Bifidobacterium longum subsp. longum strain for regulating intestinal homeostasis to relieve intractable constipation and application thereof

Abstract

The application discloses a Bifidobacterium longum subsp. longum strain for regulating intestinal homeostasis and relieving intractable constipation and an application thereof, and belongs to the field of microorganisms. The strain CCFM1319 provided by the application can shorten the time of the first black stool of intractable constipation mice, significantly enhance the colon peristalsis ability of the intractable constipation mice, increase the expression level of the characteristic marker of enteric glial cells in the colon tissue, repair the intestinal nerve damage caused by intractable constipation, reduce the expression level of inflammatory factors in the colon tissue of the intractable constipation mice, relieve the inflammation of the intestinal immune barrier, reduce the pathological damage of the colon tissue, increase the expression amount of the tight junction protein in the colon tissue, repair the damage of the intestinal mechanical barrier, avoid excessive water absorption, be beneficial to the excretion of feces, regulate the intestinal chemical barrier, improve the intestinal microecological imbalance, make the intestinal flora structure tend to be healthy, and repair the damage of the intestinal biological barrier, so as to play a role in comprehensively relieving intractable constipation.

Description

TECHNICAL FIELD

[0001] The present application relates to a long subspecies of Bifidobacterium longum for regulating intestinal homeostasis to relieve intractable constipation and an application thereof, and belongs to the field of microorganisms. BACKGROUND

[0002] Intractable constipation is a relatively serious functional constipation, and most of them have both colonic slow transit and outlet obstruction. It is difficult to treat clinically and is often seen in elderly patients with constipation. There are many reasons for intractable constipation, such as mental and psychological factors, age factors, etc., and one of the most important reasons is the abuse of laxatives. Constipation is different from other diseases, and most people think it is a symptom, so when constipation occurs, they will choose a means to relieve themselves, and stimulant laxatives are favored due to their quick effect. In addition, with the improvement of people's living standards, especially the pursuit of beauty by some young women, some weight loss preparations or detoxification and skin care preparations containing anthraquinone stimulant laxative ingredients are taken, which will eventually develop into intractable constipation.

[0003] At present, for the treatment of intractable constipation, gastrointestinal motility promoting drugs are often used in China, such as 5-hydroxytryptamine receptor 4 (5-HT4) receptor agonists mosapride, prucalopride, and secretagogue lubiprostone, combined with some methods to assist defecation. For some severe patients, such as patients with colonic melanosis, only colonic resection can be performed to relieve symptoms, which causes great psychological impact and economic pressure on patients and their families. Therefore, it is very important to find a safe and effective relief method.

[0004] Bifidobacterium is a common probiotic that is often added to food and is considered safe due to its long history of consumption. Numerous studies have shown that Bifidobacterium has a variety of probiotic functions, one of which is to moisten the intestines and relieve constipation. In previous studies, it was also found that the content of Bifidobacterium and Lactobacillus in patients with intractable constipation was significantly reduced. After restoring the intestinal microecology through fecal flora transplantation, the clinical symptoms of patients with intractable constipation can be significantly improved. Therefore, Bifidobacterium can be considered to relieve the symptoms of intractable constipation.

[0005] At present, scholars at home and abroad have studied the physiological functions related to Bifidobacterium, and the application of its constipation relief effect should be further explored. Whether it is made into bacterial powder or added to fermented milk for consumption, it has a very large application prospect, can prevent the occurrence of constipation and even relieve the symptoms of constipation. Through the study of Bifidobacterium in relieving intractable constipation, it will have a great impact on food science, microbiology, preventive medicine and other aspects, therefore, it is necessary to study Bifidobacterium in relieving intractable constipation. SUMMARY

[0006] The application provides a Bifidobacterium longum subsp.longum for regulating intestinal homeostasis and relieving intractable constipation, and application of the strain.

[0007] In view of the defects of the prior art, such as slow growth of Bifidobacterium with the effect of relieving intractable constipation, difficulty in activation, and single function, the application provides a Bifidobacterium longum subsp.longum, which can effectively improve intestinal peristalsis, shorten the time of the first black stool, increase the water content and defecation frequency of feces, increase the expression level of S100beta, a characteristic marker of enteric glial cells in colon tissue, repair intestinal nerve damage, reduce the expression level of inflammatory factors in colon tissue, relieve intestinal immune barrier inflammation, reduce colon tissue pathological damage, promote the expression of Occludin, a tight junction protein in colon tissue, repair intestinal mechanical barrier damage, reduce the expression level of AQP4, a water channel protein in colon tissue, regulate intestinal stem cells through the WNT / β-catenin signaling pathway, regulate intestinal chemical barrier, improve intestinal microecological imbalance, repair intestinal biological barrier, regulate intestinal homeostasis, and provide corresponding probiotic preparations or functional foods, thereby effectively relieving intractable constipation.

[0008] The application provides a Bifidobacterium longum subsp.longum CCFM1319, which is preserved in the Guangdong Microbial Culture Collection Center on August 4, 2023, is located at No. 59 Building, 5th Floor, Guangzhou Xianlie Middle Road 100 Courtyard, and has a preservation number of GDMCC No: 63715.

[0009] The Bifidobacterium longum subsp. longum is from the stool of a 45-year-old middle-aged male in Beijing. The strain is sequenced and analyzed. The nucleic acid sequence obtained by sequencing is subjected to nucleic acid sequence alignment in NCBI Standard Nucleotide BLAST. The result shows that the similarity with the nucleic acid sequence of the Bifidobacterium is 100%. The result shows that the strain is Bifidobacterium longum subsp. longum, which is named Bifidobacterium longum subsp. longum CCFM1319.

[0010] The Bifidobacterium longum subsp. longum CCFM1319 has the following biological characteristics:

[0011] (1) Bacterial characteristics: Gram-positive, no spore, bacterial body about 0.6-1.2 μm x 1.5-7.6 μm, multiple branches, Y and V type.

[0012] (2) Colony characteristics: about 0.3-2.4 mm in diameter, round and regular edge, convex or lenticular, slightly white, opaque, surface smooth.

[0013] (3) Growth characteristics: the optimal growth temperature of the strain is 36-38℃, and it grows well at 32-38℃, the minimum growth temperature is 15℃, and it can also grow at 45℃; the optimal initial pH is 6-7, and it grows less at pH 5.5 or below; after 20h of culture, it enters the early stationary phase, and the pH of the final culture medium is 4.0-4.8.

[0014] (4) It has good tolerance to simulated gastrointestinal fluid.

[0015] (5) It has adhesion, and can better adhere to colon cancer cells HT-29.

[0016] (6) It can significantly shorten the first black stool time of refractory constipation mice, significantly increase the fecal water content and 5h fecal particle number, promote intestinal peristalsis, increase the expression level of S100β, a characteristic marker of enteric nervous glial cells in colon tissue, repair intestinal nerve damage, reduce the expression levels of inflammatory factors IL-1β and IL-6 in colon tissue, relieve intestinal immune barrier inflammation, reduce colon tissue pathological damage, promote the expression of tight junction protein Occludin in colon tissue, repair intestinal mechanical barrier damage, reduce the expression level of water channel protein AQP4 in colon tissue, regulate intestinal stem cells through WNT / β-catenin signaling pathway, regulate intestinal chemical barrier, improve intestinal microecological imbalance, repair intestinal biological barrier, regulate intestinal homeostasis, and relieve refractory constipation.

[0017] The present application also provides a microbial preparation, wherein the microbial preparation contains the Bifidobacterium longum subsp. longum CCFM1319 or a fermentation broth thereof, or a lysate of the Bifidobacterium longum subsp. longum CCFM1319, or a freeze-dried powder of the Bifidobacterium longum subsp. longum CCFM1319.

[0018] The present application provides a composition, wherein the active ingredient of the composition comprises the Bifidobacterium longum subsp. longum CCFM1319 or a fermentation broth thereof, or a freeze-dried powder thereof, or a lysate thereof.

