Bifidobacterium longum subsp. Infantis for regulating intestinal motility and aquaporin and application thereof

Abstract

The invention discloses bifidobacterium longum subsp. Infantis for regulating intestinal motility and aquaporin and application of the bifidobacterium longum subsp. Infantis, and belongs to the technical field of microorganisms. According to the invention, the bifidobacterium longum subsp. Infantis CCFM1269 is screened, and the bifidobacterium longum subsp. Infantis CCFM1269 has the effects of intestinal motility and aquaporin, specifically, the defecation frequency is reduced, and the water content of excrement and the small intestine propulsion rate are reduced. The intestinal motility abnormality is relieved by reducing the levels of neurotransmitters 5-HT, SP and VIP, and the intestinal moisture absorption and secretion are regulated by improving the mRNA expression of aquaporin AQP4 and AQP8. Meanwhile, the intestinal flora is adjusted by improving the diversity of the intestinal flora, and the content of propionic acid and butyric acid in excrement is increased.

Description

Technical Field

[0001] This invention relates to a strain of Bifidobacterium longum infantis that regulates intestinal motility and aquaporins and its applications, belonging to the field of microbial technology. Background Technology

[0002] Diarrhea is a common digestive tract disease in infants and young children, and is the second leading cause of death in children under 5 years old worldwide. Chronic diarrhea lasts longer than 14 days, with increased frequency and altered stool consistency as the main clinical symptoms. Prolonged chronic diarrhea can lead to malnutrition, growth retardation, impaired immune function, and even death in children. Therefore, it is crucial to take appropriate measures to improve diarrhea symptoms. Due to the complex etiology and pathogenesis of chronic diarrhea, diagnosis and treatment are difficult, and the clinical prognosis is poor, making it a focus of clinical attention. Currently, clinical treatment for infant diarrhea mainly involves antidiarrheal drugs, fluid replacement, and probiotics to correct dehydration and electrolyte imbalances, but the therapeutic effects have not been entirely satisfactory.

[0003] A growing body of research indicates a close relationship between diarrheal diseases and the gut microbiota. Reports suggest that children with chronic diarrhea often exhibit an imbalanced gut microbiota, with abnormalities in intestinal motility, water absorption, and secretion. Furthermore, the levels of metabolic products such as short-chain fatty acids are abnormal. This dysbiosis and abnormal metabolic products can lead to a variety of adverse symptoms.

[0004] Probiotics are gaining increasing attention in the prevention and treatment of gastrointestinal diseases. The gut microbiota is crucial for maintaining host health and is also involved in the pathogenesis of diarrhea. Early life is a critical stage for the initial colonization of the infant's gut microbiota, during which the immune system develops and matures. These processes have long-term effects that may extend into adolescence and even adulthood. Bifidobacteria are considered dominant probiotics in infancy, and the relative abundance of Bifidobacteria in the infant gut is negatively correlated with the occurrence and development of many diseases, suggesting their potential to regulate intestinal immunity and intestinal disorders. However, research on Bifidobacteria's ability to improve chronic diarrhea in infants is limited.

