Bifidobacterium breve A3-3 with high ILA production and application thereof

Abstract

The application provides a Bifidobacterium breve A3-3 strain with high ILA yield and application thereof, the strain is preserved in the China General Microbiological Culture Collection Center, and the preservation number is CGMCC NO. 38736, and the preservation date is May 19, 2026. The strain is cultured in vitro, and the indole-3-lactic acid (ILA) yield is as high as 53.28 mg / L. Experiments show that the strain can significantly relieve the pathological symptoms of TNBS-induced colitis mice, improve the survival rate, reduce the disease activity index, restore the colon length and reduce the histopathological damage. The action mechanism comprises the following aspects: remodeling the intestinal flora homeostasis, improving the flora diversity, inhibiting conditional pathogenic bacteria and enriching beneficial bacteria; strengthening the tryptophan metabolism and enriching indole anti-inflammatory metabolites; inhibiting the IL-17 inflammatory pathway, restoring the Th17 / Treg, Th1 / Th2 and macrophage M1 / M2 immune balance; and up-regulating the expression of tight junction proteins and enhancing the intestinal barrier function. The application provides a safe and efficient new probiotic intervention scheme for inflammatory bowel disease, and has important clinical application value.

Description

Technical Field

[0001] This invention belongs to the field of microbial technology, and specifically relates to a short Bifidobacterium A3-3 that produces high levels of indole-3-lactic acid (ILA) and its applications. Background Technology

[0002] Inflammatory bowel disease (IBD) is a group of chronic idiopathic inflammatory bowel diseases affecting the ileum, rectum, and colon. It mainly includes ulcerative colitis and Crohn's disease, characterized by a long course, high relapse rate, and high risk of cancer development, severely impacting patients' quality of life and imposing a heavy medical burden. In recent years, the global incidence of IBD has continued to rise, especially in newly industrialized regions such as Asia, becoming a major public health issue. The pathogenesis of IBD is complex, involving multiple factors such as genetics, environment, gut microbiota imbalance, intestinal barrier damage, and immune disorders. Among these, gut microbiota dysbiosis and abnormal tryptophan metabolism are the core pathological links.

[0003] The gut microbiota is a key hub for regulating host immune homeostasis, and its metabolites play a crucial role in the development and progression of intestinal inflammation. In IBD patients, gut microbiota diversity is significantly reduced, with a substantial decrease in the abundance of beneficial commensal bacteria such as Bifidobacteria and Lactobacillus, while opportunistic pathogens such as Proteobacteria and adhesive invasive Escherichia coli proliferate abnormally, leading to gut microecological imbalance. Crucially, the tryptophan metabolism pathway in the gut is severely impaired in IBD patients: a reduction in indole-producing metabolic bacteria and a decrease in the abundance of aromatic lactate dehydrogenase (Aldh) genes result in insufficient ILA production. ILA is a unique tryptophan-indole pathway metabolite specific to gut microbiota and an important ligand for the aryl hydrocarbon receptor (AhR). It can activate the AhR signaling pathway, playing a key role in anti-inflammation, maintaining the intestinal barrier, and regulating immune homeostasis. Its content is significantly negatively correlated with IBD disease activity, making it a core active mediator connecting dysbiosis and intestinal inflammation.

[0004] Currently, clinical treatment for IBD mainly relies on aminosalicylic acids, glucocorticoids, and biologics. However, these treatments suffer from limitations such as limited efficacy, significant side effects, high cost, and low long-term response rates. There is an urgent need for safe, efficient, and economical new intervention methods. Probiotics, as gut microbiota regulators, are highly safe and readily accepted, showing great potential in IBD intervention. Studies have confirmed that certain Bifidobacteria and Lactobacilli can alleviate intestinal damage by regulating the gut microbiota and inhibiting inflammation. However, existing probiotics generally have shortcomings such as weak tryptophan metabolism, low ILA production, and limited anti-inflammatory effects. Summary of the Invention

[0005] In view of the problems existing in the prior art, the purpose of this invention is to provide a high-ILA-producing Bifidobacterium breve A3-3 and its application, aiming to safely and effectively prevent and / or treat inflammatory bowel disease by improving gut microbiota, increasing ILA levels, inhibiting inflammatory pathways, restoring immune balance and strengthening the intestinal barrier, and providing a novel, safe and efficient probiotic intervention solution for IBD.

[0006] The objective of this invention is achieved through the following technical solution: The first aspect of this invention provides a high-ILA-producing Bifidobacterium breve A3-3 strain, which is deposited at the China General Microbiological Culture Collection Center (CGMCC) with accession number CGMCC NO.38736 and deposit date of May 19, 2026.

[0007] Furthermore, the strain produced 53.28 mg / L of indole-3-lactic acid in vitro.

[0008] A second aspect of the present invention provides a microbial agent containing the Bifidobacterium breve A3-3 and / or the metabolites of the Bifidobacterium breve A3-3.

[0009] A third aspect of the present invention provides a pharmaceutical composition, wherein the active ingredient of the pharmaceutical composition comprises the *Bifidobacterium breve* A3-3 described in the first aspect, or the bacterial agent described in the second aspect.

[0010] Furthermore, the composition also includes a pharmaceutically acceptable carrier or excipient.

[0011] Furthermore, the dosage form of the composition includes lyophilized powder, capsules, oral liquid, tablets, or granules.

[0012] The fourth aspect of the present invention provides the use of the Bifidobacterium breve A3-3 described in the first aspect, or the bacterial agent described in the second aspect, or the pharmaceutical composition described in the third aspect in the preparation of a medicament for the prevention and / or treatment of inflammatory bowel disease.

[0013] Furthermore, the inflammatory bowel disease includes ulcerative colitis and Crohn's disease.

[0014] Furthermore, the drug is one or more of the following: (1) The drug can reshape the intestinal flora homeostasis; specifically, it can improve the diversity of intestinal flora, reduce the Firmicutes / Bacteroidetes ratio, reduce conditionally pathogenic bacteria including Proteobacteria, Escherichia coli-Shigella, and Enterococcus; enrich beneficial bacteria including Bifidobacterium and butyric acid-producing bacteria, and restore the structure of healthy flora. (2) The drug can enhance tryptophan metabolism and enrich indole anti-inflammatory metabolites; specifically, it carries a highly active Ald gene, efficiently metabolizes tryptophan to generate indole-3-lactic acid (ILA), indole-3-acetamide (IAM), etc.; increases intestinal ILA level, activates AhR pathway, directly anti-inflammatory and repairs intestinal barrier. (3) The drug can inhibit the IL-17 inflammatory pathway and restore intestinal immune balance; specifically, it can significantly downregulate pro-inflammatory factors including TNF-α, IL-1β, and IL-6, and upregulate anti-inflammatory factor IL-10; inhibit the IL-17 signaling pathway and downstream chemokines (Cxcl5, Ccl2, Lcn2); correct the imbalance of Th17 / Treg and Th1 / Th2, promote Treg differentiation, and inhibit Th17 overactivation; regulate the polarization of macrophages from pro-inflammatory M1 type to anti-inflammatory M2 type, reduce inflammatory infiltration, and promote tissue repair. (4) The drug can enhance the intestinal barrier function; specifically, it can upregulate the expression of tight junction proteins including ZO-1, Claudin-1, and Occludin, repair intestinal mucosal damage, reduce intestinal permeability, block the invasion of endotoxins and inflammatory factors, and reduce the inflammatory cascade response.

