Method for screening bifidobacterium based on tryptophan metabolic capacity and strain thereof

Abstract

This invention provides a method and strain for screening Bifidobacteria based on tryptophan metabolism. The screening method is based on the high expression of the key tryptophan metabolism gene Aldh and the production of indole-3-lactic acid (ILA). Specifically, it includes: extracting total RNA from Bifidobacterium strains, reverse transcribing the RNA into cDNA, detecting the relative expression level of Aldh by RT-qPCR, and screening candidate strains with high Aldh expression; further, anaerobic culturing the candidate strains, preparing cell-free supernatant, and quantitatively detecting the ILA content in the cell-free supernatant using HPLC, resulting in a high-ILA-producing strain—Bifidobacterium denticulatum B2-2. This strain is deposited at the China General Microbiological Culture Collection Center (CGMCC) with accession number CGMCC NO. 38737, and the deposit date is May 19, 2026. Experiments have confirmed that Bifidobacterium denticulatum B2-2 alleviates intestinal inflammation through multiple targets, including remodeling the gut microbiota, enriching ILA, inhibiting inflammatory pathways, restoring immune homeostasis, and strengthening the intestinal barrier.

Description

Technical Field

[0001] This invention belongs to the field of microbial technology, and specifically relates to a method and strain for screening Bifidobacteria based on tryptophan metabolism. Background Technology

[0002] Inflammatory bowel disease (IBD) is an idiopathic disease characterized by chronic, relapsing inflammation of the intestines and damage to the intestinal barrier. It primarily includes ulcerative colitis and Crohn's disease, severely impacting patients' quality of life and increasing the risk of cancer. In recent years, the incidence of IBD has been rising globally, particularly 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, and immune dysregulation. Among these, gut microbiota dysbiosis and abnormal tryptophan metabolism are considered key pathological links.

[0003] The gut microbiota is a core hub regulating host immune homeostasis, and its metabolites play a crucial role in the development and progression of intestinal inflammation. Studies have shown that gut microbiota diversity is significantly reduced in IBD patients, with a substantial decrease in the abundance of beneficial bacteria such as Bifidobacteria and Lactobacillus, and abnormal proliferation of Proteobacteria and opportunistic pathogens, leading to intestinal microecological imbalance. Crucially, the tryptophan metabolic pathway in the gut is severely impaired in IBD patients: a reduction in indole-producing bacteria and a decrease in the abundance of aromatic lactate dehydrogenase (Aldh) genes, resulting in insufficient production of indole-3-lactic acid (ILA). ILA is a tryptophan metabolite unique to gut microbiota and an important ligand for the aryl hydrocarbon receptor (AhR). It has functions such as anti-inflammation, maintaining the intestinal barrier, and regulating immune homeostasis. Its content is significantly negatively correlated with IBD disease activity and is a key active molecule for alleviating intestinal inflammation.

[0004] Currently, clinical treatment of IBD mainly relies on aminosalicylic acids, glucocorticoids, and biologics, which suffer from 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 intestinal microecological regulators, have advantages such as high safety and easy acceptance, 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 drawbacks such as weak tryptophan metabolism, low ILA production (usually below 20 mg / L), and limited anti-inflammatory effects. Furthermore, their functions are highly strain-specific, necessitating targeted screening for specific probiotic strains with high tryptophan metabolism and high ILA production. Summary of the Invention

[0005] To address the problems existing in the prior art, the present invention aims to provide a method and strain for screening Bifidobacteria based on tryptophan metabolism capacity.

[0006] The objective of this invention is achieved through the following technical solution: In a first aspect, the present invention provides a method for screening Bifidobacteria based on tryptophan metabolism capacity, comprising the following steps: (1) Activate the Bifidobacterium strain to be screened to obtain a logarithmic growth phase bacterial solution; (2) Collect bacterial cells, extract total RNA from Bifidobacterium strains, reverse transcribe the RNA into cDNA, use 16S rRNA as internal reference gene, use RT-qPCR to detect the relative expression level of Aldh, a key gene for tryptophan metabolism, and screen candidate strains with high Aldh expression and strong tryptophan metabolism capacity. (3) The candidate strains were cultured anaerobically to prepare cell-free supernatant; (4) The content of indole-3-lactic acid (ILA) in the cell-free supernatant was quantitatively determined by high performance liquid chromatography, and high-yielding strains with ILA yield ≥45 mg / L were screened.