[0005] The diarrhea model caused by senna leaves and restraint stress belongs to functional diarrhea, which is a type of chronic diarrhea. The core cause is intestinal dysfunction caused by drug stimulation and psychological stress. The main active ingredient of senna leaves, sennoside, is not active itself, but after being metabolized by intestinal bacteria, it is converted into rhein anthrone, which has strong irritant properties. This substance directly acts on the enteric nervous system and epithelial cells of the colon, resulting in two main effects: 1) strongly enhancing colonic peristalsis; 2) inhibiting the absorption of water and electrolytes in the intestine. The two work together to increase the water content of intestinal contents and expel them rapidly, resulting in diarrhea. The mechanism by which senna leaves cause constipation is (1) neurotoxic damage (core mechanism): long-term, high-dose exposure to anthraquinones will have a toxic effect on the myenteric plexus of the colon, leading to neuronal degeneration, apoptosis, and even a reduction in the number of neurons. This is known as the nerve damage associated with "melanosis coli". (2) Smooth muscle dysfunction: after losing effective nerve innervation, the contractile ability of the colonic smooth muscle itself will also weaken, becoming relaxed and weak. (3) Drug dependence: Due to nerve damage, the peristaltic function of the colon is severely reduced, making it impossible to defecate normally without drug stimulation. This eventually leads to laxative-dependent constipation, where defecation is impossible without medication. Chronic diarrhea requires long-term treatment targeting the underlying cause, aiming to regulate intestinal function and microecological balance. Chronic diarrhea differs significantly from other types of diarrhea in terms of pathogenesis and treatment. For example, acute diarrhea in infants and young children is mainly caused by viruses, bacteria, and parasites. Pathogenic bacteria invade the intestinal mucosal epithelial cells, causing inflammation, ulcers, and bloody stools. At the same time, pathogenic bacteria produce enterotoxins, stimulating the intestinal mucosa to secrete large amounts of water and electrolytes, leading to secretory diarrhea. Furthermore, pathogenic bacteria adhere to the intestinal mucosa, damaging the function of microvilli. Oral rehydration salts are the first choice for the treatment of acute diarrhea, as they can simultaneously replenish water, sodium, potassium, chloride, and glucose.

[0006] Because the pathogenesis of chronic and acute diarrhea is fundamentally different, and because the mechanisms of diarrhea and constipation caused by short-term and long-term senna leaf modeling differ, as well as the specificity and individualization of different probiotics, a probiotic or drug that can treat one type of acute diarrhea may not necessarily treat another type of chronic diarrhea. Different diseases require different probiotics to be tried and matched to explore their effectiveness. However, current technology has not yet discovered a probiotic that can effectively treat chronic diarrhea. Summary of the Invention

[0007] The purpose of this invention is to overcome the shortcomings of existing technologies and, through in vivo animal experiments, obtain a strain of *Bifidobacterium longum* subsp. *infantile* that can alleviate chronic diarrhea in infants and young children. This strain reduces the number of fecal particles, decreases fecal water content, and reduces small intestinal propulsion rate. It improves gastrointestinal motility abnormalities by reducing the levels of 5-hydroxytryptamine (5-HT), substance P (SP), and vasoactive intestinal peptide (VIP). It regulates intestinal water secretion and absorption by modulating the mRNA expression of aquaporins. Furthermore, it enhances gut microbiota diversity and regulates the content of short-chain fatty acids in feces.

[0008] To achieve the above objectives, the present invention provides the use of Bifidobacterium longum subsp. infantis CCFM1269 in the preparation of medicaments for the prevention and / or treatment of chronic diarrhea.

[0009] In one embodiment of the present invention, the viable count of the above-mentioned Bifidobacterium longum subsp. infantis CCFM1269 in the drug is not less than 1×10⁻⁶. 9 CFU / g or 1×10 9 CFU / mL.

[0010] In one embodiment of the present invention, the drug contains the above-mentioned Bifidobacterium longum infantis subsp. CCFM1269, a drug carrier, and / or pharmaceutical excipients.

[0011] In one embodiment of the present invention, the drug carrier includes microcapsules, microspheres, nanoparticles and / or liposomes; the drug excipients include fillers, binders, wetting agents, disintegrants, excipients and / or flavoring agents.

[0012] In one embodiment of the present invention, the drug contains one or more of the following substances: a live strain of Bifidobacterium longum subsp. infantis CCFM1269, a dried strain of Bifidobacterium longum subsp. infantis CCFM1269, metabolites of Bifidobacterium longum subsp. infantis CCFM1269, inactivated Bifidobacterium longum subsp. infantis CCFM1269, lysates of Bifidobacterium longum subsp. infantis CCFM1269, or extracts of Bifidobacterium longum subsp. infantis CCFM1269.

[0013] In one embodiment of the present invention, the drug is a microbial preparation containing Bifidobacterium longum subsp. infantis CCFM1269.

[0014] In one embodiment of the present invention, the microbial preparation includes a solid preparation, a liquid preparation, or a semi-solid preparation.

[0015] In one embodiment of the present invention, the solid dosage form includes powder or tablets.

