animal experiment.

[0057] The detection method of the expression amount of BDNF / TrkB gene involved in the following examples

[0058] The expression amount of BDNF / TrkB gene was determined by real-time fluorescence quantitative polymerase chain reaction (qRT-PCR), and RNA was first extracted from fresh tissue, and the specific method was as follows:

[0059] The fresh colon tissue 0.2 g taken out after mouse dissection was repeatedly ground in a mortar (180℃, 4 h high temperature enzyme inactivation) added with liquid nitrogen, 1 mL TrizoL reagent was further added to the mortar, and the grinding was continued. After the liquid was basically clarified, it was collected into a 1.5 mL enzyme-free centrifuge tube, and was placed at room temperature for 15 min. 200 μL of chloroform solution was added to the centrifuge tube, shaken for 15 s, and placed at room temperature for 10 min. Centrifugation was performed at 4℃, 12000 r / min for 15 min, 600 μL of upper colorless aqueous phase was taken into another enzyme-free centrifuge tube, and 500 μL of isopropanol was added. Mix well by inverting up and down, and place at room temperature for 10 min. After standing, centrifugation was performed at 4℃, 12000 r / min for 10 min, the supernatant was discarded, and the white precipitate of RNA formed at the bottom of the centrifuge tube was left. 1 mL of 75% ethanol solution prepared with DEPC water was added, vortexed to resuspend, centrifuged at 4℃, 7500 r / min for 5 min, the supernatant was discarded, and the room temperature was naturally volatilized and dried. 30 μL of RNase free water was added to the dried RNA, and the RNA concentration and purity were determined by Nanodrop after the RNA was dissolved. The quality of the RNA was detected by agarose gel electrophoresis. The total RNA extracted was used as a template, and the reverse transcription to synthesize cDNA was performed according to the operation steps of HiFiScript gDNA RemovaL RT MasterMix reverse transcription kit instruction manual. Store at -20℃.

[0060] The primers of mouse BDNF / TrkB gene and reference gene β-actin gene are shown in Table 1,

[0061] Table 1 Primer sequences of mouse BDNF / TrkB gene and β-actin gene

[0062]

[0063] qRT-PCR reaction system and conditions:

[0064] PCR amplification was performed with a Bio-Rad® CFX96™ Real-Time Fluorescent Quantitative PCR Instrument, and the fluorescent signal was read.

[0065] The reaction system of c-kit gene qRT-PCR was as follows:

[0066]

[0067] The reaction conditions of qRT-PCR were as follows:

[0068] 95℃ 30s; 95℃ 10s, 56℃ 30s, 40 cycles in total. The β-actin gene was used as an internal reference gene, and the results were analyzed by CFX96 Manager software.

[0069] The method for detecting the content of BDNF in colon tissue involved in the following examples

[0070] The expression amount of BDNF in colon tissue was quantified by ELISA. The specific method was as follows: the colon tissue was washed with pre-cooled PBS to remove residual blood, and the surrounding adipose tissue was removed. After weighing, the tissue was cut into 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 5000xg for 5-10 minutes, and the supernatant was detected. The experiment was performed according to the corresponding kit instructions, and the BDNF in the tissue was calculated according to the standard curve.

[0071] The method for detecting the expression amount of S100β gene involved in the following examples

[0072] Real-time fluorescent quantitative polymerase chain reaction (qRT-PCR) was used to determine the expression amount of S100β gene. First, RNA was extracted from fresh tissue, and the specific method was as follows:

[0073] Fresh colon tissue 0.2 g removed after dissection of mice was repeatedly ground in a mortar (180℃, 4 h high temperature enzyme inactivation) added with liquid nitrogen, then 1 mL TrizoL reagent was added to the mortar and the grinding was continued, and after the liquid was basically clarified, it was collected into a 1.5 mL enzyme-free centrifuge tube, and room temperature standing for 15 min, 200 μL chloroform solution was added to the centrifuge tube, and light shaking for 15 s, room temperature standing for 10 min, 4℃, 12000 r / min centrifugation for 15 min, 600 μL of the upper colorless aqueous phase was taken into another enzyme-free centrifuge tube, and 500 μL of isopropanol was added. Mix well by inverting up and down, stand at room temperature for 10 min, after standing, centrifuge at 4℃, 12000 r / min for 10 min, discard the supernatant, leave the white precipitate of RNA at the bottom of the centrifuge tube, add 1 mL of 75% ethanol solution prepared with DEPC water, vortex to resuspend, centrifuge at 4℃, 7500 r / min for 5 min, discard the supernatant, and dry at room temperature. Add 30 μL RNase free water to the dried RNA, and after dissolving the RNA, determine the RNA concentration and purity by Nanodrop, and detect the quality of the RNA by agarose gel electrophoresis. Total RNA extracted as a template, according to the operation steps of HiFiScript gDNA RemovaL RT MasterMix reverse transcription kit of Kangwei Century Company, cDNA was synthesized by reverse transcription, and stored at -20℃.

[0074] The primers of mouse S100β gene and reference gene β-actin gene are shown in Table 2,

[0075] Table 2 Primers of mouse S100β gene and β-actin gene

[0076]

[0077] qRT-PCR reaction system and conditions:

[0078] PCR amplification was performed using Bio-Rad CFX96TM real-time fluorescent quantitative PCR instrument, and the fluorescence signal was read.

[0079] The qRT-PCR reaction system of c-kit gene is:

[0080]

[0081] The qRT-PCR reaction conditions are:

[0082] 95℃ 30s; 95℃ 10s, 58℃ 30s, a total of 40 cycles. β-actin gene as an internal reference gene, the results were analyzed by CFX96Manager software.

[0083] The method for detecting the content of Ach / NO in the colon tissue involved in the following examples

[0084] The expression amount of Ach / NO in the colon tissue was quantified by ELISA method. The specific method is as follows: the colon tissue was washed with pre-cooled PBS to remove residual blood, and the surrounding adipose tissue was removed. After weighing, it was cut into 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 content of Ach / NO in the tissue was calculated according to the standard curve.

[0085] The method for detecting the expression amount of TLR2 / TNF-α gene involved in the following examples

[0086] Real-time fluorescent quantitative polymerase chain reaction (qRT-PCR) was used to determine the expression amount of TLR2 / TNF-α gene. First, RNA was extracted from fresh tissue. The specific method is as follows:

[0087] Fresh colon tissue 0.2 g taken from the mouse after dissection was repeatedly ground in a mortar (180℃, 4 h high temperature enzyme inactivation) added with liquid nitrogen. Then 1 mL TrizoL reagent was added to the mortar and continued to be ground. After the liquid was basically clarified, it was collected into a 1.5 mL enzyme-free centrifuge tube and room temperature for 15 min. 200 μL of chloroform solution was added to the centrifuge tube, shaken for 15 s, and room temperature for 10 min. 4℃, 12000 r / min centrifugation for 15 min, 600 μL of upper colorless water phase was taken into another enzyme-free centrifuge tube, and 500 μL of isopropanol was added. Mix well by inverting up and down, and let stand at room temperature for 10 min. After standing, 4℃, 12000 r / min centrifugation for 10 min, discard the supernatant, leave the white precipitate of RNA at the bottom of the centrifuge tube, add 1 mL of 75% ethanol solution prepared with DEPC water, vortex to resuspend, 4℃, 7500 r / min centrifugation for 5 min, discard the supernatant, and let it dry naturally at room temperature. Add 30 μL of RNase free water to the dried RNA, and measure the RNA concentration and purity with Nanodrop after dissolving the RNA. The quality of the RNA was detected by agarose gel electrophoresis. The total RNA extracted was used as a template to synthesize cDNA according to the operation steps of HiFiScript gDNA RemovaL RT MasterMix reverse transcription kit of Kangwei Century Company. Store at -20℃.

