A strain of Bifidobacterium breve and its application

Abstract

The present invention discloses a strain of Bifidobacterium breve and its application, wherein the Bifidobacterium breve comprises a 16S rDNA sequence having at least 80%, at least 85%, at least 90%, at least 94%, at least 96%, at least 98%, at least 99% or 100% identity with the nucleotide sequence shown in SEQ ID NO: 1. The Bifidobacterium breve XA-2103 provided by the present invention can significantly alleviate ulcerative colitis induced by DSS and can significantly reduce the overall inflammation level of experimental animals. At the in vitro evaluation level, the strain can significantly reduce the secretion of cellular inflammatory factors and maintain the integrity of the intestinal barrier. Therefore, the Bifidobacterium breve XA-2103 has great application prospects in the preparation of drugs for preventing and / or treating ulcerative colitis, as well as in the preparation of drugs for preventing and / or treating inflammation.

Description

Technical Field

[0001] The invention belongs to the field of microorganisms, and particularly relates to a strain of Bifidobacterium breve and application thereof. Background Art

[0002] Ulcerative colitis (UC) is a type of inflammatory bowel disease (IBD). It is an inflammatory bowel disease that can accumulate and manifests itself in patients with symptoms including abdominal pain, diarrhea, bloody stools, and fatigue. If not treated promptly and effectively, it can also lead to serious complications such as colorectal cancer, threatening human health.

[0003] While there's currently no definitive cause for ulcerative colitis and inflammatory bowel disease (IBD), research has shown that external factors, including intestinal microbiota, environment, age, and lifestyle, are all risk factors for IBD. The primary manifestation of IBD is an imbalance in the immune system, including acquired immune dysregulation (primarily involving Th cells) and innate immune dysregulation (including neutrophils and ILC-3 cells). Furthermore, IBD patients often experience damaged intestinal barrier function and thinning of the intestinal mucosa, leading to an imbalance in the intestinal microbiome and the invasion of harmful microorganisms.

[0004] Currently, there is no effective treatment for inflammatory bowel disease (IBD), and the only approach to alleviate symptoms is through methods such as suppressing inflammation and neutralizing inflammatory factors. Currently, the main treatments for IBD include glucocorticoids, anti-inflammatory drugs, immunosuppressants, and biologics. However, long-term use of these drugs may lead to immune tolerance or liver damage. Therefore, there is a strong urgency and necessity to find more effective IBD treatments that are less likely to induce immune tolerance or liver damage. Summary of the Invention

[0005] In order to solve the above problems in the prior art, the present invention proposes a strain of Bifidobacterium breve Bifidobacterium breve .

[0006] The present invention also provides a method containing the above-mentioned Bifidobacterium breve Bifidobacterium breve products.

[0007] The present invention also proposes the above-mentioned Bifidobacterium breve Bifidobacterium breve Or containing the above-mentioned Bifidobacterium breve Bifidobacterium breve application of products.

[0008] In one aspect of the present invention, a strain of Bifidobacterium breve is provided. Bifidobacterium breve , the Bifidobacterium breve Bifidobacterium breveIncluded are 16S rDNA sequences that are at least 80%, at least 85%, at least 90%, at least 94%, at least 96%, at least 98%, at least 99%, or 100% identical to the nucleotide sequence shown in SEQ ID NO: 1.

[0009] In some embodiments of the present invention, the Bifidobacterium breve Bifidobacterium breve It is deposited in the General Microbiology Center of China Culture Collection Administration, with the deposit number CGMCC No.26611 and the deposit date being February 21, 2023.

[0010] In some embodiments of the present invention, the Bifidobacterium breve Bifidobacterium breve Isolated from feces.

[0011] In a second aspect of the present invention, a product is provided, comprising at least one of (1) to (8):

[0012] (1) the above-mentioned Bifidobacterium breve;

[0013] (2) a bacterial agent containing the above-mentioned Bifidobacterium breve;

[0014] (3) a live bacterial solution containing the above-mentioned Bifidobacterium breve;

[0015] (4) a dead bacteria solution containing the above-mentioned Bifidobacterium breve;

[0016] (5) a culture product containing the above-mentioned Bifidobacterium breve;

[0017] (6) containing metabolites of the above-mentioned Bifidobacterium breve;

[0018] (7) An extract containing the above-mentioned Bifidobacterium breve;

[0019] (8) A culture supernatant containing the above-mentioned Bifidobacterium breve; the product is a medicine or a medical device.

[0020] In some embodiments of the present invention, the preparation may further contain other bacterial agents, which preferably include at least one of strains that promote colonization, other strains that have synergistic functions on the strains of the present invention, or formulated bacterial agents.

[0021] In some embodiments of the present invention, the viable count of the Bifidobacterium breve XA-2103 in the drug is ≥1×10 6 CFU.

[0022] In some embodiments of the present invention, the drug further comprises a drug carrier and / or a pharmaceutical excipient.