[0016] In one embodiment of the present invention, the preparation method of the drug is as follows: the above-mentioned Bifidobacterium longum subsp. infantis CCFM1269 is inoculated into the culture medium at an inoculation amount of 1% to 5% of the total mass of the culture medium, and cultured at 37°C for 24 h to obtain a culture solution; the culture solution is centrifuged to obtain bacterial cells; the bacterial cells are resuspended in physiological saline and then combined with a drug carrier and / or pharmaceutical excipients.

[0017] In one embodiment of the present invention, the culture medium is mMRS culture medium.

[0018] This invention also provides the application of Bifidobacterium longum infantis subspecies CCFM1269 in the preparation of health products that regulate intestinal flora.

[0019] Beneficial effects: This invention confirms that *Bifidobacterium longum* subsp. infantis CCFM1269 can reduce fecal particle count, decrease fecal water content, and improve small intestinal propulsion rate. It improves gastrointestinal motility abnormalities by reducing 5-HT, SP, and VIP levels. It regulates intestinal water secretion and absorption by modulating AQP mRNA expression. Furthermore, it enhances gut microbiota diversity and regulates the content of short-chain fatty acids in feces. It exhibits good preventative or alleviating effects on chronic diarrhea, thus it can be used to prepare probiotic drugs for the prevention and treatment of chronic diarrhea, showing very broad application prospects. Attached Figure Description

[0020] Figure 1 The change in the number of fecal particles within 10 minutes in each group indicates that *Bifidobacterium longum* subsp. infantis CCFM1269 reduced the frequency of defecation in diarrheal pups. *: p<0.05, **: p<0.01, ***: p<0.001, ****: p<0.0001.

[0021] Figure 2 The changes in fecal water content in each group indicate that *Bifidobacterium longum* subsp. infantis CCFM1269 reduced fecal water content in diarrheal young mice. In the figure, *: p < 0.05, **: p < 0.01, ***: p < 0.001, ****: p < 0.0001.

[0022] Figure 3 The figures show the changes in small intestinal propulsion rate in each group, indicating that *Bifidobacterium longum infantis* subsp. CCFM1269 reduced the small intestinal propulsion rate in diarrheal pups. In the figure, *: p < 0.05, **: p < 0.01, ***: p < 0.001, ****: p < 0.0001.

[0023] Figure 4The figures show the changes in 5-HT levels in the serum of each group, indicating that *Bifidobacterium longum infantis* subsp. CCFM1269 reduced 5-HT levels in diarrheal young mice. *: p < 0.05, **: p < 0.01, ***: p < 0.001, ****: p < 0.0001.

[0024] Figure 5 The changes in SP levels in the serum of each group indicate that *Bifidobacterium longum infantis* subsp. CCFM1269 reduced SP levels in diarrheal infant mice. In the figure, *: p<0.05, **: p<0.01, ***: p<0.001, ****: p<0.0001.

[0025] Figure 6 The changes in VIP levels in the serum of each group indicate that *Bifidobacterium longum infantis* subspecies CCFM1269 reduced VIP levels in diarrheal infant mice. In the figure, *: p < 0.05, **: p < 0.01, ***: p < 0.001, ****: p < 0.0001.

[0026] Figure 7 The figures show the changes in the relative expression level of aquaporin AQP4 mRNA in the colon of each group, indicating that Bifidobacterium longum infantis subspecies CCFM1269 reduced the relative expression level of aquaporin AQP4 mRNA in diarrheal young mice. *: p < 0.05, **: p < 0.01, ***: p < 0.001, ****: p < 0.0001.

[0027] Figure 8 The figures show the changes in the relative expression level of aquaporin AQP8 mRNA in the colon of each group, indicating that *Bifidobacterium longum* subsp. infantis CCFM1269 reduced the relative expression level of aquaporin AQP8 mRNA in diarrheal young mice. *: p < 0.05, **: p < 0.01, ***: p < 0.001, ****: p < 0.0001.

[0028] Figure 9 The changes in the Shannon index, representing the α diversity in fecal 16S rDNA sequencing of each group, indicate that Bifidobacterium longum subsp. infantis CCFM1269 can improve the α diversity of the bacterial community; in the figure, *: p<0.05, **: p<0.01, ***: p<0.001, ****: p<0.0001.