[0015] Information on strain preservation: The strain of *Bifidobacterium breve* was named A3-3 and is deposited at the China General Microbiological Culture Collection Center (CGMCC), located at No. 3, Courtyard 1, Beichen West Road, Chaoyang District, Beijing. Its classification name is *Bifidobacterium breve*, accession number is CGMCC NO. 38736, and the deposit date is May 19, 2026.

[0016] The advantages of this invention compared to the prior art are as follows: 1. The Bifidobacterium breve A3-3 provided by this invention was obtained by screening from the feces of healthy infants. It has the outstanding advantages of high production of indole-3-lactic acid (ILA) and strong tryptophan metabolism. The in vitro ILA production reached 53.28 mg / L, which is significantly higher than that of existing common Bifidobacteria (usually less than 20 mg / L). It provides a highly efficient source of endogenous anti-inflammatory metabolites for relieving intestinal inflammation. 2. The Bifidobacterium breve A3-3 described in this invention exerts significant anti-inflammatory and intestinal protective effects through multiple mechanisms: reshaping the intestinal flora structure, increasing flora diversity, inhibiting the proliferation of harmful bacteria, and enriching beneficial symbiotic bacteria; efficiently metabolizing tryptophan to generate ILA, activating the AhR pathway, and directly exerting anti-inflammatory activity; inhibiting inflammatory pathways such as IL-17, downregulating pro-inflammatory factors, upregulating anti-inflammatory factors, and restoring the immune balance between Th17 / Treg and macrophages; upregulating the expression of tight junction proteins, repairing intestinal mucosal damage, reducing intestinal permeability, and enhancing intestinal barrier function. 3. The Bifidobacterium breve A3-3 strain described in this invention has high safety, natural source, definite anti-inflammatory effect, and clear mechanism of action. It makes up for the shortcomings of existing probiotics, such as weak tryptophan metabolism, low ILA production, and limited anti-inflammatory effect. It provides a safe, efficient, and long-term applicable new probiotic intervention program for inflammatory bowel disease, and has important clinical application value and industrialization prospects. Attached Figure Description

[0017] The present invention will be further described below with reference to the accompanying drawings and embodiments: Figure 1 The expression levels of aromatic lactate dehydrogenase (Aldh) in different Bifidobacteria are shown; (A) Bifidobacterium longum; (B) Bifidobacterium bifidum; (C) Bifidobacterium pseudostrandii; (D) Bifidobacterium breve; (E) Bifidobacterium dentatum. Figure 2 The table shows the content of tryptophan metabolites in the culture supernatant of Bifidobacterium (8 strains); where (A) ILA; (B) IAM; (C) IAld; Figure 3 This demonstrates the effect of high ILA-producing Bifidobacterium on the survival rate of mice with colitis; Figure 4 The effect of high ILA-rich Bifidobacterium on pathological symptoms in colitis mice is shown; (A) survival rate; (B) DAI score; Figure 5 The effect of high-ILA-producing Bifidobacterium on colonic tissue in mice with colitis is shown; (A) colonic image; (B) colonic length; Figure 6 The effects of Bifidobacterium breve A3-3 on the histopathological damage of colon tissue in colitis mice are shown; (A) representative H&E stained colon tissue (100 μm); (B) histopathological score; Figure 7 The effect of Bifidobacterium breve A3-3 on the mRNA level of tight junction protein in the colon of colitis mice is shown; where (A) Claudin-1; (B) Occludin; (C) ZO-1; Figure 8 The effects of Bifidobacterium breve A3-3 on the expression of colonic inflammatory factors in colitis mice are shown; where (A) TNF-α; (B) IL-1β; (C) IL-6; (D) IL-10; Figure 9 The results show the α-diversity analysis of gut microbiota in mice under different treatment groups; where (A) Shannon index; (B) Sobs index; (C) Chao index; Figure 10 PCoA analysis of gut microbiota in mice under different treatment groups is shown; Figure 11 The relative abundance of gut microbiota species in mice under different treatment groups is shown; (A) at the phylum level; (B) at the genus level. Figure 12 The differences in gut microbiota genera among different treatment groups are shown (Wilcoxon rank-sum test); (A) CON group vs. TNBS group; (B) TNBS group vs. B. breve A group; Figure 13 The analysis of gut microbiota genus-level biomarkers (LEfSe) in mice under different treatment groups is shown. Figure 14 The levels of tryptophan metabolites in the intestinal contents of mice in different treatment groups are shown. Figure 15 This demonstrates the regulatory effect of Bifidobacterium breve A3-3 on colonic macrophage polarization; Figure 16 The regulatory effect of Bifidobacterium breve A3-3 on colonic macrophage polarization was demonstrated (qPCR verification). Detailed Implementation

[0018] The embodiments described are provided to better illustrate the present invention, but are not intended to limit the scope of the invention to the embodiments described. Therefore, non-essential improvements and adjustments made to the embodiments by those skilled in the art based on the above description are still within the scope of protection of the present invention.

[0019] The endpoints and any values ​​of the ranges disclosed herein are not limited to the precise ranges or values, and these ranges or values ​​should be understood to include values ​​close to these ranges or values. For numerical ranges, the endpoint values ​​of the various ranges, the endpoint values ​​of the various ranges and individual point values, and individual point values ​​can be combined with each other to obtain one or more new numerical ranges, which should be considered as specifically disclosed herein.

[0020] The present invention will be described in detail below through embodiments. It should be understood that the following embodiments are only used to exemplify and further explain and illustrate the content of the present invention, and are not intended to limit the present invention.

[0021] Example 1: Isolation, identification and screening of high-ILA-producing Bifidobacterium Fresh fecal samples were collected from four healthy infants aged 0-3 years. Suspected Bifidobacterium strains were isolated from these samples using a modified MRS medium. 16S rRNA gene sequencing successfully identified 174 Bifidobacterium strains. These strains belonged to five subspecies: 80 strains of *Bifidobacterium longum*, 38 strains of *Bifidobacterium breve*, 18 strains of *Bifidobacterium bifidum*, 26 strains of *Bifidobacterium pseudocatenulatum*, and 12 strains of *Bifidobacterium dentium*.

[0022] Further, the transcriptional level of the aromatic lactate dehydrogenase (Aldh) gene in the above 174 Bifidobacterium strains was detected by RT-qPCR to assess the molecular potential of the screened strains in producing tryptophan metabolites. The specific steps included: 1. Inoculate the strain identified as Bifidobacterium from a 4℃ storage plate onto fresh liquid MRS medium. After three generations of anaerobic culture at 37℃, take the bacterial solution and mix it with sterile glycerol and skim milk in a certain proportion, and store it in a -80℃ refrigerator for long-term storage.