[0019] In an embodiment of the present application, the composition is a food composition, a health food composition, a pharmaceutical composition, a special medical purpose food composition, a cosmetic composition, a medical device composition, or a feed composition.

[0020] In an embodiment of the present application, the number of cells of the Bifidobacterium longum subsp. longum CCFM1319 is ≥ 1 x 10 8 CFU / g or 1 x 10 8 CFU / mL.

[0021] In an embodiment of the present application, the composition is a fermented food, a dietary supplement, or a medicine.

[0022] In an embodiment of the present application, the fermented food is a fermented dairy product, a fermented soy product, and a fermented fruit and vegetable product, which are fermented using the Bifidobacterium longum subsp. longum CCFM1319.

[0023] In an embodiment of the present application, the dietary supplement comprises a powder, a capsule, a gummy, or a liquid formulation of the Bifidobacterium longum subsp. longum CCFM1319.

[0024] The present application also provides use of the Bifidobacterium longum subsp. longum CCFM1319 in the preparation of a medicine for alleviating intractable constipation, slow transit constipation, or gastrointestinal motility disorder.

[0025] In an embodiment of the present application, the alleviation of intractable constipation, slow transit constipation comprises at least one function of:

[0026] (a) shortening the first black stool time of an intractable constipation individual, increasing the fecal water content, and increasing the defecation frequency;

[0027] (b) increasing the expression level of S100P, a characteristic marker of enteric glial cells in the colon tissue of the individual with intractable constipation, repairing the damage to the enteric nerve;

[0028] (c) reducing the expression level of IL-1P, IL-6, an inflammatory factor in the colon tissue of the individual with intractable constipation, relieving the inflammation of the intestinal immune barrier;

[0029] (d) reducing the pathological damage to the colon tissue of the individual with intractable constipation, promoting the expression of Occludin, a tight junction protein in the colon tissue, and repairing the damage to the mechanical barrier of the intestinal tract;

[0030] (e) reducing the expression level of AQP4, a water channel protein in the colon tissue of the individual with intractable constipation, regulating the intestinal stem cells through the WNT / β-catenin signaling pathway, and regulating the chemical barrier of the intestinal tract;

[0031] (f) improving the intestinal microecological imbalance of the individual with intractable constipation, and repairing the damage to the biological barrier of the intestinal tract.

[0032] In an embodiment of the present application, the dosage form of the drug is granules, capsules, tablets, pills or oral liquid.

[0033] The present application also provides a microbial preparation containing the above-mentioned Bifidobacterium longum subsp. longum CCFM1319.

[0034] In an embodiment of the present application, the number of cells of the Bifidobacterium longum subsp. longum CCFM1319 is ≥ 1 x 10 8 CFU / g or 1 x 10 8 CFU / mL.

[0035] The present application provides a product containing the above-mentioned Bifidobacterium longum subsp. longum CCFM1319 or its fermentation broth, or its freeze-dried powder, or its lysate, and the product includes food, medicine, health product, fermented food, dietary supplement.

[0036] The present application also provides a food containing the above-mentioned Bifidobacterium longum subsp. longum CCFM1319 or the above-mentioned microbial preparation.

[0037] In one embodiment of the present application, the food is a fermented food, and the fermented food is produced by fermentation using Bifidobacterium longum subsp. longum CCFM1319, and the fermented food includes a solid food, a liquid food, or a semi-solid food.

[0038] In one embodiment of the present application, the Bifidobacterium longum subsp. longum is added to the food in an amount of at least 10 8 CFU / mL or 10 8 CFU / g.

[0039] In one embodiment of the present application, the fermented food includes a dairy product, a soy product, or a fruit / vegetable product.

[0040] In one embodiment of the present application, the dairy product is a fermented dairy product, including fermented milk, a fermented milk beverage, butter, cheese, or milk powder; the soy product includes soy milk, a soy milk beverage, or soy milk powder; and the fruit / vegetable product includes a fermented fruit / vegetable beverage or food fermented using Chinese cabbage, white radish, cucumber, sugar beet, yellow peach, or waxberry as a raw material.

[0041] The present application also provides a pharmaceutical product containing the above-mentioned Bifidobacterium longum subsp. longum CCFM1319 or the above-mentioned microbial agent.

[0042] In one embodiment of the present application, the pharmaceutical product contains Bifidobacterium longum subsp. longum CCFM1319 and a pharmaceutically acceptable carrier.

[0043] In one embodiment of the present application, the carrier includes one or more of a filler, a binder, a wetting agent, a disintegrating agent, a lubricant, or a flavoring agent commonly used in medicine.

[0044] In one embodiment of the present application, the pharmaceutical product is in the form of a granule, a capsule, a tablet, a pill, or an oral solution.

[0045] In one embodiment of the present application, the Bifidobacterium longum subsp. longum CCFM1319 is added to the pharmaceutical product in an amount of at least 10 8 CFU / mL or 10 8 CFU / g.

[0046] The application also provides a bacterial agent containing the Bifidobacterium longum subsp.longum CCFM1319.

[0047] In an embodiment of the application, the number of viable bacteria of the Bifidobacterium longum subsp.longum CCFM1319 in the bacterial agent is ≥10 8 cfu / g or 10 8 cfu / mL.

[0048] In an embodiment of the application, the bacterial agent is a powder obtained by drying a bacterial solution containing the Bifidobacterium longum subsp.longum CCFM1319, and the number of viable bacteria is ≥10 8 cfu / g or 10 8 cfu / mL.

[0049] In an embodiment of the application, the drying refers to vacuum freeze-drying.

[0050] Advantages

[0051] 1. The Bifidobacterium longum subsp.longum GDMCC No: 63715 has good activity, specifically:

[0052] (1) can shorten the first black stool time of intractable constipation mice by 80.5% (p<0.01), increase the fecal water content and 5h fecal particle number by 10.98% (p<0.05) and 50% (p<0.05), and significantly enhance the colon peristalsis ability of intractable constipation mice;

[0053] (2) can increase the expression level of S100β, a characteristic marker of enteric glial cells in colon tissue, by 82.4% (p<0.05), and repair the intestinal nerve damage caused by intractable constipation;

[0054] (3) can reduce the expression levels of inflammatory factors IL-1β and IL-6 in colon tissue of intractable constipation mice by 46.4% (p<0.001) and 59% (p<0.0001), respectively, and alleviate the inflammation of intestinal immune barrier;

[0055] (4) can reduce the pathological damage of colon tissue, increase the expression level of Occludin, a tight junction protein in colon tissue, by 79.12% (p<0.01), and repair the damage of intestinal mechanical barrier;

[0056] (5) can reduce the expression level of aquaporin AQP4 in colon tissue by 50.74% (p<0.05), avoid excessive absorption of water, facilitate the discharge of feces, regulate intestinal stem cells through the WNT / beta-catenin signal pathway, and regulate the intestinal chemical barrier;

[0057] (6) can improve intestinal microecological imbalance, make the intestinal flora structure tend to be healthy, repair the damage of the intestinal biological barrier, regulate the intestinal homeostasis, so as to play a role in relieving stubborn constipation.

[0058] 2, the application can be regarded as a medicine for relieving or treating stubborn constipation, and can also be applied to medicines or some fermented foods and functional foods, so as to widely play its role and have very valuable application prospect.

[0059] Biological material preservation

[0060] A strain of Bifidobacterium longum subsp.longum CCFM1319, which is taxonomically named as Bifidobacterium longum subsp.longum, has been preserved in Guangdong Microbial Culture Collection Center on August 4, 2023, with a preservation number of GDMCC No: 63715 and a preservation address of 5th Floor, No. 59 Building, Institute of Microbiology, Guangdong Academy of Sciences, 100 Middle Martyrs Road, Guangzhou. BRIEF DESCRIPTION OF DRAWINGS

[0061] Figure 1 : Schematic diagram of stubborn constipation mouse symptom relief related indexes (first black stool time, fecal water content, 5h fecal particle number, small intestine propulsion rate) of Bifidobacterium longum subsp.longum CCFM1319.