[0029] Figure 10 The values ​​represent the propionic acid content in the feces of rats in each group, indicating that *Bifidobacterium longum infantis* subsp. CCFM1269 can increase the propionic acid content in the feces of young rats. In the figure, *: p<0.05, **: p<0.01, ***: p<0.001, ****: p<0.0001.

[0030] Figure 11 The figures show the butyrate content in the feces of rats in each group, indicating that *Bifidobacterium longum infantis* subsp. CCFM1269 can increase the butyrate content in the feces of young rats. *: p<0.05, **: p<0.01, ***: p<0.001, ****: p<0.0001. Detailed Implementation

[0031] The invention will be better understood through the following examples.

[0032] In this invention, unless otherwise specified, "%" or percentage used to describe concentration or proportion refers to weight percentage.

[0033] This invention relates to the following culture media: mMRS liquid culture medium: 10 g tryptone, 10 g beef extract, 5 g yeast powder, 20 g glucose, 2 g diammonium citrate, 5 g sodium acetate, 2 g dipotassium hydrogen phosphate, 0.5 g magnesium sulfate heptahydrate, 0.25 g manganese sulfate monohydrate, 1 mL Tween 80, 0.5 g / L cysteine ​​amino acid, add water to 1000 mL.

[0034] mMRS solid medium is obtained by adding 1.5% agar based on the total weight of the liquid medium to the above.

[0035] The *Bifidobacterium longum* subsp. *infantica* CCFM1269 mentioned in the following examples is disclosed in patent CN115820488B. The senna leaf extract mentioned in the following examples was purchased from Xi'an Xuhuang Biotechnology Co., Ltd.

[0036] Example 1: Culture of Bifidobacterium longum subsp. infantis CCFM1269 (1) Activation culture: The culture was carried out in mMRS solid medium (containing 0.05 g / 100 mL cysteine) and anaerobic culture at 37 ℃.

[0037] Culture objective: To activate Bifidobacterium longum subsp. infantis CCFM1269 by taking cryopreserved bacterial cells, inoculating them into mMRS solid medium, and incubating them statically in an anaerobic incubator at 37 ℃ for about 48 h. (2) Primary training: The culture was carried out in MRS liquid medium (containing 0.05 g / 100 mL cysteine) and anaerobic at 37 °C.

[0038] Culture objective: To transfer activated cultured Bifidobacterium longum subsp. infantis CCFM1269 to mMRS liquid medium (containing 0.05 g / 100 mL cysteine) at an inoculum volume of 2% and passage it for two generations.

[0039] (3) Secondary cultivation: The culture medium and culture conditions are the same as those of the first-stage culture.

[0040] Bifidobacterium longum subsp. infantis CCFM1269, after primary culture, was transferred at an inoculum volume of 2% to 1 L mMRS liquid medium (containing 0.05 g / 100 mL cysteine). The culture was then anaerobic at 37 °C for approximately 24 h, and the cells were collected. The cells were washed twice with physiological saline, resuspended in 30% sucrose solution, and stored at -80 °C until use. The viable count was 6 × 10⁻⁶. 10 CFU / mL. For subsequent experiments, centrifuge to discard the 30% sucrose solution, wash once with physiological saline, and then resuspend in physiological saline.

[0041] Example 2: Effects of Bifidobacterium longum infantis subsp. CCFM1269 on defecation parameters in young mice with chronic diarrhea 1. Laboratory animals SPF-grade SD pregnant rats were purchased from Beijing Vital River Laboratory Animal Technology Co., Ltd. The rats were housed in polypropylene cages, one pregnant rat per cage, with food and water provided. The temperature (22 ℃) and relative humidity (50% ± 10%) were controlled, with free access to water. They were fed standard feed. Male pups were used for experiments after birth.

