Bifidobacterium bifidum WP139 for relieving irritable bowel syndrome, product and application

Abstract

The invention relates to bifidobacterium bifidum WP139 for relieving irritable bowel syndrome, a product and application. The bifidobacterium bifidum WP139 is preserved in the China General Microbiological Culture Collection Center (CGMCC), and the preservation number is CGMCC NO.35956. The invention also relates to a preparation method of the bifidobacterium bifidum WP139. The compound can tolerate the gastrointestinal tract environment, can be effectively colonized in the intestinal tract, comprehensively relieves multiple core symptoms of IBS through specific mechanisms such as anti-inflammation, barrier enhancement and flora adjustment, has excellent performance in the aspects of improving the intestinal barrier function, adjusting immune balance and relieving visceral hypersensitivity, provides a new solution for intervention of IBS, and has a wide application prospect. And a foundation is laid for the development of a targeted probiotic preparation.

Description

Technical Field

[0001] This invention belongs to the field of microbial technology, specifically relating to a Bifidobacterium bifidum WP139 for relieving irritable bowel syndrome, the product, and its application. Background Technology

[0002] Irritable bowel syndrome (IBS) is a common functional gastrointestinal disorder with a high global prevalence. Its main clinical features include recurrent abdominal pain and bloating, accompanied by changes in bowel habits (such as diarrhea, constipation, or alternating bowel movements) and stool characteristics. The pathophysiological mechanisms of IBS are complex and not fully understood, but it is generally believed to be closely related to multiple factors, including visceral hypersensitivity, abnormal gastrointestinal motility, brain-gut axis dysfunction, intestinal mucosal immune inflammation, impaired intestinal barrier function, and gut microbiota dysbiosis. In recent years, with the advancement of microbiome research, gut microbiota dysbiosis has been confirmed as one of the key links in the occurrence and development of IBS. IBS patients typically exhibit reduced gut microbial diversity, decreased abundance of beneficial bacteria (such as Bifidobacteria and Lactobacillus), and an increased proportion of potentially pathogenic bacteria. This microecological imbalance may lead to a reduction in beneficial metabolites such as short-chain fatty acids, disruption of intestinal barrier integrity, low-grade inflammation activation, and abnormal nerve signal transduction, thereby exacerbating IBS symptoms. Therefore, using probiotics to correct gut microbiota imbalance and restore microecological balance has become a highly promising strategy for preventing and alleviating IBS.

[0003] Bifidobacteria ( Bifidobacterium Bifidobacteria, as a core beneficial flora in the human gut, have been extensively studied and applied in probiotic products. They have been proven to possess various beneficial properties, including enhancing intestinal barrier function, regulating immune responses, inhibiting pathogenic bacterial growth, and regulating intestinal motility. Currently, several strains on the market claim to be beneficial to gastrointestinal health. However, the efficacy of probiotics exhibits high strain-specificity. This means that different strains, and even different strains of the same species, may have significant differences in physiological function, tolerance, and practical application effects; not all Bifidobacterium strains possess the same ability to alleviate IBS symptoms.

[0004] Although there are some probiotic products for relieving gastrointestinal discomfort in the prior art, the mechanism of action of most of the products is unclear, the clinical evidence is insufficient, or the effect varies from person to person, and there is a lack of stability and universality. In particular, for IBS, a complex syndrome with complex causes, it is urgent to screen new specific strains with clear scientific verification and high efficiency in relieving symptoms, which can better tolerate the gastrointestinal environment (such as gastric acid and bile acid salt), effectively colonize the intestinal tract, and comprehensively relieve multiple core symptoms of IBS through specific mechanisms (such as anti-inflammatory, barrier enhancement, and regulation of flora), to fill the gap in the prior art and provide a safe, efficient and stable microbial intervention scheme for IBS patients.

[0005] And Bifidobacterium bifidum (Bifidobacterium bifidum) Bifidobacterium bifidum ) is one of the core probiotics in the human intestinal tract. The strong colonization ability and high safety of Bifidobacterium bifidum have been widely used in food and clinical practice, and no adverse reactions have been reported. However, different Bifidobacterium bifidum strains have significant differences in function. Therefore, screening new Bifidobacterium bifidum strains that can better tolerate the gastrointestinal environment (such as gastric acid and bile acid salt), effectively colonize the intestinal tract, and comprehensively relieve multiple core symptoms of IBS through specific mechanisms (such as anti-inflammatory, barrier enhancement, and regulation of flora) is of great significance for the development of targeted probiotic preparations. SUMMARY

[0006] The purpose of the present application is to overcome the defects in the prior art and provide a Bifidobacterium bifidum WP139 for relieving irritable bowel syndrome, a product and an application. The Bifidobacterium bifidum WP139 performs excellently in improving intestinal barrier function, regulating immune balance and relieving visceral hypersensitivity, and provides a new solution for the intervention of IBS.

[0007] To achieve the above-mentioned purpose, the technical solutions adopted by the present application are as follows: A Bifidobacterium bifidum WP139 for relieving irritable bowel syndrome, which is preserved in the China General Microbiological Culture Collection Center with a preservation number of CGMCC NO. 35956.

[0008] As a further technical solution, it is isolated from infant feces.

[0009] As a further technical solution, the Bifidobacterium bifidum WP139 is obtained by static culture at 37 DEG C based on LMRS culture.

[0010] As a further technical solution, the LMRS culture medium is MRS culture medium added with 0.05% (w / v) L-cysteine hydrochloride.

[0011] As a further technical solution, the Bifidobacterium bifidum WP139 has bacteriostatic property to Citrobacter rodentium.

[0012] A targeted probiotic preparation comprising the Bifidobacterium bifidum WP139.

[0013] Use of the Bifidobacterium bifidum WP139 or the targeted probiotic preparation in the preparation of a functional food, health product or drug for relieving irritable bowel syndrome.