[0062] Figure 2 : Schematic diagram of changes in expression amount of intestinal neuroglia cell characteristic marker S100β in colon tissue of stubborn constipation mouse after intervention of Bifidobacterium longum subsp.longum CCFM1319.

[0063] Figure 3 : Schematic diagram of changes in expression amount of interleukin IL-1β and IL-6 in colon tissue of stubborn constipation mouse after intervention of Bifidobacterium longum subsp.longum CCFM1319.

[0064] Figure 4: Schematic diagram of pathological changes in colon tissue sections of intractable constipation mice after intervention of Bifidobacterium longum subsp. longum CCFM1319.

[0065] Figure 5 : Schematic diagram of changes in the expression of tight junction protein Occludin in the colon tissue of intractable constipation mice after intervention of Bifidobacterium longum subsp. longum CCFM1319.

[0066] Figure 6 : Schematic diagram of changes in the expression of water channel protein AQP4 in the colon tissue of intractable constipation mice after intervention of Bifidobacterium longum subsp. longum CCFM1319.

[0067] Figure 7 : Schematic diagram of changes in the expression of intestinal stem cell marker Lgr5 gene in the colon tissue of intractable constipation mice after intervention of Bifidobacterium longum subsp. longum CCFM1319.

[0068] Figure 8 : Schematic diagram of changes in the expression of intestinal stem cell marker Lgr5 protein in the colon tissue of intractable constipation mice after intervention of Bifidobacterium longum subsp. longum CCFM1319.

[0069] Figure 9 : Schematic diagram of changes in the expression of WNT / β-catenin pathway related genes (WNT3a, TCF4, c-Myc) in the colon tissue of intractable constipation mice after intervention of Bifidobacterium longum subsp. longum CCFM1319.

[0070] Figure 10 : Schematic diagram of changes in intestinal flora of intractable constipation mice after intervention of Bifidobacterium longum subsp. longum CCFM1319; wherein, (a) α diversity (b) β diversity (c) door level (d) genus level;

[0071] Note: The symbols above the column chart represent the data significance level, * represents p < 0.05, ** represents p < 0.01, *** represents p < 0.001, **** represents p < 0.0001 (compared with the CC group), # represents p < 0.05, ## represents p < 0.01, ### represents p < 0.001, and #### represents p < 0.0001 (compared with the NC group). DETAILED DESCRIPTION

[0072] Example 1: Screening, identification and culture of Bifidobacterium longum subsp. longum CCFM1319

[0073] (I) Isolation and screening of strains:

[0074] (1) A disposable sterile sampling device was used to collect the feces of a 45-year-old middle-aged man in Beijing. The fecal sample was enriched in a liquid culture medium containing fructooligosaccharides and L-cysteine hydrochloride (0.05%-0.1% by mass) in an anaerobic incubator (N2:CO2:H2=80:10:10) for 12 h;

[0075] (2) The fecal sample was gradient diluted with sterile normal saline and then plated on solid plates containing sterile 100 μg / mL mupirocin and 50 U / mL nystatin, and L-cysteine hydrochloride (0.05%-0.1% by mass) in MRS. The plates were incubated for 24-48 h;

[0076] (3) Single colonies that met the basic morphology of Bifidobacterium were selected for plate streaking and purification to isolate the selected Bifidobacterium strains;

[0077] (4) The single colonies were cultured in liquid MRS + L-cysteine (0.05%-0.1% by mass) culture medium for 24 h, and then subjected to Gram staining. Gram-positive Bifidobacterium longum was selected for subsequent tests.

[0078] (II) Preliminary identification of Bifidobacterium: fructose-6-phosphate phosphoketolase assay

[0079] (1) The Bifidobacterium strains selected in step (I) were cultured in liquid MRS + L-cysteine (0.05%-0.1% by mass) culture medium for 24 h, and then 1 mL of the culture was centrifuged at 8000 rpm for 2 min;

[0080] (2) Washed twice with 0.05M KH2PO4 solution containing 0.05% (by mass) cysteine at pH 6.5;

[0081] (3) Resuspended in 200 μL of the above phosphate buffer containing 0.25% (by mass) Triton X-100;

[0082] (4) Add 50 μL of a mixture of sodium fluoride and sodium iodoacetate at concentrations of 6 mg / mL and 10 mg / mL, respectively, and 50 μL of fructose-6-phosphate at a concentration of 80 mg / mL, and incubate at 37°C for 1 h;

[0083] (5) Add 300 μL of light amine hydrochloride at a concentration of 0.139 g / mL and pH 6.5, and leave at room temperature for 10 min;

[0084] (6) Add 200 μL of 15% (mass percentage) trichloroacetic acid and 4M HCl, respectively;

[0085] (7) Add 200 μL of 0.1M HCl containing 5% (mass percentage) ferric trichloride. If the system rapidly turns red, it is F6PPK positive, and it can be preliminarily determined to be a Bifidobacterium.

[0086] (Three) Molecular biological identification of Bifidobacterium

[0087] (1) Take 1 mL of the bacterial body of step (two) that has been screened and activated for 3 generations (cultured for 12-48 h) for strain identification, centrifuge at 6000 r / min for 3 min, discard the supernatant, and obtain the bacterial body.

[0088] (2) After washing the bacterial body by adding 1 mL of sterile water and blowing, centrifuge at 10000 r / min for 1 min, discard the supernatant, add 500 μL of sterile water to resuspend, and use as a bacterial liquid template.

[0089] (3) 16S rDNA PCR system:

[0090] A. Bacterial 16S rDNA, 20 μL PCR reaction system: 27F, 0.5 μL; 1492R, 0.5 μL; Taq enzyme, 1 μL; template, 1 μL; ddH20, 8 μL.

[0091] B. PCR conditions: 94°C for 5 min; 94°C for 30 s; 55°C for 30 s; 72°C for 2 min; 72°C for 10 min; step 2-4 30x; 12°C for 2 min.

[0092] (4) Prepare 1% agarose gel, then mix the PCR product with 10000x Loading buffer, load 2 μL, run at 120V for 30 min, and then perform gel imaging;

[0093] (5) The PCR product of 16S rDNA was sequenced and analyzed, and the obtained sequence results were searched and similarity aligned in GenBank using BLAST, and the sequencing results were selected, which showed that the strain was Bifidobacterium longum subsp. longum, which was named Bifidobacterium longum subsp. longum CCFM1319, and was preserved at -80°C for standby.

[0094] The Bifidobacterium longum subsp. longum CCFM1319 has the following biological characteristics:

[0095] (1) Cell characteristics: Gram-positive, spherical cells, diameter 0.8-1.0 μm, no flagella, no spores;

[0096] (2) Colony characteristics: colony is milky white, edge is neat, spherical, convex, opaque, surface is wet and smooth;

[0097] (3) Growth characteristics: the minimum growth temperature of the strain is 15°C, the maximum growth temperature is 45°C, the optimal growth temperature is 35-37°C, the optimal growth pH is 6.5, and after 16h of culture, it enters the late logarithmic phase or the early stationary phase.

[0098] (Four) Preparation of Bifidobacterium longum subsp. longum CCFM1319 bacterial suspension and survival rate of frozen storage

[0099] Bifidobacterium longum subsp. longum CCFM1319 was inoculated into MRS solid culture medium and cultured at 37°C for 72h to obtain single colonies, and the prepared single colonies were inoculated into MRS liquid culture medium and cultured at 37°C for 24h for activation;

[0100] The bacterial liquid after 3 generations of activation was inoculated into 1L MRS liquid culture medium at an inoculation amount of 2%, and after shaking and mixing, it was cultured at 37°C in an anaerobic incubator for 24h. Centrifuged at 8000g / min, 4°C for 15min, washed twice with sterile physiological saline (containing 0.05%-0.1% L-cysteine hydrochloride), and similarly centrifuged under the same conditions, after removing the supernatant, resuspended with 30% glycerol, obtained the bacteria liquid for gavage, and stored in a refrigerator at -80°C for one week.