[0042] 2. Experimental grouping and drug administration Eight mice were placed in the control group without treatment, while the remaining 24 mice were randomly divided into three groups: the model group, the *Bifidobacterium longum* subsp. infantis CCFM1269 treatment group, and the positive control drug (montmorillonite powder) group. Intervention was administered from day 7 to 28. The *Bifidobacterium longum* subsp. infantis CCFM1269 treatment group received 2 × 10⁻⁶ mice via gavage. 9 CFU / 0.2 mL / mouse / day; the positive control group was administered 0.2 mL (300 mg / kg) of montmorillonite powder dissolved in physiological saline by gavage; the control group and model group were administered the same volume of physiological saline by gavage. Modeling was performed from days 14 to 28. Except for the control group, each group of young mice was administered 0.1 mL (500 mg / kg) of senna leaf extract by gavage daily and restrained in a transparent plastic bottle for 3 hours. They were separated into different cages on day 21.

[0043] Chronic diarrhea can lead to increased bowel movements, increased stool water content, and abnormal intestinal motility in children. In severe cases, it can cause malnutrition, stunted growth, and other problems, affecting the patient's quality of life. Bowel movement frequency, stool water content, and small intestinal propulsion rate are important indicators for alleviating the condition.

[0044] Appendix Figure 1The results showed that the number of fecal particles in the control group was significantly lower than that in the model group (p<0.05). Compared with the model group, both the *Bifidobacterium longum* subsp. infantis CCFM1269 group and the montmorillonite powder group significantly reduced the number of fecal particles in diarrheal mice (p<0.05). The number of fecal particles in the control group was 2.5 particles / 10 minutes, the model group was 4.50 particles / 10 minutes, the montmorillonite powder group was 3.00 particles / 10 minutes, and the *Bifidobacterium longum* subsp. infantis CCFM1269 group was 3.50 particles / 10 minutes.

[0045] Appendix Figure 2 The results showed that the fecal water content in the control group was significantly lower than that in the model group (p<0.05). Compared with the model group, both the *Bifidobacterium longum* subsp. infantis CCFM1269 group and the montmorillonite powder group significantly reduced the fecal water content in diarrheal mice (p<0.05). The fecal water content was 55.23% in the control group, 74.60% in the model group, 67.93% in the montmorillonite powder group, and 69.68% in the *Bifidobacterium longum* subsp. infantis CCFM1269 group.

[0046] Appendix Figure 3 The results showed that the small intestinal propulsion rate in the control group was significantly lower than that in the model group (p<0.05). Compared with the model group, both the *Bifidobacterium longum* subsp. infantis CCFM1269 group and the montmorillonite powder group significantly reduced the small intestinal propulsion rate in diarrheal mice (p<0.05). The small intestinal propulsion rate was 63.53% in the control group, 79.58% in the model group, 68.49% in the montmorillonite powder group, and 71.35% in the *Bifidobacterium longum* subsp. infantis CCFM1269 group.

[0047] According to Chinese patent CN117821305A, mice treated with Bifidobacterium bifidum CCFM1359 showed a significant reduction in intestinal transit time (98.5 hours) by 18.26% (p < 0.01); a 200% increase in fecal particle count within 5 hours (22.5 particles) (p < 0.01); and a 31.92% increase in small intestinal propulsion rate (73.15%) (p < 0.01). This indicates that oral administration of Bifidobacterium bifidum CCFM1359 can repair enteric nerve damage caused by senna leaves in mice and accelerate intestinal motility, without slowing down intestinal motility in mice treated with senna leaves.

[0048] Example 3: Bifidobacterium longum infantis subsp. CCFM1269 reduced serum 5-HT, SP, and VIP levels in young mice with chronic diarrhea. See Examples 1 and 2 for strain culture and animal experiments.

[0049] The levels of 5-HT, SP, and VIP in the serum of diarrheal young mice were detected using enzyme-linked immunosorbent assay (ELISA).

[0050] Diarrhea irritates or damages the intestinal mucosa. This stimulation activates enterochromaffin cells to release large amounts of 5-HT and activates the enteric nervous system, leading to increased excitability of neurons releasing SP and VIP. Abnormally elevated levels of 5-HT, SP, and VIP can cause accelerated intestinal motility and significant loss of water and electrolytes, thus resulting in diarrhea.