[0023] In some embodiments of the present invention, the drug carrier comprises a microcapsule, a microsphere, a nanoparticle and / or a liposome.

[0024] In some embodiments of the present invention, the pharmaceutical excipients include solvents, propellants, solubilizers, cosolvents, emulsifiers, colorants, adhesives, disintegrants, fillers, lubricants, wetting agents, osmotic pressure regulators, stabilizers, glidants, flavoring agents, preservatives, suspending agents, coating materials, fragrances, anti-adhesives, integrities, penetration enhancers, pH regulators, buffers, plasticizers, surfactants, foaming agents, defoamers, thickeners, inclusion agents, humectants, absorbents, diluents, flocculants and deflocculating agents, filter aids and / or release retardants.

[0025] In some embodiments of the present invention, the dosage form of the drug is powder, ointment, drops, gel, lozenge, granules, suspension, syrup, patch, capsule, spray, tablet, pill, injection, gel or oral solution.

[0026] In some embodiments of the present invention, the powder includes a powder and a lyophilized powder.

[0027] In some embodiments of the present invention, the cream comprises an emulsion or a cream.

[0028] In some embodiments of the present invention, the medical device is a gel.

[0029] In a third aspect of the present invention, a food or health product is provided, comprising at least one of (1) to (8):

[0030] (1) the above-mentioned Bifidobacterium breve;

[0031] (2) a bacterial agent containing the above-mentioned Bifidobacterium breve;

[0032] (3) a live bacterial solution containing the above-mentioned Bifidobacterium breve;

[0033] (4) a dead bacteria solution containing the above-mentioned Bifidobacterium breve;

[0034] (5) a culture product containing the above-mentioned Bifidobacterium breve;

[0035] (6) containing metabolites of the above-mentioned Bifidobacterium breve;

[0036] (7) An extract containing the above-mentioned Bifidobacterium breve;

[0037] (8) A culture supernatant containing the above-mentioned Bifidobacterium breve.

[0038] In some embodiments of the present invention, the food comprises one of a special medical food, a health product and a functional beverage.

[0039] In some embodiments of the present invention, the food further comprises a food additive.

[0040] In some embodiments of the present invention, the food additive includes at least one of a protective agent, a nutrient, an antioxidant, an enzyme preparation, a flavor enhancer, a preservative, and a sweetener.

[0041] In some embodiments of the present invention, the protective agent comprises glycerol, L-cysteine ​​hydrochloride, inulin, lysine, maltodextrin and / or trehalose.

[0042] In some embodiments of the present invention, the nutrient comprises glucose, lactose, galactose, fructose, N-acetylglucosamine, mannose, sucrose, maltotriose, pectin, raffinose, and / or stachyose.

[0043] In the fourth aspect of the present invention, the above-mentioned Bifidobacterium breve is provided. Bifidobacterium breve Or the use of the product proposed in the second aspect above, wherein the use is in the preparation of a product for treating and / or preventing inflammation.

[0044] In some embodiments of the present invention, the application is application in the preparation of a product for treating and / or preventing intestinal diseases.

[0045] In some embodiments of the present invention, the intestinal disease includes at least one of ulcerative colitis, bacterial diarrhea, viral diarrhea, antibiotic-associated diarrhea, irritable bowel syndrome, and functional dyspepsia.

[0046] In some embodiments of the present invention, the application is application in preparing a product for repairing intestinal barrier damage.

[0047] In some embodiments of the present invention, the product is a pharmaceutical product.

[0048] In some embodiments of the present invention, the application is application in the preparation of inflammatory factor regulators.

[0049] In some embodiments of the present invention, the inflammatory factors include but are not limited to TNF-α, IL-6 and IL-10.

[0050] According to some embodiments of the present invention, at least the following beneficial effects are achieved: The present invention provides Bifidobacterium breve XA-2103, which can significantly alleviate DSS-induced ulcerative colitis and significantly reduce overall inflammation levels in experimental animals. In in vitro evaluation, this strain significantly reduces the secretion of inflammatory factors and maintains the integrity of the intestinal barrier. Therefore, Bifidobacterium breve XA-2103 has great application prospects in the preparation of drugs for the prevention and / or treatment of ulcerative colitis and inflammation. Furthermore, Bifidobacterium breve XA-2103 is a probiotic, listed on the national "List of Bacteria Acceptable for Food Use," and is less likely to induce immune tolerance or liver damage. BRIEF DESCRIPTION OF THE DRAWINGS

[0051] The present invention will be further described below with reference to the accompanying drawings and embodiments, in which:

[0052] Figure 1 This is the effect of Bifidobacterium breve XA-2103 in Example 2 of the present invention on the body weight changes of UC mice induced by dextran sulfate sodium salt;

[0053] Figure 2 This is a graph showing the effect of Bifidobacterium breve XA-2103 in Example 2 of the present invention on the body weight change of DSS-induced UC mice on day 10, where "*" represents p < 0.05 and "***" represents p < 0.001;