[0014] As a further technical solution, the irritable bowel syndrome is induced by Citrobacter rodentium in combination with water avoidance stress.

[0015] As a further technical solution, relieving irritable bowel syndrome includes one or more of relieving visceral hypersensitivity symptoms, relieving diarrhea, improving inflammation, restoring intestinal mucosal permeability and barrier function, and regulating intestinal flora structure and improving intestinal flora imbalance.

[0016] As a further technical solution, the visceral hypersensitivity symptoms include one or more of reducing pain response and reducing tryptase content.

[0017] As a further technical solution, the improvement of inflammation includes one or more of improving inflammatory infiltration, inhibiting the secretion of pro-inflammatory factors, and promoting the secretion of anti-inflammatory factors.

[0018] As a further technical solution, the pro-inflammatory factors include one or more of TNF-α, IL-6, and IL-1β.

[0019] As a further technical solution, the anti-inflammatory factor is IL-10.

[0020] As a further technical solution, the restoration of intestinal mucosal permeability and barrier function includes one or more of inhibiting the rise of diamine oxidase, inhibiting the rise of D-lactic acid, and reducing the expression level of Occludin protein.

[0021] As a further technical solution, the improvement of flora imbalance includes one or more of increasing the relative abundance of Lactobacillus salivarius, Lactobacillus casei, Akkermansia, and Parabacteroides, and inhibiting the increase in abundance of Shigella, Desulfovibrio, Zurichia, and Bacteroides.

[0022] A drug for relieving irritable bowel syndrome, comprising at least one active ingredient, the active ingredient being the Bifidobacterium bifidum WP139 and / or a fermentation product of the Bifidobacterium bifidum WP139.

[0023] A functional food for relieving irritable bowel syndrome, comprising at least one active ingredient, the active ingredient being the Bifidobacterium bifidum WP139 and / or a fermentation product of the Bifidobacterium bifidum WP139.

[0024] 1. The *Bifidobacterium bifidum* WP139 isolated and screened in this invention exhibits excellent acid resistance and adhesion, demonstrating a strong colonization ability in the intestine. It can reduce pain response, decrease trypsin levels, and alleviate visceral hypersensitivity symptoms; it can restore colon length in IBS-D mice, alleviate hyperactivity and diarrhea in IBS-D mice; it can improve inflammatory infiltration in IBS-D mice, maintain colon tissue integrity in model mice, inhibit the secretion of pro-inflammatory factors TNF-α, IL-6, and IL-1β, promote the secretion of anti-inflammatory factor IL-10, and reduce inflammation. It can inhibit the rise of diamine oxidase and D-lactic acid in IBS-D mice, reduce the expression level of Occludin protein in IBS-D mice, restore intestinal mucosal permeability and barrier function; it can improve the richness and diversity of intestinal microbiota, increase the relative abundance of Lactobacillus salivarius, Lactobacillus salivarius, Akkermansia salivarius, and Alternaria salivarius, and inhibit the increase of abundance of Shigella salivarius, Desulfovibrio salivarius, Bacteroides salivarius, and Bacteroides salivarius, regulate the structure of intestinal microbiota, alleviate the intestinal microbiota imbalance caused by irritable bowel syndrome, and improve intestinal barrier function and immune homeostasis.

[0025] 2. This invention is one of the core probiotics of the human gut, and has the characteristic of high safety.

[0026] In summary, the novel Bifidobacterium bifidum WP139 strain screened in this application is tolerant to the gastrointestinal environment, can effectively colonize the intestine, and comprehensively alleviates multiple core symptoms of IBS through specific mechanisms (such as anti-inflammation, barrier enhancement, and microbiota regulation). It shows excellent performance in improving intestinal barrier function, regulating immune balance, and alleviating visceral hypersensitivity, providing a new solution for IBS intervention and laying the foundation for the development of targeted probiotic preparations. Attached Figure Description

[0027] Figure 1 Figure 1 shows the effect of Bifidobacterium bifidum WP139 on visceral hypersensitivity symptoms in mice under different atmospheric pressures. exist Figure 1 Medium, A: 20mmHg; B: 30mmHg; C: 40mmHg; Figure 2 The effect of Bifidobacterium bifidum WP139 on the content of trypsin-like enzymes in the colon of mice is shown in the figure. Figure 3 Figure showing the effect of Bifidobacterium bifidum WP139 on colon length in mice; Figure 4 The effect of Bifidobacterium bifidum WP139 on inflammatory infiltration of the colon in mice; Figure 5 The effect of Bifidobacterium bifidum WP139 on inflammatory factors in mouse serum; exist Figure 5Middle, A: TNF-α; B: IL-6; C: IL-1β; D: IL-10; Figure 6 Figure showing the effect of Bifidobacterium bifidum WP139 on intestinal permeability in mice; exist Figure 6 In the diagram, A: plasma diamine oxidase; B: D-lactic acid; Figure 7 Figure showing the effect of Bifidobacterium bifidum WP139 on the expression level of Occludin protein in mouse colon tissue; exist Figure 7 In the image, A: Electrophoresis diagram; B: Protein expression level diagram; Figure 8 The effect of Bifidobacterium bifidum WP139 on the principal components, ACE index, and Shannon index of the intestinal microbiota in mice is shown in the figure. exist Figure 8 In the diagram, A: Non-metric multidimensional scaling analysis plot; B: ACE index plot; C: Shannon index plot; Figure 9 This is a diagram showing the effect of Bifidobacterium bifidum WP139 on the structure of the intestinal microbiota in mice at the phylum level. exist Figure 9 In the diagram, A: Species distribution at the phylum level; B: Relative abundance of Firmicutes; C: Relative abundance of Bacteroidetes; D: Actinobacteria; E: Firmicutes / Bacteroidetes; Figure 10 Figure showing the effect of Bifidobacterium bifidum WP139 on the structure of the intestinal flora in mice at the genus level; exist Figure 10 In the text, A: *Lactobacillus salivarius*; B: *Alternaria*; C: *Ackermania*; E: *Lactobacillus*. F: Enterobacter spp. Shigella; G: Desulfovibrio spp.; H: Lactobacillus spp.; I: Bacteroides spp. Detailed Implementation

[0028] The technical solution of the present invention will be clearly and completely described below with reference to specific embodiments. Obviously, the described embodiments are only some embodiments of the present invention, not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention.

