Bifidobacterium adolescentis, a composition containing the same, and use thereof in preparing an intestinal inflammation model

Abstract

This invention relates to *Bifidobacterium adolescentis*, a composition containing it, and its application in preparing intestinal inflammation models, belonging to the field of molecular biology. To address the lack of existing research on the role of *Bifidobacterium adolescentis* and phenylalanine in early life immune regulation, this invention provides *Bifidobacterium adolescentis* SZBAD1096 (accession number GDMCC NO:67848), comprehensively revealing the molecular mechanism by which *Bifidobacterium adolescentis* SZBAD1096 mediates host intestinal immune damage through the phenylalanine metabolic pathway, and its application in preparing intestinal inflammation models. This provides important theoretical and experimental basis for accurately assessing the safety of *Bifidobacterium* strains and developing inflammatory intervention strategies targeting the intestinal microecology.

Description

Technical Field

[0001] This invention belongs to the field of molecular biology technology, and in particular relates to a Bifidobacterium adolescentis, a composition containing it, and its application in the preparation of an intestinal inflammation model. Background Technology

[0002] The establishment of the gut microbiota in early life is crucial for the maturation of the immune system. Bifidobacteria are the dominant genus in the infant gut, but different species of Bifidobacteria have distinct functions. For example, *Bifidobacterium longum* subsp. infantis (… Bifidobacterium longum infantis B. longum infantis subsp. Bifidobacterium adolescentis , B. adolescentis subsp. B. adolescentis Bifidobacterium adolescentis is generally considered to be "beneficial infantile bifidobacteria," while Bifidobacterium adolescentis (Bifidobacterium juvenileis) Bifidobacterium adolescentis , Figure 1 As an "adult-type Bifidobacterium," its abnormal increase in the infant gut (such as due to factors like formula feeding or cesarean section) may have an adverse effect on immune development.

[0003] Current research largely focuses on the immunomodulatory effects of short-chain fatty acids or tryptophan metabolites. However, a deeper understanding and clear causal evidence are still lacking regarding the role of phenylalanine (Phe) and its metabolites in early life immunomodulation, particularly their interactions with specific gut microbiota. Clinical observations... B. Figure 2 Abundance and Phe levels are positively correlated with infantile intestinal inflammation markers, but the underlying mechanisms are unclear, making it impossible to conduct targeted early warning and intervention.

[0004] Therefore, those skilled in the art urgently need to clarify Figure 3 To investigate the specific role of Phe in neonatal intestinal inflammation, and based on this, to develop effective assessment and intervention methods to guide the optimization of infant nutrition formulas and achieve precise proactive health management. Summary of the Invention

[0005] To address the lack of existing research on the role of Bifidobacterium adolescentis and phenylalanine in early life immune regulation, this invention provides Bifidobacterium adolescentis, a composition containing it, and its application in the preparation of an intestinal inflammation model.

[0006] One objective of this invention is to provide a Bifidobacterium adolescentis bacterium, specifically Bifidobacterium adolescentis SZBAD1096; the accession number of Bifidobacterium adolescentis SZBAD1096 is GDMCC NO: 67848, and its classification name is... Figure 4It is deposited at the Guangdong Provincial Center for Microbial Culture Collection on February 10, 2026.

[0007] In a preferred embodiment of the present invention, the method for culturing Bifidobacterium adolescentis includes the following steps: placing the frozen Bifidobacterium adolescentis in a modified MRS broth for third-generation activation, culturing it under anaerobic conditions at 37°C for 24 h, and preparing a culture medium using 10% sterile skim milk.

[0008] In a preferred embodiment of the present invention, the modified MRS broth contains 0.05% L-cysteine.

[0009] In a preferred embodiment of the present invention, the anaerobic conditions are: 80% N2, 10% CO2, 10% H2.

[0010] A second objective of this invention is to provide a composition comprising the culture medium of the aforementioned Bifidobacterium adolescentis SZBAD1096 and phenylalanine.