[0023] 2. Bifidobacterium RNA extraction and Aldh expression detection (1) Extraction of total bacterial RNA Take 1 mL of bacterial culture in the logarithmic growth phase, centrifuge at 10,000 rpm for 1 min to collect the bacterial cells, and discard the supernatant. Wash the bacterial pellet once with 1 mL of sterile physiological saline, add 100 μL of TE buffer containing 3 mg / mL lysozyme, resuspend repeatedly by pipetting, and incubate at 37°C for 10 min. Add 3-5 glass beads and grind in a cryogenic grinder for 2 min (-10°C, 50 Hz).

[0024] Subsequent RNA extraction was performed according to the instructions of the Tiangen Bacterial Total RNA Extraction Kit. Once total bacterial RNA was extracted, reverse transcription was immediately performed.

[0025] (2) RNA reverse transcription Dilute the RNA to an appropriate concentration, and perform reverse transcription using the HiScript IV All-in-One Ultra RT SuperMix for qPCR kit. The reaction volume is as follows:

[0026] After preparing the reaction mixture, vortex it thoroughly. After brief centrifugation, place the sample into a gene amplification instrument for reverse transcription. The reaction procedure is as follows:

[0027] The obtained cDNA was stored at -20°C for later use.

[0028] (3) Real-time quantitative PCR Using cDNA synthesized by reverse transcription as a template, RT-qPCR was performed using the SYBR Green method. The 20 μL reaction mixture is as follows:

[0029] The two-step PCR amplification system is as follows:

[0030] This embodiment uses 16S rRNA as an internal reference gene. The method was used to determine the relative expression level of the target gene. The primer sequences are as follows:

[0031] The detection results for 143 strains of bacteria from 5 subspecies (Bifidobacterium breve n=37, Bifidobacterium bifidum n=17, Bifidobacterium dentatum n=7, Bifidobacterium longum n=63, and Bifidobacterium pseudostreptomyces n=19) are as follows: Figure 1 As shown, different species of Bifidobacteria exhibit varying tryptophan metabolic potential. Specifically, the vast majority of strains of *Bifidobacterium breve*, *Bifidobacterium bifidum*, and *Bifidobacterium dentatum* (100%, 100%, and 71%, respectively) showed significantly higher Aldh expression levels than the control strain; conversely, 90% of *Bifidobacterium longum* and all *Bifidobacterium pseudostrandii* showed significantly lower expression levels than the control strain. We selected eight strains with the highest Aldh expression levels from the three subspecies of *Bifidobacterium bifidum*, *Bifidobacterium dentatum*, and *Bifidobacterium breve* for subsequent HPLC analysis, and their renumbered strains are shown in Table 6.

[0032]

[0033] 3. HPLC quantitative analysis of tryptophan metabolites Furthermore, the content of tryptophan metabolites (IAM, ILA, IAld) in the cell-free supernatant of the above eight Bifidobacterium strains was quantitatively detected using the external standard method. Specifically, this included: (1) Preparation of cell-free supernatant Eight strains of Bifidobacterium were inoculated at a 1% (v / v) inoculum into 400 ml of MRS liquid medium and anaerobically cultured at 37°C for 20 h until the stationary phase. The bacterial suspension was aliquoted into 50 mL sterile centrifuge tubes and centrifuged at 4500 rpm for 10 min at 4°C to remove cells, collecting the supernatant. The supernatant was concentrated 10-fold, resuspended in sterile water, sterilized at 121°C, and finally centrifuged at 10000 rpm for 10 min to remove denatured proteins. The supernatant was then filtered through a 0.22 μm sterile filter to obtain cell-free supernatant (CFS). The CFS was transferred to new centrifuge tubes, lyophilized, and stored at -80°C until use.

[0034] (2) Determination of ILA content by high performance liquid chromatography 2.1 Sample Pretreatment Take the lyophilized cell-free supernatant (CFS) powder into a 1.5 mL centrifuge tube, add 1.0 mL of ultrapure water (Watsons) to reconstitute, and vortex for 30 s to completely dissolve the sample. Transfer 100 μL of the reconstituted solution to a new 1.5 mL centrifuge tube, add 400 μL of methanol extraction buffer (protein precipitant), and vortex for 30 s. Place the mixture in a cryogenic sonicator and sonicate at 5 °C and 40 kHz for 30 min. After sonication, immediately transfer the sample to a -20 °C freezer and let it stand for 30 min to allow for complete protein precipitation. Then centrifuge at 13,000 rcf for 15 min at 4 °C, and carefully collect 350 μL of the supernatant into a new 1.5 mL centrifuge tube, taking care to avoid aspirating the lower layer of protein precipitate.

[0035] The supernatant was freeze-dried in a freeze dryer until completely dry (approximately 12 h). 100 μL of ultrapure water was added to the dried residue for reconstitution, and the mixture was vortexed for 30 s. The solution was then sonicated again at 5 °C and 40 kHz for 15 min to ensure complete dissolution. The reconstituted solution was centrifuged at 13,000 rcf for 15 min at 4 °C. The supernatant was carefully transferred to a brown vial with an inner tube and stored at 4 °C protected from light for later use. Analytical analysis was performed within 24 h.

[0036] 2.2 Chromatographic parameters ILA content was determined using a Shimadzu Prominence LC-20AT high-performance liquid chromatography system, equipped with a DGU-20A5R online degasser, a SIL-20A autosampler, and a CTO-20A column oven. The chromatographic column was an Agilent C18 reversed-phase column (4.6 mm × 150 mm, 2.7 µm). Mobile phase preparation: Mobile phase A was ultrapure water containing 0.1% (v / v) trifluoroacetic acid (chromatographic grade); mobile phase B was acetonitrile containing 0.1% trifluoroacetic acid (chromatographic grade). Both were prepared fresh and degassed by sonication for 15 min before use. Gradient elution program: As shown in Table 7, flow rate 0.40 mL / min, column temperature 40℃, injection volume 10 μL, detection wavelength 280 nm, sampling frequency 2.5 Hz. The mobile phase was equilibrated with the initial mobile phase for at least 60 min until baseline stability before each analysis. A blank sample (ultrapure water) is inserted every 10 samples to exclude system drift and residue. The single-needle analysis time is 56 min to ensure good separation between the target peak and impurity peaks.

