Bacteroides vulgaris highly utilizing stachyose and application of bacteroides vulgaris in relieving irritable bowel syndrome

Abstract

The invention discloses bacteroides vulgaris highly utilizing stachyose and application of the bacteroides vulgaris in relieving irritable bowel syndrome, and belongs to the technical field of microorganisms and the technical field of medicine. The common bacteroides CCFM1468 obtained through screening can make high use of stachyose and can relieve the irritable bowel syndrome at the same time, and particularly, the common bacteroides CCFM1468 is high in growth rate and short in generation time in an mBHI culture medium with stachyose as a carbon source; (2) stachyose is highly utilized by the bacteroides vulgaris CCFM1468, so that the diarrhea symptom of a mouse with the irritable bowel syndrome is relieved, related indexes of the irritable bowel syndrome are adjusted, the intestinal function is maintained, and the intestinal health is improved; therefore, the common bacteroides CCFM1468 has a huge application prospect in preparation of a product for improving the health of a host.

Description

Technical Field

[0001] The present invention relates to a common Bacteroides strain capable of highly utilizing stachyose and application thereof in alleviating irritable bowel syndrome, belonging to the technical fields of microorganisms and medicine. Background Art

[0002] Irritable bowel syndrome (IBS) is a chronic functional intestinal disorder characterized by abdominal pain or discomfort accompanied by changes in bowel habits or stool consistency. These symptoms can be persistent or intermittent, severely impacting patients' daily lives. IBS-related symptoms include diarrhea, constipation, or a combination of the two. Current research suggests that the mechanisms underlying IBS include abnormalities in the brain-gut axis, disruption of the intestinal mucosal barrier, dysbiosis of the intestinal flora, and abnormalities in related factors and cells.

[0003] Numerous studies have demonstrated that gut microbes play a crucial role in IBS. As the importance of the gut microbiome in human health grows, interest in interventions that can modulate the gut microbiome and its interactions with the host is growing. Besides diet and fecal microbiota transplantation, probiotics are the best-known and most widely recognized agents for modulating the human gut microbiome. Bacteroides vulgaris is a common Bacteroides species in the gut microbiome of healthy adults. Studies have shown that Bacteroides vulgaris can modulate the gut microbiome and regulate related cytokines, thereby reducing intestinal damage and inflammation in mice. Several studies have demonstrated potential therapeutic benefits for Bacteroides vulgaris in conditions such as cardiovascular disease, intestinal inflammation, and obesity. However, existing studies have not shown that Bacteroides vulgaris can alleviate irritable bowel syndrome. Therefore, Bacteroides vulgaris holds broad promise as a potential probiotic for alleviating irritable bowel syndrome.

[0004] Studies have found that with the onset of disease or aging, the content of probiotics in the body gradually decreases. Increasing probiotic colonization has become a research focus in recent years. Administration of prebiotics is one strategy for modulating the composition and metabolic activity of the intestinal microbiota. Prebiotics are defined as "partially or completely undigested and absorbed by the host, resulting in specific changes in the composition and / or activity of the gastrointestinal microbiota, thereby benefiting host health." Commercially available prebiotics include inulin, oligofructose, and stachyose. Stachyose is a prebiotic traditionally extracted from plants, such as vegetables. Studies have shown that stachyose can reduce colonic tissue damage and lower levels of proinflammatory cytokines in mice with colitis, while also restoring intestinal bacterial dysbiosis. However, it remains unclear whether stachyose can alleviate irritable bowel syndrome (IBS), nor has a microorganism been identified that can synergistically enhance the relief of IBS symptoms. Therefore, developing a Bacteroides vulgaris strain that can efficiently utilize stachyose and synergistically enhance the IBS relief effect has become a research hotspot and challenge. Summary of the Invention

[0005] [Technical Issues]

[0006] The technical problem to be solved by the present invention is to provide a common Bacteroides strain (Bacteroides vulgatus) with strong ability to utilize stachyose and its application in alleviating diarrhea symptoms of irritable bowel syndrome.

[0007] [Technical solution]

[0008] To solve the above problems, the present invention provides a strain of Bacteroides vulgatus CCFM1468, which is deposited in Guangdong Provincial Microbial Culture Collection Center with a deposit number of GDMCC No: 65606 and a deposit date of December 11, 2024.

[0009] In one embodiment of the present invention, the Bacteroides vulgatus CCFM1468 was sequenced and analyzed, and the sequence obtained by sequencing was compared with the nucleic acid sequence of the Bacteroides vulgatus species of the genus Bacteroides. The results showed that the similarity with the nucleic acid sequence of the Bacteroides vulgatus species was 100%; the results showed that the strain was Bacteroides vulgatus, and it was named Bacteroides vulgatus CCFM1468.

[0010] In one embodiment of the present invention, the colonies of Bacteroides vulgatus CCFM1468 grown on a BHI solid culture medium supplemented with 0.05% cysteine ​​are round protrusions with a smooth and translucent surface and a diameter of 1 to 2 mm.