[0051] Appendix Figure 4 The results showed that the serum 5-HT level in the blank group was 17.58 ng / mL, in the model group it was 21.33 ng / mL, in the montmorillonite powder group it was 18.02 ng / mL, and in the Bifidobacterium longum subsp. infantis CCFM1269 group it was 18.85 ng / mL. The serum 5-HT level in the blank group was significantly lower than that in the model group (p<0.05). Compared with the model group, the serum 5-HT level in the montmorillonite powder group decreased by 15.53%, and in the Bifidobacterium longum subsp. infantis CCFM1269 group it decreased by 11.65%.

[0052] Appendix Figure 5 The results showed that the serum SP level in the blank group was 2.12 ng / mL, in the model group it was 2.83 ng / mL, in the montmorillonite powder group it was 2.25 ng / mL, and in the Bifidobacterium longum subsp. infantis CCFM1269 group it was 2.30 ng / mL. The serum SP level in the blank group was significantly lower than that in the model group (p<0.05). Compared with the model group, the serum SP level in the montmorillonite powder group decreased by 20.60%, and in the Bifidobacterium longum subsp. infantis CCFM1269 group it decreased by 18.56%.

[0053] Appendix Figure 6 The results showed that the serum VIP level in the blank group was 245.42 pg / mL, in the model group it was 334.15 pg / mL, in the montmorillonite powder group it was 280.31 pg / mL, and in the Bifidobacterium longum subsp. infantis CCFM1269 group it was 296.76 pg / mL. The serum VIP level in the blank group was significantly lower than that in the model group (p<0.05). Compared with the model group, the serum VIP level in the montmorillonite powder group decreased by 16.11%, and in the Bifidobacterium longum subsp. infantis CCFM1269 group it decreased by 11.12%.

[0054] Example 4: Bifidobacterium longum infant subspecies CCFM1269 regulates the mRNA expression of aquaporins in the colon of young mice with chronic diarrhea. See Examples 1 and 2 for strain culture and animal experiments.

[0055] Total RNA was extracted from a portion of the colon using the Trizol method, reverse transcribed into cDNA, and then the relative expression level of aquaporin mRNA was detected using quantitative PCR (qPCR).

[0056] Diarrhea is essentially caused by reduced water absorption and / or excessive water secretion in the intestines, and aquaporins are key to water absorption in the intestines. Many types of diarrhea are directly or indirectly related to impaired function or downregulated expression of aquaporins.

[0057] Appendix Figure 7 The results showed that the expression of aquaporin AQP4 in the control group was significantly higher than that in the model group (p<0.05). Compared with the model group, both the *Bifidobacterium longum* subsp. infantis CCFM1269 group and the montmorillonite powder group significantly increased the mRNA expression of aquaporin AQP4 in the colon of diarrheal young mice (p<0.05). The mRNA expression levels of colonic AQP4 in the model group, the montmorillonite powder group, and the *Bifidobacterium longum* subsp. infantis CCFM1269 group were 0.54-fold, 0.76-fold, and 0.88-fold higher than those in the control group, respectively.

[0058] Appendix Figure 8 The results showed that the expression of aquaporin AQP8 in the control group was significantly higher than that in the model group (p<0.05). Compared with the model group, both the *Bifidobacterium longum* subsp. infantis CCFM1269 group and the montmorillonite powder group significantly increased the mRNA expression of aquaporin AQP8 in the colon of diarrheal young mice (p<0.05). The mRNA expression levels of colonic AQP8 in the model group, the montmorillonite powder group, and the *Bifidobacterium longum* subsp. infantis CCFM1269 group were 0.68-fold, 0.89-fold, and 0.92-fold higher than those in the control group, respectively.

[0059] Example 5: Bifidobacterium longum infantis subspecies CCFM1269 improves fecal microbiota diversity in young mice with chronic diarrhea. See Examples 1 and 2 for strain culture and animal experiments.

[0060] Feces from young mice were collected the day before sacrifice and stored at -80 °C for subsequent experiments. Fecal DNA was extracted using a rapid DNA extraction kit and then sequenced as 16S rDNA to analyze changes in the gut microbiota.

[0061] Maintaining a stable gut microbiota is crucial, and children with chronic diarrhea often suffer from gut microbiota dysbiosis, primarily characterized by reduced gut microbiota diversity. Restoring gut microbiota homeostasis can effectively alleviate chronic diarrhea.

