Bifidobacterium adolescentis capable of relieving constipation and application of bifidobacterium adolescentis

Abstract

The invention relates to bifidobacterium adolescentis capable of relieving constipation and application of the bifidobacterium adolescentis, and belongs to the technical field of biological medicines. The invention provides a strain of bifidobacterium adolescentis XA-1434. The preservation number of the strain is CGMCC (China General Microbiological Culture Collection Center) No.31382; the strain can efficiently degrade arabinoxylan and grow by taking an arabinoxylan degradation product as a unique carbon source, and genomic sequencing and arabinoxylan degradation gene exploration results prove that the strain contains an arabinoxylanase GH8 gene Contig129, so that the strain can relieve constipation by efficiently degrading arabinoxylan; meanwhile, animal experiments prove that the strain can remarkably increase the small intestine propulsion rate of constipation model mice and increase the six-hour defecation amount of the constipation model mice, and it is further proved that the strain can effectively relieve constipation. Therefore, the strain has great application prospects in preparation of drugs for preventing and / or treating constipation.

Description

Technical Field

[0001] The invention relates to a strain of Bifidobacterium adolescentis capable of relieving constipation and application thereof, belonging to the technical field of biomedicine. Background Art

[0002] Constipation is a functional disease characterized by persistent difficulty in defecation, decreased defecation frequency, and dry and hard stools. It is one of the common gastrointestinal diseases. At present, there are more than 500 million constipation patients in the world, accounting for about 3-26% of the total population. In recent years, with the changes in people's dietary structure, the influence of psychological and social factors, and the intensification of the aging trend of the population, the incidence of constipation has been increasing year by year.

[0003] As a complex disease caused by multiple factors, constipation can seriously affect the patient's quality of life. In addition, constipation can significantly increase the mortality rate of hypertension, cardiovascular disease and ischemic stroke. At the same time, studies have shown that constipation is closely related to the incidence of colorectal cancer. Therefore, timely and effective treatment of constipation is very important for maintaining the life, health and quality of life of patients with constipation.

[0004] At present, traditional treatments for constipation mainly rely on various laxatives (including gastrointestinal prokinetics such as mosapride and itopride, as well as laxatives such as bisacodyl, lactulose, mannitol, senna, phenolphthalein tablets and aloe capsules). However, long-term use of laxatives can lead to significant side effects (for example, electrolyte disorders and intestinal inflammation, etc., and even colon cancer in severe cases). Surgery can also be used to relieve the symptoms of constipated patients, but most constipated patients have limited acceptance of surgical treatments. Therefore, it is urgent to find a constipation treatment drug that is effective, has few side effects, and can be taken for a long time. Summary of the invention

[0005] To solve the above problems, the present invention provides a strain of Bifidobacterium adolescentis (Bifidobacterium adolescentis) XA-1434, which is deposited in the General Microbiological Center of China Microbiological Culture Collection Administration, with a deposit number of CGMCC No.31382 and a deposit date of July 22, 2024.

[0006] The Bifidobacterium adolescentis XA-1434 was derived from fecal samples of healthy adults in Shenzhen. The strain was sequenced and analyzed, and its 16S rDNA sequence was shown in SEQ ID NO: 1. The sequence obtained was compared with the nucleic acid sequence using the Blastn program of NCBI, and the results showed that the strain was Bifidobacterium adolescentis.

[0007] The bifidobacterium adolescentis XA-1434 contains a gene cluster capable of degrading arabinoxylan and converting it into xylose, and its nucleotide sequence carries a gene cluster consisting of three GH43 genes connected in series.

[0008] The present invention also provides the use of the above-mentioned Bifidobacterium adolescentis XA-1434 in the preparation of a drug, wherein the drug has any of the following functions:

[0009] (a) prevention and / or treatment of constipation;

[0010] (b) Improve intestinal barrier function.

[0011] In one embodiment of the present invention, the prevention and / or treatment of constipation comprises degrading arabinoxylan, increasing the small intestinal propulsion rate of constipated patients, increasing the water content of feces of constipated patients and / or increasing the stool volume of constipated patients.

[0012] In one embodiment of the present invention, the medicine contains live bacteria, inactivated bacteria and / or metabolites of the above-mentioned Bifidobacterium adolescentis.

[0013] In one embodiment of the present invention, the drug further contains a pharmaceutically acceptable excipient.

[0014] In one embodiment of the present invention, the pharmaceutically acceptable excipient includes a drug carrier.

[0015] In one embodiment of the present invention, the drug carrier comprises microcapsules, microspheres, nanoparticles and / or liposomes.

[0016] In one embodiment of the present invention, the pharmaceutically acceptable excipients also include solvents, propellants, solubilizers, cosolvents, emulsifiers, colorants, adhesives, disintegrants, fillers, lubricants, wetting agents, osmotic pressure regulators, stabilizers, glidants, flavoring agents, preservatives, suspending agents, coating materials, fragrances, anti-adhesives, integrities, penetration enhancers, pH regulators, buffers, plasticizers, surfactants, foaming agents, defoamers, thickeners, inclusion agents, humectants, absorbents, diluents, flocculants and deflocculating agents, filter aids and / or release retardants.