[0029] 1. “Above” and “below” in this article include the original number.

[0030] 2. The term "Bifidobacterium bifidum" in this article refers to... Bifidobacterium bifidum WP139", "Bifidobacterium bifidum ( Bifidobacterium bifidum) Bifidobacterium bifidum WP139” “Bifidobacterium bifidum WP139” “WP139” “WP139 strain” “strain WP139” all refer to Bifidobacterium bifidum with the preservation number of CGMCC NO. 35956 Bifidobacterium bifidum ) WP139.

[0031] 3、The culture medium and reagents used in the experimental examples of the present application are as follows: MRS culture medium: 10 g / L of proteose peptone, 5 g / L of beef infusion powder, 5 g / L of yeast powder, 20 g / L of glucose, 5 g / L of anhydrous sodium acetate, 2 g / L of diammonium hydrogen citrate, 1 g / L of Tween 80, 2 g / L of K2HPO4, 0.2 g / L of MgSO4, 0.05 g / L of MnSO4, pH = 6.80 ± 0.20, 121℃, sterilization for 20 min.

[0032] LMRS culture medium: 0.05% (w / v) of L-cysteine hydrochloride is added to the MRS culture medium, pH = 6.80 ± 0.20, 121℃, sterilization for 20 min.

[0033] LB culture medium: 10 g / L of tryptone, 5 g / L of yeast extract, 10 g / L of sodium chloride (NaCl), pH = 7.00 ± 0.20, 121℃, sterilization for 20 min.

[0034] 4、The activation of the strain and the preparation method of the bacterial suspension in the present application are as follows: Bifidobacterium bifidum activation culture: the strain is taken out from the -80℃ refrigerator, inoculated in the LMRS anaerobic tube liquid medium at a inoculation amount of 2% (v / v), and cultured at 37℃ to obtain the first generation of Bifidobacterium bifidum; then the second generation of Bifidobacterium bifidum WP139 bacterial liquid is obtained by inoculating the LMRS anaerobic tube liquid medium at a inoculation amount of 2% (v / v) and culturing at 37℃ for 16 h.

[0035] Bifidobacterium bifidum WP139 bacterial suspension preparation: Bifidobacterium bifidum WP139 is activated for 2 generations at a inoculation amount of 2%, the cultured Bifidobacterium bifidum bacterial liquid is centrifuged at 10000 r / min for 1 min, the supernatant is poured out, the bacterial body is washed twice with the same volume of PBS, and then resuspended with the same volume of PBS to obtain the Bifidobacterium bifidum WP139 bacterial suspension for standby use.

[0036] 5、The raw materials used in the present application are all commercially available raw materials unless otherwise specified.

[0037] Example 1: Isolation and identification of Bifidobacterium bifidum (1) Isolation and preliminary screening of Bifidobacterium bifidum The collected infant feces were placed in sterile sampling tubes and transported in ice boxes. Under sterile conditions, they were diluted with 0.85% physiological saline, and the appropriate dilution gradient was selected for coating on LMRS+Li agar plates and incubated at 37°C for 48-72 hours. The suspected single colonies were picked by observing their colony morphology with the naked eye, and preliminary screening and purification culture were performed. After purification, the LMRS liquid anaerobic tube was used for 37°C culture for 8-12 hours. After centrifugation to remove the supernatant, the bacteria were resuspended in sterile 25% glycerol water solution and stored in the strain library of Wuhan Micro-Kang Bifidobacterium Research Institute.

[0038] (2) Identification of Bifidobacterium bifidum: The target strains were subjected to liquid expansion culture, and the bacterial cells were collected. The genomic DNA was extracted, and the 16S rDNA fragments were amplified using the universal primers 27F and 1492R as described in paragraph 58 of Chinese invention patent ZL202210478937.4. The PCR amplification products were detected by agarose gel electrophoresis, and the PCR products were sequenced. The PCR reaction system: 10x buffer 10 μL, 10 mM dNTP 2 μL, 1 μL of upper and lower primers, 2 μL of DNA template, 0.5 μL of Taq enzyme, and 34 μL of dd H2O. 95°C pre-denaturation for 10 min; then 94°C for 30 s, 60°C for 30 s, 72°C for 1 min for a total of 35 cycles, and then 72°C extension for 5 min. After gel electrophoresis detection, the PCR products were sent to Wuhan Jin Kai Rui Biological Engineering Co., Ltd. for sequencing. The identified gene sequence was compared in the NCBI database using the BLAST tool. According to the molecular biology identification results, the Latin name of the strain category is Bifidobacterium bifidum , and three strains were determined to be Bifidobacterium bifidum, designated as Bifidobacterium bifidum 1, Bifidobacterium bifidum 2, and Bifidobacterium bifidum 3.

[0039] Example 2: Screening of Bifidobacterium bifidum (Study on the antibacterial ability of Citrobacter rodentium) (1) Preparation of Bifidobacterium bifidum: From the -80°C strain, inoculate into the LMRS anaerobic tube at a 10% inoculation amount, and incubate at 37°C overnight to obtain the first generation of bacterial liquid. Inoculate the first generation of bacterial liquid into the LMRS anaerobic tube at a 5% inoculation amount, and incubate at 37°C for 6-8 hours to obtain the second generation of bacterial liquid. Inoculate the second generation of bacterial liquid into the LMRS anaerobic tube at a 2% inoculation amount, and incubate at 37°C for 18 hours to obtain the third generation of bacterial liquid for standby.