[0037]

[0038] 2.3 Establishment of Standard Curve and Quantitative Analysis Accurately weigh 5.00 mg of 3-indoleacetamide (IAM), ILA, and IAld standards into a 1.5 mL centrifuge tube. Add 1.0 mL of methanol-water mixture (50:50, v / v), vortex for 60 s (3000 rpm), and sonicate for 5 min (25℃) to prepare a 5.0 mg / mL standard stock solution. Aliquot the stock solution into 100 μL / tube into 1.5 mL brown centrifuge tubes and store immediately at -80℃. Immediately before use, serially dilute with mobile phase to prepare working standard solutions of concentrations (0.625, 1.25, 2.5, 5, 10, 20 μg / mL). Establish a standard curve using the external standard method. Inject the seven working standard solutions in ascending order of concentration, repeating the injection three times and taking the average peak area. Using the standard concentration X (μg / mL) as the x-axis and the peak area Y (mAU·min or integral unit) as the y-axis, a linear regression was performed using the weighted least squares method to establish the regression equation Y = aX + b, and the correlation coefficient was... ≥ 0.998.

[0039] The indole content in the sample is calculated using the following formula: Indole content (mg / L) = (C × V × DF) / Where C is the concentration calculated from the standard curve (μg / mL), V is the reconstitution volume (0.1 mL), and DF is the dilution factor during sample pretreatment. The original sample volume (based on the initial CFS volume). Three technical replicates were set for each sample, and results are expressed as mean ± standard deviation.

[0040] The standard curve equations and correlation coefficients for each substance are shown in the table below.

[0041]

[0042] HPLC quantitative analysis results showed that ( Figure 2 Within the same species, different strains exhibited significant differences in tryptophan metabolism. Within *Bifidobacterium breve*, strain 93-10 showed low metabolic activity and no bias; strains E1-4, D3-1, and A3-3 tended towards ILA synthesis. Among them, strain A3-3 demonstrated dual metabolic advantages, with a yield of 33.98 mg / L in the IAM pathway and an even higher yield of 53.28 mg / L in the ILA pathway, significantly superior to the other three *Bifidobacterium breve* strains. Within *Bifidobacterium bifidum*, the two strains showed completely different metabolic preferences; strain D1-6 favored the IAM synthesis pathway, with an IAM yield of 19.60 mg / L, and its downstream product IAld yield of 5.47 mg / L, significantly higher than the other *Bifidobacterium bifidum* strain; strain E2-3 favored the ILA synthesis pathway, with an ILA yield of 32.83 mg / L, showing significant intraspecific metabolic diversity. Within *Bifidobacterium dentata*, the two strains showed the same metabolic preference. Compared with B1-2, B2-2 has significant advantages in the IAM and ILA synthesis pathways, with an ILA yield of 48.88 mg / L, an IAM yield of 20.69 mg / L, and an IALd yield of 6.74 mg / L.

[0043] By comparing the tryptophan metabolism characteristics of eight candidate Bifidobacterium strains, *Bifidobacterium breve* A3-3 and *Bifidobacterium dentatum* B2-2 were selected for further functional validation. The selection criteria were as follows: the ILA yields of the two strains were 53.28 mg / L and 48.88 mg / L, respectively, which were significantly higher than those of common Bifidobacterium strains (usually below 20 mg / L).

[0044] Example 2: The alleviating effect of high-ILA-producing Bifidobacterium on colitis in mice This embodiment uses a 2,4,6-trinitrobenzenesulfonic acid (TNBS)-induced mouse colitis model to investigate whether two high-ILA-producing Bifidobacterium strains (Bifidobacterium breve A3-3 and Bifidobacterium dentata B2-2) can alleviate intestinal inflammation.

[0045] 1. Experimental materials Strains: The strains used in this example are the high-ILA-producing Bifidobacterium strains obtained in Example 1: Bifidobacterium breve A3-3 and Bifidobacterium dentata B2-2. After activation, the strains were inoculated into MRS medium and cultured anaerobically at 37°C until the stationary phase (approximately 20 h). The culture medium was centrifuged to collect the bacterial cells, washed three times with sterile PBS, and resuspended. CFU / mL and The concentration of CFU / mL was verified by plate counting and then used in the gavage experiment.

[0046] Animals: This study was approved by the Animal Care and Use Committee of Beijing Technology and Business University and strictly followed laboratory animal welfare and ethical guidelines. Sixty SPF-grade male Balb / c mice (6 weeks old, weighing 20±2 g) were purchased from Beijing Spefol Biotechnology Co., Ltd. The animals were housed at the SPF-grade laboratory animal platform of China Agricultural University, with the ambient temperature controlled at 22±2℃, relative humidity at 50%–60%, and a 12-hour light / 12-hour dark cycle. Mice had free access to food and water. All animals underwent a 3-day acclimatization period before the experiment.

[0047] 2. Experimental Methods (1) Animal grouping and treatment Balb / c mice were randomly divided into 6 groups of 10 mice each. After acclimatization for 3 days, the experiment began. An acute colitis model was induced in mice via enema with 2.5% TNBS ethanol solution. The experiment lasted a total of 13 days. The experimental animal groups are as follows: (a) Control group (CON): Abdominal hair removal was performed immediately after the adaptation period, followed by sensitization treatment with 100% ethanol solution; on day 7, 100% ethanol solution was administered via enema. Normal saline was administered via gavage starting from the end of the adaptation period.

[0048] (b) Model group (TNBS): Abdominal hair removal was performed immediately after the adaptation period, followed by sensitization treatment with 1% TNBS ethanol solution; on day 7, an enema with 2.5% TNBS ethanol solution was administered. Oral administration of physiological saline was initiated after the end of the adaptation period.

[0049] (c) High- and low-dose Bifidobacterium breve A3-3 intervention groups (HB. breve A; LB. breve A): Abdominal hair removal was performed immediately after the adaptation period, followed by sensitization treatment with 1% TNBS ethanol solution; on day 7, a 2.5% TNBS ethanol solution enema was administered. Gavage was initiated after the end of the adaptation period: the gavage concentration for the HB. breve A group was [missing information]. CFU / mL, LB. breve concentration in group A administered via gavage was [missing value]. CFU / mL.

[0050] (d) High- and low-dose Bifidobacterium dentium B2-2 intervention groups (HB. dentium B; LB. dentium B): Abdominal hair removal was performed immediately after the adaptation period, followed by sensitization treatment with 1% TNBS ethanol solution; on day 7, a 2.5% TNBS ethanol solution enema was administered. Gavage was initiated after the end of the adaptation period: the HB. dentium group received a gavage concentration of [missing information]. CFU / mL, the gavage concentration in the LB. dentium group was [missing value]. CFU / mL.

[0051] The specific procedures for establishing a TNBS-induced mouse colitis model are as follows: On day 0 of the experiment, mice were lightly anesthetized with isoflurane, and the abdominal hair (approximately 2 cm × 2 cm) was removed. Each group was then sensitized by applying either 100% ethanol or 1% TNBS ethanol solution. On day 7 of the experiment, after fasting for 12 hours but allowing free access to water, mice were deeply anesthetized with isoflurane. A lubricated rectal administration tube was slowly inserted approximately 4 cm into the colon through the anus. Each group was then injected with either 100% ethanol or 2.5% TNBS ethanol solution (75 μL / mouse). Note that after injection, the mouse's head should be kept upside down for approximately 60 seconds to prevent leakage, until the mouse awakens from anesthesia.