[0054] Figure 3 This is a graph showing the test results of the effect of Bifidobacterium breve XA-2103 in Example 2 of the present invention on the colon length ratio of DSS-induced UC mice;

[0055] Figure 4 This is a graph showing the results of testing the effect of Bifidobacterium breve XA-2103 in Example 2 of the present invention on the changes in DAI (Disease Activity Index) scores of DSS-induced UC mice;

[0056] Figure 5 This is a graph showing the effect of Bifidobacterium breve XA-2103 in Example 2 of the present invention on the DAI scores of each group of DSS-induced UC mice on the 10th day, where "**" means p < 0.01;

[0057] Figure 6 This is a graph showing the effect of Bifidobacterium breve XA-2103 in Example 2 of the present invention on the serum IL-6 (interleukin-6) concentration in DSS-induced UC mice, where "*" means p < 0.05;

[0058] Figure 7 This is a graph showing the results of testing the effect of Bifidobacterium breve XA-2103 in Example 2 of the present invention on the serum TNF-α (tumor necrosis factor-α) concentration in DSS-induced UC mice;

[0059] Figure 8 This is a graph showing the effect of Bifidobacterium breve XA-2103 in Example 2 of the present invention on colon pathology in DSS-induced UC mice;

[0060] Figure 9This is a graph showing the effect of Bifidobacterium breve XA-2103 in Example 3 of the present invention on the expression of IL-10 (interleukin-10) in the supernatant of PBMC (peripheral blood mononuclear cells) induced by LPS, wherein "*" represents p < 0.05, "**" represents p < 0.01, and "***" represents p < 0.001;

[0061] Figure 10 This is a graph showing the ratio of IL-6 / IL-10 in the supernatant of PBMCs induced by LPS by Bifidobacterium breve XA-2103 in Example 3 of the present invention;

[0062] Figure 11 This is a graph showing the ratio of Bifidobacterium breve XA-2103 to LPS-induced PBMC supernatant TNF-α / IL-10 in Example 3 of the present invention, where "*" indicates p < 0.05 and "**" indicates p < 0.01;

[0063] Figure 12 This is a graph showing the test results of the effect of Bifidobacterium breve XA-2103 in Example 4 of the present invention on the FITC-dextran transmittance in a confluent monolayer epithelial injury model;

[0064] Figure 13 This is a graph showing the effect of Bifidobacterium breve XA-2103 in Example 4 of the present invention on the transmembrane electrical resistance of a monolayer confluent epithelial injury model, where "****" means p < 0.0001;

[0065] Figure 14 This is a graph showing the results of testing the effect of Bifidobacterium breve XA-2103 in Example 4 of the present invention on the content of tight junction protein ZO-1 in a monolayer confluent epithelial injury model. DETAILED DESCRIPTION

[0066] The following will clearly and completely describe the concept and technical effects of the present invention in conjunction with the embodiments to fully understand the purpose, features and effects of the present invention. Obviously, the embodiments described are only part of the embodiments of the present invention, not all of them. Based on the embodiments of the present invention, other embodiments obtained by those skilled in the art without creative work are all within the scope of protection of the present invention.

[0067] The culture medium and its preparation method involved in the following examples are as follows:

[0068] BHI liquid medium: Weigh 37 g of BHI broth powder and add it to 1000 mL of ultrapure water. Add 1 g of L-cysteine ​​hydrochloride and 0.01 g of hemin. Once completely dissolved, sterilize at 121°C for 15 min. Once the medium has cooled to 60°C, add 1 mg of vitamin K1 and mix thoroughly to prepare BHI liquid medium.

[0069] BHI solid medium: Weigh 37 g of BHI broth powder and add it to 1000 mL of ultrapure water. Add 1 g of L-cysteine ​​hydrochloride, 0.01 g of hemin, and 2% (m / v) agar powder. Once completely dissolved, sterilize at 121°C for 15 min. When the medium cools to 60°C, add 1 mg of vitamin K1, mix thoroughly, pour onto a plate, and let stand on an anaerobic bench for 16 hours to obtain BHI solid medium.

[0070] mGAM liquid medium: Weigh 41.7 g of mGAM broth powder (purchased from Haibo Biotechnology) and 10 mg of hemin, dissolve in 1000 mL of ultrapure water, and sterilize at 121°C for 20 minutes after aliquoting. After cooling to room temperature, add 2 μL of vitamin K1 per 200 mL of medium.

[0071] mGAM solid medium: Weigh 41.7 g of mGAM broth powder and 10 mg of hemin, dissolve in 1000 mL of ultrapure water, and add 2% (m / v) agar powder. Sterilize at 121°C for 20 minutes. After cooling to room temperature, add 2 μL of vitamin K1 per 200 mL of medium, mix thoroughly, pour into a plate, and let stand in an anaerobic workbench for 16 hours to obtain BHI solid medium.