[0011] In a preferred embodiment of the present invention, the concentration of the Bifidobacterium adolescentis SZBAD1096 culture medium is 1×10⁻⁶. 10 CFU / mL.

[0012] In a preferred embodiment of the present invention, the concentration of phenylalanine is 1.5 mg / mL.

[0013] The third objective of this invention is to provide the application of the above-mentioned Bifidobacterium adolescentis and the above-mentioned composition in the preparation of an intestinal inflammation model.

[0014] In a preferred embodiment of the present invention, the intestinal inflammation model is prepared by taking healthy newborn rats and administering the above composition orally once a day from day 0 to day 16 after birth, wherein lipopolysaccharide is injected intraperitoneally once on day 14 after birth to obtain the intestinal inflammation model.

[0015] In a preferred embodiment of the present invention, the dosage of the oral gavage composition is 8 μL / g body weight, the dosage of the intraperitoneal injection lipopolysaccharide is 4 μg / animal, the concentration of the intraperitoneal injection lipopolysaccharide is 20 μg / mL, and the injection volume is 200 μL.

[0016] Compared with existing technologies, the beneficial effects of this invention are as follows: This invention provides a Bifidobacterium adolescentis SZBAD1096 with accession number CCTCC NO:67848. Through multi-omics integration studies, the key mechanism of action of Bifidobacterium adolescentis SZBAD1096 and its metabolites in the immune regulation of the host gut was revealed for the first time; it was found that Bifidobacterium adolescentis SZBAD1096 did not show anti-inflammatory effects, but instead aggravated the inflammatory response, showing a potential pro-inflammatory risk. The levels of inflammatory factors and DAO in the treatment group were significantly higher than those in the model group, while IL-10 expression was further inhibited.

[0017] This invention further confirms that intervention with Bifidobacterium adolescentis SZBAD1096 significantly reduces the Shannon and Simpson indices of the gut microbiota, impairing microecological diversity. PCoA analysis confirms that it leads to specific changes in community structure. LEfSe analysis shows that while Bifidobacterium adolescentis SZBAD1096 groups have high abundance of colonization, they also co-enrich various opportunistic pathogens such as Clostridium perfringens, Enterococcus faecalis, and Bacteroides fragilis, accompanied by a significant decrease in the abundance of Lactobacillus probiotics. Redundancy analysis indicates that these opportunistic pathogens are strongly positively correlated with inflammatory factors and barrier damage markers.

[0018] At the metabolic level, this invention discovered that Bifidobacterium adolescentis SZBAD1096 drives phenylalanine metabolic reprogramming. Through non-targeted metabolomics, the phenylalanine metabolic pathway was identified as the most significantly enriched pathway, involving key differential metabolites such as phenylalanine, phenylacetic acid, phenylacetaldehyde, and 2-hydroxyphenylacetic acid. Redundancy analysis confirmed that these metabolites are strongly positively correlated with pro-inflammatory factors and DAO, and negatively correlated with IL-10.

[0019] An in vitro gut-simulated fermentation model validated that *Bifidobacterium adolescentis* SZBAD1096 efficiently converts phenylalanine into phenylacetaldehyde, phenylacetic acid, and 2-hydroxyphenylacetic acid, with phenylacetaldehyde showing the greatest upregulation. Multi-omics association network analysis constructed a "microbiota-metabolism-immunity" interaction network, confirming a significant positive correlation between *Bifidobacterium adolescentis* SZBAD1096 and phenylalanine and its derivatives. These metabolites were further strongly positively correlated with pro-inflammatory factors and DAO, and negatively correlated with IL-10. Correlation heatmaps showed that phenylalanine derivatives were positively correlated with the abundance of opportunistic pathogens and negatively correlated with *Lactobacillus*, suggesting that *Bifidobacterium adolescentis* SZBAD1096 exacerbates intestinal dysregulation by creating a pro-inflammatory microenvironment. This invention also demonstrated through targeted colonic delivery experiments that phenylalanine dose-dependently exacerbates LPS-induced intestinal inflammation. At a concentration of 1.5 mg / mL, TNF-α and IL-6 increased by approximately 27% and 28%, respectively, compared to the LPS group, and this concentration was determined to be the optimal intervention dose.