[0011] In one embodiment of the present invention, after the administration of stachyose for in vitro simulated fermentation, the absolute abundance of Bacteroides vulgatus CCFM1468 in the original host intestine can be significantly increased.

[0012] In one embodiment of the present invention, the growth rate of the Bacteroides vulgatus CCFM1468 is significantly increased and the generation time is significantly shortened in an mBHI liquid culture medium using stachyose as a carbon source in vitro.

[0013] In one embodiment of the present invention, the Bacteroides vulgatus CCFM1468, when co-administered with stachyose, reduced fecal water content, defecation frequency, and gastrointestinal transit rate in mice with diarrhea-predominant irritable bowel syndrome, effectively alleviating diarrhea symptoms.

[0014] In one embodiment of the present invention, the Bacteroides vulgatus CCFM1468, when used in combination with stachyose, reduced serotonin levels in the serum and colon tissue of mice with diarrhea-predominant irritable bowel syndrome, increased aquaporin 3 levels in colon tissue, and simultaneously reduced inducible nitric oxide synthase levels in colon tissue. This can alleviate diarrhea symptoms by improving intestinal motility and water absorption.

[0015] The present invention also provides a microbial preparation containing the above-mentioned Bacteroides vulgatus CCFM1468.

[0016] In one embodiment of the present invention, the number of cells of Bacteroides vulgatus CCFM1468 in the microbial preparation is not less than 1×10 6 CFU / mL or 1×10 6 CFU / g.

[0017] The present invention also provides a product, which contains the above-mentioned Bacteroides vulgatus CCFM1468 or the above-mentioned microbial preparation.

[0018] In one embodiment of the present invention, the product further contains stachyose.

[0019] In one embodiment of the present invention, the number of Bacteroides vulgaris cells in the product is not less than 1×10 6 CFU / mL or 1×10 6 CFU / g.

[0020] In one embodiment of the invention, the product comprises a food product.

[0021] In one embodiment of the present invention, the food is a health food.

[0022] In one embodiment of the present invention, the food is a dairy product, soy product or fruit and vegetable product produced using the above-mentioned Bacteroides vulgatus CCFM1468 or a starter culture of the above-mentioned microbial preparation.

[0023] In one embodiment of the present invention, the food is a beverage or snack containing the above-mentioned Bacteroides vulgatus CCFM1468 or the above-mentioned microbial preparation.

[0024] In one embodiment of the present invention, the preparation method of the starter is as follows: the above-mentioned Bacteroides vulgatus CCFM1468 is inoculated into the culture medium according to an inoculum amount of 2 to 4% of the total mass of the culture medium, and anaerobically cultured at 37°C for 48 hours to obtain a culture solution; the culture solution is centrifuged to obtain bacterial cells; the bacterial cells are washed three times with physiological saline and then resuspended with a freeze-drying protective agent to obtain a resuspension; the resuspension is freeze-dried by a vacuum freezing method to obtain a starter.

[0025] In one embodiment of the present invention, the mass ratio of the lyoprotectant to the bacterial cells is 2:1.

[0026] In one embodiment of the present invention, the lyoprotectant comprises 130 g / L skim milk powder.

[0027] In one embodiment of the present invention, the pH of the culture medium is 6.8.

[0028] In one embodiment of the present invention, the product is a medicine for relieving irritable bowel syndrome.

[0029] In one embodiment of the present invention, the medicine contains the above-mentioned Bacteroides vulgatus CCFM1468, a drug carrier and / or a pharmaceutical excipient.

[0030] In one embodiment of the present invention, the drug carrier comprises microcapsules, microspheres, nanoparticles and / or liposomes.

[0031] In one embodiment of the present invention, the pharmaceutical excipient comprises an excipient and / or an additive.

[0032] In one embodiment of the present invention, the excipient comprises a binder, a filler, a disintegrant and / or a lubricant.

[0033] In one embodiment of the present invention, the additive comprises a solubilizer, a co-solvent, a latent solvent and / or a preservative.

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

[0035] The present invention also provides the use of the Bacteroides vulgaris CCFM1468 or the microbial preparation in the preparation of medicines for alleviating irritable bowel syndrome.

[0036] In one embodiment of the present invention, the medicine further contains stachyose.

[0037] Beneficial effects

[0038] The present invention provides a strain of Bacteroides vulgatus CCFM1468, which has the ability to efficiently utilize stachyose and alleviate diarrhea in irritable bowel syndrome model mice, specifically:

[0039] (1) The Bacteroides vulgaris CCFM1468 screened in the present invention has good growth characteristics in BHI liquid culture medium with stachyose as the carbon source, with a generation time of 1.044±0.04h.