[0017] In one embodiment of the present invention, the dosage form of the drug is powder, granules, capsules, tablets, pills or oral liquid.

[0018] In one embodiment of the present invention, the content of live bacteria of the above-mentioned Bifidobacterium adolescentis in the drug is not less than 1×10 6 CFU / mL or 1×10 6 CFU / g.

[0019] The present invention also provides a medicine for preventing and / or treating constipation, wherein the medicine contains the above-mentioned Bifidobacterium adolescentis.

[0020] In one embodiment of the present invention, the constipation treatment comprises degrading arabinoxylan, increasing the small intestinal propulsion rate of constipated patients, increasing the water content of feces of constipated patients and / or increasing the stool volume of constipated patients.

[0021] In one embodiment of the present invention, the medicine contains live bacteria, inactivated bacteria and / or metabolites of the above-mentioned Bifidobacterium adolescentis.

[0022] In one embodiment of the present invention, the drug further contains a pharmaceutically acceptable excipient.

[0023] In one embodiment of the present invention, the pharmaceutically acceptable excipient includes a drug carrier.

[0024] In one embodiment of the present invention, the drug carrier comprises microcapsules, microspheres, nanoparticles and / or liposomes.

[0025] In one embodiment of the present invention, the pharmaceutically acceptable excipients also include solvents, propellants, solubilizers, cosolvents, emulsifiers, colorants, adhesives, disintegrants, fillers, lubricants, wetting agents, osmotic pressure regulators, stabilizers, glidants, flavoring agents, preservatives, suspending agents, coating materials, fragrances, anti-adhesives, integrities, penetration enhancers, pH regulators, buffers, plasticizers, surfactants, foaming agents, defoamers, thickeners, inclusion agents, humectants, absorbents, diluents, flocculants and deflocculating agents, filter aids and / or release retardants.

[0026] In one embodiment of the present invention, the dosage form of the drug is powder, granules, capsules, tablets, pills or oral liquid.

[0027] In one embodiment of the present invention, the content of live bacteria of the above-mentioned Bifidobacterium adolescentis in the drug is not less than 1×10 6 CFU / mL or 1×10 6 CFU / g.

[0028] The present invention also provides a medicine for improving intestinal barrier function, which contains the above-mentioned Bifidobacterium adolescentis.

[0029] In one embodiment of the present invention, the medicine contains live bacteria, inactivated bacteria and / or metabolites of the above-mentioned Bifidobacterium adolescentis.

[0030] In one embodiment of the present invention, the drug further contains a pharmaceutically acceptable excipient.

[0031] In one embodiment of the present invention, the pharmaceutically acceptable excipient includes a drug carrier.

[0032] In one embodiment of the present invention, the drug carrier comprises microcapsules, microspheres, nanoparticles and / or liposomes.

[0033] In one embodiment of the present invention, the pharmaceutically acceptable excipients also include solvents, propellants, solubilizers, cosolvents, emulsifiers, colorants, adhesives, disintegrants, fillers, lubricants, wetting agents, osmotic pressure regulators, stabilizers, glidants, flavoring agents, preservatives, suspending agents, coating materials, fragrances, anti-adhesives, integrities, penetration enhancers, pH regulators, buffers, plasticizers, surfactants, foaming agents, defoamers, thickeners, inclusion agents, humectants, absorbents, diluents, flocculants and deflocculating agents, filter aids and / or release retardants.

[0034] In one embodiment of the present invention, the dosage form of the drug is powder, granules, capsules, tablets, pills or oral liquid.

[0035] In one embodiment of the present invention, the content of live bacteria of the above-mentioned Bifidobacterium adolescentis in the drug is not less than 1×10 6 CFU / mL or 1×10 6 CFU / g.

[0036] The present invention also provides the use of the Bifidobacterium adolescentis XA-1434 in the preparation of an arabinoxylan degrading agent.

[0037] The present invention also provides an arabinoxylan degrading agent, which contains the live bacteria, inactivated bacteria and / or metabolites of the above-mentioned Bifidobacterium adolescentis.

[0038] The technical solution of the present invention has the following advantages:

[0039] The present invention provides a strain of Bifidobacterium adolescentis XA-1434, the deposit number of which is CGMCC No.31382; arabinoxylan is one of the main components of dietary fiber, and the degradation of arabinoxylan helps to improve constipation symptoms, and arabinoxylan utilization experiments have shown that the Bifidobacterium adolescentis XA-1434 can efficiently degrade arabinoxylan and use the arabinoxylan degradation product as the sole carbon source for growth, and genome sequencing and arabinoxylan degradation gene exploration results have shown that the Bifidobacterium adolescentis XA-1434 contains arabinoxylanase GH8 gene Contig_1_29, which can be seen , this Bifidobacterium adolescentis XA-1434 can relieve constipation by efficiently degrading arabinoxylan; at the same time, animal experiments have shown that this Bifidobacterium adolescentis XA-1434 can significantly increase the small intestinal propulsion rate of constipation model mice, and increase the 6-hour stool volume of constipation model mice (the number of stool particles and stool weight are significantly increased), which further confirms that this Bifidobacterium adolescentis XA-1434 can effectively relieve constipation; in addition, Bifidobacterium adolescentis belongs to intestinal probiotics and is included in the "List of Bacteria that can be used in Food", and long-term use is safe and harmless. Therefore, this Bifidobacterium adolescentis XA-1434 has a significant effect on treating constipation, and has the advantages of small side effects and long-term use, and has great application prospects in the preparation of drugs for preventing and / or treating constipation.