[0072] The detection method of the viable cell count involved in the following examples is as follows:

[0073] The national standard "GB 4789.35-2016 National Food Safety Standard Food Microbiology Testing Lactic Acid Bacteria Testing" was used for testing.

[0074] The specific preparation method of the Bifidobacterium breve XA-2103 bacterial suspension involved in the following examples is as follows:

[0075] Bifidobacterium breve XA-2103 was streaked onto mGAM solid medium and cultured statically at 37°C in an anaerobic workstation (Electrotek AW500TG) for 24 h to obtain a single colony; a single colony was picked and inoculated into mGAM liquid medium and cultured in an anaerobic workstation (Electrotek AW500TG) at 37°C for 18 h for activation to obtain an activation solution; the activation solution was inoculated into mGAM liquid medium at a 4% (v / v) inoculation volume and cultured in an anaerobic workstation (Electrotek AW500TG) at 37°C for 18 h to obtain a bacterial solution; the bacterial solution was centrifuged at 4000×g for 5 min to obtain Bifidobacterium breve cells; the Bifidobacterium breve cells were washed with physiological saline and resuspended in physiological saline to a bacterial concentration of 1×10 10 CFU / mL, and obtain Bifidobacterium breve suspension.

[0076] Example 1 Strain isolation and identification

[0077] 1. Bifidobacterium breve Bifidobacterium breve Isolation of strain XA-2103

[0078] The XA-2103 strain was isolated from a stool sample. The specific isolation method is as follows:

[0079] In an anaerobic workbench, 1 g of fresh fecal sample from healthy people in Shenzhen was taken and dissolved in 10 mL of sterile PBS buffer (purchased from Sangon Biotech Co., Ltd., pH 7.2-7.4), and the mixture was shaken to obtain a fecal suspension. The fecal suspension was diluted 10-fold in a stepwise gradient using sterile PBS buffer to obtain 10 -1 , 10 -2 , 10 -3 , 10 -4 , 10 -5 , 10 -6 Dilution; draw 10 -4 diluent, 10 -5 dilution and 10 -6 The dilutions were spread on BHI solid culture medium at a coating amount of 100 μL / plate and incubated anaerobically at 37°C for 48 h. A single colony was picked from a BHI solid culture medium with a colony count between 30 and 500 using an inoculating loop, and the culture was re-streaked onto a new BHI solid culture medium and incubated anaerobically at 37°C for 48 h. A single colony on the new BHI solid culture medium was picked using an inoculating loop and inoculated into a BHI liquid culture medium, and incubated anaerobically at 37°C for 24 h to obtain a bacterial solution. The strain corresponding to this bacterial solution was named XA-2103.

[0080] 2. Identification of strain XA-2103

[0081] 1. Biochemical identification of strain XA-2103

[0082] The prepared Bifidobacterium breve XA-2103 was streaked onto mGAM solid medium and cultured anaerobically at 37°C for 48 h, after which the cells were collected. The cells were resuspended in a 0.45% (m / v) sodium chloride aqueous solution, and the McFarland turbidity was adjusted to 2.6 to obtain a bacterial suspension. The bacterial suspension was used as a sample to detect the physiological and biochemical properties of Bifidobacterium breve XA-2103 using a BioMérieux VITEK2 biochemical analyzer and an ANC anaerobic bacteria test card. The test results are shown in Table 3.

[0083] Table 1

[0084]

[0085] Explanation of symbols: “+”, positive; “-”, negative.

[0086] The results are shown in Table 1. As can be seen, Bifidobacterium breve XA-2103 exhibited positive results (marked with +) for carbon sources such as sucrose (SAC), maltotriose (MTE), D-glucose (dGLU), and D-galactose (dGAL), indicating that the strain can utilize these substances as carbon sources. However, the strain exhibited negative results (marked with -) for arginine (ARG) and α-L-arabinoside (AARAF), indicating that the strain cannot utilize these substances. Overall, this strain has a wide range of carbon sources and can utilize most common carbon sources, making it easy to cultivate.

[0087] 2. 16s rRNA sequence identification

[0088] 2 μL of XA-2103 bacterial solution was used as a template, and the strain was identified by 16S rRNA sequence PCR amplification and sequencing. The PCR product was confirmed by nucleic acid electrophoresis analysis and sent to BGI for sequencing. The returned sequencing results were spliced ​​(the spliced ​​16S rDNA sequence is shown in SEQ ID NO.1) and sequence alignment was performed using the NCBI database. The results showed that XA-2103 was Bifidobacterium breve , named Bifidobacterium breve Bifidobacterium breve XA-2103.

[0089] The PCR reaction system was prepared according to Table 2. The PCR reaction conditions were: step 1, 98°C, 60 s; step 2, 98°C, 10 s; step 3, 58°C, 20 s; step 4, 72°C, 90 s; step 5, 72°C, 5 min; steps 2 through 4 were repeated 30 times. The primer sequences used for PCR are shown in Table 3.