[0088] The primers of mouse TLR2 / TNF-α gene and reference gene β-actin gene are shown in Table 3.

[0089] Table 3. Primer sequences of mouse TLR2 / TNF-a gene and β-actin gene

[0090]

[0091] qRT-PCR reaction system and conditions:

[0092] PCR amplification was performed using Bio-Rad CFX96TM real-time fluorescent quantitative PCR instrument, and the fluorescence signal was read.

[0093] The qRT-PCR reaction system of c-kit gene was:

[0094]

[0095] The qRT-PCR reaction conditions were:

[0096] 95°C for 30s; 95°C for 10s, 60°C for 30s, for a total of 40 cycles. The β-actin gene was used as an internal reference gene, and the results were analyzed by CFX96 Manager software.

[0097] The detection method of the expression amount of IL-10 involved in the following examples

[0098] Real-time fluorescent quantitative polymerase chain reaction (qRT-PCR) was used to determine the expression amount of IL-10. First, RNA was extracted from fresh tissue, and the specific method was as follows:

[0099] Fresh colon tissue 0.2 g removed after dissection of mice was repeatedly ground in a mortar (180℃, 4 h high temperature enzyme inactivation) added with liquid nitrogen, then 1 mL TrizoL reagent was added to the mortar and the grinding was continued, and after the liquid was basically clarified, it was collected into a 1.5 mL enzyme-free centrifuge tube, and room temperature was placed for 15 min, 200 μL chloroform solution was added to the centrifuge tube, and it was shaken for 15 s, and room temperature was placed for 10 min, and 4℃, 12000 r / min centrifugation for 15 min, 600 μL of the upper colorless aqueous phase was taken into another enzyme-free centrifuge tube, and 500 μL of isopropanol was added. Mix well by inverting up and down, and let stand at room temperature for 10 min, then centrifuge at 4℃, 12000 r / min for 10 min, discard the supernatant, and leave the white precipitate of RNA at the bottom of the centrifuge tube, add 1 mL of 75% ethanol solution prepared with DEPC water, vortex to resuspend, centrifuge at 4℃, 7500 r / min for 5 min, discard the supernatant, and dry at room temperature. Add 30 μL RNase free water to the dried RNA, and measure the RNA concentration and purity with Nanodrop after dissolving the RNA, and detect the quality of the RNA by agarose gel electrophoresis. Reverse transcription to synthesize cDNA was performed according to the operation steps of HiFiScript gDNA RemovaL RT MasterMix reverse transcription kit of Kangwei Century Company, and the cDNA was stored at -20℃.

[0100] The primers of mouse IL-10 and reference gene β-actin gene are shown in Table 4,

[0101] Table 4 Primers of mouse IL-10 gene and β-actin gene

[0102]

[0103] qRT-PCR reaction system and conditions:

[0104] PCR amplification was performed with Bio-Rad CFX96TM real-time fluorescent quantitative PCR instrument, and the fluorescence signal was read.

[0105] The qRT-PCR reaction system of c-kit gene is:

[0106]

[0107] The qRT-PCR reaction conditions are:

[0108] 95℃ 30s; 95℃ 10s, 60℃ 30s, a total of 40 cycles. The β-actin gene was used as the reference gene, and the results were analyzed by CFX96Manager software.

[0109] Example 1: Bifidobacterium longum (CCFM1359) Bifidobacterium bifidum Obtaining of Bifidobacterium longum (CCFM1359)

[0110] 1. Isolation and screening of Bifidobacterium longum

[0111] (l) Collect the fecal samples of pregnant women in Guangzhou using disposable sterile sampling device, and place them in anaerobic environment for 37°C culture for 72 hours; observe and record the colony morphology, pick the colonies and streak for purification; in MRS liquid medium, 37°C culture for 48 hours, and perform gram staining on the obtained colonies, and record the colony morphology. Discard the gram-negative bacterial strains and gram-positive cocci in the colonies, and select the gram-positive bacilli.