[0040] In addition, experiments have shown that this Bifidobacterium adolescentis XA-1434 can effectively repair the intestinal barrier. It can be seen that this Bifidobacterium adolescentis XA-1434 also has the effect of improving the intestinal barrier function and has great application prospects in the preparation of drugs for improving the intestinal barrier.

[0041] Biomaterial Deposit

[0042] A strain of Bifidobacterium adolescentis XA-1434, taxonomically named Bifidobacterium adolescentis, was deposited in the General Microbiology Center of China Microorganism Culture Collection Administration on July 22, 2024, with the deposit number CGMCC No.31382, and the deposit address is No. 3, Yard 1, Beichen West Road, Chaoyang District, Beijing. BRIEF DESCRIPTION OF THE DRAWINGS

[0043] Figure 1 : Growth results of arabinoxylan utilization experiment of Bifidobacterium adolescentis XA-1434.

[0044] Figure 2 : Growth results of arabinoxylan utilization experiment of Bifidobacterium dentate XA-2067.

[0045] Figure 3 :Growth results of arabinoxylan utilization experiment of Bifidobacterium longum standard strain ATCC 15707.

[0046] Figure 4 : Growth results of arabinoxylan utilization experiment of Bifidobacterium pseudocatenulatum standard strain ATCC 27919.

[0047] Figure 5 : Phylogenetic analysis of Bifidobacterium adolescentis XA-1434 and the reference genome.

[0048] Figure 6 : Effects of Bifidobacterium adolescentis XA-1434 on small intestinal propulsion rate in constipation model mice (the marker line is the average value of the corresponding index in the model group; *, p<0.05; **, p<0.01; ***, p<0.001; ****, p<0.0001).

[0049] Figure 7 : Effects of Bifidobacterium adolescentis XA-1434 on the number of stool particles in 6-hour constipation model mice (the marker line is the average value of the corresponding index in the model group; *, p<0.05; **, p<0.01; ***, p<0.001; ****, p<0.0001).

[0050] Figure 8 : Effects of Bifidobacterium adolescentis XA-1434 on 6-hour stool weight in constipation model mice (the marker line is the average value of the corresponding index in the model group; *, p<0.05; **, p<0.01; ***, p<0.001; ****, p<0.0001).

[0051] Fig. 9 :Transmembrane resistance values ​​of cell monolayers in different groups of monolayer epithelial inflammatory injury models. DETAILED DESCRIPTION

[0052] The following examples are provided for a better understanding of the present invention, but are not intended to limit the best mode of implementation, nor to limit the content and protection scope of the present invention. Any product identical or similar to the present invention obtained by anyone under the inspiration of the present invention or by combining the features of the present invention with other prior arts shall fall within the protection scope of the present invention.

[0053] If no specific experimental steps or conditions are specified in the following examples, the conventional experimental steps or conditions described in the literature in the field can be used. If no manufacturer is specified for the reagents or instruments used, they are all conventional reagent products that can be purchased commercially.

[0054] The method for detecting the number of viable bacteria involved in the following embodiments is: using the national standard "GB 4789.35-2016 National Food Safety Standard Food Microbiology Detection Lactic Acid Bacteria Detection".

[0055] Experimental Example 1: Acquisition and Identification of Bifidobacterium adolescentis XA-1434

[0056] The specific steps are as follows:

[0057] 1. Strain Isolation

[0058] A large number of healthy donors were recruited in Shenzhen to obtain fresh stool samples for the isolation of probiotic strains. The isolation operation process of donor sample WL29, from which strain XA-1434 was isolated, is as follows:

[0059] In the anaerobic operating table, 1 g of fecal sample from the donor was uniformly suspended in 10 mL of PBS buffer (pH 7.4 ± 0.1, 1×, purchased from Corning cellgro, catalog number 21-040-CV) to obtain 10 -1 Dilution: Use PBS buffer to dilute 10 -1 The dilution was serially diluted to 10 -6 , we get 10 -2 , 10 -3 , 10 -4 , 10 -5 , 10 -6 Dilution. Pipette 100mL of 10 -6 The dilution was placed on an MRS agar plate (purchased from Solebao) and evenly spread with a disposable sterile coating stick, and cultured anaerobically at 37°C for 3 days. After 3 days of culture, a single clone with a surface colony diameter of 1.0 to 1.5 mm, slightly raised, round, mucous surface, and milky white color was selected. A single clone was picked with a sterile loop and streaked on an MRS agar plate (purchased from Solebao), and cultured anaerobically at 37°C for 3 days to obtain a purified single colony. The strain corresponding to the purified single colony was recorded as bacteria XA-1434.