[0040] (2) After one week of administration of the present invention's combination of Bacteroides vulgaris CCFM1468 and stachyose to mice with diarrhea-type irritable bowel syndrome, the water content in the mice's feces and the frequency of defecation were significantly reduced. In addition, 5-hydroxytryptamine, aquaporin, inducible nitric oxide synthase, and other factors associated with irritable bowel syndrome also showed significant changes after intervention. Compared with the use of Bacteroides vulgaris CCFM1468 and stachyose alone, the combined use of Bacteroides vulgaris CCFM1468 and stachyose can play a synergistic role in alleviating the symptoms of irritable bowel syndrome.

[0041] Therefore, the compound formula of Bacteroides vulgaris CCFM1468 and stachyose of the present invention has great application prospects in the preparation of medicines that can relieve the diarrhea symptoms of irritable bowel syndrome and improve host health.

[0042] Biomaterial Deposit

[0043] A strain of Bacteroides vulgatus CCFM1468, taxonomically named Bacteroides vulgatus, was deposited in the Guangdong Provincial Microbial Culture Collection on December 11, 2024, with the deposit number GDMCC No: 65606. The deposit address is Guangdong Institute of Microbiology, 5th Floor, Building 59, No. 100 Xianlie Middle Road, Guangzhou. BRIEF DESCRIPTION OF THE DRAWINGS

[0044] Figure 1 : Colony morphology characteristics of Bacteroides vulgatus CCFM1468 on mBHI solid medium supplemented with 0.05% cysteine.

[0045] Figure 2Figure 3. Growth of three Bacteroides vulgaris strains in mBHI liquid medium with stachyose as the carbon source. These strains are Bacteroides vulgaris CCFM1468, Bacteroides vulgaris FTJS10K3, and Bacteroides vulgaris FSDLZ51K1. (A) Generation time, (B) Growth curve. "#" indicates comparison with Bacteroides vulgaris FTJS10K3, and "*" indicates comparison with Bacteroides vulgaris FSDLZ51K1.

[0046] Figure 3 : Grouping and treatment methods of animal experiments, where the symbol “ / ” means no treatment.

[0047] Figure 4 : Effects of stachyose and Bacteroides vulgatus CCFM1468 on the apparent defecation indicators of irritable bowel syndrome model mice (* indicates comparison with the model group, # indicates comparison with the blank group);

[0048] Among them, (A) defecation frequency of mice (*P<0.05, **P<0.01, ***P<0.001, ns indicates no significant difference (P>0.05));

[0049] (B) Fecal water content (*P<0.05, **P<0.01, ***P<0.001, ns indicates no significant difference (P>0.05));

[0050] (C) Time to first black stool excretion (gastrointestinal transit time) (*P < 0.05, **P < 0.01, ***P < 0.001, ns indicates no significant difference (P > 0.05));

[0051] (D) Gastrointestinal transit rate (*P<0.05, **P<0.01, ***P<0.001, ns indicates no significant difference (P>0.05)).

[0052] Figure 5 : Effects of stachyose and Bacteroides vulgatus CCFM1468 on irritable bowel syndrome-related indicators (* indicates comparison with the model group, # indicates comparison with the blank group);

[0053] Among them, (A) serotonin content in colon tissue (*P<0.05, **P<0.01, ***P<0.001, ns indicates no significant difference (P>0.05));

[0054] (B) Serum serotonin content (*P<0.05, **P<0.01, ***P<0.001, ns indicates no significant difference (P>0.05));

[0055] (C) Aquaporin-3 content in colon tissue (*P < 0.05, **P < 0.01, ***P < 0.001, ns indicates no significant difference (P > 0.05));

[0056] (D) Inducible nitric oxide synthase levels in colon tissue (*P<0.05, **P<0.01, ***P<0.001, ns indicates no significant difference (P>0.05)). DETAILED DESCRIPTION

[0057] The present invention will be further described below with reference to specific embodiments.

[0058] Stachyose involved in the following examples was purchased from Shanghai Yuanye Biotechnology Co., Ltd. Bacteroides vulgaris CCFM1468, Bacteroides vulgaris FTJS10K3, and Bacteroides vulgaris FSDLZ51K1 involved in the following examples were strains screened during the same period and deposited in the in-house strain bank of the Biotechnology Center of the School of Food Science and Engineering, Jiangnan University.

[0059] The culture medium involved in the following examples is as follows:

[0060] Bacteroides-specific screening medium: Dissolve 43.1 g of Brucella medium powder in 1 L of water, add 0.01‰ hemin and 0.01‰ vitamin K1, and sterilize at 121°C for 15 min. Once cooled to 50°C, add 0.1‰ kanamycin, 0.0075‰ vancomycin, and 5% sheep blood. Mix thoroughly and pour into a sterile Petri dish.

[0061] mBHI liquid medium: peptone 10.0 g / L, dehydrated calf brain extract powder 12.5 g / L, dehydrated ox heart extract powder 5.0 g / L, sodium chloride 5.0 g / L, glucose 2.0 g / L, disodium hydrogen phosphate 2.5 g / L, hemin 10 mg / L, vitamin K 2 mg / L, L-cysteine ​​0.5 g / L; pH 6.8.