[0052] (2) Disease Activity Index (DAI) detection From the day of enema treatment (day 7) until sacrifice (day 10), the mice's weight changes, fecal characteristics, and bloody stools were observed and recorded at fixed times each day. The DAI scoring criteria are shown in Table 9. The overall DAI score was calculated using the following formula: DAI = (Weight loss score + Fecal characteristics score + Bloody stool score) / 3.

[0053]

[0054] (3) Sample collection and processing On day 10 of the experiment, mice were fasted for 12 hours but allowed free access to water. After weighing, they were deeply anesthetized with isoflurane, and their eyes were removed to collect whole blood. Blood samples were allowed to stand at room temperature for 30 minutes, then centrifuged at 4°C and 4000 rpm for 15 minutes to separate serum, which was then aliquoted and stored at -80°C for later use. Immediately after blood collection, mice were euthanized by cervical dislocation. The spleen, colon, small intestine, and cecum were dissected and completely separated. The spleen was weighed and recorded. The colon tissue was completely removed from the terminal cecum to the anus, fat and connective tissue were removed, and the length (cm) was measured and photographed. The colon tissue was longitudinally dissected, gently rinsed with pre-cooled PBS to remove contents, blotted dry with filter paper, and divided into three segments: the proximal colon was fixed in 4% paraformaldehyde solution for histopathological analysis; the mid-colon was placed in TRIzol reagent and stored at -80°C for RNA extraction and inflammatory factor expression detection; and the distal colon was cryopreserved at -80°C for subsequent transcriptomics analysis. Simultaneously, colon contents were collected, flash-frozen in liquid nitrogen, and stored at -80°C for analysis of intestinal flora and metabolites.

[0055] 3. Experimental Results (1) Effect of high-ILA-producing Bifidobacterium on the survival rate of mice with colitis TNBS-induced acute colitis can cause a severe inflammatory response, leading to significant weight loss, diarrhea, or bloody stools in mice, and in severe cases, death. Figure 3 As shown, compared with the CON group, the survival rate of the TNBS group was significantly lower (P < 0.01), at 54.17%. The survival rates of all intervention groups improved to varying degrees. For the two high-ILA-producing Bifidobacterium strains, the overall survival rate of the Bifidobacterium breve A3-3 group was higher than that of the Bifidobacterium dentata B2-2 group, and the survival rate of the high-dose group was higher than that of the low-dose group; among them, the survival rate of the high-dose Bifidobacterium breve A3-3 group was significantly higher than that of the TNBS group (P < 0.05), at 90.90%.

[0056] The above results indicate that intervention with high-ILA-producing Bifidobacterium can reduce the mortality rate of mice with TNBS-induced acute colitis to varying degrees, and this effect is dose-dependent.

[0057] (2) Effects of high-ILA-producing Bifidobacterium on the pathological state of colitis mice The mice exhibited symptoms such as persistent weight loss, reduced activity, altered fecal characteristics, and bloody stools, indicating the successful establishment of a TNBS-induced colitis model. Among these, weight change was the most common indicator reflecting disease activity.

[0058] like Figure 4As shown in Figure A, the fasting treatment before TNBS enema in all six groups of mice on day 7 resulted in a significant decrease in body weight in all groups on day 8. On days 9-10, during the initial recovery period after modeling, except for the TNBS group which continued to show a decreasing trend in body weight, the other groups showed varying degrees of weight recovery, with the CON group showing the most significant recovery (P < 0.001). On day 11, with the return to normal feeding and the inflammatory response induced by TNBS modeling, all six groups showed a decreasing trend in body weight. The high-dose Bifidobacterium breve A3-3 group and the high-dose Bifidobacterium dentate B2-2 group showed the smallest decrease in body weight, while the TNBS group showed a continuous decreasing trend in body weight after modeling. This indicates that intervention with high-dose Bifidobacterium breve A3-3 and Bifidobacterium dentate B2-2 can alleviate the sustained weight loss caused by TNBS-induced colitis.

[0059] Compared to the single indicator of weight change mentioned above, the DAI index can more comprehensively reflect IBD disease activity by integrating weight, fecal characteristics, and bloody stools. On day 8, after TNBS modeling, mouse weight, fecal characteristics, and occult blood were recorded daily. Starting from day 9, mice in the TNBS group developed loose stools, which became more severe over the next two days, with some mice experiencing bloody stools. Compared to the TNBS group, the onset and severity of loose and bloody stools were delayed in all intervention groups. The DAI index for each group was calculated. Figure 4 (B) After modeling, the DAI scores of both the TNBS group and each intervention group showed an upward trend. On the last day of the experiment, compared with the CON group, the DAI score of the TNBS group was significantly higher (P < 0.001), reaching 3.20; while after intervention with high-ILA-producing Bifidobacterium, the DAI scores of each group decreased to varying degrees compared with the TNBS group. Among them, the intervention effects of high-dose Bifidobacterium breve A3-3 group and high-dose Bifidobacterium dentata B2-2 group were the best, with DAI scores decreasing to 1.44 and 1.60, respectively. In conclusion, high-ILA-producing Bifidobacterium can reduce the DAI score of TNBS-induced colitis mice in a dose-dependent manner.

[0060] (3) Effects of high-ILA-producing Bifidobacterium on colonic tissue of mice with colitis Colonic shortening is a macroscopic indicator for assessing the degree of intestinal inflammation. In IBD mice, shortened colonic length is associated with intestinal wall edema, fibrosis, and muscle contraction. For example... Figure 5 As shown in Figure A, the colon tissue of mice in the CON group was intact, normal in color, and relatively long. The colon tissue of mice in the TNBS group showed congestion, edema, thickening of the intestinal wall, and shortening in length. All intervention groups alleviated congestion, edema, and shortening to varying degrees, with the high-dose short-dose Bifidobacterium A3-3 group showing the best effect. Furthermore, the high-dose group of the same strain provided better relief than the low-dose group.

[0061] Statistical analysis based on the recorded colon length shows that ( Figure 5 Compared with the CON group, the colon length in the TNBS group was significantly reduced (P < 0.001); compared with the TNBS group, the colon length in the HB. breve A group and HB. dentium B group was significantly increased (P < 0.01 or P < 0.01), while the colon length in the LB. breve A group and LB. dentium B group showed an increasing trend, but the difference was not statistically significant. These results indicate that high-yielding ILA-producing Bifidobacteria have a good protective effect against TNBS-induced colonic tissue damage, and high doses are more effective than low doses.

[0062] Example 3: Effects of Bifidobacterium breve A3-3 on intestinal barrier function and inflammatory factors in TNBS-induced colitis mice This embodiment aims to further verify the intestinal protective effect of the optimal strain selected in Example 2—Bifidobacterium breve A3-3—on TNBS-induced colitis mice, and to preliminarily explore its mechanism of action, focusing on its effects on colonic histopathology, intestinal barrier function, and inflammatory factors.