[0101] Before the animal experiment, the bacteria liquid stored in the refrigerator was taken out, centrifuged at 6000r / min for 5min, washed twice with sterile physiological saline, resuspended with 10% skim milk, and the number of viable bacteria before and after freezing for one week was determined by plate pouring method.

[0102] The formula of MRS liquid medium is as follows: 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 0.8 g, pH is adjusted to 6.8±0.2, and the volume is made up to 1 L.

[0103] MRS solid medium: 2% agar powder is added to the MRS liquid medium. High pressure sterilization at 115°C for 20 min.

[0104] Experimental results: the initial viable cell count was 6×10 9 cfu / mL, and the viable cell count was 3.4×10 9 cfu / mL after being frozen for 1 week, and the order of magnitude did not change, indicating that freezing the bacterial solution would not affect the experiment and could be used for animal experiments.

[0105] Example 2: Relief effect of B. longum subsp. longum CCFM1319 on refractory constipation mice

[0106] (1) Preparation of bacterial suspension

[0107] B. longum subsp. longum CCFM1319 and B. longum subsp. longum CCFM1112 (the strain is described in Chinese patent CN113943681B) were taken out from the-80°C refrigerator, streaked on MRS solid medium, and cultured at 37°C for 48 h. Single colonies were picked and cultured in MRS liquid medium at 37°C for 20 h to prepare seed solutions.

[0108] The prepared seed solutions were inoculated into new MRS liquid medium at a concentration of 2% (v / v) and cultured at 37°C for 18 h. The same process was repeated to prepare B. longum subsp. longum CCFM1319 fermentation broth and CCFM1112 fermentation broth.

[0109] The prepared B. longum subsp. longum CCFM1319 fermentation broth and CCFM1112 fermentation broth were centrifuged at 6000 r / min and 4°C for 5 min, and then resuspended with 10% (m / v) skim milk to prepare 5×10 9 CFU / mL bacterial suspension for animal experiments.

[0110] (2) Take 40 healthy male C57BL / 6J mice of 8 weeks old, adapt to the environment for 1 week, and randomly divide them into 5 groups: control group (normal), model group (model), prucalopride group, Bifidobacterium longum subsp. longum CCFM1319 group and Bifidobacterium longum subsp. longum CCFM1112 group, each containing 8 mice. The dose of the bacterial suspension for gavage is 5x10 9 CFU / mL, and gavage is started at 9 am every day, with 0.2 mL each time.

[0111] The grouping and treatment methods of experimental animals are shown in Table 1:

[0112] Table 1 Grouping and treatment methods of experimental animals

[0113]

[0114]

[0115] After gavage at the 15th week, the mice are placed in cages padded with absorbent paper one by one, and the feces are collected, weighed as wet weight, and freeze-dried as dry weight. The water content of the feces is calculated according to the following formula.

[0116] Fecal water content (%) = (fecal wet weight - fecal dry weight) / fecal wet weight x 100%;

[0117] The mice are placed in cages padded with absorbent paper one by one, and the time of placement is recorded. The feces are collected for 5 hours, and the fecal particle count of each mouse is recorded, which is the 5h fecal particle count.

[0118] The first black fecal particle time is evaluated by the Evans blue experiment. The mice are fasted for 12 hours before measurement, and free water is provided during this period. At the start of the measurement, the mice are gavaged with 0.2 ml of Evans blue semi-liquid solution (2.5% Evans blue and 1% methyl cellulose). The time of gavage and the time of the first blue fecal particle are recorded for each mouse, and the difference between the two is the first black fecal particle time.

[0119] Before the mice are sacrificed, each mouse is given 0.2 ml of ink, and 30 minutes later, they are sacrificed and dissected. The upper end from the lower end of the pylorus to the cecum is cut, and the total length of the small intestine is measured as "total length of the small intestine". The ink front is measured from the pylorus to the ink front, and the small intestine propulsion rate is calculated according to the following formula.

[0120] Small intestine propulsion rate = (ink propulsion length (cm) / total length of small intestine (cm)) x 100%.

[0121] The experimental results of fecal water content, 5h fecal particle count, first black fecal particle time, and small intestine propulsion rate are shown in Figure 1 Figure 1 ​Compared with the control group, the first black stool time of the model group was prolonged to 1.77 times of the normal group (p<0.0001), the fecal water content decreased from 50.09% to 47.32% (p<0.01), the 5h fecal particle number decreased from an average of 10 per mouse to 6 (p<0.001), and the small intestinal propulsion rate had no significant change.

[0122] After modeling, there was no significant difference in the small intestinal propulsion rate of the mice, but the first black stool time was significantly prolonged, that is, the whole intestinal transport capacity was significantly decreased, indicating that the model group mice had obvious colon transport disorder, and the fecal water content was significantly decreased. The success of modeling of the model group mice indicates that the model group mice have colon transport disorder, and the refractory constipation model is successful.

[0123] Compared with the constipation model group, the first black stool time of the constipation mice (161.1 minutes) was shortened by 80.5% (p<0.01) by gavage with Bifidobacterium longum subsp. longum CCFM1319, the fecal water content (52.52%) was increased by 10.98% (p<0.05) compared with the model group (47.32%), and the 5h fecal particle number (9) was increased by 50% (p<0.05) compared with the model group (6), significantly enhancing the colon peristalsis capacity of the refractory constipation mice. Gavage with Bifidobacterium longum subsp. longum CCFM1112 only had a significant effect on the first black stool time of the constipation mice, and had no significant change in the fecal water content and fecal particle number of the constipation mice, indicating that gavage with Bifidobacterium longum subsp. longum CCFM1319 has a better effect on the relief of refractory constipation than Bifidobacterium longum subsp. longum CCFM1112.

[0124] Example 3: Bifidobacterium longum subsp. longum CCFM1319 can increase the expression level of enteric glial cell marker S100β in colon tissue

[0125] Enteric glial cells (EGCs) are important auxiliary cells of the enteric nervous system, and play an important role in the production and development of neurons, submucosal extension, and signal transmission. More and more studies have shown that EGCs also play an important role in intestinal homeostasis. The activity of breaking EGCs in vivo can block the neural control of intestinal peristalsis, causing constipation in mice.

[0126] The grouping, modeling and treatment methods of C57BL / 6J mice were the same as in Example 2. Real-time fluorescent quantitative polymerase chain reaction (RT-qPCR) was used to determine the expression amount of enteric glial cell marker S100β gene in colon tissue. First, RNA was extracted from fresh colon tissue, and the specific method was as follows:

[0127] 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 at room temperature, and it was placed for 10 min, and it was centrifuged at 4℃, 12000 r / min for 15 min, 600 μL of the upper colorless aqueous phase was taken into another enzyme-free centrifuge tube, and 500 μL of isopropyl alcohol was added. Mix well by inverting up and down, and let 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 formed 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 the RNA is dissolved, 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 HiScript III RTSuperMix for qPCR Reverse Transcription Kit of Novozyme Biotech Co., Ltd. Reverse transcription to synthesize cDNA, store at -20℃.

[0128] The primers of mouse S100β gene and reference gene mGAPDH gene are shown in Table 2,

[0129] Table 2: Primer sequences of mouse S100β gene and mGAPDH gene

[0130]

[0131] qRT-PCR reaction system and conditions: PCR amplification was performed using CFX96TM real-time fluorescence quantitative PCR instrument, and the fluorescence signal was read. The S100β gene qRT-PCR reaction system was:

[0132]

[0133] The qRT-PCR reaction conditions were: 95℃ for 30 s; 95℃ for 10 s, 60℃ for 30 s, a total of 40 cycles. The mGAPDH gene was used as the reference gene, and the results were analyzed by CFX96Manager software. The experimental results are shown in Figure 2 .