[0062] Appendix Figure 9 This indicates that the Shannon index in the model group was significantly lower than that in the control group (p<0.05). This suggests that the microbial diversity in the model group was significantly reduced. Bifidobacterium longum subsp. infantis CCFM1269 can improve microbial diversity, while montmorillonite powder has no such effect.

[0063] Example 6: Bifidobacterium longum infantis subspecies CCFM1269 improves short-chain fatty acid content in young mice with chronic diarrhea. See Examples 1 and 2 for strain culture and animal experiments.

[0064] Feces from young mice were collected the day before sacrifice and stored at -80 °C for subsequent experiments. After sample pretreatment, the content of short-chain fatty acids in the feces was measured using GC-MS.

[0065] Short-chain fatty acids (SCFAs) are among the most important metabolites of gut microbiota that benefit host health. They are not only an energy source for the gut but also key signaling molecules regulating systemic immunity, metabolism, and endocrine function. Multiple studies have shown that abnormalities in SCFAs and other metabolites in the feces of children with chronic diarrhea can help alleviate diarrhea symptoms by restoring SCFA levels to normal.

[0066] Appendix Figure 10 The results showed that, compared with the control group, the propionic acid content in the feces of the model group was significantly reduced (p<0.05), and *Bifidobacterium longum* subsp. infantis CCFM1269 could increase the propionic acid content in the feces (p<0.05). The propionic acid content in the feces of the control group, model group, montmorillonite powder group, and *Bifidobacterium longum* subsp. infantis CCFM1269 group were 7.84 μmol / g, 3.42 μmol / g, 6.12 μmol / g, and 8.12 μmol / g, respectively. Compared with the model group, the propionic acid content in the montmorillonite powder group and the *Bifidobacterium longum* subsp. infantis CCFM1269 group increased by 79.13% and 137.62%, respectively. *Bifidobacterium longum* subsp. infantis CCFM1269 was more effective than montmorillonite powder in increasing propionic acid levels.

[0067] Appendix Figure 11 The results showed that, compared with the control group, the fecal butyrate content in the model group was significantly reduced (p<0.05). *Bifidobacterium longum* subsp. infantis CCFM1269 increased the fecal butyrate content (p<0.05), while montmorillonite powder had no such effect. The fecal propionic acid content in the control group, model group, montmorillonite powder group, and *Bifidobacterium longum* subsp. infantis CCFM1269 group were 10.32 μmol / g, 6.36 μmol / g, 7.27 μmol / g, and 10.59 μmol / g, respectively. Compared with the model group, the montmorillonite powder group and the *Bifidobacterium longum* subsp. infantis CCFM1269 group increased by 14.35% and 66.44%, respectively. *Bifidobacterium longum* subsp. infantis CCFM1269 was more effective than montmorillonite powder in increasing butyrate content, and the butyrate content reached a level comparable to the control group.

[0068] The above experimental results indicate that *Bifidobacterium longum* subsp. infantis CCFM1269 can alleviate chronic diarrhea symptoms in young mice, specifically by reducing defecation frequency, fecal water content, and small intestinal propulsion rate. It alleviates intestinal motility abnormalities by reducing the levels of neurotransmitters 5-HT, SP, and VIP, and regulates intestinal water absorption and secretion by increasing the mRNA expression of aquaporins AQP4 and AQP8. Simultaneously, it regulates the gut microbiota by increasing gut microbiota diversity and increases the content of propionic and butyric acids in feces.

[0069] In conclusion, Bifidobacterium longum subsp. infantis CCFM1269 can significantly improve chronic diarrhea in young mice, and it can be used to prepare drugs for the prevention and treatment of chronic diarrhea.

[0070] Example 7: Preparation of tablets containing Bifidobacterium longum subsp. infantis CCFM1269 bacterial agent The basic steps of the specific production process are as follows: strain activation → scale-up culture → cell collection → bacterial suspension preparation → freeze drying → total mixing → tableting. 1. Activation of bacterial strain: Bifidobacterium longum subsp. infantis CCFM1269 was inoculated at 1% of the culture medium volume in mMRS liquid medium and statically cultured in an anaerobic workstation at 37°C for two consecutive generations.