[0090] Table 2 PCR reaction system for bacterial identification

[0091]

[0092] Table 3 Primer names and sequences

[0093]

[0094] Bifidobacterium breve Bifidobacterium breve The sequence of 16s rDNA of XA-2103 is shown below:

[0095]

[0096] The strain was deposited in the General Microbiology Center of China Culture Collection Administration (CGMCC, address: No. 3, Yard 1, Beichen West Road, Chaoyang District, Beijing) on ​​February 21, 2023, with the deposit number CGMCC No. 26611.

[0097] Experimental Example 2 Effects of Bifidobacterium breve XA-2103 on DSS-induced ulcerative colitis in mice

[0098] This experimental example provides an experiment on the effect of Bifidobacterium breve XA-2103 on DSS-induced ulcerative colitis in mice. The experimental process is as follows:

[0099] The experimental animals were obtained from Guangdong Weitong Lihua and were SPF-grade C57BL / 6 female mice, aged 6 to 8 weeks and weighing 18 to 20 g. The experimental animals were randomly divided into three groups: control group, DSS-induced group, and XA-2103-treated group, with 9 mice in each group. The experimental animals were housed for 10 days. The control group mice drank sterile water and were gavaged daily with 200 μL of 0.9% (m / v) saline. The DSS-induced group mice had their drinking water replaced with 2.5% (m / L) DSS aqueous solution from day 1 to day 7 of the experiment, and were gavaged daily with 200 μL of 0.9% (m / v) saline. The XA-2103-treated group drank 2.5% (m / v) DSS aqueous solution from day 1 to day 7 of the experiment, and were gavaged daily with 200 μL of 2×10 9 CFU of Bifidobacterium breve XA-2103 (solvent: physiological saline). Days 8 and 9 of the experiment served as a rest period, during which all mice in each group received sterile water and gavage was discontinued. Day 10 marked the end point of the experiment, with all mice euthanized and assessed for inflammation according to the evaluation criteria.

[0100] The evaluation criteria for inflammation are as follows:

[0101] From the first day to the end of the experiment, the weight of mice in all groups was recorded and statistically analyzed daily. The statistical results are shown in Figure 1 .

[0102] The body weight at the end of the experiment was counted, and the differences in body weight changes among the groups are shown in Figure 2 .

[0103] At the end of the experiment, mice were euthanized and the colon length and total intestinal length of all groups of mice were measured. The ratio of colon length to total intestinal length was calculated. The statistical results are shown in Figure 3 .

[0104] After the experiment started, the feces of the mice were collected on the 1st, 4th, 7th and 10th days, the fecal characteristics were observed and the blood in the stool was detected. The DAI score was calculated based on the weight changes. The score results are shown in Figure 4 .

[0105] On the end day of the experiment, the feces of the mice were collected and DAI scores were calculated. The statistical differences in DAI scores among the groups on that day are shown in Figure 5 .

[0106] The DAI scoring system is as follows: The DAI score follows Murthy's scoring system and includes three aspects: weight change, blood in stool, and stool consistency (the specific scoring criteria are shown in Table 4). During the modeling period, mice were weighed daily, and blood in stool and stool consistency were tested. Scores were calculated according to Table 4. The DAI score is the sum of the three scores divided by 3: DAI score = (weight change score + blood in stool score + stool consistency score) / 3. Fecal occult blood was determined using a fecal occult blood (OB) reagent (Pyramide hole semi-quantitative test method) (purchased from Zhuhai Beso). If reddish-brown or bright red blood is visible in the stool, it is considered grossly bloody. Stool consistency is categorized into three levels: normal, loose, and loose. Normal mouse feces are formed and granular; loose feces are characterized by increased viscosity and easy dispersion, but do not adhere to the anus; and loose feces are characterized by unformed or watery feces that adhere to the anus.

[0107] Table 4 DAI scoring criteria

[0108]

[0109] After euthanasia at the end of the experiment, blood was quickly collected from all groups of mice and serum was separated. The cytokine content in the serum was detected using a flow cytometry multi-factor detection kit (purchased from Biolegend). The statistical results of serum IL-6 (interleukin-6) are shown in Figure 6 , the statistical results of serum TNF-α (tumor necrosis factor-α) are shown in Figure 7 .

[0110] At the end of the experiment, mice were euthanized and colon length was measured. A 0.5 cm segment of colon near the cecum was cut and fixed with 4% (m / v) paraformaldehyde. After fixation, paraffin sections were prepared and H&E staining was performed to evaluate intestinal pathology. The evaluation results are shown in the table. Figure 8 .