[0040] (2) Preparation of Citrobacter rodentium bacterial liquid: inoculate the strain into 50 mL of LB liquid medium at a 2% inoculation amount, and incubate at 37°C on a shaker for 18 hours to adjust the bacterial liquid concentration to 10 8 CFU / mL for standby.

[0041] 3) Antagonism experiment: After washing *Citrobacter murineis* twice, resuspend it in PBS and adjust the OD value to approximately 0.4 (10⁻⁶). 8 (CFU / mL) for later use; 1 mL of Citrobacter muscarinicus suspension was added to 100 mL of LB solid culture medium and mixed well before being poured into a plate containing Oxford cups. After the medium solidified, the Oxford cups were removed, and 150 mL of probiotic solution was added to the wells. The plates were incubated at 37°C for 24 h. The diameter of the inhibition zone was measured. Three replicates were performed for each strain. The results are shown in Table 1. Table 1. Results of Bifidobacterium bifidum inhibiting pathogen growth

[0042] Where a, b, and c are p Significance at the 0.05 level; *Citrobacter rodentium*, a Gram-negative bacterium, is a mucosal pathogen in mice. It shares several pathogenic mechanisms with enteropathogenic *Escherichia coli* (EPEC) and enterohemorrhagic *Escherichia coli* (EHEC). *Citrobacter rodentium* has been used to study mucosal immunology, including the dysregulation of intestinal inflammatory responses during bacterial-induced colitis and the role of the gut microbiota in mediating resistance to intestinal pathogen colonization. Screening for strains that preliminarily inhibit *Citrobacter rodentium* has certain guiding significance for post-infectious diarrhea-predominant irritable bowel syndrome. The results of the inhibition experiment on *Citrobacter rodentium* are shown in Table 1. *Bifidobacterium bifidum* 3 showed the strongest antibacterial activity and was selected as the initial screening strain for further research.

[0043] Example 3: Preservation of bacterial strains The nucleotide sequence of the 16S rDNA of Bifidobacterium bifidum 3 obtained by the method described in Example 1 is shown in SEQ ID: OM403393.1; Bifidobacterium bifidum 3 was named Bifidobacterium bifidum WP139 and deposited for preservation. Its preservation information is as follows: Accession number: CGMCC NO.35956; Classification and nomenclature: Bifidobacterium bifidum Bifidobacterium bifidum ; Deposit date: October 23, 2025; Preservation institution: China General Microbiological Culture Collection Center; Address: Institute of Microbiology, Chinese Academy of Sciences, No. 3, Courtyard 1, Beichen West Road, Chaoyang District, Beijing.

[0044] Example 4: Evaluation of the gastric juice tolerance of Bifidobacterium bifidum WP139 (1) Reagent preparation: Artificial gastric juice preparation: 0.5% physiological saline was prepared, 0.3% pepsin was added, and 1 moL / L HCl was used to adjust the pH to 3. After complete dissolution, 0.22 μm microporous filter membrane was used for filtration and sterilization for standby use; (2) Experimental strain preparation: According to the preparation method of Bifidobacterium bifidum WP139 bacterial suspension, Bifidobacterium bifidum WP139 bacterial suspension was obtained, and according to the previous live bacteria counting results, the live bacteria number of the bacterial solution was adjusted to 10 8 CFU / mL for the experiment.

[0045] (3) Artificial gastric juice resistance experiment: 0.1 mL of bacterial suspension was added into 0.9 mL of artificial gastric juice (pH=3) respectively, and incubated at 37℃ for 3 h; Gradient dilution was performed on the treated liquid at 0 h and 3 h, and then plated on LMRS plates for viable cell counting. The survival rate of the strain in artificial gastric juice and bile salt was calculated according to the following formula, and the results are shown in Table 2;

[0046] Wherein, N1 represents the number of viable bacteria in the strain system after treatment, CFU / mL; N0 represents the initial number of viable bacteria in the strain system, i.e. the number of viable bacteria measured at 0 h, CFU / mL; Table 2. Bifidobacterium bifidum artificial gastric juice (pH=3) tolerance results

[0047] The experimental results of Table 2 show that the survival rate of Bifidobacterium bifidum WP139 after 3 h treatment in artificial gastric juice (pH=3) is 93.52%, indicating that it has good gastric juice tolerance, indicating that Bifidobacterium bifidum WP139 can survive in a low-acid gastric juice environment.

[0048] Example 5: Adhesion ability of Bifidobacterium bifidum WP139 to Caco-2 cells (1) Caco-2 cell culture: After 24 h of cell recovery culture, replace the DMEM cell culture medium with 10% fetal bovine serum, and incubate at 37℃ with 5% CO2 until the cells cover 80% of the cell culture bottle.

[0049] (2) Cell passage: aspirate the culture medium, add 2 mL PBS buffer solution for rinsing, discard, add 1 mL trypsin in a T75 culture bottle, shake to make the trypsin completely cover the bottom, aspirate part of the trypsin (about 0.5 mL), digest the cell digestion solution for about 5 min, add 3 mL culture medium to terminate digestion, and gently blow the cells with a pipette to completely detach and disperse them into multiple new cell culture bottles (plates), and continue to culture at 37℃ with 5% CO2.

[0050] (3) Caco-2 cell adhesion experiment Liquid culture of experimental strains: 2nd generation bacteria liquid, 2% inoculation amount 7 mL LMRS medium / anaerobic tube, 37°C, static culture for 15 h, and then the bacteria liquid was obtained for standby.