[0063] The animal model, bacterial strain culture, gavage method, and TNBS induction protocol used in this embodiment are the same as in Example 2. The intervention group with the best effect from Example 2 was selected, namely the high-dose Bifidobacterium breve A3-3 intervention group (HB. breve A, gavage concentration of...). The mice were divided into a control group (CON) and a model group (TNBS) with a concentration of CFU / mL. The number of animals in each group and the treatment method were the same as in Example 2. After the experiment, mouse colon tissue was collected according to the method described in Example 2 for histopathological analysis, RNA extraction, and detection of inflammatory factor expression.

[0064] 1. The effect of Bifidobacterium breve A3-3 on improving the pathological damage of colon tissue in colitis mice. To visually evaluate the protective effect of Bifidobacterium breve A3-3 against colon tissue damage, mouse colon tissue was stained with hematoxylin and eosin (H&E) and its morphological changes were observed under an optical microscope. At the same time, a double-blind method was used to score the histopathological findings.

[0065] Experimental results: like Figure 6As shown in Figure A, the colonic tissue structure of mice in the CON group was intact, with continuous mucosal epithelium, clear layers, and neatly arranged crypts, and no obvious inflammatory cell infiltration was observed. In contrast, the colonic tissue of mice in the TNBS group showed severe damage, characterized by extensive inflammatory cell infiltration in the mucosal and submucosal layers, destruction of crypt structures, significant loss of goblet cells, and obvious epithelial ulceration. Compared with the TNBS group, intervention with high-dose Bifidobacterium breve A3-3 significantly improved the pathological damage of the mouse colonic tissue: inflammatory cell infiltration was significantly reduced, crypt structures were partially restored, epithelial integrity was improved, and the areas of mucosal erosion and ulceration were significantly reduced.

[0066] Histopathological score (e.g.) Figure 6 The findings (shown in Figure B) further confirmed the above observations. The pathological score of the TNBS group was significantly higher than that of the CON group (P < 0.001), while the pathological score of the Bifidobacterium breve A3-3 intervention group was significantly lower than that of the TNBS group (P < 0.01). These results indicate that Bifidobacterium breve A3-3 can effectively alleviate TNBS-induced pathological damage to colonic tissue.

[0067] 2. Effects of Bifidobacterium breve A3-3 on the expression of intestinal barrier function-related proteins in colitis mice Tight junction proteins (such as Claudin-1, Occludin, and ZO-1) are key structures for maintaining the integrity of the intestinal mechanical barrier. In this study, the mRNA expression levels of these three tight junction proteins in mouse colon tissue were detected by RT-qPCR.

[0068] like Figure 7 As shown, compared with the control group, the mRNA levels of Claudin-1, Occludin, and ZO-1 in the colon tissue of mice in the TNBS group were significantly decreased. This indicates that TNBS disrupts the integrity of the intestinal barrier in mouse colon tissue, leaving the intestine in a damaged state. In contrast, compared with the TNBS group, the mRNA expression of Claudin-1, Occludin, and ZO-1 in colitis mice supplemented with high-dose Bifidobacterium breve A3-3 was significantly increased. These results indicate that supplementation with Bifidobacterium breve A3-3 can upregulate the expression of tight protein, improve TNBS-induced epithelial damage, and restore and maintain the intestinal barrier function in colitis mice.

[0069] 3. The regulatory effect of Bifidobacterium breve A3-3 on the expression of inflammatory factors in the colonic tissue of colitis-infected mice. Impaired intestinal barrier function can trigger an excessive immune response, leading to the release of large amounts of pro-inflammatory factors. In this study, the mRNA expression levels of key pro-inflammatory factors (TNF-α, IL-1β, and IL-6) and anti-inflammatory factor (IL-10) in mouse colon tissue were detected by RT-qPCR.

[0070] like Figure 8 As shown, compared with the CON group, the mRNA expression levels of pro-inflammatory factors TNF-α, IL-1β, and IL-6 in the colon tissue of mice in the TNBS group were significantly increased (P < 0.05), and the expression level of anti-inflammatory factor IL-10 also showed an upward trend, indicating that TNBS-induced intestinal inflammation was severe, leading to compensatory anti-inflammatory responses. After intervention with high-dose Bifidobacterium breve A3-3, the expression levels of pro-inflammatory factors were significantly reduced (P < 0.05), while the expression level of anti-inflammatory factor IL-10 was significantly increased (P < 0.01). These results indicate that Bifidobacterium breve A3-3 alleviates intestinal inflammation by regulating the pro-inflammatory / anti-inflammatory balance.

[0071] Example 4: Mechanism of action of Bifidobacterium breve A3-3 in alleviating colitis This embodiment further explores the underlying mechanism by which Bifidobacterium breve A3-3 alleviates TNBS-induced colitis. From four levels—intestinal flora structure, tryptophan metabolite content, colonic transcriptome changes, and immune cell balance—the mechanism by which this strain exerts its anti-inflammatory and intestinal protective effects through the "flora-metabolism-immunity" axis is systematically elucidated.

[0072] 1. The effect of Bifidobacterium breve A3-3 on the remodeling of intestinal flora in colitis mice Colonic contents were collected from the control group (CON), model group (TNBS), and high-dose Bifidobacterium breve A3-3 intervention group (HB.breve A) mice (n=4 randomly selected from each group) as described in Example 2, and total genomic DNA of the microbial community was extracted. The V3-V4 variable region of the 16S rRNA gene was amplified by PCR. After amplification, the results were detected by 2% agarose gel electrophoresis, and the PCR products were purified using a DNA gel recovery and purification kit. The purified PCR products were used to construct libraries using the NEXTFLEX Rapid DNA-Seq Kit and sequenced using the Illumina Nextseq2000 platform. ASV clustering was performed using UPARSE software with 97% similarity, and species annotation was performed using the Silva (v138) database. Differences in gut microbiota among the groups were assessed using α-diversity (Shannon index, Sobs index, Chao index), β-diversity (PCOA analysis), species composition analysis (phylum and genus levels), and LEfSe analysis.

[0073] Experimental results: (1) α-diversity analysis like Figure 9As shown, compared with the CON group, the TNBS group showed a significant decrease in the Shannon, Sobs, and Chao indices (P < 0.001), indicating that TNBS-induced acute colitis significantly reduced the species richness and diversity of the intestinal microbiota in mice. After intervention with Bifidobacterium breve A3-3, the Shannon, Sobs, and Chao indices of the mouse intestinal microbiota were significantly higher than those in the TNBS group (P < 0.001 or P < 0.01), and the Shannon index recovered to approximately 84.4% of that in the CON group. These results indicate that intervention with Bifidobacterium breve A3-3 significantly restored the species richness and community diversity of the intestinal microbiota in mice with TNBS-induced acute colitis.