[0111] The experimental results are as follows:

[0112] like Figure 1 and Figure 2As shown, by day 8 of the experiment, the weight of mice in the control group remained largely unchanged throughout the experiment. However, the weight of mice in the DSS-induced group began to decline from day 5, reaching its lowest point on day 9 (a 6% decrease compared to the initial weight), and then showed a slight recovery on day 10 (a 5.5% decrease compared to the initial weight). After oral administration of Bifidobacterium breve XA-2103, the weight loss trend in the XA-2103-treated group was alleviated compared to the DSS-induced group. By day 10, the weight of mice in the treated group had decreased by only 2%, a significant increase compared to the DSS-induced group. This suggests that Bifidobacterium breve XA-2103 can significantly mitigate the weight loss caused by DSS.

[0113] like Figure 3 As shown, after mice were given DSS-treated water, the proportion of colon length to the total tract decreased slightly (by 1.4%). However, after oral administration of Bifidobacterium breve XA-2103, the proportion of colon length to the total tract increased (by 1.2% compared to the disease group). These results suggest that Bifidobacterium breve can mitigate the DSS-induced tendency for colon shortening.

[0114] like Figure 4 and Figure 5 As shown, the DAI scores of the control group mice remained low (0-0.5) over the course of the experiment. However, the DAI scores of the other two groups increased, but the DAI scores of the DSS-induced group were higher than those of the XA-2103-treated group. At the end of the experiment (day 10), the DAI scores of the DSS-induced group approached 1.5, while those of the XA-2103 group were less than 0.5, representing a significant decrease compared to the DSS model group.

[0115] like Figure 6 As shown in the results, compared with the control group, the IL-6 concentration in the serum of inflammatory mice in the DSS-induced group increased from 373 pg / mL to 442 pg / mL. However, after the mice received oral gavage treatment with Bifidobacterium breve XA-2103, the IL-6 concentration decreased significantly compared with the DSS modeling group, reaching only 324 pg / mL.

[0116] like Figure 7 As shown in the results, compared with the control group, the serum TNF-α concentration in the DSS-induced inflammatory mice increased from 515 pg / mL to 566 pg / mL. However, after mice were treated with Bifidobacterium breve XA-2103 by oral gavage, the TNF-α concentration decreased compared with the DSS-induced group, reaching 545 pg / mL. Both IL-6 and TNF-α stimulate immune responses and exacerbate IBD symptoms. These results suggest that Bifidobacterium breve XA-2103 significantly reduced the overall level of inflammation in mice, playing a suppressive role.

[0117] like Figure 8 As shown, the colonic tissue of mice in the control group was normal, with intact mucosal epithelial cells, normal crypts, neatly arranged glands, and virtually no immune cell infiltration. However, DSS caused severe damage to the colonic mucosa, with ulceration of the mucosal layer, and virtually no orderly glandular arrangement, while immune cell infiltration was clearly observed. Compared with the DSS-induced group, the colonic epithelium of mice in the XA-2103-treated group was intact, with only a few ulcerations, visible glandular arrangement, and significantly reduced immune cell infiltration. These results demonstrate that Bifidobacterium breve XA-2103 intervention maintained colonic integrity and reduced colonic inflammation.

[0118] The above results show that Bifidobacterium breve XA-2103 can reduce the inflammation level of mice in many aspects, maintain the structural integrity of the mice's intestines, and significantly alleviate the symptoms of ulcerative colitis caused by DSS.

[0119] Experimental Example 3 Effect of Bifidobacterium breve XA-2103 on the expression of inflammatory factors in PBMC cells

[0120] This experimental example provides an experiment on the effect of Bifidobacterium breve XA-2103 on the expression of inflammatory factors in PBMC cells induced by LPS. The experimental process is as follows:

[0121] PBMC cells (purchased from Shanghai Aoneng Company) were taken out of the liquid nitrogen tank and thawed in a 37°C water bath with shaking for 3 minutes to obtain thawed PBMC cells; the thawed PBMC cells were resuspended in 5 mL of PBS buffer preheated to 37°C, centrifuged at 400×g for 10 minutes, and the supernatant was discarded to obtain precipitate A; precipitate A was washed with 5 mL of cell culture medium (90% RPMI The cells were resuspended in RPMI 1640 medium + 10% fetal bovine serum + 50 μg / mL double-antibody, where % refers to volume ratio; RPMI 1640 medium was purchased from Gibco, fetal bovine serum was purchased from Solebol, and double-antibody was purchased from Gibco), and cell counting and viability detection were performed to obtain resuspension A; 8 mL of cell culture medium was added to resuspension A, and the cells were cultured at 5% (v / v) CO2 and 37°C for 6 h to obtain culture medium; the culture medium was centrifuged at 400 × g for 10 min, the supernatant was discarded, and the cells were counted to obtain precipitate B; the precipitate B was resuspended in cell culture medium to a concentration of 1 × 10 6 cells / mL, and resuspension B was obtained; first, resuspension B was added to a 96-well plate at a volume of 100 μL per well, and then lipopolysaccharide (LPS, purchased from Sigma-Aldrich) was added to the 96-well plate at a volume of 1 μg / mL, and then 1×10 7A bacterial solution of Bifidobacterium breve XA-2103 (solvent: physiological saline) with a concentration of 10 μL / well was added to a 96-well plate, and the 96-well plate was anaerobically incubated at 37°C for 2 h. After incubation, the live Bifidobacterium breve XA-2103 bacteria in the 96-well plate were washed away with PBS buffer containing 10% (m / v) bispecific antibody, and new cell culture medium was added to the 96-well plate at a concentration of 125 μL. The cells were cultured at 5% (v / v) CO2 and 37°C for 22 h. After culture, the supernatant was collected by centrifugation, and the cytokine content in the supernatant was detected using a flow cytometry multifactor detection kit (purchased from Biolegend).