[0051] Preparation of bacterial suspension of experimental strains: take the cultured target strain bacteria liquid, centrifuge at 10000 r / min at room temperature for 1 min to collect the bacteria, and wash twice with sterile PBS, and finally resuspend in DMEM medium to make the bacterial suspension concentration 1×10 8 CFU / mL; Preparation of single cell layer: Caco-2 cells were inoculated into DMEM medium added with 10% (v / v) fetal bovine serum, transferred to 12-well cell culture plates, and the cell addition amount was 1 mL of 4.0×10 5 cells / mL per well, 5% CO2, 37°C constant temperature culture, and the single cell layer was obtained after 93 h of culture (the medium was changed after 24 h of culture, and then the culture was continued for 24 h, and then the medium was changed, and then the culture was continued for 24 h, and then the medium was changed, and then the culture was continued until the single layer was obtained); Incubation of probiotics and Caco-2 cells: the culture medium of the prepared Caco-2 cell single cell layer was aspirated, rinsed with PBS buffer for 2 times, the buffer was aspirated, and 1 mL of prepared bacterial suspension was added per well, and then mixed, and then incubated at 5% CO2, 37°C for 2 h; the culture supernatant was carefully removed, and sterile PBS was added for rinsing 5 times respectively to remove the unadhered bacteria. Digestion treatment: 0.5 mL of trypsin cell digestion solution was added per well, and after 5 min of digestion, 0.5 mL of PBS was added to terminate the digestion, and the cells were blown off from the cell culture plate well wall with a gun, and the collected solution was the sample. Gradient dilution and viable bacteria counting were performed on the collected sample; the results are shown in Table 3; The adhesion ability was calculated according to the following formula: Adhesion ability (CFU / cell) = number of bacteria adhered to cells (CFU) / number of cells in the well (cells); (4) Lactobacillus rhamnosus LGG was used as a control to determine the adhesion ability.

[0052] Table 3. Adhesion ability of Bifidobacterium bifidum WP139 to human colon cancer cells (Caco-2)

[0053] From the results of the adhesion ability experiment of Bifidobacterium bifidum WP139 to human colon cancer cells in Table 3, the adhesion ability of Bifidobacterium bifidum WP139 to Caco-2 cells was 25.28 ± 2.23 CFU / cell, which was significantly higher than that of the comparative strain Lactobacillus rhamnosus LGG. The experimental results showed that Bifidobacterium bifidum WP139 could adhere well to human colon cancer cells.

[0054] Example 6: Verification of the effect of Bifidobacterium bifidum WP139 in relieving enteritis induced by Citrobacter rodentium combined with water avoidance stress in mice All mice were placed in a controllable temperature (22-24℃) and humidity (40-70%) environment, and the light was cycled according to a 12 h light-dark cycle, and they could freely eat commercial standard diet and distilled water. The pathogenesis of IBS is the result of the combined action of multiple factors such as physiology and psychology, and mild inflammation, visceral hypersensitivity and intestinal flora disorder are the basic clinical features of IBS. Citrobacter rodentium is a pathogenic bacterium that colonizes the colon of rodents and can cause acute intestinal mucosal inflammation, intestinal tissue hyperplasia and intestinal barrier dysfunction. WAS is considered a chronic psychological stress, and repeated performance can cause intestinal flora disorder and visceral pain. Citrobacter rodentium combined with WAS stress is considered a classic IBS model, which is usually modeled in mice and can effectively simulate the physiological and psychological pathogenesis and clinical symptoms of IBS. Therefore, in this study, Citrobacter rodentium infection combined with WAS stress was used to establish a mouse IBS model.

[0055] I. Modeling of intestinal irritable bowel syndrome after infection in mice 30 mice were randomly divided into 3 groups, 10 mice in each group. After the mice were adapted for 7 days, the experiment started, and the experimental time was 30 days. On the first day of the experiment, the mice in the blank group were gavaged with 0.2 mL of sterile normal saline, and the mice in the other groups were gavaged with 0.2 mL of Citrobacter rodentium DBS 100 (5×10 11CFU). From day 2 to day 8, all mice were injected subcutaneously with 0.5 mL of Lactated Ringer's solution to prevent dehydration caused by diarrhea. From day 18 to day 30, all mice were subjected to water avoidance stress (WAS). The WAS device was a water bucket with a diameter of 27 cm and a height of 33 cm. The bottom of the bucket was equipped with a dry platform with a diameter of 4 cm and a height of 9 cm. The water bucket was filled with water, and the water level was 1 cm lower than the platform. The mice were placed on the platform for 1 hour every day, and if the mice fell into the water, they were immediately fished out and dried with a towel. The blank group mice were placed on the platform for 1 hour without water. In addition to the above treatments, from day 2 to day 29, the control group mice and the model group mice were given sterile normal saline (0.2 mL / each / d) by gavage. The Bifidobacterium WP139 group mice were given 0.2 mL of Bifidobacterium WP139 bacterial suspension (5 x 10 10 CFU).

[0056] At the end of the experiment, the mice were sacrificed under carbon dioxide anesthesia. Blood samples were collected and centrifuged at 3500 x g for 20 min at 4°C to collect serum, which was stored at -80°C. The colon of the mice was collected, a portion was fixed with paraformaldehyde, and the remaining portion was stored at -80°C. The cecum was collected and stored at -80°C.

[0057] II. Determination of each index and results 1. Effect of Bifidobacterium WP139 on visceral hypersensitivity symptoms in mice 1) Method: The abdominal withdrawal reflex (AWR) method was used to determine the visceral sensitivity of the colon in mice. The mice in each group were fasted for 16 h before the experiment. After the start of the experiment, the mice were anesthetized with 4% isoflurane and continuously anesthetized with 2% isoflurane. A child urinary catheter (6 Fr, 2 mm outer diameter) was lubricated with liquid paraffin and inserted into the descending colon of the mouse from the anus. The urinary catheter was fixed to the tail of the mouse with tape, and the mouse was placed in a gas-permeable plastic box. After 30 min of adaptation in the plastic box, the colon and rectum were distended by gradually increasing the pressure. The pressure was gradually increased from 10 mmHg, 20 mmHg to 30 mmHg, and each pressure distension lasted for 20 s. The gas was emptied when the pressure was changed, and there was a 5 min rest time after each 20 s distension. After the evaluation was completed, the balloon was deflated and withdrawn.