[0060] 2. Strain sequencing and identification

[0061] In an anaerobic operating table, a single clone was picked from the purified single colony of the bacteria XA-1434 itself by splitting and propagating with a sterile inoculation loop and put into 5 mL of lactic acid bacteria liquid culture medium (formula see Table 3), and cultured anaerobically at 37°C for 24 hours to obtain a bacterial solution. 1 mL of the bacterial solution was taken, and the strain DNA was extracted from the bacterial solution using a DNA extraction kit (purchased from QIAGEN), and the strain gene fragment was PCR amplified using universal primers 27F (SEQ ID NO: 2, AGAGTTTGATCMTGGCTCA, where M is a degenerate base, M = A or C) / 1492R (SEQ ID NO: 3, TGTACGGYTACCTTGTTACGACTT, where Y is a degenerate base, Y = C or T), and the amplified product was gel-cleaved and purified, and the recovered fragment was extracted using a SanPrep column DNA gel recovery kit (REF: 518131-0100). The recovered fragments were sequenced, and the 16S ribosomal RNA gene sequence (16SrDNA) of the bacteria XA-1434 obtained by sequencing was shown in SEQ ID NO: 1. The 16S rDNA of the strain XA-1434 was compared with the NCBI Blastn program for nucleic acid sequence alignment, and the results showed that the strain was Bifidobacterium adolescentis, and was named Bifidobacterium adolescentis XA-1434. The 50 μL PCR amplification reaction system used for PCR amplification is shown in Table 1, and the reaction conditions used for PCR amplification are shown in Table 2.

[0062] Table 1 PCR amplification reaction system

[0063] Volume / μl Reagents 25 TaqPCRMix premix (2X, containing blue dye) 2 27F 2 1492R 3 XA-1434 DNA (concentration: 38.1 ng / μL) 18 <![CDATA[Deionized water (ddH2O)]]>

[0064] Table 2 PCR amplification reaction conditions

[0065]

[0066] 3. Strain preservation

[0067] The bacterial solution of Bifidobacterium adolescentis XA-1434 was placed in a 20% (v / v) glycerol aqueous solution (the volume ratio of the glycerol aqueous solution to the bacterial solution was 1:1) and preserved in the General Microbiological Center of the China Microbiological Culture Collection Administration, with the preservation number CGMCC No. 31382. Subsequently, Bifidobacterium adolescentis XA-1434 was further identified by genome sequencing and analysis, and it was confirmed to be Bifidobacterium adolescentis.

[0068] Experimental Example 2: Verification of the arabinoxylan degradation performance of Bifidobacterium adolescentis XA-1434

[0069] Arabinoxylan is a polypentose, which is a polysaccharide chain formed by xylose connected by β-1,4 glycosidic bonds. Arabinoxylan is the main hemicellulose component in plant cells, accounting for 35% of the dry weight of plant cells, and is one of the main components of dietary fiber. Arabinoxylan cannot be directly digested by the human body, but can be degraded by human colon bacteria. Its degradation products include oligoxylose and arabinose. Both oligoxylose and arabinose can promote the proliferation of beneficial intestinal bacteria (including bifidobacterium and lactic acid bacteria), increase intestinal motility, improve intestinal peristalsis and maintain intestinal water retention, all of which help relieve constipation. On this basis, clinical studies have shown that taking arabinoxylan can significantly improve the constipation symptoms of patients with constipation (see literature: et al., 2012; Kamiya et al., 2014). Clinical studies have also shown that the intestinal microbiome and metabolome of patients taking arabinoxylan have undergone significant changes. Specifically, the abundance of Bifidobacterium bacteria that can degrade arabinoxylan in the feces has increased significantly, and the content of xylose, a degradation product of arabinoxylan in the feces has also increased significantly (see literature: Chen et al., 2019; Mendis et al., 2016; Nie et al., 2022). Based on the above, it can be inferred that Bifidobacterium bacteria that can degrade arabinoxylan may help improve constipation symptoms.

[0070] Based on the above inference, this experimental example uses Bifidobacterium dentate XA-2067 (recorded in the patent application text with publication number CN116036135A), Bifidobacterium longum standard strain ATCC15707 (purchased from ATCC) and Bifidobacterium pseudoselocarbazine standard strain ATCC27919 (purchased from ATCC) as controls, and uses arabinoxylan as the sole carbon source to culture Bifidobacterium adolescentis XA-1434, Bifidobacterium dentate XA-2067, Bifidobacterium longum standard strain ATCC15707 and Bifidobacterium pseudoselocarbazine standard strain ATCC 27919 to explore whether strains XA-1434, strains XA-2067, strains ATCC15707 and strains ATCC27919 can degrade arabinoxylan. The experimental process is as follows:

[0071] The purified single colonies of XA-1434, Bifidobacterium dentate XA-2067, Bifidobacterium longum standard strain ATCC15707 and Bifidobacterium pseudomicrocystis standard strain ATCC27919 obtained in Example 1 were inoculated in 10 mL of lactic acid bacteria culture medium (see Table 3 for culture medium formula), and cultured anaerobically at 37 ° C for 8 h to obtain the resuscitation and activation bacterial liquid of strains XA-1434, strain XA-2067, strain ATCC15707 and strain ATCC27919. The resuscitation and activation bacterial liquids were all inoculated in 10 mL of fresh lactic acid bacteria culture medium, and cultured anaerobically at 37 ° C for 16 h until the bacterial liquid OD 600 >1, and the seed liquid of strain XA-1434, strain XA-2067, strain ATCC15707 and strain ATCC27919 was obtained. The seed liquid was inoculated into lactic acid bacteria medium (MRS medium), basal medium (Basal medium, MRS-Glu) and arabinoxylan medium (Xylan medium, MRS-Glu + 2% Arb) at an inoculum of 5% (v / v) (each medium was set up with 3 parallels), and anaerobically cultured at 37°C for 40h. During the culture period, the culture liquid was sampled every 1h for OD 600 According to OD 600 The growth curves of strain XA-1434, strain XA-2067, strain ATCC15707 and strain ATCC27919 were drawn to evaluate whether strain XA-1434, strain XA-2067, strain ATCC15707 and strain ATCC27919 can degrade arabinoxylan for growth. The experimental results are shown in Figure 1 to Figure 4 shown.

[0072] Figure 1 to Figure 4 The experimental results showed that XA-1434 grew well in the arabinoxylan medium with arabinoxylan as the sole carbon source, compared with the other three groups with no obvious growth (OD 600 XA-1434 reached a plateau 25 hours after inoculation, OD 600 Compared with the growth curve of XA-1434 in lactic acid bacteria culture medium, although the hysteresis period is longer in arabinoxylan culture medium, the OD reached in the plateau period is 600 It can be seen that among all the strains in this experiment, only strain XA-1434 can decompose arabinoxylan and use its decomposition products as the sole carbon source for good growth.

[0073] Table 3 Culture medium formula

[0074]

[0075] Experimental Example 3: Genome sequencing of Bifidobacterium adolescentis XA-1434 and exploration of arabinoxylan degradation genes

[0076] Arabinoxylan is a complex polysaccharide, and its main sugar chain skeleton is formed by xylose connected by β-1,4 glycosidic bonds. Therefore, the degradation of arabinoxylan by organisms requires arabinoxylanase (Xylanase) that can hydrolyze the β-1,4 glycosidic bonds of xylose. At present, according to the records of the carbohydrate active enzyme database (CAZymes), arabinoxylanase mainly exists in the 8th class (GH8) and the 10th class (GH10) of glycoside hydrolases. Therefore, in this embodiment, the genome of Bifidobacterium adolescentis XA-1434 is sequenced to obtain the carbohydrate active enzyme gene of the strain predicted by the genome sequence, so as to determine whether the strain XA-1434 has arabinoxylanase, and then verify the genetic basis of the strain's ability to degrade arabinoxylan. The experimental process is as follows:

[0077] 1. Sequencing and assembly of bacterial genomes

[0078] The DNA of Bifidobacterium adolescentis XA-1434 was extracted by SDS (sodium dodecyl sulfate polyacrylamide) method, the quality of the extracted DNA was tested by agarose gel electrophoresis, and the quality of the extracted DNA was tested by fluorescence spectrophotometer. 2.0 was used to quantify the concentration of extracted DNA. Bacterial genome sequencing was performed at Novogene Co., Ltd. A 350 bp small fragment library was constructed using strain DNA, and then the second-generation sequencing of the bacterial genome was performed using the Illumina platform. The bacterial genome was assembled using SPAdes software to obtain the draft genome of strain XA-1434.

[0079] 2. Species identification based on genome analysis

[0080] The reference genome of Bifidobacterium was downloaded, and the orthofinder software was used to perform comparative genome analysis and core genome construction. Then, the core genome of Bifidobacterium strains was phylogenetically analyzed using the iqtree software to determine the species information of strain XA-1434.

[0081] 3. Identification of arabinoxylanase gene

[0082] Prodigal software was used to predict the coding genes of the assembled draft genome of Bifidobacterium adolescentis XA-1434. The dbcan software was used to predict the carbohydrate-active enzyme genes of the genome, and to infer which carbohydrate-active enzymes related to arabinoxylanase existed in the genome of strain XA-1434, and to predict the arabinoxylan degradation potential of XA-1434.

[0083] The genome assembly results of strain XA-1434 showed that the draft genome of XA-1434 consisted of 21 contigs. The total length of the genome was 2,128,665 bp, and the GC content was 59.19%. The genome encodes 1,687 genes, the total length of the gene coding region bases is 1.87 Mbp, the average number of gene bases is 1,111 bp, and the total length of the coding region accounts for 88.05% of the whole genome. The phylogenetic analysis results of strain XA-1434 based on the genome are as follows Figure 5 As shown, the genome of strain XA-1434 clustered with that of the standard strain ATCC15703 of the species Bifidobacterium adolescentis, and therefore, XA-1434 was further identified as Bifidobacterium adolescentis.