[0122] Test results such as Figures 9-11 As shown in the figure, LPS induction can reduce the IL-10 content in the cell supernatant while increasing the IL-6 / IL-10 and TNF-α / IL-10 ratios. These results indicate that LPS can stimulate inflammatory responses in cells. However, when the cells interacted with Bifidobacterium breve XA-2103, the IL-10 content increased significantly, while the IL-6 / IL-10 and TNF-α / IL-10 ratios decreased significantly. These results demonstrate that Bifidobacterium breve XA-2103 has the ability to suppress inflammation.

[0123] Experimental Example 4: Repair Function of Bifidobacterium breve XA-2103 in Confluent Epithelial Monolayer Damage

[0124] This experimental example provides an experiment on the effect of Bifidobacterium breve XA-2103 on LPS-induced CACO-2 epithelial monolayer confluent injury. The experimental process is as follows:

[0125] CACO-2 cells (introduced from the Shanghai Institute of Cell Biology, Chinese Academy of Sciences) and HT29-MTX-E12 cells (purchased from ATCC) in the logarithmic growth phase were seeded into the upper chamber of the Transwell chamber at a ratio of 9:1, with a total seeding volume of 2.5 × 10 5 The cells were placed in a 100 μL and 600 μL cell culture medium were added to the upper and lower chambers, respectively, and cultured at 5% (v / v) CO2 and 37°C for 24 hours. After culture, the cell culture medium was replaced with fresh medium in the upper and lower chambers, and the cells were cultured at 5% (v / v) CO2 and 37°C. Transmembrane resistance was measured using a cell resistance meter. Once the resistance value stabilized, the cells were considered fully confluent, resulting in a confluent CACO-2 / HT29-MTX-E12 epithelial monolayer. After obtaining a confluent epithelial monolayer, the culture medium in the upper and lower chambers was replaced with cell culture medium containing 100 μg / mL LPS, 40 ng / mL TNF-α, and 100 ng / mL IL-1β. The cells were cultured at 5% (v / v) CO2 and 37°C for 24 hours to establish a monolayer epithelial inflammatory injury model.

[0126] After obtaining the monolayer epithelial inflammation injury model, the culture medium in the upper and lower chambers was removed and replaced with new stimulant-free culture medium after washing. 7 A bacterial solution of Bifidobacterium breve XA-2103 (solvent: physiological saline) was added to the upper chamber of the Transwell at a volume of 10 μL per well and incubated anaerobically at 37°C for 2 hours. After the incubation, the culture medium in the upper and lower chambers was removed, and the Transwell chamber was washed once with PBS buffer. 100 μL and 600 μL of DMEM high-glucose cells without double antibodies were added to the upper and lower chambers of the Transwell chamber, respectively, and cultured in a cell culture incubator at 37°C and 5% (v / v) CO2 for 22 hours. During this period, the transmembrane electrical resistance (TEER, unit: Ω*cm2) of the confluent epithelial monolayer was measured using a cell resistance meter at -24 (before modeling), 0, 3, 6, and 12 hours of culture. The test results are shown in the table. Figure 12 .

[0127] Add 20 μL of FITC-dextran (4 KDa, 100 μg / mL) to the upper chamber and incubate in a cell culture incubator at 37°C and 5% (v / v) CO2 for 1 hour. Collect 60 μL of the culture medium in the lower chamber and use a multifunctional microplate reader to measure the fluorescence intensity of the substances in the culture medium in the lower chamber under the conditions of excitation light at 490 nm and emission light at 520 nm. The cell membrane permeability of FITC-dextran is calculated. The calculation results are shown in Figure 2. Figure 13 . Remove the culture medium from the upper and lower chambers, wash the Transwell chamber three times with PBS buffer, add 100 μL of 4% (w / v) paraformaldehyde pre-cooled to 4°C, and fix at 4°C for 25 minutes. After washing with PBS buffer, add 200 μL of PBS buffer containing 0.2% (w / v) Triton X-100 to each well and let it stand on ice for 10 minutes. After washing with PBS buffer, add 300 μL of PBS buffer containing 5% (w / v) BSA to each well and block at room temperature for 1 hour. Add anti-ZO-1 antibody according to the instructions. Incubate at 4°C for 16 hours; after washing with PBS buffer, add FITC-labeled secondary antibody and incubate at room temperature in the dark for 2 hours; after washing with PBS buffer, carefully peel off the membrane of the Transwell chamber, add a mounting medium containing DAPI, place it in a glass-bottomed culture dish and observe with a fluorescence microscope. The observation results are shown in the figure. Figure 14 .