[0112] (2) After catalase analysis, discard the catalase-positive strains and retain the catalase-negative strains.

[0113] 2. Molecular biology identification of Bifidobacterium longum

[0114] (l) Single bacterial genome extraction: culture the catalase-negative strains selected in step 1 overnight, take 1 mL of the overnight cultured bacterial suspension in a 1.5 mL centrifuge tube, centrifuge at 10000 rpm for 2 min, discard the supernatant to obtain the bacterial body; blow the bacterial body with 1 mL of sterile water, then centrifuge at 10000 rpm for 2 min, discard the supernatant to obtain the bacterial body; add 200 μL of SDS lysis solution, and incubate at 80°C for 30 min; add 200 μL of phenol-chloroform solution to the bacterial lysis solution, wherein the composition and volume ratio of the phenol-chloroform solution are Tris-saturated phenol: chloroform: isopropyl alcohol = 25:24:1, mix well by inverting, then centrifuge at 12000 rpm for 5-10 min, take 200 μL of the supernatant; add 400 μL of ice ethanol or ice isopropyl alcohol to 200 uL of the supernatant, and stand at -20°C for 1 h, centrifuge at 12000 rpm for 5-10 min, discard the supernatant; resuspend the precipitate with 500 μL of 70% (volume percentage) ice ethanol, centrifuge at 12000 rpm for 1-3 min, discard the supernatant; dry in a 60°C oven or air dry; resuspend the precipitate with 50 μL of ddH2O for PCR;

[0115] (2) 16S rDNA PCR

[0116] A. Bacterial 16S rDNA 50 μL 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.

[0117] B. PCR condition: 94℃ 5 min; 94℃ 30 s, 52℃ 30 s, 72℃ 30 s; step 2-4 30x; 72℃ 10 min; 12℃ 2 min;

[0118] C. Prepare 1% agarose gel, then mix the PCR product with 10000x loading buffer, load 2 μL, run at 120V for 30 min, then gel imaging;

[0119] D. Send the obtained PCR product to a professional sequencing company, search and similarity comparison in GeneBank using BLAST, perform nucleic acid sequence comparison of the obtained 16S rDNA (SEQ ID NO. 1) sequence in NCBI Standard Nucleotide BLAST, the results show that the strain is Bifidobacterium bifidum, which is named Bifidobacterium bifidum (CCFM1359) and preserved at -80℃ for standby. Bifidobacterium bifidum

[0120] The 16S rDNA (SEQ ID NO. 1) sequence is as follows:

[0121] ​

[0122] Example 2: Bifidobacterium bifidum (CCFM1359) can improve the content of BDNF in colon tissue of mice with impaired enteric nerves Bifidobacterium bifidum

[0123] The specific steps are as follows:

[0124] (1) Preparation of Bifidobacterium bifidum (CCFM1359) bacterial suspension Bifidobacterium bifidum

[0125] After taking the Bifidobacterium bifidum CCFM1359 strain out of the -80°C refrigerator, it was streaked on MRS solid culture medium and cultured at 37°C for 48 h. Single colonies were picked and cultured in MRS liquid medium at 37°C for 24 h to prepare the seed liquid.

[0126] The prepared seed liquid was inoculated into new MRS liquid medium at an inoculation amount of 2% (v / v) and cultured at 37°C for 18 h. The same method was used to culture another generation to prepare the Bifidobacterium bifidum CCFM1359 fermentation broth.

[0127] Then the prepared Bifidobacterium bifidum CCFM1359 fermentation broth was centrifuged at 8000 r / min and 4°C for 10 min, and then resuspended with 10% skim milk to prepare the bacterial suspension for animal experiments.