[0062] Sugar-free mBHI liquid medium: Based on mBHI liquid medium, glucose is removed.

[0063] mBHI solid medium: Add 20 g / L agar to mBHI (BHI containing L-cysteine) liquid medium.

[0064] The detection methods involved in the following embodiments are as follows:

[0065] Method for detecting viable bacteria count: adopt the national standard "GB 4789.35-2016 National Food Safety Standard Food Microbiology Detection Lactic Acid Bacteria Detection".

[0066] The preparation method of the common Bacteroides cells involved in the following examples is as follows:

[0067] The common Bacteroides was streaked on mBHI solid culture medium and cultured under anaerobic conditions at 37°C for 48 hours to obtain a single colony; a single colony was picked and inoculated into 5 mL of mBHI liquid culture medium, and cultured under anaerobic conditions at 37°C for 18 hours for activation, and two generations of activation were continuously activated to obtain an activation solution; the activation solution was inoculated into mBHI liquid culture medium at an inoculum rate of 2% (v / v), and cultured under anaerobic conditions at 37°C for 18 hours to obtain a bacterial solution; the bacterial solution was centrifuged at 5000 r / min for 15 minutes, and the supernatant culture medium was discarded to obtain common Bacteroides cells.

[0068] Example 1: Screening and identification of Bacteroides vulgatus CCFM1468

[0069] The specific steps are as follows:

[0070] 1. Sample collection

[0071] A stool sample was collected from a patient with irritable bowel syndrome in Zhangye, Gansu Province. The sample was placed in a stool tube filled with 30% glycerol and stored in an insulated box with ice packs. After being brought back to the laboratory, it was quickly placed in a -80℃ refrigerator for separation and screening.

[0072] 2. Isolation and purification of bacteria

[0073] (1) Dilution coating: Take about 0.5g of the contents stored in 30% glycerol and add it to a 10mL centrifuge tube filled with 4.5mL of physiological saline under a sterile environment to obtain 10 -1 Dilution, repeat the above dilution steps to obtain 10 -2 , 10 -3 , 10 -4 , 10 -5 , 10 -6 diluent;

[0074] (2) Spreading culture: Take 100 μL of the above 10 -4 , 10 -5 , 10 -6 Three gradient dilutions were evenly spread on a specific screening medium for Bacteroides isolated and screened from the human intestine using a spreading rod, and cultured under anaerobic conditions at 37°C for 48 hours to obtain a dilution spread plate;

[0075] (3) Primary purification culture: Take a dilution plate with a colony count between 30 and 300, and randomly select 10 milky white or white single colonies with smooth surface, neat edges, and different sizes for each sample and streak them on mBHI solid medium. Incubate at 37°C under anaerobic conditions for 48 h to obtain single colonies;

[0076] (4) Secondary purification culture: Take a single colony from the streaked plate in step (3) and inoculate it into mBHI liquid culture medium, and culture it at 37°C under anaerobic conditions for 20 hours to obtain a secondary purification culture medium.

[0077] 3. Strain preservation and identification

[0078] (1) Preservation of bacterial strains:

[0079] The secondary purified culture medium obtained in step 2 (4) was mixed, and the bacterial cells (anaerobic culture at 37°C for 16-20h) were taken into 2mL clean culture storage tubes respectively, and 5 parallel portions were prepared. 4 of the portions were resuspended by adding 750μL bacterial solution and 750μL 60% glycerol, and then placed in a -80°C refrigerator after standing for 30 minutes; 1 portion was added with 1mL bacterial solution for bacterial identification, centrifuged at 6000r / min for 3min, and the supernatant was discarded to obtain the bacterial cells.

[0080] (2) Identification of bacterial species:

[0081] Step 3 (1) Add 1 mL of sterile water to the preservation tube for bacterial identification, blow and wash the bacteria, centrifuge at 10,000 rpm for 1 min, discard the supernatant, and resuspend the bacteria in 500 μL of sterile water to serve as a bacterial liquid template;

[0082] The system and primers for 16S rDNA PCR are shown in Tables 1 and 2 , respectively;

[0083] Conditions for 16S rDNA PCR: Step 1: 94°C, 5 min; Step 2: 94°C, 30 s; Step 3: 55°C, 30 s; Step 4: 72°C, 2 min; Step 5: 72°C, 10 min. Steps 2 to 4 were repeated for 30 cycles.

[0084] Table 1 Bacterial species identification 25 μL 16S rDNA PCR reaction system

[0085]

[0086] Table 2 Primer names

[0087]

[0088] After identification, the strain obtained by screening was Bacteroides vulgaris ( Figure 1 ), named Bacteroides vulgatus CCFM1468, has been deposited in Guangdong Provincial Microbial Culture Collection with the deposit number GDMCC No: 65606.

[0089] In addition, Bacteroides vulgaris FTJS10K3 and Bacteroides vulgaris FSDLZ51K1 were screened and used in subsequent experiments.