[0084] The results of searching for carbohydrate active enzyme genes in the genome are shown in Table 4. The results show that the genome of Bifidobacterium adolescentis XA-1434 has the characteristic amino acid arrangement of arabinoxylanase GH8, and the similarity E value with the GH8 enzyme domain sequence in the database is 9.5e-76, which is much smaller than the E value of 1e-10 recommended by dbcan software. Therefore, the genetic basis of the arabinoxylan degradation activity of strain XA-1434 is that it contains the arabinoxylanase GH8 gene Contig_1_29.

[0085] Table 4 GH8 gene alignment results of Bifidobacterium adolescentis XA-1434 genome

[0086]

[0087] Experimental Example 4: Effects of Bifidobacterium adolescentis XA-1434 on constipation model mice

[0088] This experiment provides an experiment on the effect of Bifidobacterium adolescentis XA-1434 on constipation model mice. This experiment has been reviewed by the Animal Care and Use Committee of Shenzhen Jingtuo Technology Co., Ltd. The experimental process is as follows:

[0089] 1. Preparation of bacterial solution

[0090] The purified single colony of XA-1434 obtained in Example 1 was picked and inoculated into 10 mL of lactic acid bacteria culture medium (the culture medium formula is shown in Table 3), and anaerobically cultured at 37° C. for 24 h to obtain a bacterial solution of Bifidobacterium adolescentis XA-1434.

[0091] 2. Model establishment and drug administration

[0092] Loperamide

[0093] Bisacodyl is a kind of irritant laxative, mainly acts on large intestine, and its mechanism of action is mainly by directly contacting intestinal mucosa, stimulating sensory nerve endings, causing intestinal reflex peristalsis to increase, thereby causing defecation. In addition, bisacodyl can also stimulate local axon reflex and segmental reflex, produce extensive colon peristalsis, and can suppress the absorption of sodium, chlorine and moisture in the colon, make the intestinal content volume increase, further promote defecation. Clinically used for acute and chronic constipation, suppository rectal medication can be used for cleaning intestines before colonoscopy, intestinal X-ray examination or abdominal surgery. However, because bisacodyl has direct stimulation to the digestive tract, easily cause abdominal discomfort, for example, abdominal pain and cramps etc., therefore, can not be taken for a long time, otherwise can cause a series of untoward reactions such as intestinal spasm, diarrhea and electrolyte disturbance. This experiment uses bisacodyl as the therapeutic effect of XA-1434 that positive drug verification embodiment 1 obtains to constipation.

[0094] Thirty-two SPF-grade Balb / c mice (male, 8-9 weeks old, purchased from Hunan Slake Jingda Experimental Animal Co., Ltd.) were randomly divided into 4 groups, with 8 mice in each group. The 4 groups were: control group (Control), model group (Model), probiotic group (XA-1434) and positive drug group (Bisacodyl).

[0095] All mice were adaptively housed in separate cages in an SPF animal room for 1 week, with a room temperature of (24±2)℃, alternating day and night every 12 hours, and free access to water. After adaptive feeding, the mice were randomly grouped according to their body weight and began to model the constipation model and administer the drug. The specific operation was as follows: starting from the first day of the experiment, the mice were gavaged every day for 8 consecutive days. All groups except the control group were gavaged with loperamide hydrochloride (solvent was 200μL saline) at a dose of 20mg / kg for modeling. Correspondingly, the control group was gavaged with 200μL saline solution; 1h later, the probiotic group was gavaged with 1.0×10 9 The positive drug group was intragastrically administered with 1.0 mg / kg bisacodyl ( The solvent was 200 μL PBS buffer) for intervention, and the control group and model group were gavaged with 200 μL PBS buffer (the experimental grouping and drug administration design are shown in Table 5).

[0096] On the 7th day after intragastric administration, the weight and number of feces discharged by the mice within 6 hours were measured. The experimental results are shown in Figure 7-Figure 8 On the 8th day of intragastric administration, the experiment was terminated and the mice were subjected to a small intestinal propulsion rate test. The experimental results are shown in Figure 6 The data were compared between groups using one-way ANOVA, and multiple significant differences between the two groups were corrected using Tukey's method. If p < 0.05 between the two groups, it was considered to be statistically significant.

[0097] Among them, the method for determining the stool weight and number of stool particles in 6 hours is as follows:

[0098] After all gavage operations were completed on the morning of the 7th day of gavage, the mice in each group were placed individually in an IVC cage box covered with clean filter paper to collect the feces of each mouse. The collected feces were placed in a sterile 1.5 mL EP tube, which was then placed in an ice box for storage. The feces weight and number of feces pellets of each mouse were recorded during the collection period.