[0134] From Figure 2It can be seen that after modeling, the expression of S100β, a marker of enteric glial cells in the colon tissue of the intractable constipation mice (0.6727) decreased to 44.9% of that of the normal group (1.221) (p<0.05), indicating that the intractable constipation mice had obvious intestinal nerve damage. After the intractable constipation mice were administered B. longum subsp. longum CCFM1319 by gavage, the expression of S100β (1.227) of the intractable constipation mice increased by 82.40% (p<0.05) compared with that of the model group (0.6727). The expression of S100β (0.9287) of the prucalopride group increased by 38.05% (p>0.05) compared with that of the model group. The expression of S100β (0.9915) of the CCFM1112 group increased by 47.39% (p>0.05) compared with that of the model group.

[0135] Therefore, B. longum subsp. longum CCFM1319 can significantly repair the intestinal nerve damage of the intractable constipation mice, and the effect is better than that of the drug group and the B. longum subsp. longum CCFM1112 group.

[0136] Example 4: B. longum subsp. longum CCFM1319 can reduce the expression levels of inflammatory factors IL-1β and IL-6 in the colon tissue

[0137] Interleukin IL-1β is an important pro-inflammatory cytokine involved in innate immunity. It can synergize with antigens to promote B cell growth and differentiation, promote the antigen presentation ability of APCs such as monocyte-macrophages, and cause the release of inflammatory mediators. IL-6 is reported to have pro-inflammatory or anti-inflammatory properties, depending on its concentration and combination with other inflammatory cytokines. High concentrations of IL-1β combined with IL-6 can reduce neurogenesis.

[0138] The C57BL / 6J mice were grouped, modeled, and treated according to the method of Example 2. Real-time fluorescent quantitative polymerase chain reaction (RT-qPCR) was used to determine the expression of IL-1β and IL-6 genes. The RNA extraction and reverse transcription methods were the same as those of Example 3.

[0139] The primers of the mouse IL-1β, IL-6 genes and the internal reference gene mGAPDH gene are shown in Table 3,

[0140] Table 3: Primer sequences of mouse IL-1β, IL-6 genes and mGAPDH gene

[0141]

[0142] qRT-PCR reaction system and conditions: 10 μL of the reaction system was prepared according to the instructions of the SYBR® Premix Ex TaqTM kit. PCR amplification was performed using a CFX96TM real-time fluorescent quantitative PCR instrument, and the fluorescent signal was read.

[0143] IL-1β, IL-6 gene qRT-PCR reaction system is:

[0144]

[0145] qRT-PCR reaction conditions: 95℃ 30s; 95℃ 10s, 60℃ 30s, a total of 40 cycles. The mGAPDH gene was used as an internal reference gene, and the results were analyzed by CFX96 Manager software. The experimental results are shown in Figure 3 .

[0146] It can be seen from Figure 3 that after modeling, the expression of IL-1β and IL-6 in the colon tissue of the intractable constipation mice increased to 1.61 times (p<0.01) and 3.19 times (p<0.001) of the normal group, indicating that the intractable constipation mice had severe inflammatory damage. After intragastric administration of B. longum subsp. longum CCFM1319, the expression of IL-1β and IL-6 in the intractable constipation mice (0.8680 and 1.309, respectively) was significantly reduced by 46.4% (p<0.001) and 59% (p<0.0001) compared with the model group (1.619 and 3.201, respectively). The expression of IL-1β and IL-6 in the colon of the drug prucalopride group (1.253 and 1.906, respectively) was reduced by 22.6% (p<0.05) and 40.4% (p<0.01) compared with the model group. The expression of IL-1β and IL-6 in the colon of the CCFM1112 group (0.8688 and 1.942, respectively) was reduced by 46.4% (p<0.001) and 39.3% (p<0.01) compared with the model group.

[0147] Therefore, B. longum subsp. longum CCFM1319 can significantly alleviate the inflammatory response of the colon tissue of intractable constipation mice, and the effect is better than that of the drug group and the B. longum subsp. longum CCFM1112 group, and improves the barrier damage of the intestinal tract.

[0148] Example 5: B. longum subsp. longum CCFM1319 improves the pathological damage of colon tissue caused by intractable constipation

[0149] The grouping, modeling, and treatment methods for C57BL / 6J mice were the same as in Example 1. After dissection, fresh colon tissue was taken from the mice and soaked in paraformaldehyde fixative, then rinsed overnight. The samples were dehydrated sequentially by 70%, 80%, and 90% ethanol solutions for 30 minutes each, followed by two 20-minute immersions in 95% and 100% ethanol solutions. The samples were then immersed in a mixture of equal parts pure ethanol and xylene for 15 minutes, followed by 15 minutes of xylene pre-wash and 15 minutes of xylene post-wash until clear. Finally, the samples were immersed in a 1:1 mixture of xylene and paraffin for 15 minutes, followed by 50-60 minutes of paraffin I and paraffin II permeation to remove the clearing agent. The treated samples were then embedded, sectioned, spread, baked, stained with H&E, and mounted.

[0150] Observe the pathological sections of mouse colon tissue, such as Figure 4 As shown, compared with the normal group, the colonic tissue of mice in the model group exhibited a thinner mucus layer, a reduction in goblet cells, and increased inflammatory infiltration. After gavage administration of *Bifidobacterium longum* subsp. *longum* CCFM1319, the mucus layer in the mouse colonic tissue thickened, the boundaries of goblet cells became clearer, and inflammatory infiltration significantly improved. This effect was superior to the drug group and the *Bifidobacterium longum* subsp. *longum* CCFM1112 group. Therefore, the results indicate that *Bifidobacterium longum* subsp. *longum* CCFM1319 can alleviate the pathological damage to colonic tissue caused by refractory constipation.

[0151] Example 6: Bifidobacterium longum subsp. CCFM1319 can increase the expression level of the tight junction protein Occludin in colon tissue.

[0152] Tight junction proteins are important structures connecting intestinal epithelial cells, affecting intestinal epithelial permeability and playing a crucial role in preventing the entry of toxic macromolecules. The grouping, modeling, and treatment methods for C57BL / 6J mice were the same as in Example 2. The expression level of the tight junction protein Occludin in colon tissue was determined using real-time quantitative polymerase chain reaction (qRT-PCR). RNA extraction and reverse transcription methods were the same as in Example 3. Primers for the mouse Occludin gene and the internal reference gene mGAPDH are shown in Table 4.

[0153] Table 4 Primer sequences for mouse Occludin and mGAPDH genes

[0154]

[0155] qRT-PCR reaction system and conditions: using The CFX96TM real-time quantitative PCR instrument was used for PCR amplification and the fluorescence signal was read.

[0156] The qRT-PCR reaction system for the Occludin gene is as follows:

[0157]

[0158] The qRT-PCR reaction conditions for the Occludin gene were: 95℃ for 30 s; 95℃ for 10 s, 60℃ for 30 s, for a total of 40 cycles. The mGAPDH gene was used as an internal control gene, and the results were analyzed using CFX96Manager software.

[0159] Depend on Figure 5 It was found that after modeling, the expression level of Occludin in the colonic tissue of mice with refractory constipation decreased, indicating that modeling led to the destruction of tight junction structures in mice with refractory constipation. After gavage administration of Bifidobacterium longum subsp. CCFM1319, the expression level of Occludin in mice with refractory constipation (1.400) was significantly increased by 79.12% compared with the model group (0.7816) (p<0.01). The expression levels of Occludin in the colonic tissue of the drug prucalopride group and the Bifidobacterium longum subsp. CCFM1112 group (1.124 and 1.093, respectively) were 43.8% (p>0.05) and 39.84% (p>0.05) higher than those of the model group, respectively, with no significant difference. That is, the effect of Bifidobacterium longum subsp. CCFM1319 was better than that of the drugs prucalopride and Bifidobacterium longum subsp. CCFM1112.