[0090] Example 2: Growth characteristics of Bacteroides vulgatus CCFM1468 in mBHI liquid medium with stachyose as a carbon source

[0091] The specific steps are as follows:

[0092] (1) Bacteroides vulgaris CCFM1468, Bacteroides vulgaris FTJS10K3, and Bacteroides vulgaris FSDLZ51K1 were streaked onto mBHI solid medium and cultured anaerobically at 37°C for 48 h to obtain single colonies;

[0093] A single colony was picked and inoculated into mBHI liquid culture medium, cultured at 37°C for 18 h for activation, and activated for two generations to obtain activation liquid.

[0094] (2) Take 1 mL of activation solution in a sterile centrifuge tube, centrifuge at 5000 r / min for 15 min, discard the upper culture medium to obtain bacterial sludge, resuspend the bacterial sludge with 1 mL of sterile saline, and then inoculate it with 2% (v / v) inoculum in sugar-free mBHI liquid medium supplemented with 2.0 g / L stachyose. The initial OD 600 The OD value of the culture medium was 0.098±0.003 (intestinal microbial in vitro simulation culture method), placed in a microplate reader, and cultured anaerobically at 37°C for 16 hours. During the culture process, the OD value of the culture medium was measured every 2 hours. 600 .

[0095] The results showed that the growth curves of the three common Bacteroides were as follows: Figure 2 (B) shows the generation time of 3 strains of Bacteroides vulgaris. Figure 2 (A) shows that the generation time refers to the time required for each cell division; the shorter the generation time, the faster the growth rate; the generation time of each strain in the logarithmic phase can reflect the speed of its growth rate. The formula for the generation time in the logarithmic phase is as follows: generation time = (t2-t1) / 3.322(OD2-OD1); where t2 is the time at the end of the logarithmic growth phase, t1 is the time just entering the logarithmic growth phase, and OD2 and OD1 are the absorbance values ​​of the bacterial suspension measured at t2 and t1.

[0096] Depend on Figure 2 (A) As can be seen, Bacteroides vulgaris CCFM1468 has a shorter generation time, meaning it takes less time for the bacteria to reproduce. This result also indicates that Bacteroides vulgaris CCFM1468 has a strong ability to utilize stachyose, using it for its own growth and reproduction, resulting in a faster growth rate and a shorter generation time. Bacteroides vulgaris FSDLZ51K1 has the longest generation time, indicating that it has the poorest ability to utilize stachyose.

[0097] Depend on Figure 2(B) It can be seen that Bacteroides vulgaris CCFM1468 has a faster growth rate in the logarithmic growth phase and has the maximum growth amount when it reaches the growth stationary phase. 600 was 0.74±0.007, while the OD of Bacteroides vulgaris FTJS10K3 and Bacteroides vulgaris FSDLZ51K1 in the stable period 600 The values ​​were 0.605±0.006 and 0.56±0.012, respectively. This result indicates that Bacteroides vulgaris CCFM1468 has a strong ability to utilize stachyose and is the most adaptable to the mBHI medium environment with stachyose as a carbon source. However, Bacteroides vulgaris FSDLZ51K1 has the slowest growth rate and the smallest growth amount when it reaches the growth stationary phase.

[0098] Example 3: Effects of Stachyose and Bacteroides vulgatus CCFM1468 on Diarrhea Symptoms in Irritable Bowel Syndrome Model Mice

[0099] The specific steps are as follows:

[0100] Bacteroides vulgaris CCFM1468 was inoculated into mBHI liquid medium and cultured anaerobically at 37°C for 18 h. The bacterial sludge was collected by centrifugation at 5000 rpm for 15 min at 4°C, washed with physiological saline, and resuspended in physiological saline. The bacterial concentration was 1×10 9 CFU / mL was used to obtain a bacterial suspension, which was prepared daily and used immediately, with 1 mL administered orally each time. Stachyose was administered at a dose of 1 g / kg daily by oral gavage. The negative control for stachyose was raffinose, administered at a dose of 1 g / kg.

[0101] Sixty-four SPF-grade C57BL / 6J male mice weighing 20-22 g were randomly divided into 8 groups, with 8 mice in each group, namely: (1) blank group: blank control; (2) model group: given normal saline during the intervention period; (3) raffinose group: given raffinose solution during the intervention period; (4) stachyose group: given stachyose solution by gavage during the intervention period; (5) vulgaris group: given vulgaris CCFM1468 bacterial suspension by gavage during the intervention period; (6) stachyose + vulgaris group: given stachyose and vulgaris CCFM1468 bacterial suspension during the intervention period.

[0102] The experiment lasted for 7 weeks: the first week was the adaptation period for mice, during which no treatment was done and mice had free access to food and water; the second and third weeks were the modeling period; the fourth week was the adaptation period, during which no treatment was done; and the fifth week was the intervention period.