[0128] (2) Treatment of senna extract

[0129] The purchased senna extract was prepared into different concentrations of senna extract solution with sterilized water for use.

[0130] (3) 24 healthy 8-week-old male C57BL / 6J mice were used, and they were adapted to the environment for 1 week (week 0). They were randomly divided into 5 groups: blank control group, model group, 7,8-DHF group, Bifidobacterium bifidum intervention group CCFM1359, and M203F02M632 group (Bifidobacterium bifidum obtained at the same time), each containing 6 mice. The dose of bacterial suspension for gavage was 5×10 9 CFU / mL, and gavage was started at 9 am every day, with 0.2 mL each time.

[0131] The grouping and treatment method of experimental animals are shown in Table 5:

[0132] Table 5: Grouping of experimental animals

[0133]

[0134] Real-time fluorescent quantitative polymerase chain reaction (qRT-PCR) was used to determine the relative expression of BDNF gene in colon tissue, and ELISA method was used to quantify the BDNF protein level in colon tissue.​​

[0135] Brain-derived neurotrophic factor (BDNF) is an important member of the neurotrophic factor family. It is abundant in the enteric nervous system, intestinal mucosal epithelial cells, and interstitial cells, and can regulate intestinal motility by influencing the structure and function of the enteric nervous system. In humans, BDNF accelerates colonic emptying and increases defecation frequency; in animal experiments, BDNF enhances gastrointestinal electromyographic activity and strengthens mucosal stimulation-induced colonic motility reflexes. Results are as follows... Figure 1 As shown.

[0136] The results show:

[0137] Depend on Figure 1 As shown in a, the relative expression level of BDNF in the colon of the enteric nerve injury model mice (0.577) was downregulated by 43.87% compared with the blank group (1.028). However, after gavage administration of CCFM1359, the BDNF level in the colon (0.769) was increased by 33.28% compared with the model group.

[0138] Depend on Figure 1 As shown in b, the concentration of BDNF in the colon of the enteric nerve injury model mice (7.493 ng / g protein) was downregulated by 33.87% compared to the BDNF concentration in the control group (11.330 ng / g protein). However, after gavage administration of CCFM1359, the BDNF concentration in the colon (12.088 ng / g protein) was significantly increased by 61.32% compared to the model group. p <0.05).

[0139] The results indicate that senna leaf extract modeling causes a downregulation of BDNF levels in the colon, while Bifidobacterium bifidum CCFM1359 can increase BDNF expression, repair the enteric nervous system, and accelerate intestinal motility.

[0140] Example 3: Bifidobacterium bifidum ( Bifidobacterium bifidum CCFM1359 can increase the expression level of TrkB, a downstream receptor of BDNF, in the colon tissue of mice with enteric nerve damage.

[0141] The grouping, modeling and treatment methods of C57BL / 6J mice were the same as those in Example 2. Real-time fluorescent quantitative polymerase chain reaction (qRT-PCR) was used to determine the expression level of BDNF high-affinity receptor protein tyrosine kinase receptor B (TrkB) gene in the colon tissue. After specific binding of BDNF and TrkB, TrkB is phosphorylated into p-TrkB, activates downstream signal transduction, and thus plays a role in increasing intracellular Ca 2+ The results are shown in Table 2. Figure 2

[0142] The results are shown in Table 2.

[0143] It can be seen from Table 2 that after senna modeling, the relative expression level of TrkB (0.541) was significantly decreased by 45.9% (P < 0.01) compared with the expression level of TrkB (1) in the blank group, and the expression level of TrkB (0.684) after CCFM1359 gavage was up-regulated by 26.43% compared with the model group. Figure 2 p <0.01), and the expression level of TrkB (0.684) after CCFM1359 gavage was up-regulated by 26.43% compared with the model group.

[0144] It is inferred that the CCFM1359 strain repairs intestinal nerves, promotes intestinal movement, or depends on the BDNF-TrkB signaling pathway.