[0099] The method for measuring small intestinal propulsion rate is as follows:

[0100] On the 8th day of intragastric administration of mice, mice in each group were fasted but not watered for 16 hours on the evening of the 7th day. Then, the model group, probiotic group and positive drug group were intragastricly administered with loperamide saline solution according to body weight, and the control group was administered with the same volume of normal saline; 30 minutes later, the probiotic group and positive drug group were intragastricly administered with ink containing the corresponding dose of the test sample, and the control group and model group were intragastricly administered with the same amount of ink; 30 minutes after intragastric administration of ink, the mice were killed by cervical dislocation, the abdominal cavity was opened, the mesentery was separated, the intestinal tube from the pylorus at the upper end and the ileocecal part at the lower end was cut, and placed on a tray, the small intestine was gently pulled into a straight line, and the length of the intestinal tube was measured as the "total length of the small intestine", and the length from the pylorus to the front edge of the ink was the "ink propulsion length", and the propulsion rate of the small intestine was calculated according to the formula: small intestine propulsion rate (%) = (ink propulsion length / total length of the small intestine) × 100% (the units of ink propulsion length and total length of the small intestine were both cm).

[0101] Figure 6 to Figure 8The results showed that the probiotic group (XA-1434) mice were significantly higher than the model group mice in terms of small intestinal propulsion rate, stool weight and number of stool particles (p<0.05). Among them, the small intestinal propulsion rate of the probiotic group (XA-1434) mice was about 60%, which was about 1.3 times that of the model group mice. The number of stool particles of the probiotic group (XA-1434) mice was 12, which was 50% higher than that of the model group mice. The stool weight of the probiotic group (XA-1434) mice was about 0.2g, which was about 0.7g higher than that of the model group mice. According to the result judgment criteria of the "Health Food Function Test and Evaluation Method (2023 Edition)", Bifidobacterium adolescentis XA-1434 helps to smooth bowel movements and relieve constipation, and the experimental results are positive. A comprehensive comparison of the various indicators of the probiotic group and the positive drug group showed that the small intestinal propulsion rate of the probiotic group mice was significantly higher than that of the model group mice, but there was no significant difference between the positive drug group mice and the model group mice. In addition, the statistically significant p value (p<0.01) of the probiotic group mice and the model group mice in terms of the two indicators of stool weight and stool particle number was lower than the p value (p<0.05) of the positive drug group mice and the model group mice. It can be seen that the various indicators of the probiotic group mice were better than those of the positive drug group mice. In summary, the effect of Bifidobacterium adolescentis XA-1434 in improving constipation is better than that of the positive drug bisacodyl.

[0102] Table 5 Experimental grouping and drug administration design

[0103]

[0104]

[0105] In Table 5, Vehicle solution: PBS buffer; PO: oral gavage administration; QD: administration every other day.

[0106] Experimental Example 5: Intestinal barrier repair function of Bifidobacterium adolescentis XA-1434

[0107] This experimental example provides the effect of Bifidobacterium adolescentis XA-1434 on the monolayer epithelial inflammatory injury cell model. The experimental process is as follows:

[0108] 1. Construction of a monolayer epithelial inflammatory injury cell model

[0109] The 0.4 μm pore size Transwell chamber was incubated with DMEM-high glucose complete medium for 2 h to obtain the pretreated Transwell chamber. CaCO-2 cells (purchased from ATCC) and HT-29-MTX-E12 cells in the logarithmic growth phase were inoculated into the upper chamber of the pretreated Transwell chamber at a cell number ratio of 9:1, with a total inoculation amount of 2.5×10 6cells, and added 100 μL and 600 μL of complete culture medium (purchased from Thermo Fisher Scientific) to the upper chamber and lower chamber of the Transwell chamber respectively, and cultured in a cell culture incubator at 37°C and 5% (v / v) CO2. Fresh complete culture medium was replaced every other day, and the cell transmembrane resistance at different time points was measured with an EVOM3 cell resistance meter to draw a resistance value curve. After the cells were completely fused, the resistance value was measured three times in a row and stabilized to obtain the CaCO-2 / HT-29-MTX-E12 cell monolayer model, which can be used for subsequent experiments. After the CaCO-2 / HT-29-MTX-E12 cell monolayer model was constructed, the culture medium in the upper and lower chambers was removed, and the monolayer cell epithelium treated with DMEM high glucose medium (purchased from Thermo Fisher) containing 10% (v / v) inactivated FBS was used as a blank control. 100 μL and 600 μL of DMEM high glucose medium containing 100 ng / mL IL-1β, 40 ng / mL TNFα, 100 μg / mL LPS (lipopolysaccharide) and 10% (v / v) inactivated FBS were added to the upper and lower chambers of the Transwell chamber, respectively, and cultured in a cell culture incubator at 37°C and 5% (v / v) CO2 for 24 hours to treat the monolayer cell epithelium, and a monolayer epithelial inflammatory injury cell model was obtained, which can be used for subsequent experiments. The transepithelial electrical resistance (TEER) of the cell monolayer can be used as an evaluation parameter for the density of the CaCO-2 cell monolayer.