[0103] Modeling method: First, a pseudo-germ-free mouse model was established by gavage with quadruple antibiotics for 10 days, and then fecal microbiota transplantation was performed by gavage with fecal microbiota suspension from patients with irritable bowel syndrome for 3 days.

[0104] Preparation of quadruple antibiotics: Made of vancomycin, neomycin, metronidazole and ampicillin, the oral dose is vancomycin (100 mg / kg), neomycin (200 mg / kg), metronidazole (200 mg / kg) and ampicillin (200 mg / kg).

[0105] Fecal microbial suspension preparation: Fecal samples from patients with irritable bowel syndrome (IBS) collected from the outpatient department of gastroenterology at the Affiliated Hospital of Jiangnan University were prepared as a 100 mg / mL suspension in PBS. Antibiotic-treated mice were used as recipients of the microbiota, with an oral gavage dose of 500 mg / kg. The use of human samples was approved by the Ethics Committee of the Affiliated Hospital of Jiangnan University (LS2024278).

[0106] To prepare the ink: Mix 100g of gum arabic with 800mL of water and boil until the solution is transparent. Add 50g of activated carbon and boil three times to make an activated carbon solution. After the solution cools, dilute it to 1000mL with water and store at 4°C. Shake well before use.

[0107] For detailed experimental groups and treatments, see Figure 3 .

[0108] After the intervention, the time of the first black stool was detected, and the frequency of defecation was tested and feces were collected. After the experiment, before the mice were killed, the small intestinal propulsion rate was first tested: fasting overnight before dissection. The mice were gavaged with ink, anesthetized after 15 minutes, and the blood of the mice was collected through the orbital plexus. All mice were humanely killed by cervical dislocation. The abdominal cavity was opened, the mesentery was separated, and the intestinal tube from the upper end to the cardia to the lower end to the ileocecum was cut. The small intestine was gently pulled into a straight line, and the length of the stomach and intestinal tube was measured as the "total gastrointestinal length". The length from the cardia to the ink front was the "ink propulsion length", and the gastrointestinal propulsion rate was calculated. Then, the blood sample was centrifuged at 3000g for 30 minutes to collect serum. The colon tissue was quickly fixed in liquid nitrogen.

[0109] In previous studies, defecation frequency, fecal water content, and gastrointestinal transit rate were considered as apparent indicators of diarrhea in mice (Zhai, L., Huang, C., Ning, Z., Zhang, Y., Zhuang, M., Yang, W., Wang, X., Wang, J., Zhang, L., Xiao, H., Zhao, L., Asthana, P., Lam, YY, Chow, CFW, Huang, J., Yuan, S., Chan, KM, Yuan, C.-S., Lau, JY-N., … Bian, Z. (2023). Ruminococcus gnavus plays a pathogenic role in diarrhea-predominant irritable bowel syndrome by increasing serotonin biosynthesis. Cell Host & Microbe, 31(1), 33-44.e5.Q1. https: / / doi.org / 10.1016 / j.chom.2022.11.006). Therefore, this embodiment measures the fecal indicators and diarrhea appearance indicators of mice. The results are as follows Figure 4 shown.

[0110] The results showed that stachyose and Bacteroides vulgaris, whether used alone or in combination, significantly reduced bowel movement frequency and fecal water content in IBS model mice, whereas raffinose, which has a similar structure to stachyose, had no significant effect. This suggests that these interventions can effectively regulate intestinal function and alleviate diarrheal symptoms. Furthermore, the gastrointestinal transit time of the intervention group mice was significantly reduced compared to that of the IBS model group mice. This result may reflect improved gastrointestinal motility, which promotes nutrient absorption and water recovery by prolonging the residence time of feces in the intestine, further alleviating diarrheal symptoms. Specifically, when stachyose and Bacteroides vulgaris CCFM1468 were used together, the number of stool particles in two hours was 14.86±0.9863, which was 97.8% of the control group. The fecal water content was 43.23±1.464%, which was 92.2% of the control group. The time to first black stool was 150.9±5.911 minutes, which was 105.6% of the control group. The gastrointestinal transit rate was 65.10±4.08%, which was 102.4% of the control group. Compared with the use of stachyose and Bacteroides vulgaris alone, the combined use of stachyose and Bacteroides vulgaris had a more precise regulatory effect on the above indicators, bringing the indicators of the treated mice closer to the state of the control group mice, indicating that Bacteroides vulgaris CCFM1468 and stachyose play a synergistic role in improving the symptoms of irritable bowel syndrome.