[0145] Example 4: Bifidobacterium bifidum CCFM1359 increases the number of enteric glial cells in the colon tissue of mice with damaged intestinal nerves

[0146] The grouping, modeling and treatment methods of C57BL / 6J mice were the same as those in Example 2. Real-time fluorescent quantitative polymerase chain reaction (qRT-PCR) was used to determine the expression level of S100 β gene in the colon tissue.

[0147] Senna extract, as a stimulant laxative, mainly plays a role by stimulating the enteric nervous system. Mucosal glial cells are mainly involved in epithelial barrier function, and glial cells in the ganglion mainly act on nerve repair, closely interact with neurons, support the differentiation of these cells and glial cells, and participate in the occurrence and formation of nerves. The number of glial cells can well reflect the health status of intestinal nerves. S100 calcium binding protein beta (S100 calcium binding protein beta, S100 β) is a marker of intestinal glial cells, reflecting the number of enteric glial cells. By detecting the gene expression level of S100 β in the colon tissue, the influence of senna and probiotics can be characterized. The results are shown in Table 3. Figure 3

[0148] The results are shown in Table 3.

[0149] It can be seen from Table 3 that after senna modeling, the relative expression level of S100 β (0.541) was significantly decreased by 45.9% (P < 0.01) compared with the expression level of S100 β (1) in the blank group, and the expression level of S100 β (0.684) after CCFM1359 gavage was up-regulated by 26.43% compared with the model group.​​​Figure 3 It can be seen that the expression of S100β in the intestinal nerve damaged mice (0.423) decreased by 57.68% compared with the expression of S100β in the blank group (1) p <0.001), indicating that the number of intestinal nerve glial cells decreased and the intestinal nerve was damaged. After 7,8-DHF treatment, there was an upward trend, but there was no significant difference (expression: 0.530). After gavage with CCFM1359, the expression (0.735) increased by 73.76% compared with the model group p <0.001), and the effect was better than that of the 7,8-DHF positive control group, indicating that the intestinal nerve was reconstructed. It can be seen that gavage with CCFM1359 can restore the health of the intestinal nerve, thereby promoting the colon movement and improving the colon motility.

[0150] Example 5: Bifidobacterium bifidum (Bifidobacterium longum) Bifidobacterium bifidum CCFM1359 can increase the content of excitatory neurotransmitter ACh and down-regulate the content of inhibitory neurotransmitter NO in the colon tissue of intestinal nerve damaged mice.

[0151] The 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 of excitatory neurotransmitter Ach and the expression of inhibitory neurotransmitter nNOS in the colon tissue. Excitatory neurotransmitters can cause local depolarization of the postsynaptic membrane, making the postsynaptic neurons excited, thereby transmitting excitatory signals to the smooth muscle and promoting intestinal movement. Inhibitory neurotransmitters can cause hyperpolarization of the postsynaptic membrane, which is manifested as inhibition of postsynaptic neuron activity, thereby causing relaxation of smooth muscle cells and inhibiting intestinal movement. Among them, NO is chemically active and can be easily oxidized in vivo, so here the change of NO level is represented by detecting Neuronal nitric oxide synthase (nNOS). The results are shown in Figure 4

[0152] The results show that, as Figure 4 can be seen, senna modeling caused the concentration of Ach in the colon of mice (19.238 pmol / g protein) to decrease by 30.21% compared with the blank group (concentration: 27.566 pmol / g protein) p <0.05), and the nNOS level (concentration: 0.468 pmol / g protein) in the model group increased by 82.81% compared with the blank group (concentration: 0.256 pmol / g protein) p ​<0.01). When CCFM1359 was administered (Ach concentration: 29.521 pmol / g protein), the concentration of Ach was significantly increased by 53.45% compared to the model group (Ach concentration: 19.238 pmol / g protein) p <0.001), which was almost the same as the 7,8-DHF positive control group (concentration: 30.040 pmol / g protein). In addition, after CCFM1359 was administered, the concentration of NO (concentration: 0.288 pmol / g protein) was significantly reduced by 38.49% ( p <0.01), which was better than the 7,8-DHF positive control group (concentration: 0.347 pmol / g protein).