[0058] The AWR scoring criteria are as follows: 0 points-no obvious reaction; 1 point-short head shaking and then stopping; 2 points-abdominal muscle contraction; 3 points-abdominal lifting; 4 points-arching back and lifting the pelvis. The scores were made by two experimenters who did not know the detailed grouping of the mice, and the results are shown in Table 1. Figure 1 .

[0059] 2) Results and analysis: For IBS patients, the direct manifestation of visceral hypersensitivity is the enhancement of visceral pain sensation, i.e. the enhanced response of patients to painful stimuli and the pain response to painless stimuli. The experimental results are shown in Figure 1 1) In the abdominal withdrawal reflex experiment, the pain sensation of the model group mice was indeed more severe than that of the blank group mice. 2) Under the same gas pressure, the model group mice showed head shaking, abdominal contraction and arching back and other pain responses. This shows that the mice have visceral hypersensitivity symptoms and also proves the successful establishment of the IBS model. 3) In the Bifidobacterium WP139 group, the pain response of the mice was reduced under 10 mmHg gas pressure, 20 mmHg gas pressure and 30 mmHg gas pressure, indicating that Bifidobacterium WP139 can alleviate the visceral hypersensitivity symptoms of IBS-D mice.

[0060] 2, Effect of Bifidobacterium WP139 on trypsin-like enzyme content in diarrhea-predominant irritable bowel syndrome mice 1) Method: Visceral hypersensitivity is essentially a disorder of the peripheral and central nervous system. In the gastrointestinal tract, mast cells release trypsin-like enzymes after being activated, which in turn activate the PAR-2 on the surface of intestinal nerve cells, ultimately causing a sustained nerve excitement state feedback to the gastrointestinal tract, i.e. causing gastrointestinal motility disorders. Therefore, in this embodiment, the trypsin-like enzyme content in the colon tissue of diarrhea-predominant irritable bowel syndrome mice was determined, and the results are shown in Figure 2 .

[0061] Among them, the determination method of trypsin-like enzyme is as follows: after the mice are dissected, the colon tissue of the mice is collected, and the trypsin-like enzyme Elisa kit is used to determine the trypsin-like enzyme content in the colon tissue.

[0062] Results and analysis: From Figure 2It can be seen that the trypsin-like enzyme content of the control group mice was 22.76 ± 6.44 pg / mg; the trypsin-like enzyme content of the IBS-D group mice was 98.62 ± 17.47 pg / mg, and the content of the Bifidobacterium bifidum WP139 intervention group was 52.18 ± 4.74 pg / mg. The experimental results showed that the intervention of Bifidobacterium bifidum WP139 could effectively inhibit the release of trypsin-like enzyme from activated mast cells, thereby weakening the PAR-2 on the surface of intestinal nerve cells, ultimately relieving the persistent nerve excitement state, thereby relieving the visceral sensitivity of IBS-D mice. The results can be mutually verified with the results in the above "Effect of Bifidobacterium bifidum WP139 on the visceral hypersensitivity of mice", which again shows that the intervention of Bifidobacterium bifidum WP139 can effectively relieve the visceral sensitivity of IBS-D mice.

[0063] 3、Bifidobacterium bifidum WP139 on the colon length of mice 1) Method: Studies have shown that the IBS-D model mainly reflects the level of intestinal inflammation, motor function status and mucosal structural integrity, which is one of the intuitive indicators for evaluating the severity of the disease and the effect of treatment. One of the core features of IBS-D is intestinal hyperperistalsis and diarrhea. Colon shortening may be related to overcontraction of intestinal smooth muscle or high motility state, reflecting the acceleration of intestinal transit speed. Therefore, the length of the colon can be used as an indicator to reflect the movement and diarrhea of the intestine. In this embodiment, after the mice were dissected, the colon from the cecum to the anus of the mice was collected, and the length of the colon was measured. The results are shown in Figure 3 ; 2) Results and analysis: From Figure 3 it can be seen that compared with the blank control group, the colon length of the IBS-D mice (MC group) was significantly shortened, and after the intervention of Bifidobacterium bifidum WP139 (WP139 group), the colon length of the IBS-D mice was restored close to that of the blank control mice (NC group); this shows that Bifidobacterium bifidum WP139 effectively relieves the excessive movement of the intestinal tract of IBS-D mice and relieves diarrhea.

[0064] 4、Bifidobacterium bifidum WP139 on the colon inflammation infiltration of diarrhea-predominant irritable bowel syndrome mice 1) Method: In this embodiment, after the intervention, the mice were sacrificed, the colon tissue sections were stained with hematoxylin-eosin, and then histopathological analysis was performed. The mouse colon tissue sections are shown in Figure 4 .

[0065] 2) Results and analysis: From Figure 4The results can be seen: the control group mice colon tissue normal, IBS-D model group mice colon tissue exists inflammatory infiltration phenomenon, Bifidobacterium WP139 intervention group mice intestinal state significantly improved, almost no inflammatory infiltration symptoms. Visible Bifidobacterium WP139 intervention can maintain the colon tissue integrity of the model mice, reduce inflammation.

[0066] 5. Determination of inflammatory factors in serum of diarrhea-predominant irritable bowel syndrome mice by Bifidobacterium WP139 1) Method: To further understand the effect of Bifidobacterium WP139 on intestinal inflammation of diarrhea-predominant irritable bowel syndrome mice, blood was taken and the mice were sacrificed after modeling, the mouse orbital blood was taken, centrifuged at 3000 rpm / min x 15 min, the upper serum was collected, and the levels of TNF-α, IL-6, IL-1β and IL-10 were determined by enzyme-linked immunosorbent assay kit, and the results are shown in Figure 5 .