[0128] The calculation formula of FITC-dextran paracellular transient permeability is:

[0129] FITC-dextran paracellular transient permeability P = (test well / blank well) * %.

[0130] The experimental results are as follows Figure 12-14 As shown, from Figure 12 As can be seen in the figure, in the absence of LPS stimulation, the transmembrane resistance did not change significantly, indicating that the fused epithelium maintained good integrity. However, under stimulation with LPS, TNF-α, and IL-1β, the fused epithelium was damaged, causing a continuous decrease in transmembrane resistance (by approximately 37%). However, after the addition of Bifidobacterium breve XA-2103, the transmembrane resistance decreased slightly before recovering. At the end of the experiment, the transmembrane resistance of the XA-2103 interaction group was essentially the same as that of the control group.

[0131] from Figure 13 As can be seen in the figure, at the end of the experiment, the FITC-Dextran permeability increased significantly (by approximately 2.25 times) after the stimulant was added to the cells compared to the control group. However, after the interaction with Bifidobacterium breve XA-2103, the FITC-Dextran permeability decreased significantly (to 49% of the stimulant group), remaining essentially the same as the control group.

[0132] from Figure 14 It can be seen that a large amount of tight junction protein ZO-1 is distributed on the cell membrane of the control group, while ZO-1 protein is almost unobservable on the epithelium of the treated group. However, the ZO-1 content in the monolayer epithelial injury model treated with Bifidobacterium breve XA-2013 can be significantly restored.

[0133] The above experiment on repairing damaged monolayer epithelium showed that Bifidobacterium breve XA-2103 could alleviate epithelial damage caused by irritants, repair epithelial barrier and maintain the integrity of epithelial structure.

[0134] While the embodiments of the present invention have been described in detail above with reference to the accompanying drawings, the present invention is not limited to the embodiments described above. Various modifications may be made within the scope of knowledge possessed by a person skilled in the art without departing from the spirit of the present invention. Furthermore, the embodiments of the present invention and the features thereof may be combined with one another unless there is a conflict.

Claims

1. A strain of Bifidobacterium breve Bifidobacterium breve , characterized in that, Bifidobacterium breve Bifidobacterium breve The breve Bifidobacterium Bifidobacterium breve The 16S rDNA sequence is shown in SEQ ID NO:

1.

2. A product, characterized in that Including at least one of (1) to (4): (1) The Bifidobacterium breve according to claim 1 Bifidobacterium breve ; (2) Containing the Bifidobacterium breve according to claim 1 Bifidobacterium breve of microbial agents; (3) Containing the Bifidobacterium breve according to claim 1 Bifidobacterium breve of live bacterial liquid; (4) Containing the Bifidobacterium breve according to claim 1 Bifidobacterium breve The culture product is a medicine.

3. A food or health product, characterized in that: Include at least one of (1) to (4): (1) The Bifidobacterium breve according to claim 1 Bifidobacterium breve ; (2) Containing the Bifidobacterium breve according to claim 1 Bifidobacterium breve of microbial agents; (3) Containing the Bifidobacterium breve according to claim 1 Bifidobacterium breve of live bacterial liquid; (4) Containing the Bifidobacterium breve according to claim 1 Bifidobacterium breve culture products.

4. The product according to claim 2, characterized in that The dosage form of the medicine is powder, ointment, drops, gel, lozenge, granule, suspension, syrup, patch, capsule, spray, tablet, pill, injection, gel or oral solution.

5. The product according to claim 4, characterized in that The medicine also includes a drug carrier and / or pharmaceutical excipients.

6. The product according to claim 5, characterized in that The drug carrier comprises microcapsules, microspheres, nanoparticles and / or liposomes.

7. The product according to claim 5, characterized in that The pharmaceutical excipients include solvents, propellants, solubilizers, cosolvents, emulsifiers, colorants, adhesives, disintegrants, fillers, lubricants, wetting agents, osmotic pressure regulators, stabilizers, glidants, flavoring agents, preservatives, suspending agents, coating materials, fragrances, anti-adhesives, integrators, penetration enhancers, pH regulators, buffers, plasticizers, surfactants, foaming agents, defoaming agents, thickeners, inclusion agents, humectants, absorbents, diluents, flocculants and deflocculating agents, filter aids and / or release retardants.

8. The Bifidobacterium breve according to claim 1 Bifidobacterium breve Or use of the product according to claim 2 in the preparation of a product for treating and / or preventing ulcerative colitis.

9. The Bifidobacterium breve according to claim 1 Bifidobacterium breve Or the use of the product according to claim 2 in the preparation of a product for repairing LPS-induced intestinal barrier damage.

Images