[0110] 2. Experiment on the interaction between Bifidobacterium adolescentis XA-1434 and monolayer epithelial validation cell model

[0111] After the monolayer epithelial inflammatory injury cell model was constructed, the culture medium in the upper and lower chambers was removed, and the Transwell chamber was washed once with phosphate buffer. DMEM high-glucose cell culture medium without bispecific antibody and containing 10% (v / v) FBS was added to the lower chamber, and a sodium butyrate solution with a final concentration of 2 μM (the solvent was DMEM high-glucose cell culture medium) was used as the sodium butyrate control, and DMEM high-glucose cell culture medium was used as the solvent control. In the upper chamber, 100 μL of Bifidobacterium adolescentis XA-1434 (XA-1434 group) and Bifidobacterium adolescentis XA-1 (XA-1 group, Bifidobacterium adolescentis XA-1 was screened from fresh fecal samples in the same batch as Bifidobacterium adolescentis XA-1434) were added at an infection multiplicity (MOI) of 10:1. Bifidobacterium bacteria obtained), Bifidobacterium breve XA-1267 (XA-1267 group, Bifidobacterium breve XA-1267 is recorded in the patent application text with publication number CN117431190A), and Bifidobacterium breve XA-2103 (XA-2103 group, Bifidobacterium breve XA-2103 is recorded in the patent application text with publication number CN118185785A), were allowed to stand for 2 hours at 37°C in an anaerobic table; after the interaction, the culture medium in the upper and lower chambers was removed, and the Transwell chamber was washed once with phosphate buffer, and 100 μL and 600 μL of DMEM high-glucose cells without double antibodies were added to the upper and lower chambers of the Transwell chamber, respectively, and cultured in a cell culture incubator at 37°C and 5% (v / v) CO2 for 22 hours. After the culture was completed, the cell monolayer transmembrane electrical resistance (TEER) of each group of monolayer epithelial validation cell models was measured. The method for determining the transmembrane electrical resistance (TEER) of the cell monolayer is as follows: 20 μL FITC-dextran (4KDa, 100 μg / mL) was added to the upper chamber, and the transmembrane electrical resistance (TEER) of the confluent epithelial monolayer was measured after incubation at 37°C and 5% (v / v) CO2 for 0, 3, 6, 12 or 24 hours. The results are shown in Table 1. Fig. 9 shown.

[0112] like Fig. 9As shown, compared with the blank control group (24h TEER was 91.69% of the initial TEER), the cell monolayer transmembrane resistance of the solvent control group gradually decreased (24h TEER dropped to 67.03% of the initial TEER). After XA-1434 treatment, the TEER of the CaCO-2 / HT-29-MTX-E12 cell monolayer model was restored (24h TEER recovered to 89.57% of the initial TEER), and the recovery was much better than that of the XA-1 group (24h TEER recovered to 88.45% of the initial TEER), the XA-1267 group (24h TEER recovered to 83.44% of the initial TEER) and the XA-2103 group (24h TEER recovered to 71.17% of the initial TEER). The above results show that Bifidobacterium adolescentis XA-1434 has the function of intestinal barrier repair.

[0113] Obviously, the above embodiments are merely examples for the purpose of clear explanation, and are not intended to limit the implementation methods. For those skilled in the art, other different forms of changes or modifications can be made based on the above description. It is not necessary and impossible to list all the implementation methods here. The obvious changes or modifications derived therefrom are still within the scope of protection of the invention.

Claims

1. A strain of Bifidobacterium adolescentis, characterized in that The Bifidobacterium adolescentis is deposited in the General Microbiological Center of China Microbiological Culture Collection Administration, with the deposit number being CGMCC No.31382.

2. The use of Bifidobacterium adolescentis according to claim 1 in the preparation of medicines, characterized in that: The drug has any of the following functions: (a) prevention and / or treatment of constipation; (b) Improve intestinal barrier function.

3. The use according to claim 2, characterized in that The prevention and / or treatment of constipation comprises degrading arabinoxylan, improving the small intestinal propulsion rate of patients with constipation, increasing the water content of feces of patients with constipation and / or increasing the stool volume of patients with constipation.

4. The use according to claim 2 or 3, characterized in that The medicine contains live bacteria, inactivated bacteria and / or metabolites of Bifidobacterium adolescentis according to claim 1.

5. The use according to any one of claims 2 to 4, characterized in that: The medicine also contains pharmaceutically acceptable excipients.

6. A drug for preventing and / or treating constipation, characterized in that: The medicine contains the Bifidobacterium adolescentis according to claim 1.

7. The drug according to claim 6, characterized in that The constipation treatment comprises degrading arabinoxylan, improving the small intestinal propulsion rate of constipated patients, increasing the fecal water content of constipated patients and / or increasing the stool volume of constipated patients.

8. A drug for improving intestinal barrier function, characterized in that: The medicine contains the Bifidobacterium adolescentis according to claim 1.

9. Use of the Bifidobacterium adolescentis according to claim 1 in the preparation of an arabinoxylan degrading agent.

10. An arabinoxylan degradation agent, characterized in that: The arabinoxylan degrading agent contains live bacteria, inactivated bacteria and / or metabolites of the Bifidobacterium adolescentis described in claim 1.

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