[0074] (2) β-diversity analysis Principal Coordinate Analysis (PCoA) is performed based on the Bray-Curtis distance metric. The visualization results of the principal coordinate analysis are shown below. Figure 10 The percentage change in principal coordinate 1 (PC1) was 36.56%, and the percentage change in principal coordinate 2 (PC2) was 15.81%. The CON group samples mainly clustered in the left region of the PCoA plot, while the TNBS group samples were significantly biased towards the right region, showing a significant separation between the two groups (R=0.5278, p=0.012). The B. breve A group samples were distributed between the CON and TNBS groups, and were closer to the CON group. These results indicate that TNBS modeling significantly alters the gut microbiota structure in mice, and that intervention with Bifidobacterium breve A3-3 can regulate the gut microbiota structure, enabling it to recover to a healthy state.

[0075] (3) Species composition analysis (phylum and genus levels) like Figure 11 As shown in Figure A, at the phylum level, the gut microbiota of the three groups of mice mainly consisted of Firmicutes (Bacillota), Bacteroidetes, Pseudomonadota, and Actinomycetota. After TNBS modeling, the gut microbiota structure of mice was disordered: Firmicutes abundance increased, Bacteroidetes abundance decreased, and the Firmicutes / Bacteroidetes (F / B) ratio significantly increased; the abundance of Pseudomonadota also significantly increased. After intervention with Bifidobacterium breve A3-3, the abundance of Firmicutes decreased to 58.2%, the abundance of Bacteroidetes recovered to 18.6%, the F / B ratio tended to normalize, and the abundance of Pseudomonadota decreased to 4.2%.

[0076] like Figure 11As shown in Figure B, at the genus level, the CON group had a relatively balanced microbiota composition, with high abundance of genera including *Ligilactobacillus*, *Candidatus_Saccharimonas*, and *Lachnospiraceae_NK4A136_group*, which are related to maintaining intestinal homeostasis. The TNBS group had a monotonous microbiota composition: *Ligilactobacillus* abundance was abnormally high at 61.2%, and potentially pathogenic bacteria *Escherichia-Shigella* (11.2%) and *Enterococcus* (8.7%) were also abundant. The *Bifidobacterium breve* A3-3 intervention group showed a more balanced microbiota composition: *Ligilactobacillus* abundance decreased to 16.8%, and *Escherichia-Shigella* and *Enterococcus* abundance decreased to 2.1% and 1.5%, respectively. Notably, the abundance of *Bifidobacterium* and *Lactobacillus* increased in the *Bifidobacterium breve* A3-3 intervention group.

[0077] (4) LEfSe analysis was used to assess the differences in gut microbiota among the groups. Wilcoxon rank-sum test ( Figure 12 The results showed that, compared with the CON group, the relative abundance of potential pathogens Escherichia-Shigella and Enterococcus was significantly increased in the TNBS group (P < 0.05), while the abundance of beneficial symbiotics Candidatus_Saccharimonas, Adlercreutzia, and butyric acid-producing bacteria Lachnospiraceae_NK4A136_group was significantly decreased (P < 0.05). Compared with the TNBS group, the abundance of conditionally pathogenic bacteria Enterococcus was significantly decreased in the Bifidobacterium breve A3-3 intervention group (P < 0.05), while the abundance of butyric acid-producing bacteria Lachnospiraceae_NK4A136_group, [Eubacterium]_siraeum_group, and norank_o_Clostridia_UCG-014 was significantly increased (P < 0.05).

[0078] LEfSe analysis ( Figure 13Further analysis showed that the characteristic microbiota of the CON group was mainly composed of symbiotic bacteria that produce short-chain fatty acids (such as Candidatus_Saccharimonas, Adlercreutzia, Roseburia, Lachnospiraceae_NK4A136_group, and Blautia, etc.); the characteristic microbiota of the TNBS group was mainly composed of conditionally pathogenic bacteria (Escherichia-Shigella, Enterococcus, and Ligilactobacillus); and the characteristic microbiota of the Bifidobacterium breve A3-3 intervention group was mainly composed of butyric acid-producing bacteria (Clostridia_UCG-014, [Eubacterium]_siraeum_group, Acutalibacter, Christensenellaceae, and UCG-005, etc.).

[0079] The above results indicate that intervention with Bifidobacterium breve A3-3 can not only inhibit the proliferation of opportunistic pathogens, but also promote the recovery of beneficial bacteria such as butyric acid-producing bacteria, thus reshaping the gut microbiota homeostasis.

[0080] 2. Effects of Bifidobacterium breve A3-3 on tryptophan metabolites in the intestines of mice with colitis High-performance liquid chromatography (HPLC) was used to quantitatively detect the contents of tryptophan metabolites (IAM, ILA, IAld, IAA) in the colonic contents of mice in the control group (CON), model group (TNBS), and high-dose Bifidobacterium breve A3-3 intervention group (HB.breve A). The chromatographic conditions were the same as in Example 1, and the standard curve equations are shown in Table 10.

[0081]

[0082] Experimental results: HPLC detection results are as follows Figure 14 As shown, compared with the CON group, the IAM content in the colonic contents of the Bifidobacterium breve A3-3 intervention group increased from 4.58 mg / kg to 6.70 mg / kg, the ILA content increased from 2.50 mg / kg to 3.87 mg / kg, and the IAld content also showed an increasing trend; the IAA content did not differ significantly between the two groups. These results indicate that Bifidobacterium breve A3-3 may promote the production of various indole metabolites in vivo by activating the tryptophan-indole metabolic pathway. These metabolites collectively constitute the metabolic basis for inhibiting TNBS-induced excessive inflammatory responses.

[0083] 3. Regulation of the transcriptome and IL-17 signaling pathway in colon tissue of colitis mice by Bifidobacterium breve A3-3 Total RNA was extracted from colon tissues of mice in the control group (CON), model group (TNBS), and high-dose Bifidobacterium breve A3-3 intervention group (HB.breve A) for transcriptome sequencing. Differentially expressed genes (|log2 fold change (FC)| > 1 and corrected p-value (p-adj) < 0.05) were screened using DESeq2 software, and KEGG pathway enrichment analysis was performed using KOBAS software. The infiltration levels of 12 immune cell types were quantitatively analyzed using the ssGSEA method. Key differentially expressed genes were validated using RT-qPCR.

[0084] Experimental results: (1) Screening of differentially expressed genes Compared with the CON group, the TNBS group identified 3,596 differentially expressed genes, of which 2,324 were upregulated and 1,272 were downregulated. Compared with the TNBS group, the Bifidobacterium breve A3-3 intervention group identified 3,977 differentially expressed genes, of which 1,801 were upregulated and 2,176 were downregulated. The number of downregulated genes exceeded the number of upregulated genes, suggesting that one of the main mechanisms by which Bifidobacterium breve A3-3 exerts its anti-inflammatory effect is by inhibiting the expression of pro-inflammatory genes.

[0085] (2) KEGG pathway enrichment analysis Compared with the CON group, the differentially regulated genes in the TNBS group were significantly enriched in the cytokine-cytokine receptor interaction pathway, PI3K-Akt signaling pathway, MAPK signaling pathway, TNF signaling pathway, and IL-17 signaling pathway. Compared with the TNBS group, the differentially regulated genes after Bifidobacterium breve A3-3 intervention were mainly enriched in the same pathways, indicating that this strain can effectively inhibit TNBS-induced excessive inflammatory response and tissue damage, and exert a direct anti-inflammatory effect.