[0153] It is speculated that the CCFM1359 strain regulates intestinal motility or is related to the regulation of neurotransmitter expression.

[0154] Example 6: Bifidobacterium longum (B. longum) CCFM1359 down-regulates the gene expression levels of TLR2 and TNF-α, up-regulates the gene expression level of IL-10, reduces the inflammation level caused by sennoside, and speeds up intestinal motility. Bifidobacterium bifidum

[0155] 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 TLR2, TNF-α, and IL-10 genes in the colon tissue.

[0156] Intestinal inflammation level is usually directly related to intestinal barrier damage, and severe cases are accompanied by intestinal dysfunction. Tumor necrosis factor alpha (TNF-α) can inhibit intestinal motility through the central nervous system. Interleukin-10 (IL-10) as an anti-inflammatory factor reflects the level of intestinal inflammation. In addition, pattern recognition receptors are commonly used to identify foreign substances or bacteria in the intestine, which can initiate and regulate inflammatory responses and complete the body's immune response. The activation of TLR2 can promote the proliferation of cholinergic neurons and has a regulatory effect on intestinal motility, as shown in Figure 5

[0157] As shown in a of Figure 5 , the gene expression level of TNF-α in the model mice (1.664) was significantly increased by 66.40% compared to the blank group (expression level: 1) p ​​<0.05), indicating that the central nervous system of mice with enteric nerve damage inhibited intestinal motility. After gavage administration of CCFM1359, the expression level of TNF-α gene (1.006) was significantly downregulated by 39.54%. p <0.001), which is better than the 7,8-DHF positive control group (expression level is 1.197).

[0158] Depend on Figure 5 As shown in b, after senna leaf modeling, the TLR2 level in the colon of mice (expression level: 1.231) was upregulated by 23.10% compared with the blank group (expression level: 1), while after gavage administration of CCFM1359, its expression level (expression level: 0.835) was significantly downregulated by 32.17%. p <0.01), which is better than the 7,8-DHF positive control group (expression level: 1.007).

[0159] Depend on Figure 5 As shown in c, senna leaf modeling induced a 50.30% significant downregulation of the excitatory neurotransmitter IL-10 in the mouse colon (expression level: 0.497) compared to the control group (expression level: 1). p <0.001), after oral administration of CCFM1359, its level (expression level: 0.920) was significantly upregulated by 85.11% compared to the model group. p <0.001), which is better than the 7,8-DHF positive control group (expression level: 0.836).

[0160] Therefore, the results show that Bifidobacterium bifidum CCFM1359 can alleviate intestinal inflammation and regulate intestinal motility.

[0161] Example 7: Bifidobacterium bifidum ( Bifidobacterium bifidum CCFM1359 alleviates symptoms of sennoside-induced intestinal motility disorders.

[0162] The grouping and treatment methods for experimental animals are shown in Table 5, the same as in Example 1. After gavage in week 16, 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 dry weight was obtained, and the water content of the feces was calculated according to the following formula.

[0163] Fecal moisture content = (fecal wet weight - fecal dry weight) / fecal wet weight × 100%.

[0164] Each mouse was administered 0.2 mL of ink by gavage, and the mice were sacrificed 30 min later. The mice were dissected and the section from the stomach to the terminal ileum was taken. The length from the pylorus to the terminal ileum was taken as the "total length of the small intestine", and the distance from the pylorus to the tip of the ink was taken as the "distance of ink propulsion". The propulsion rate of the small intestine was calculated according to the following formula (Zhou et al., 2022).

[0165] Small intestinal propulsion rate % (w / v) = (Ink propulsion distance (cm)) / (Total small intestinal length (cm)) × 100%;

[0166] 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.