[0067] 2) Results and analysis: As shown in Figure 5 A, the content of pro-inflammatory factor TNF-α in the serum of the control group mice was 75.88±15.44 pg / mg, the content of pro-inflammatory factor TNF-α in the serum of the IBS-D model group mice was 345.34±21.24 pg / mg, and the content of pro-inflammatory factor TNF-α in the serum of the WP139 intervention group mice was 168.86±10.02 pg / mL. It can be seen that the content of pro-inflammatory factor TNF-α in the serum of the IBS-D model group mice is significantly higher than that of the control group, and the content of pro-inflammatory factor TNF-α in the serum of the mice after WP139 intervention is significantly decreased compared with the model group.

[0068] As shown in Figure 5 B, the content of pro-inflammatory factor IL-6 in the serum of the control group mice was 57.48±8.25 pg / mg, the content of pro-inflammatory factor IL-6 in the serum of the IBS-D model group mice was 261.21±10.88 pg / mg, and the content of pro-inflammatory factor IL-6 in the serum of the WP139 intervention group mice was 138.86±10.02 pg / mg. It can be seen that the content of pro-inflammatory factor IL-6 in the serum of the IBS-D model group mice is significantly higher than that of the control group, and the content of pro-inflammatory factor IL-6 in the serum of the mice after WP139 intervention is significantly decreased compared with the model group, and is close to that of the control group.

[0069] As shown in Figure 5As shown in Figure C, the serum IL-1β level in the control group was 103.81±19.39 pg / mg, the serum IL-1β level in the IBS-D model group was 496.05±59.30 pg / mg, and the serum IL-1β level in the Bifidobacterium WP139 intervention group was 250.45±19.38 pg / mg. The intervention of Bifidobacterium WP139 significantly reduced the serum IL-1β level in mice and was able to restore it to normal levels, which were lower than those in the control group.

[0070] like Figure 5 As shown in Figure D, the serum IL-10 level in the control group was 501.18 ± 70.10 pg / mg, the serum IL-10 level in the IBS-D model group was 113.19 ± 13.05 pg / mg, and the serum IL-10 level in the BC99 intervention group was 288.70 ± 14.29 pg / mg. Compared with the IBS-D model group, the IL-10 level showed an increasing trend after intervention with Bifidobacterium bifidum WP139, indicating that Bifidobacterium bifidum WP139 can promote the production of the anti-inflammatory factor IL-10 and enhance its anti-inflammatory effect.

[0071] 6. Effects of Bifidobacterium bifidum WP139 on intestinal permeability in mice with irritable bowel syndrome 1) Method: Plasma diamine oxidase (DAO) activity is significantly correlated with intestinal mucosal lesions and integrity. The accumulation of D-lactate in plasma reflects intestinal mucosal membrane permeability and barrier function. Therefore, plasma DAO activity and D-lactate concentration can indirectly evaluate intestinal permeability. Thus, in this example, plasma DAO activity and D-lactate concentration were measured, and the results are shown below. Figure 6 ; The methods for measuring diamine oxidase and D-lactic acid are as follows: After modeling, blood was collected from mice and the mice were sacrificed. Blood from the orbital rim was collected, centrifuged at 3000 rpm / min × 15 min, and the supernatant serum was collected. The levels of diamine oxidase and D-lactic acid were measured using an enzyme-linked immunosorbent assay (ELISA) kit. The results are shown in […]. Figure 6 .

[0072] 2) Results and Analysis: like Figure 6 As shown in (A), the activity of diamine oxidase (DAO) in the plasma of IBS-D mice was significantly increased. Intervention with Bifidobacterium bifidum WP139 effectively inhibited the increase of diamine oxidase in IBS-D mice and alleviated the lesions of the intestinal mucosa. Experimental results are as follows Figure 6As shown in (B), D-lactic acid (D-LA) levels in the plasma of mice in the IBS-D group increased significantly. Intervention with Bifidobacterium bifidum WP139 effectively inhibited the increase of D-lactic acid (D-LA) and restored intestinal mucosal permeability and barrier function.

[0073] 7. Effects of Bifidobacterium bifidum WP139 on the intestinal barrier in mice with irritable bowel syndrome 1) Method: The intestinal barrier is composed of a physical barrier formed by tightly packed intestinal epithelial cells, a mucus layer, and the intestinal immune system. Tight junction proteins are crucial structures that maintain the tight junctions between intestinal epithelial cells and ensure the integrity of the intestinal barrier. Occludin, as a key component of the intestinal barrier, is one of the major tight junction proteins and plays a central role in maintaining intestinal barrier function and resisting external invasive substances. Therefore, this embodiment used Western blotting to determine the expression level of ocludin, and the results are shown below. Figure 7 .

[0074] 2) Results and Analysis: like Figure 7 As shown, Western blot results indicated that, compared with the blank control group, the expression level of Occludin protein in the colon tissue of the model group mice was significantly downregulated ( p <0.01. However, the expression of Occludin protein in the group treated with Bifidobacterium bifidum WP139 via gavage showed a significant increase compared to the model group. p <0.01), and close to the normal level.

[0075] 8. Effects of Bifidobacterium bifidum WP139 on gut microbiota in mice with irritable bowel syndrome 1) Method: After the experiment, the mice were euthanized and dissected, and the cecal contents were collected. The 16S rRNA from the cecal contents was then sequenced and analyzed. The results are shown below. Figure 8-9 ; 2) Results and Analysis: Figure 8 The results showed that the intestinal flora in mice with irritable bowel syndrome induced by citric acid infection combined with water avoidance stress was disordered. Compared with the normal group, the ACE index and Shannon index in the model group were significantly reduced. p < 0.05). The ACE index and Shannon index of the Bifidobacterium bifidum WP139 intervention group increased significantly ( p < The results above (0.01) indicate that supplementation with Bifidobacterium bifidum WP139 can improve the richness and diversity of the gut microbiota.