[0160] Example 7: *Bifidobacterium longum* subsp. CCFM1319 can increase the expression level of aquaporin AQP4 in colonic tissue. AQPs are transport proteins in the colonic epithelium that control the rapid transport of water. AQP4 is the main AQP subtype expressed in the mouse colon. Under normal circumstances, the osmotic pressure of the colonic lumen is much lower than that of the body, requiring rapid water transport through aquaporins to achieve water reabsorption and thus form feces. However, excessive water absorption can cause the stool to become dry and hard, making it difficult to pass. Studies have shown that abnormal expression of AQPs exists in constipated mice, which may be one of the reasons for the dry stool in constipated mice.

[0161] The grouping, modeling, and treatment methods for C57BL / 6J mice were the same as in Example 2. The expression level of the tight junction protein AQP4 in colon tissue was determined using real-time quantitative polymerase chain reaction (qRT-PCR). RNA extraction and reverse transcription methods were the same as in Example 2.

[0162] Example 3

[0163] The primers for the mouse AQP4 gene and the internal reference gene mGAPDH are shown in Table 5.

[0164] Table 5 Primer sequences for mouse AQP4 and mGAPDH genes

[0165]

[0166] qRT-PCR reaction system and conditions: use PCR amplification was performed by CFX96TM real-time fluorescence quantitative PCR instrument, and the fluorescence signal was read.

[0167] The AQP4 gene qRT-PCR reaction system is:

[0168]

[0169] The AQP4 gene qRT-PCR reaction conditions are: 95°C for 30s; 95°C for 10s, 60°C for 30s, a total of 40 cycles. The mGAPDH gene is used as an internal reference gene, and the results are analyzed by CFX96 Manager software.

[0170] From Figure 6 It can be seen that after modeling, the expression of AQP4 in the colon tissue of the intractable constipation mice increased to 1.48 times that of the normal group (p<0.05), indicating that the expression level of AQP4 in intractable constipation mice increased, and there was excessive absorption of water, which led to dry and hard feces and difficulty in excretion. After intragastric administration of Bifidobacterium longum subsp. CCFM1319, the expression of AQP4 in intractable constipation mice (0.7133) was significantly reduced by 50.74% (p<0.05) compared with the model group (1.448), the expression of AQP4 in the CCFM1112 group (0.6928) was reduced by 52.15% (p<0.05) compared with the model group, and the drug prucalopride had no significant effect on the expression of AQP4 in the colon tissue. Therefore, the regulation of water channel protein in the colon tissue by Bifidobacterium longum subsp. CCFM1319 is equivalent to that of Bifidobacterium longum subsp. CCFM1112, and the effect is better than that of the drug prucalopride.

[0171] Example 8: Bifidobacterium longum subsp. CCFM1319 can regulate the gene expression level of intestinal stem cell marker Lgr5

[0172] Lgr5 is an important marker of intestinal stem cells. The dynamic balance of intestinal stem cell proliferation and differentiation plays a crucial role in the maintenance of intestinal structure and function and the regulation of intestinal homeostasis. The human intestine has tens of thousands of microorganisms, and studies have shown that intestinal microbial metabolites have a regulatory effect on intestinal stem cells.

[0173] The C57BL / 6J mice were grouped, modeled and treated as in Example 2. Real-time fluorescence quantitative polymerase chain reaction (qRT-PCR) was used to determine the expression of intestinal stem cell marker Lgr5. The extraction and reverse transcription of RNA were the same as in Example 3.

[0174] The primers for the mouse Lgr5 gene and the internal reference gene mGAPDH are shown in Table 6.

[0175] Table 6: Primer sequences for mouse Lgr5 and mGAPDH genes

[0176]

[0177] qRT-PCR reaction system and conditions: using The CFX96TM real-time quantitative PCR instrument was used for PCR amplification and the fluorescence signal was read.

[0178] The qRT-PCR reaction system for the Lgr5 gene is as follows:

[0179]

[0180] The qRT-PCR reaction conditions for the Lgr5 gene were: 95℃ for 30 s; 95℃ for 10 s, 60℃ for 30 s, for a total of 40 cycles. The mGAPDH gene was used as an internal reference gene, and the results were analyzed using CFX96Manager software.

[0181] Depend on Figure 7 It was found that after modeling, the expression level of Lgr5 in the colon tissue of mice with refractory constipation (1.871) increased by 87.3% compared with the normal group (1.001) (p<0.01), indicating that modeling led to abnormal expression of intestinal stem cells in constipated mice, resulting in excessive proliferation of intestinal stem cells, which may lead to adverse consequences such as excessive proliferation of intestinal epithelium or carcinogenesis. After gavage administration of Bifidobacterium longum subsp. CCFM1319, it can be seen that the expression level of Lgr5 in constipated mice (1.210) was significantly reduced by 66.43% compared with the model group (p<0.01), and tended to be similar to that of the normal group. Its effect was better than that of the drug prucalopride (1.342) (a decrease of 53.2% p<0.01), while gavage administration of Bifidobacterium longum subsp. CCFM1112 (1.498) had no effect on the regulation of intestinal stem cells.

[0182] Therefore, Bifidobacterium longum subsp. CCFM1319 can effectively regulate the level of intestinal stem cells in the colon tissue of mice with refractory constipation and maintain intestinal homeostasis.

[0183] Example 9: Bifidobacterium longum subsp. CCFM1319 can regulate the protein expression level of the intestinal stem cell marker Lgr5.

[0184] The grouping, modeling, and treatment methods for C57BL / 6J mice were the same as in Example 2. Western blotting was used to determine the protein expression level of the intestinal stem cell marker Lgr5.

[0185] Take 20 mg of colon tissue in a 1.5 ml centrifuge tube, add 30 times the volume of RIPA lysis buffer, grind thoroughly, then centrifuge, transfer the supernatant to a new 1.5 ml centrifuge tube. According to the instructions of the Biyun Tian BCA kit, the protein concentration is determined and uniformly quantified, then 5x loading buffer is added to the metal bath at 95°C for 5 min to denature the protein, and the protein sample is obtained.

[0186] 20 μg of protein sample was separated by 10% SDS-PAGE electrophoresis and transferred to a PVDF membrane, blocked with blocking solution (5% BSA in TBST solution) for 2 h, then incubated with primary antibody at 4°C overnight, washed with TBST (3 times, 5 min each time) after incubation, then incubated with secondary antibody at room temperature for 2 h, washed with TBST (3 times, 5 min each time) after incubation, finally added developing solution, developed for 20 s in the dark, and observed under an imaging instrument.

[0187] The results are shown in Table 6. Figure 8 As shown in Table 6, after modeling, the protein expression level of Lgr5 in the colon tissue of the intractable constipation mice increased by 7.45% (p<0.05) compared with the normal group, which was consistent with the above-mentioned gene expression results, thus indicating that modeling would cause abnormal expression of intestinal stem cells and imbalance of intestinal homeostasis, and after gavage with CCFM1319, the protein expression level of Lgr5 in the colon tissue decreased compared with the model group, and tended to the expression level of the normal group, which was also consistent with the above-mentioned expression results of Lgr5 at the gene level, therefore, B. longum subsp. longum CCFM1319 can regulate the level of intestinal stem cells of intractable constipation mice and maintain the intestinal homeostasis.

[0188] Example 10: B. longum subsp. longum CCFM1319 regulates the level of intestinal stem cells through WNT / β-catenin pathway

[0189] Under physiological and pathological conditions, ISCs activity is strictly regulated by multiple niche signals to regulate intestinal homeostasis. Wnt signal plays a crucial role in ISCs regulation, among which the classical Wnt signaling pathway, i.e. Wnt / β-catenin signaling pathway, plays a major role.