[0111] Example 4: Effects of Stachyose and Bacteroides vulgatus CCFM1468 on Intestinal Motility and Water Absorption-Related Factors in Irritable Bowel Syndrome Model Mice

[0112] The specific implementation method is the same as Example 3, and the colon tissue is quickly fixed in liquid nitrogen after the mouse is dissected. The total RNA in the colon tissue is extracted using the FastPure Cell / Tissue Total RNA Isolation Kit. In addition, HiScriptIVAll-in-One Ultra RT SuperMix for qPCR is used to synthesize complementary DNA (cDNA). Both kits were purchased from Nanjing Novozymes Biotechnology Co., Ltd. The mRNA expression levels of Htr4 and AQP3 were analyzed by RT-PCR. The sequences of the qPCR primers used in this study are shown in Table 1. The corresponding ELISA kits were purchased from Nanjing Senbega Biotechnology Co., Ltd. and were used to detect the levels of 5-HT, AQP3 and iNOS in serum and colon tissue. The BCA protein assay kit was purchased from Biyuntian Biotechnology Co., Ltd. and was used to detect the total protein content of all colon tissues. The results are as shown in the figure. Figure 5 shown.

[0113] In colonic tissue, both the stachyose and Bacteroides vulgaris CCFM1468 groups significantly reduced 5-HT levels. In serum, 5-HT levels were significantly reduced in the stachyose and Bacteroides vulgaris CCFM1468 combination groups, while there was a trend toward a decrease in 5-HT in the stachyose group, but this was not significant. Specifically, when stachyose and Bacteroides vulgaris CCFM1468 were used together, 5-HT levels in colonic tissue were 47.35±5.751 ng / mg protein, recovering to 136.9% of that in the blank group; and in serum, 5-HT levels were 129.5±0.8309 ng / mL, recovering to 103.8% of that in the blank group. Decreased 5-HT in colonic tissue may directly affect intestinal motility and secretory function, while changes in serum 5-HT may further influence intestinal sensory function through feedback mechanisms in the central nervous system. Furthermore, when stachyose and Bacteroides vulgaris CCFM1468 were used together, AQP3 levels in colonic tissue were significantly higher than in the model group, reaching 224.1±20.29 pg / mg protein, and returning to 113.5% of the blank group. This increased expression may lead to increased intestinal water absorption, which in turn promotes a decrease in intestinal water content and alleviates diarrheal symptoms. iNOS levels in colonic tissue were significantly lower than in the model group, reaching 1.723±0.1349 nmol / mg protein, and returning to 129.8% of the blank group. iNOS is an important enzyme that catalyzes nitric oxide synthesis and is typically expressed more strongly in response to inflammatory stimulation and visceral hypersensitivity. This significant decrease in protein levels may reflect a reduction in inflammatory responses and intestinal sensitivity.

[0114] Example 5: Application of Bacteroides vulgatus CCFM1468

[0115] Bacteroides vulgaris CCFM1468 can be used to prepare bacterial powder. The specific preparation process of bacterial powder is as follows:

[0116] Common Bacteroides CCFM1468 was streaked on mBHI solid medium and cultured anaerobically at 37°C for 48 hours to obtain a single colony. A single colony was picked and inoculated into mBHI liquid medium, and activated by anaerobically cultured at 37°C for 18 hours. After two generations of continuous activation, an activation solution was obtained. The activation solution was inoculated into the culture medium at a 2% (v / v) inoculation rate, and cultured anaerobically at 37°C for 18 hours to obtain a bacterial solution. The bacterial solution was centrifuged at 5000 rpm for 15 minutes to obtain bacterial mud. After washing the bacterial mud three times with physiological saline, it was resuspended with a protective agent to a concentration of 1×10 10 CFU / mL, and a bacterial suspension was obtained. The bacterial suspension was incubated at 37°C for 60 minutes and then freeze-dried to obtain Bacteroides vulgaris CCFM1468 bacterial powder.

[0117] The culture medium is prepared by dissolving 10% enzymatically hydrolyzed skim milk, 0.5% glucose, 1.5% tryptone and 0.3% yeast extract in 87.7% water based on the total weight of the culture medium, and then adjusting the pH to 6.8 to obtain the culture medium.

[0118] The ingredients of the protective agent include: 130g / L skimmed milk powder.

[0119] Example 6: Application of Bacteroides vulgatus CCFM1468

[0120] Bacteroides vulgaris CCFM1468 can be used to prepare capsule products. The specific preparation process of the capsule products is as follows:

[0121] Bacteroides vulgaris CCFM1468 was streaked on mBHI solid medium and cultured anaerobically at 37°C for 48 hours to obtain a single colony; a single colony was picked and inoculated into mBHI liquid medium and cultured anaerobically at 37°C for 18 hours for activation, and two generations of activation were performed to obtain an activated solution; the activated solution was inoculated into the medium at an inoculum volume of 2% (v / v) and cultured anaerobically at 37°C for 18 hours to obtain a bacterial solution; the bacterial solution was centrifuged at 5000 r / min for 15 minutes to obtain a bacterial sludge; the bacterial sludge was washed three times with physiological saline and resuspended with a protective agent to a concentration of 1×10 10 CFU / mL, and obtain bacterial suspension; the bacterial suspension is added to a 30g / L sodium alginate solution to a concentration of 2×10 9CFU / mL, stirring thoroughly to uniformly disperse the cells of Bacteroides vulgaris CCFM1468 in the sodium alginate solution to obtain a mixed solution; extruding the mixed solution into a calcium chloride solution with a concentration of 20 g / L to form micelles; after the formed micelles were allowed to solidify for 30 minutes, filtering and collecting the micelles; freeze-drying the collected micelles for 48 hours to obtain a powder; and filling the powder into a medicinal capsule to obtain a capsule product.