[0076] Figure 9 The results showed that, at the phylum level, compared with the normal group, the Firmicutes and Actinobacteria phyla decreased in the model group, while the Bacteroidetes phylum significantly increased. p <0.01, p <0.01), after supplementing with Bifidobacterium bifidum WP139, the abundance of Firmicutes and Actinobacteria increased, while the abundance of Bacteroidetes decreased, indicating that supplementing with Bifidobacterium bifidum WP139 can regulate the intestinal flora structure and alleviate the intestinal flora imbalance caused by irritable bowel syndrome.

[0077] Figure 10 The results showed that the relative abundance of genera and species in the cecal contents of mice in each group was significantly increased compared with the model group in the Bifidobacterium WP139 treatment group. p <0.05). The abundance of Shigella, Desulfovibrio, Zurich bacillus, and Bacteroides was significantly increased in the model group (MC), but intervention with Bifidobacterium bifidum WP139 could reduce their relative abundance.

[0078] Increased abundance of *Lactobacillus salivarius* may alleviate intestinal inflammation and permeability abnormalities, thus improving diarrhea symptoms in IBS-D. *Lactobacillus* spp. ( Lacticaseibacillus Similar to *Lactobacillus salivarius*, it is a newly classified genus within the *Lactobacillus* family. It can increase the relative abundance of beneficial bacteria and decrease the relative abundance of harmful bacteria, regulating gut microbiota balance, improving gut-brain axis function (e.g., reducing stress response), and indirectly alleviating intestinal dysfunction induced by water avoidance stress. *Alternaria* genus ( Alistipes It participates in bile acid metabolism and tryptophan breakdown, and may affect intestinal inflammation and nerve signal transduction. Increased abundance may be associated with improved bile acid metabolism and reduced bile acid malabsorption diarrhea in IBS-D.

[0079] Shigella ( Escherichia Some strains of *Bifidobacterium bifidum* produce enterotoxins or pro-inflammatory lipopolysaccharides (LPS), disrupting barrier function and activating the TLR4 pathway, triggering low-grade inflammation. Intervention with *Bifidobacterium bifidum* WP139 can reduce its abundance, thereby lowering LPS levels and alleviating intestinal inflammation and osmotic diarrhea. *Desulfovibrio* spp. Desulfovibrio The reduction of sulfate produces hydrogen sulfide (H2S), which damages the function of mitochondria in the intestinal epithelium, leading to barrier dysfunction. Decreased abundance may reduce the toxic effects of H2S on the intestine.

[0080] In summary, the results of gut microbiota analysis showed that B. bifidum WP139 intervention improved intestinal barrier function and immune homeostasis by increasing beneficial bacteria (such as Lactobacillus salivarius and Lactobacillus casei), reducing pro-inflammatory bacteria (such as Shigella and Bacteroides), and reducing toxic metabolites such as LPS / H2S. It may alleviate stress-induced intestinal symptoms through the microbiota-bile acid-neural signaling pathway.

[0081] The above-described embodiments are merely preferred embodiments of the present application and are not exhaustive of the possible implementations of the present application. Any obvious modifications made by those skilled in the art without departing from the principles and spirit of the present application should be considered to be within the scope of protection of the claims of the present application.

Claims

1. A Bifidobacterium bifidum that alleviates irritable bowel syndrome (IBS) Bifidobacterium bifidum WP139, characterized in that, It is deposited at the China General Microbiological Culture Collection Center, with accession number CGMCC NO.35956.

2. The Bifidobacterium bifidum according to claim 1 ( Bifidobacterium bifidum WP139, characterized in that, Obtained from infant feces.

3. The Bifidobacterium bifidum according to claim 1 ( Bifidobacterium bifidum WP139, characterized in that, It has antibacterial properties against Citrobacter musculoskeletonis.

4. A method comprising Bifidobacterium bifidum WP139 as described in any one of claims 1-3 ( Bifidobacterium bifidum ) targeted probiotic formulations.

5. The Bifidobacterium bifidum as described in any one of claims 1-3 ( Bifidobacterium bifidum The use of the targeted probiotic preparation described in WP139 or claim 4 in the preparation of functional foods, health products or medicines for relieving irritable bowel syndrome.

6. The application according to claim 5, characterized in that, Relieving irritable bowel syndrome includes relieving visceral hypersensitivity symptoms, relieving diarrhea, improving inflammation, restoring intestinal mucosal permeability and barrier function, regulating intestinal flora structure, and improving intestinal flora imbalance, among one or more of these.

7. The application according to claim 6, characterized in that, The relief of visceral hypersensitivity symptoms includes reducing pain response and reducing trypsin levels; The improvement of inflammation includes one or more of the following: improving inflammatory infiltration, inhibiting the secretion of pro-inflammatory factors, and promoting the secretion of anti-inflammatory factors; The restoration of intestinal mucosal permeability and barrier function includes inhibiting the rise of diamine oxidase, inhibiting the rise of D-lactic acid, and reducing the expression level of Occludin protein. The improvement of dysbiosis includes increasing the relative abundance of Lactobacillus salivarius, Lactobacillus salivarius, Akkermansia salivarius, and Alternaria salivarius, while inhibiting the increase in abundance of Shigella salivarius, Desulfovibrio salivarius, Zurich bacillus, and Bacteroides salivarius.

8. A drug for relieving irritable bowel syndrome, characterized in that, It includes at least one active ingredient, said active ingredient being Bifidobacterium bifidum as described in any one of claims 1-3. Bifidobacterium bifidum WP139 and / or its fermentation products.

9. A functional food for relieving irritable bowel syndrome, characterized in that, It includes at least one active ingredient, said active ingredient being Bifidobacterium bifidum as described in any one of claims 1-3. Bifidobacterium bifidum WP139 and / or its fermentation products.

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