[0007] Furthermore, in step (1), the activation conditions are as follows: inoculate 1% into MRS liquid medium and anaerobic culture at 37°C until OD600 = 0.8~1.0.

[0008] Further, in step (2), 16S rRNA-F: 5'-GGTGTAACGGTGGAATGT-3', 16S rRNA-R: 5'-CTCCTCAGCGTCAGTAAC-3'; and Aldh-F: 5'-CGGCGTAATCGTCATGGTT-3', Aldh-R: 5'-GGTCTTGAGGCGTGAGGTA-3' were used as primers, and the following steps were performed: The method calculates the relative expression level of the target gene Aldh.

[0009] Furthermore, in step (2), the RT-qPCR reaction system includes: 10 μL of 2× real-time quantitative PCR premix, 0.5 μL each of upstream and downstream primers, and 1 μL of cDNA template. 8 μL; Reaction program: 95℃ pre-denaturation for 30 s; 95℃ denaturation for 5 s, 60℃ annealing for 30 s, 40 cycles.

[0010] Furthermore, in step (4), the quantitative detection conditions for high-performance liquid chromatography include: A C18 reversed-phase column was used with gradient elution of water containing 0.1% trifluoroacetic acid as mobile phase A and acetonitrile containing 0.1% trifluoroacetic acid as mobile phase B. The detection wavelength was 280 nm, the column temperature was 40 ℃, and the flow rate was 0.40 mL / min.

[0011] In a second aspect, the present invention provides a strain of Bifidobacterium dentium B2-2, which is deposited at the China General Microbiological Culture Collection Center (CGMCC) with accession number CGMCC NO.38737 and deposit date of May 19, 2026; the Bifidobacterium dentium B2-2 is obtained by screening using the method described in the first aspect.

[0012] A third aspect of the invention provides the use of the Bifidobacterium denticulatum B2-2 described in the first aspect in the preparation of products for alleviating inflammatory bowel disease.

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

[0014] Furthermore, the Bifidobacterium densiflorum B2-2 exerts its effects by improving intestinal flora, increasing indole-3-lactic acid content, enhancing tryptophan metabolism, inhibiting inflammatory factor expression, restoring intestinal immune balance, and strengthening intestinal barrier function.

[0015] In a fourth aspect, the present invention provides a probiotic preparation comprising the Bifidobacterium denticulatum B2-2 described in the first aspect and a pharmaceutically acceptable carrier or excipient.

[0016] Information on strain preservation: The strain of Bifidobacterium dentium is named B2-2 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 dentium, its accession number is CGMCC NO. 38737, and its deposit date is May 19, 2026.

[0017] The advantages of this invention compared to the prior art are as follows: 1. This screening method uses Aldh, a key gene for tryptophan metabolism, as a molecular target. It first screens at the gene expression level, and then combines high performance liquid chromatography to quantitatively screen the indole-3-lactic acid (ILA) content. This enables targeted, precise, and efficient screening of Bifidobacteria that produce high levels of ILA. It overcomes the shortcomings of traditional random screening, such as blindness, low efficiency, and long screening cycle, and significantly improves the screening accuracy and the rate of obtaining target strains. It provides a standardized technical path for targeted screening of functional probiotics. 2. This application obtained a probiotic strain with high ILA production from the feces of healthy infants. This strain was identified as Bifidobacterium dentatum by preservation and identification, with preservation number CGMCC NO. 38737, and classified as Bifidobacterium dentatum B2-2. The in vitro ILA production of this strain is as high as 48.88 mg / L, with outstanding tryptophan metabolism capacity, which is much higher than that of ordinary Bifidobacterium. Animal experiments show that Bifidobacterium dentatum B2-2 can significantly improve the pathological symptoms of colitis in mice, reduce the level of inflammation, protect colon tissue, and has a clear alleviating effect on inflammatory bowel disease. Attached Figure Description

[0018] 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 dendriticum. 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) colon image; (B) colon length. Detailed Implementation

[0019] The implementation of this invention is not limited to the embodiments described below. Any modifications and / or alterations made to this invention will fall within the scope of protection of this invention. In this invention, unless otherwise specified, all equipment and raw materials are commercially available or commonly used in the industry. Unless otherwise specified, the methods used in the embodiments are techniques generally applicable in the field.

[0020] For numerical ranges, the endpoint values ​​of each range, the endpoint values ​​of each range 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.