[0071] 2. Expanded culture: The activated Bifidobacterium longum subsp. infantis CCFM1269 was transferred to 1L mMRS liquid medium at an inoculation rate of 1% of the culture medium volume for expanded culture, and then incubated statically in an anaerobic workstation at 37℃ for 24h.

[0072] 3. Collect bacterial cells and prepare bacterial suspension: After the expansion culture is completed, collect the bacterial cells by centrifugation at 4°C, wash twice with PBS (pH 7.4), and then prepare a 10-fold suspension using a 13% skim milk aqueous solution (by weight). 9 CFU / mL bacterial suspension. 4. Freeze-drying: Prepare bacterial powder using conventional freeze-drying processes.

[0073] 5. Mixing: Add 2% stearic acid (by total weight of bacterial powder) as a lubricant and 3% CMC-Na as a binder, and mix thoroughly. 6. Tableting: Compress the tablets using a tableting machine according to standard tableting procedures. Example 8: Preparation of a powder containing Bifidobacterium longum subsp. infantis CCFM1269 bacterial agent The basic steps of the specific production process are as follows: strain activation → scale-up culture → collection of bacterial cells → preparation of bacterial suspension → freeze drying The steps of strain activation, large-scale culture, collection of bacterial cells, and preparation of bacterial suspension are as described above.

[0074] Freeze-drying: Freeze-dried bacterial powder is prepared according to conventional freeze-drying process.

[0075] Although the present invention has been disclosed above with reference to preferred embodiments, it is not intended to limit the present invention. Anyone skilled in the art can make various modifications and alterations without departing from the spirit and scope of the present invention. Therefore, the scope of protection of the present invention should be determined by the claims.

Claims

1. Bifidobacterium longum subsp. infantis (B. longum subsp. infantis) Bifidobacterium longum subsp. infantis ) CCFM1269 for use in the preparation of a medicament for the prevention and / or treatment of chronic diarrhea.

2. Use according to claim 1, wherein The drug has at least one of the following effects: (1) reducing the frequency of defecation; (2) reducing the water content of feces; (3) reducing the small intestine propulsion rate; (4) reducing the contents of 5-hydroxytryptamine, substance P and vasoactive intestinal peptide; (5) regulating the mRNA expression of aquaporin; (6) improving the intestinal flora diversity; (7) regulating the content of intestinal short-chain fatty acids.

3. Use according to claim 2, characterized in that, The number of viable bacteria of Bifidobacterium longum subsp. infantis CCFM1269 in the drug product is not less than 1 x 10 9 CFU / g or 1 x 10 9 CFU / mL.

4. The use according to claim 3, wherein the compound is ###0002### The drug contains the Bifidobacterium longum subsp. infantis CCFM1269, a drug carrier and / or a pharmaceutical excipient.

5. The use according to claim 4, wherein the compound is ###0002### The drug carrier includes microcapsules, microspheres, nanoparticles and / or liposomes.

6. The use according to claim 5, wherein the compound is ###0002### The pharmaceutical excipient includes fillers, binders, wetting agents, disintegrants, excipients and / or flavorings.

7. Use according to claim 6, wherein The dosage form of the drug includes solid preparations, liquid preparations or semi-solid preparations; the solid preparations include powders or tablets.

8. Use according to claim 7, wherein the compound is ###0002### The drug contains one or more of the following substances: live strains of the Bifidobacterium longum subsp. infantis CCFM1269, dry strains of the Bifidobacterium longum subsp. infantis CCFM1269, metabolites of the Bifidobacterium longum subsp. infantis CCFM1269, inactivated Bifidobacterium longum subsp. infantis CCFM1269, lysates of the Bifidobacterium longum subsp. infantis CCFM1269 or extracts of the Bifidobacterium longum subsp. infantis CCFM1269.

9. Use according to claim 8, wherein the compound is ###0002### The drug is a microbial preparation containing the Bifidobacterium longum subsp. infantis CCFM1269.

10. Use of the Bifidobacterium longum subsp. infantis CCFM1269 in the preparation of health products for regulating intestinal flora.

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