[0086] (3) Differential gene clustering analysis of KEGG pathway enrichment Based on the KEGG pathway enrichment results, differentially expressed genes enriched in the IL-17 signaling pathway were selected for cluster analysis, and RT-qPCR was performed to validate the key differentially expressed genes based on the clustering results. The results showed that compared with the CON group, the TNBS group exhibited significantly upregulated expression of the core IL-17 pathway factor Il17a (P < 0.05), significantly increased expression of downstream chemokines Cxcl5 and Ccl2 (P < 0.01), and a significantly increased expression of the neutrophil activation marker Lcn2 (P < 0.05). After intervention with Bifidobacterium breve A3-3, the expression levels of all four genes significantly decreased (P < 0.05). These results indicate that Bifidobacterium breve A3-3 may exert its anti-inflammatory effect by regulating the IL-17 signaling pathway.

[0087] (4) Analysis of immune cell infiltration To investigate the effects of Bifidobacterium breve A3-3 on the intestinal immune microenvironment, this study used the ssGSEA method to quantitatively analyze the infiltration levels of 12 immune cell types. The results showed that, compared to the CON group, TNBS mice exhibited a combination of inflammation and repair in the late acute inflammation phase: Th1 cells were significantly reduced (P < 0.001), Th17 cells remained at a high level, the proportion of Treg cells was low (P < 0.05), M1 macrophages were higher than normal (P < 0.05), and neutrophil infiltration persisted (P < 0.001). After intervention with Bifidobacterium breve A3-3, Th1 cells significantly recovered to levels close to those of the CON group (P < 0.01), Th17 cells decreased to levels similar to those of the CON group, Treg cells increased, M1 macrophages significantly decreased (P < 0.05), and neutrophil infiltration significantly decreased (P < 0.01). The results showed that intervention with Bifidobacterium breve A3-3 could effectively correct the imbalance between Th17 / Treg and Th1 / Th2, inhibit excessive infiltration of innate immune cells, and promote the restoration of the intestinal immune microenvironment to homeostasis.

[0088] 4. Regulation of intestinal immune cell balance in colitis mice by Bifidobacterium breve A3-3 Flow cytometry was used to detect the infiltration and differentiation of macrophages in the lamina propria of the mouse colon. Lymphocytes were extracted from the lamina propria and stained with multiple colors using flow cytometry. Antibodies included CD45+, CD11b+, F480+, CD80+ (M1 marker), and CD163+ (M2 marker). Data were acquired using a BD FACSCantoII flow cytometer and analyzed using FlowJo v10 software. First, lymphocyte populations were delineated using forward scatter (FSC) and side scatter (SSC) light. Then, cell viability, CD45+ immune cells, and CD11b+F480+ macrophages were delineated sequentially. The proportions of CD80+M1 and CD163+M2 macrophages among CD11b+F480+ macrophages were analyzed.

[0089] Experimental results: like Figure 15As shown, the total number of CD11b+F4 / 80+ macrophages in the lamina propria of the colon in the TNBS group was significantly higher than that in the CON group (P < 0.05), while the total number of CD11b+F4 / 80+ macrophages in the B. breve A group was lower than that in the TNBS group. This indicates that TNBS induces inflammation and a large accumulation of macrophages; after intervention with Bifidobacterium breve A3-3, the inflammatory response was significantly reduced. Analysis of macrophage polarization status revealed that compared with the CON group, the M1 / M2 ratio in the TNBS group was significantly increased (P < 0.001), consistent with the pathological state of acute colitis. Compared with the TNBS group, the M1 / M2 ratio in the B. breve A group was significantly decreased (P < 0.05), indicating that Bifidobacterium breve A3-3 can improve macrophage polarization balance, alleviate intestinal inflammation, and promote tissue repair.

[0090] Macrophage-related biomarkers were validated using RT-qPCR technology. Results are as follows: Figure 16 As shown, compared with the CON group, the expression of CD11b, iNOS, and the chemokine CCL4 in the colon of mice in the TNBS group was significantly increased (P < 0.05 or P < 0.01), and the M2 markers CD206 and Arg1 were also significantly upregulated, while IRF5 showed an increasing trend. Compared with the TNBS group, the expression of CD11b, iNOS, CCL4, and IRF5 in the B. breve A group was significantly decreased (P < 0.05 or P < 0.01), while the expression of CD206 and Arg1 was significantly increased.

[0091] In summary, Bifidobacterium breve A3-3 can effectively inhibit the recruitment of macrophages to the intestine, correct the M1 / M2 polarization imbalance, and promote the transformation of macrophages to an anti-inflammatory and repair phenotype, thereby alleviating colitis.

[0092] Finally, it should be noted that the above description is only used to illustrate the technical solutions of the present invention and is not intended to limit it. Although the present invention has been described in detail with reference to preferred embodiments, those skilled in the art should understand that modifications or equivalent substitutions can be made to the technical solutions of the present invention without departing from the spirit and scope of the present invention.

Claims

1. A Bifidobacterium breve A3-3 strain with high ILA production, characterized in that, This strain is deposited at the China General Microbiological Culture Collection Center (CGMCC) with accession number CGMCC NO. 38736 and deposit date of May 19, 2026.

2. The high ILA-producing Bifidobacterium breve A3-3 according to claim 1, characterized in that, The strain produced 53.28 mg / L of indole-3-lactic acid in vitro.

3. An inoculant characterized in that, The bacterial agent contains Bifidobacterium breve A3-3 as described in claim 1 or 2 and / or the metabolites of Bifidobacterium breve A3-3 as described in claim 1 or 2.

4. A pharmaceutical composition, characterized by, The active ingredient of the pharmaceutical composition includes Bifidobacterium breve A3-3 as described in claim 1 or 2, or the bacterial agent as described in claim 3.

5. The pharmaceutical composition of claim 4, wherein, The composition also includes a pharmaceutically acceptable carrier or excipient.

6. The pharmaceutical composition of claim 4, wherein, The dosage forms of the composition include lyophilized powder, capsules, oral liquid, tablets or granules.

7. The use of the Bifidobacterium breve A3-3 according to claim 1 or 2, or the bacterial agent according to claim 3, or the pharmaceutical composition according to any one of claims 4 to 6 in the preparation of a medicament for the prevention and / or treatment of inflammatory bowel disease.

8. Use according to claim 7, characterized in that, The inflammatory bowel disease mentioned includes ulcerative colitis and Crohn's disease.

9. Use according to claim 7, characterized in that, The drug is one or more of the following: (1) The drug can reshape the gut microbiota homeostasis; (2) The drug can enhance tryptophan metabolism and enrich indole anti-inflammatory metabolites; (3) The drug can inhibit the IL-17 inflammatory pathway and restore intestinal immune balance; (4) The drug can enhance the intestinal barrier function.

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