[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 dendriticum 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 dendriticum* (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 dendriticum*, 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, with a correlation coefficient... ≥ 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. Notably, strain A3-3 demonstrated dual metabolic advantages, producing 33.98 mg / L in the IAM pathway and a significantly higher 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, producing 19.60 mg / L of IAM, while its downstream product IAld produced 5.47 mg / L, significantly higher than the other *Bifidobacterium bifidum* strain; strain E2-3 favored the ILA synthesis pathway, producing 32.83 mg / L of ILA, demonstrating significant intraspecific metabolic diversity. Within *Bifidobacterium densiflorum*, 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 denticulatum* 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 denticulatum 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 denticulatum* 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, an enema of 2.5% TNBS ethanol solution was administered. Gavage was initiated after the end of the adaptation period: the gavage concentration for the HB. dentium group was [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 denticulatum 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, as the mice resumed normal eating and due to 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 dendriticum 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 dendriticum 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. However, 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 denticulatum 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] In summary, both high and low doses of Bifidobacterium breve A3-3 and Bifidobacterium denticulatum B2-2 can significantly alleviate the pathological symptoms of colitis in a TNBS-induced colitis mouse model.

[0063] 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 method for screening Bifidobacteria based on tryptophan metabolism capacity, characterized in that, The method includes the following steps: (1) Activate the Bifidobacterium strain to be screened to obtain a logarithmic growth phase bacterial solution; (2) Collect bacterial cells, extract total RNA from Bifidobacterium strains, reverse transcribe the RNA into cDNA, use 16S rRNA as internal reference gene, use RT-qPCR to detect the relative expression level of Aldh, a key gene for tryptophan metabolism, and screen candidate strains with high Aldh expression and strong tryptophan metabolism capacity. (3) The candidate strains were cultured anaerobically to prepare cell-free supernatant; (4) The content of indole-3-lactic acid (ILA) in the cell-free supernatant was quantitatively determined by high performance liquid chromatography, and high-yielding strains with ILA yield ≥45 mg / L were screened.

2. The method according to claim 1, characterized in that, In step (1), the activation conditions are: inoculate 1% into MRS liquid medium and culture anaerobically at 37°C until OD600 = 0.8~1.

0.

3. The method according to claim 1, characterized in that, In step (2), 16S rRNA-F: 5'-GGTGTAACGGTGGAATGT-3', 16S rRNA-R: 5'-CTCCTCAGCGTCAGTAAC-3'; and Aldh-F: 5'-CGGCGTAATCGTCATGGTT-3', Aldh-R: 5'-GGTCTTGAGGCGTGAGGTA-3' were used as primers, and the following methods were employed: The method calculates the relative expression level of the target gene Aldh.

4. The method according to claim 1, characterized in that, In step (2), the RT-qPCR reaction system includes: 10 μL of 2× real-time quantitative PCR premix, 0.5 μL each of upstream and downstream primers, and 1 μL of cDNA template. 8 μL; Reaction program: 95℃ pre-denaturation for 30 s; 95℃ denaturation for 5 s, 60℃ annealing for 30 s, 40 cycles.

5. The method according to claim 1, characterized in that, In step (4), the quantitative detection conditions for high performance liquid chromatography include: A C18 reversed-phase column was used with gradient elution of water containing 0.1% trifluoroacetic acid as mobile phase A and acetonitrile containing 0.1% trifluoroacetic acid as mobile phase B. The detection wavelength was 280 nm, the column temperature was 40 ℃, and the flow rate was 0.40 mL / min.

6. A strain of Bifidobacterium dentium B2-2, characterized in that, This strain is deposited at the China General Microbiological Culture Collection Center (CGMCC) with accession number CGMCC NO. 38737 and deposit date of May 19, 2026; the Bifidobacterium denticulatum B2-2 was obtained by screening according to any one of claims 1 to 5.

7. The use of Bifidobacterium densiflorum B2-2 as described in claim 6 in the preparation of products for relieving inflammatory bowel disease.

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

9. The application according to claim 7, characterized in that, The Bifidobacterium densiflorum B2-2 exerts its effects by improving intestinal flora, increasing indole-3-lactic acid content, enhancing tryptophan metabolism, inhibiting the expression of inflammatory factors, restoring intestinal immune balance, and strengthening intestinal barrier function.

10. A probiotic preparation, characterized in that, It comprises the Bifidobacterium denticulatum B2-2 as described in claim 6 and a pharmaceutically acceptable carrier or excipient.

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