[0190] 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 level of WNT / β-catenin pathway related genes (WNT3a, TCF4, c-Myc) in the colon tissue of intractable constipation mice. The RNA extraction and reverse transcription methods were the same as in Example 3.

[0191] The primers of mouse WNT3a, TCF4, c-Myc genes and internal reference gene mGAPDH gene are shown in Table 7,

[0192] Table 7: Primer sequences for mouse WNT3a, TCF4, c-Myc, and mGAPDH genes

[0193]

[0194] qRT-PCR reaction system and conditions:

[0195] use The CFX96TM real-time quantitative PCR instrument was used for PCR amplification and the fluorescence signal was read.

[0196] The qRT-PCR reaction system for WNT3a, TCF4, and c-Myc genes is as follows:

[0197]

[0198]

[0199] The qRT-PCR reaction conditions for WNT3a, TCF4, and c-Myc genes were: 95℃ for 30 s; 95℃ for 10 s, 60℃ for 30 s, for a total of 40 cycles. The mGAPDH gene was used as an internal reference gene, and the results were analyzed using CFX96Manager software.

[0200] Depend on Figure 9 It is known that modeling leads to overactivation of the Wnt / β-catenin signaling pathway, which is consistent with the increase in ISC expression caused by modeling. After gavage administration of Bifidobacterium longum subsp. CCFM1319, the expression levels of WNT3a, TCF4, and c-Myc genes decreased by 24.4% (p<0.05), 13.5% (p<0.05), and 28.65% (p<0.01) respectively compared with the model group, approaching the expression levels of the normal group. However, intervention in the Bifidobacterium longum subsp. CCFM1112 group did not cause any changes in this pathway, which is also consistent with the aforementioned ISC gene expression results. Therefore, Bifidobacterium longum subsp. CCFM1319 can regulate the level of intestinal stem cells through the WNT / β-catenin pathway, thereby maintaining the dynamic balance of intestinal stem cells and maintaining intestinal homeostasis.

[0201] Example 11: Bifidobacterium longum subsp. CCFM1319 improves intestinal flora imbalance and restores intestinal microecology.

[0202] The grouping, modeling, and treatment methods for C57BL / 6J mice were the same as in Example 2. Total DNA was extracted from the colon contents of mice using MP's fecal kit to detect the intestinal flora.

[0203] The specific method is as follows: 200 mg of mouse colon contents is taken out from the -80℃ refrigerator, and macrogenomic DNA is obtained by extraction according to the relevant instructions of the Fast DNA Spin Kit for Feces (MP Biomedicals, catalog No. 6570200) kit, and the obtained DNA is subjected to PCR.

[0204] 50 μL of the PCR system is:

[0205]

[0206] The PCR reaction conditions are:

[0207] 95℃ 5 min; 95℃ 30 s, 52℃ 30 s, 72℃ 30 s, a total of 40 cycles; 72℃ 7 min; 12℃ 5 min.

[0208] After PCR, electrophoresis is performed in a 4% nucleic acid dye, 1.5% agarose gel, and the obtained band is divided into 2 ml EP tubes, and purified and recovered using a DNA Gel / PCR Purification Miniprep Kit (Biomiga, BW-DC3511-01) according to the relevant instructions. The concentration of the extracted DNA is determined by NanoDrop, the samples are mixed at equal concentration, and the corresponding library is sequenced using a MiSeq sequencer (Illumina, Santiago, CA, USA).

[0209] After sequencing, sequences with a length <200 bp, primer sequences, and single sequences that cannot be spliced are removed, and the sequences are spliced according to the standard of overlapping bases >10 bp and no mismatches. Sequences with a similarity greater than 97% are defined as one classification unit (Operational Taxonomic Unit, OTU), and the species are determined by Ribosomal Database Project (RDP) Bayes classifier. The alpha diversity of the sample is calculated to evaluate the richness and diversity of the bacterial flora of the sample. Principal coordinate analysis (PCoA) is performed by Bray-Curtis distance to reflect the beta diversity of the sample, which is used to evaluate the similarity of the intestinal flora of the mouse.

[0210] The alpha diversity is characterized by Chao1 index and shannon index. The Chao1 index reflects the richness of the flora, and the larger the index, the greater the richness of the community. The shannon index reflects the diversity of the flora, and the larger the index, the higher the diversity of the community.

[0211] The gut microbiota is composed of tens of thousands of microorganisms residing in the intestines. It is localized in the gut and develops alongside the host, participating in a series of physiological processes. Both human and animal studies have shown that constipation leads to gut microbiota dysbiosis, disrupting the normal gut microecology.

[0212] Depend on Figure 10 It can be seen that after modeling, the intestinal flora of mice with refractory constipation was disordered. The Chao1 index and Shannon index (255 and 4.737, respectively) were reduced by 27.25% (p < 0.0001) and 18.9% (p < 0.0001) compared to the normal group (350.5 and 5.840, respectively), indicating a decrease in the richness and diversity of the intestinal flora in mice with refractory constipation. Significant differences were also observed in the β-diversity of the intestinal flora; the model group exhibited a different intestinal flora structure than the normal group, with a large difference in flora similarity (p < 0.01). Changes also occurred at the phylum and genus levels, manifested as a decrease in the relative abundance of beneficial bacteria and an increase in the relative abundance of harmful bacteria. *Alistipes* is an important genus of beneficial bacteria in the gut, capable of producing short-chain fatty acids and reducing intestinal inflammation. After modeling, the relative abundance of Alistipes in the model group (0.05459) was 72.15% lower than that in the normal group (0.196) (p < 0.0001). Escherichia-Shigrlla is a pathogen-carrying bacterium and belongs to harmful bacteria. The results showed that the abundance of this harmful bacterium was significantly increased in the model group (0.0003451), which was 88.6% higher than that in the normal group (3.935e-005) (p < 0.0001), indicating that the intestinal microecology of mice with refractory constipation was disrupted.

[0213] After gavage administration of Bifidobacterium longum subsp. CCFM1319, the Chao1 and Shannon indices of the gut microbiota in mice with refractory constipation (371.3 and 5.509, respectively) were significantly increased by 45.6% (p<0.01) and 16.3% (p<0.01) compared with the model group. In the CCFM1112 group, the Chao1 and Shannon indices (291.9 and 5.716, respectively) were increased by 14.47% (p>0.05) and 20.67% (p<0.01) compared with the model group. In contrast, the Chao1 and Shannon indices of the drug prucalopride (271.6 and 6.019, respectively) were increased by 6.5% (p>0.05) and 27.06% (p<0.001) compared with the model group. Therefore, the effect of Bifidobacterium longum subsp. CCFM1319 on regulating the α-diversity of gut microbiota in mice with refractory constipation is better than that of Bifidobacterium longum subsp. CCFM112 and the drug prucalopride.

[0214] β-diversity is an indicator for evaluating the similarity of gut microbiota, by analyzing... Figure 10 (b) PCoA analysis showed that while prucalopride could relieve constipation, its effect on regulating the intestinal microecology was not as good as that of *Bifidobacterium longum* subsp. *longum* CCFM1319 and CCFM1112, and its microbiota structure was more similar to the model group. Compared with *Bifidobacterium longum* subsp. *longum* CCFM1112, *Bifidobacterium longum* subsp. *longum* CCFM1319 had a microbiota structure more similar to the normal group. Therefore, in conclusion, *Bifidobacterium longum* subsp. *longum* CCFM1319 can regulate the intestinal microbiota dysbiosis in constipated mice, making its microbiota structure more similar to the normal group, and its effect is better than that of *Bifidobacterium longum* subsp. *longum* CCFM112 and prucalopride.