[0122] The culture medium is prepared by dissolving 10% enzymatically hydrolyzed skim milk, 0.5% glucose, 1.5% tryptone and 0.3% yeast extract in 87.7% water based on the total weight of the culture medium, and then adjusting the pH to 6.8 to obtain the culture medium.

[0123] Example 7: Application of Bacteroides vulgatus CCFM1468

[0124] Bacteroides vulgaris CCFM1468 can be used to prepare tablets. The specific preparation process of the tablets is as follows:

[0125] Common Bacteroides CCFM1468 was streaked on mBHI solid medium and cultured anaerobically at 37°C for 48 hours to obtain a single colony. A single colony was picked and inoculated into mBHI liquid medium, and anaerobically cultured at 37°C for 18 hours for activation. Two generations of activation were continuously activated to obtain an activated solution. The activated solution was inoculated into the culture medium at an inoculum volume of 2% (v / v), and then anaerobically cultured at 37°C for 18 hours to obtain a bacterial solution. The bacterial solution was centrifuged at a speed of 5000r / min for 15 minutes to obtain bacterial mud. After the bacterial mud was washed 3 times with physiological saline, it was resuspended with a protective agent to a concentration of 1×10 10 CFU / mL to obtain a bacterial suspension. The bacterial suspension was incubated at 37°C for 60 minutes and then freeze-dried to obtain Bacteroides vulgaris CCFM1468 bacterial powder.

[0126] The culture medium is prepared by dissolving 10% enzymatically hydrolyzed skim milk, 0.5% glucose, 1.5% tryptone and 0.3% yeast extract in 87.7% water based on the total weight of the culture medium, and then adjusting the pH thereof to 6.8 to obtain the culture medium.

[0127] The ingredients of the protective agent include 130g / L skimmed milk powder.

[0128] 25.7 parts by weight of Bacteroides vulgaris CCFM1468 powder, 55.0 parts by weight of starch, 4.5 parts by weight of a cellulose derivative, 12.0 parts by weight of sodium carboxymethyl starch, 0.8 parts by weight of talc, 1.0 parts by weight of sucrose, and 1.0 parts by weight of water were weighed to obtain raw materials. The raw materials were mixed to obtain wet granules. Subsequently, the wet granules were compressed using a tablet press from Zhongnan Pharmaceutical Machinery Factory and dried using a small drug dryer from Qingzhou Yikang Traditional Chinese Medicine Machinery Co., Ltd. to obtain tablets.

[0129] Although the present invention has been disclosed above in terms of preferred embodiments, it is not intended to limit the present invention. Anyone familiar with this technology can make various changes and modifications without departing from the spirit and scope of the present invention. Therefore, the scope of protection of the present invention should be based on the definition of the claims.

Claims

1. A strain of Bacteroides vulgatus CCFM1468, characterized in that: The common Bacteroides CCFM1468 is deposited in the Guangdong Provincial Microbial Culture Collection Center with the deposit number GDMCC No: 65606 and the deposit date of December 11, 2024.

2. A microbial preparation containing the common Bacteroides CCFM1468 described in claim 1.

3. The microbial preparation according to claim 2, characterized in that In the microbial preparation, the number of Bacteroides vulgaris CCFM1468 is not less than 1×10 6 CFU / mL or 1×10 6 CFU / g.

4. A product, characterized in that The product contains the common Bacteroides CCFM1468 described in claim 1 or the microbial preparation described in claim 2 or 3.

5. The product according to claim 4, characterized in that The product also contains stachyose.

6. The product according to claim 4 or 5, characterized in that In the product, the number of Bacteroides vulgaris CCFM1468 is not less than 1×10 6 CFU / mL or 1×10 6 CFU / g.

7. The product according to any one of claims 4 to 6, characterized in that: The product is a food; the food is a health food; or the food is a dairy product, a bean product or a fruit and vegetable product produced using a starter containing the common Bacteroides CCFM1468 described in claim 1, or the microbial preparation described in claim 2 or 3.

8. The product according to any one of claims 4 to 6, characterized in that: The product is a medicine; the medicine contains the common Bacteroides CCFM1468 as described in claim 1, a drug carrier and / or a pharmaceutical excipient; the dosage form of the medicine is powder, granules, capsules, tablets, pills or oral liquid.

9. Use of the Bacteroides vulgaris CCFM1468 described in claim 1, or the microbial preparation described in claim 2 or 3, in the preparation of a medicine for relieving irritable bowel syndrome.

10. The use according to claim 9, characterized in that: The medicine also contains stachyose.

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