[0167] The results of fecal moisture content, intestinal transit time, small intestinal propulsion rate, and number of fecal particles within 5 hours are shown in Figure 6.

[0168] The results showed that the fecal water content of the model mice treated with senna leaves was significantly reduced to 55.03%. p The concentration of fecal particles within 5 hours was significantly lower than that in mice administered saline via gavage (58.99 in the control group). Furthermore, the number of fecal particles within 5 hours was 7.5. p <0.01) decreased significantly, and the total intestinal transit time in mice (to: 120.5) was significantly reduced ( p <0.01) prolonged, small intestinal propulsion rate (55.45%) significantly decreased ( p <0.01).

[0169] The above results indicate that senna leaf treatment leads to prolonged intestinal transit time and enhanced intestinal water absorption in mice, resulting in symptoms of slowed intestinal motility, indicating that the model was successfully established.

[0170] Compared to the model group, mice treated with Bifidobacterium bifidum CCFM1359 via gavage showed a significantly shorter intestinal transit time (98.5 minutes) by 18.26%. p <0.01); the number of fecal particles (22.5) increased by 200% within 5 hours. p <0.01), and the effect was similar to that of the 7,8-DHF group (intestinal transit time: 86.67, number of fecal particles within 5 hours: 24.17); the small intestinal propulsion rate (73.15%) increased by 31.92%. p <0.01). This indicates that oral administration of Bifidobacterium bifidum CCFM1359 can repair enteric nerve damage in mice caused by senna leaves and accelerate intestinal motility in mice.

[0171] 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 longum (B. longum) CCFM1359 characterized by, Bifidobacterium bifidum ) CCFM1359, characterized by, The Bifidobacterium bifidum was deposited with the Guangdong Microbial Culture Collection Center on November 9, 2023, and the deposit number is GDMCC No: 63992.

2. A microbial inoculant, characterized in that, The microbial agent contains the Bifidobacterium bifidum CCFM1359 or the fermentation liquor thereof, or the freeze-dried powder thereof, or the culture thereof according to claim 1.

3. The microbial inoculant of claim 2, wherein, The Bifidobacterium bifidum CCFM1359 is added in the microbial inoculant in an amount of at least 10 8 CFU / mL or 10 8 CFU / g.

4. A product characterized by, The product contains the Bifidobacterium bifidum CCFM1359 according to claim 1 or the microbial agent according to claim 2, and the product is a medicine.

5. The product of claim 4, wherein, The Bifidobacterium bifidum CCFM1359 is added in the product in an amount of at least 10 8 CFU / mL or 10 8 CFU / g.

6. Use of the Bifidobacterium bifidum CCFM1359 according to claim 1 in the preparation of a medicine capable of repairing damaged enteric nerves or promoting intestinal movement in patients with intestinal motility disorders.

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

8. Use according to claim 7, characterized in that, The Bifidobacterium is added in the medicine in an amount of at least 10 8 CFU / mL or 10 8 CFU / g.

9. Use according to claim 8, characterized in that, The medicine contains Bifidobacterium bifidum CCFM1359 and carriers allowed in the pharmaceutical field.

10. Use according to any one of claims 6 to 9, characterized in that, The medicine has at least one of the following effects: (a) significantly increasing the protein level of brain-derived neurotrophic factor (BDNF) in the colon and the expression of its receptor TrkB gene; (b) significantly increasing the number of enteric glial cells in patients, up-regulating excitatory neurotransmitters, down-regulating inhibitory neurotransmitters, and repairing the enteric nervous system; (c) down-regulating the gene expression levels of TLR2 and TNF-α, up-regulating the gene expression level of IL-10, and reducing the level of inflammation in the colon; (d) significantly shortening the intestinal transit time of the host, increasing the fecal particle count within 5 hours, improving the small intestinal propulsion rate, and promoting intestinal movement.

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