[0215] At the phylum and genus levels, after intervention with *Bifidobacterium longum* subsp. *longum* CCFM1319, the ratio of posterior wall bacteria to Bacteroidetes (F / B) (1.433) increased by 36.6% compared to the model group (1.049) (p < 0.01), while *Bifidobacterium longum* subsp. *longum* CCFM112 and the drug prucalopride had no significant effect. Studies have shown that the ratio of posterior wall bacteria to Bacteroidetes is closely related to maintaining host health, and a decrease in this ratio is associated with inflammation levels, consistent with the previously mentioned increase in inflammation levels caused by modeling. *Bifidobacterium longum* subsp. *longum* CCFM1319 significantly upregulated this ratio, bringing it closer to the normal group. This indicates that it can effectively improve the gut microbiota structure and restore the gut microecology. At the genus level, *Bifidobacterium longum* subsp. *CCFM1319* significantly upregulated the relative abundance of the beneficial genus *Alistipes* (0.132), an increase of 141.8% compared to the model group (p < 0.001), and significantly downregulated the relative abundance of the harmful genus *Escherichia-Shigrlla* (4.053e-005), a decrease of 88.26% compared to the model group (p < 0.01). The drug prucalopride had no significant effect on either of these genera. *Bifidobacterium longum* subsp. *CCFM1112* did not significantly upregulate the relative abundance of *Alistipes*, but it significantly downregulated the relative abundance of *Escherichia-Shigrlla* (0.0001403), a decrease of 59.35% compared to the model group (p < 0.05). Therefore, at both the genus and phylum levels, *Bifidobacterium longum* subsp. *CCFM1319* has a significant effect and can effectively regulate intestinal flora imbalance.

[0216] Therefore, in summary, the intestinal flora of mice with refractory constipation is severely disordered, and intervention with Bifidobacterium longum subsp. CCFM1319 can effectively improve the intestinal flora structure, making it more healthy, maintaining flora homeostasis, and thus relieving constipation.

[0217] Example 12: Application of Bifidobacterium longum subsp. longum CCFM1319

[0218] The specific steps are as follows:

[0219] Bifidobacterium longum subsp. longum CCFM1319 can be used to prepare tablets, and the specific preparation process of the tablets is as follows:

[0220] The single colony of Bifidobacterium longum subsp. longum CCFM1319 obtained in Example 1 was inoculated into MRS liquid medium and cultured at 37°C for 24h to obtain an activation liquid; the activation liquid was inoculated into MRS liquid medium at an inoculation amount of 1% (v / v) and cultured at 37°C for 24h to obtain a first-stage seed liquid; the first-stage seed liquid was inoculated into MRS liquid medium at an inoculation amount of 1% (v / v) and cultured at 37°C for 24h to obtain a second-stage seed liquid; the second-stage seed liquid was inoculated into MRS liquid medium at an inoculation amount of 1% (v / v) and cultured at 37°C for 24h to obtain a bacterial liquid; the bacterial liquid was centrifuged at 6000g for 15min to collect the precipitate; the precipitate was washed twice with PBS buffer solution with a pH of 7.4, and then centrifuged at 6000g for 10min to obtain bacterial bodies; the bacterial bodies of Bifidobacterium longum subsp. longum CCFM1319 were resuspended to a cell concentration of 1x1010CFU / mL with a protective agent solution containing 130g / L skim milk, 20g / L trehalose and 20g / L sucrose to obtain a Bifidobacterium longum subsp. longum CCFM1319 bacterial liquid; the Bifidobacterium longum subsp. longum CCFM1319 bacterial liquid was freeze-dried to obtain Bifidobacterium longum subsp. longum CCFM1319 bacterial powder; 10% of the freeze-dried bacterial powder accounted for the total share, followed by 2% stearic acid as a lubricant, 3% CMC-Na, 15.5% oligogalactose, 7.8% oligoxylan, 7.8% inulin, lactitol, erythritol, xylitol, and other auxiliary materials such as starch were added in sequence according to the total weight, and then tableting was performed to obtain tablets.

[0221] Taking 1g of the above tablet by gavage to intractable constipation mice every day for four weeks, the symptoms of intractable constipation mice can be effectively relieved, the time of the first black stool of intractable constipation mice is significantly shortened, the water content and 5h fecal particle number of feces are significantly increased, and the colon peristalsis is significantly promoted; the expression level of S100β, the characteristic marker of enteric glial cells in colon tissue, can be increased, and the intestinal nerve injury can be repaired; the expression levels of IL-1β and IL-6, the inflammatory factors in colon tissue, can be reduced, and the intestinal immune barrier inflammation can be relieved; the pathological injury of colon tissue can be reduced, the expression of Occludin, the tight junction protein in colon tissue, can be promoted, and the intestinal mechanical barrier injury can be repaired; the expression level of AQP4, the water channel protein in colon tissue, can be reduced, the intestinal stem cells can be regulated through the WNT / β-catenin signal pathway, and the intestinal chemical barrier can be adjusted; the intestinal microecological imbalance can be improved, the intestinal flora structure tends to be healthy, the intestinal biological barrier injury can be repaired, and the fermented food can regulate the intestinal homeostasis and relieve intractable constipation.

[0222] Although the present application has been disclosed with reference to the preferred embodiments, it is not intended to limit the present application, and any person skilled in the art can make various modifications and modifications without departing from the spirit and scope of the present application, and therefore the protection scope of the present application should be defined by the claims.

Claims

1. A strain of Bifidobacterium longum subsp. ( Bifidobacterium longum subsp. longum CCFM1319 was deposited at the Guangdong Provincial Center for Microbial Culture Collection on August 4, 2023, with accession number GDMCC No: 63715.

2. A microbial preparation, characterized in that, The microbial preparation contains the Bifidobacterium longum subsp. longum CCFM1319 or the fermentation broth thereof according to claim 1, or contains the freeze-dried powder of the Bifidobacterium longum subsp. longum CCFM1319 according to claim 1.

3. A composition characterized in that, The active ingredient of the composition includes the Bifidobacterium longum subsp. longum CCFM1319 or the fermentation broth thereof according to claim 1, or the freeze-dried powder thereof.

4. The composition of claim 3, wherein, The number of cells of Bifidobacterium longum subsp. longum CCFM1319 is > 1 x 10 8 CFU / g or 1 x 10 8 CFU / mL.

5. A product comprising the Bifidobacterium longum subsp. longum CCFM1319 of claim 1 or a fermentation broth thereof, or a lyophilized powder thereof, characterized in that, The product is a pharmaceutical product.

6. The product of claim 5, wherein, The dosage form of the pharmaceutical product includes granules, capsules, tablets, pills or oral liquid.

7. Use of the Bifidobacterium longum subsp. longum CCFM1319 according to claim 1 or the microbial preparation according to claim 2 in the preparation of a medicament for alleviating intractable constipation, slow transit constipation or gastrointestinal motility disorder.

8. Use according to claim 7, characterized in that, The slow transit constipation includes a laxative colon.

9. Use according to claim 8, characterized in that, The use includes at least one of the following functions: (a) shortening the first black stool time of intractable constipation individuals, increasing the water content of feces, and increasing the defecation frequency; (b) increasing enteric glial cell marker expression in colonic tissue of a constipated individual S100β expression levels, repair enteric nerve damage; (c) Reduce inflammatory factors in the colonic tissue of individuals with refractory constipation IL-1β , IL-6 Expression level, alleviating inflammation of the intestinal immune barrier; (d) reducing colonic histopathological damage, promoting tight junction proteins in colonic tissue of individuals with refractory constipation Occludin expression, repair of intestinal mechanical barrier damage; (e) decreasing aquaporin in colonic tissue of a constipated individual AQP4 expression levels, regulation of intestinal stem cells by the WNT / beta-catenin signaling pathway, regulation of the intestinal chemical barrier (f) improving the intestinal microecological imbalance of intractable constipation mammals and repairing the damage of intestinal biological barrier.

10. Use according to claim 9, characterized in that, The dosage form of the medicament is granules, capsules, tablets, pills or oral liquid.

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