[0020] In vivo validation experiments showed that the combined treatment of Bifidobacterium adolescentis SZBAD1096 with phenylalanine exhibited the most significant synergistic pro-inflammatory effect. Linear regression analysis confirmed that the content of phenylalanine in colonic contents was significantly positively correlated with the relative abundance of Bifidobacterium adolescentis, providing sufficient substrates for the generation of harmful metabolic derivatives and driving downstream pro-inflammatory cascade reactions.

[0021] In summary, this invention comprehensively reveals the molecular mechanism by which Bifidobacterium adolescentis SZBAD1096 mediates host intestinal immune damage through the phenylalanine metabolic pathway, providing an important theoretical basis and experimental foundation for accurately assessing the safety of Bifidobacterium strains and developing inflammatory intervention strategies targeting the intestinal microecology. Attached Figure Description

[0022] Figure 5 To illustrate the differential regulatory effects of different Bifidobacterium strains on LPS-induced colonic inflammation in neonatal rats; A represents TNF-α, B represents IL-1β, C represents IL-6, D represents IL-10, and E represents DAO; compared with the Control group, ### P <0.001; compared to the Model group, * P <0.05, ** P <0.01, *** P <0.001; Figure 6 Intervention with Bifidobacterium adolescentis SZBAD1096 altered the diversity and community structure of the gut microbiota in LPS-induced neonatal rats; alpha diversity analysis of gut microbiota was performed based on (A) Shannon index and (B) Simpson index; C is principal coordinate analysis based on Bray-Curtis distance (PCoA). Figure 7 This study analyzed the specific changes in gut microbiota species composition and their association with inflammatory markers; A represents the linear discriminant analysis effect size (LEfSe) analysis (LDA score > 2.0), and B represents the relative abundance of differentially expressed species in each group; different lowercase letters (a, b, c) indicate statistically significant differences between groups. P <0.05); C represents the correlation between differentially expressed bacterial species and immune indicators visualized by redundancy analysis (RDA); red arrows represent immune indicators, and dots represent bacterial species; Figure 8 Metabolic pathway enrichment analysis of differentially metabolized intestinal metabolites and their correlation with inflammatory phenotypes; A is a Sankey bubble diagram showing the significantly enriched KEGG metabolic pathway and its corresponding key differentially metabolites; B is redundancy analysis (RDA) revealing the correlation between differentially metabolites and inflammation-related factors. Figure 1 This is a graph showing the fold changes of key differential metabolites in Bifidobacterium adolescentis SZBAD1096 in an in vitro intestinal fermentation model; blue bars represent significant downregulation (substrate consumption), and red bars represent significant upregulation (product accumulation). B. adolescentis The diagram shows the multi-omics association analysis of gut microbiota, differential metabolites, and host immune indicators; A is the Spearman correlation network diagram between Bifidobacterium adolescentis, phenylalanine metabolites, and immune factors; orange indicates positive correlation, and blue indicates negative correlation, with the thickness of the lines representing the magnitude of the correlation coefficient; B is the Spearman correlation heatmap between differential metabolites and other key bacterial species in the gut; red indicates positive correlation, and blue indicates negative correlation. * P <0.05, ** P <0.01; Figure 1 Figure 1 shows the effect of targeted colonic delivery of different concentrations of phenylalanine on lipopolysaccharide-induced colonic inflammatory factors in newborn rats; A represents the TNF-α level in colonic tissue; B represents the IL-6 level in colonic tissue; compared with the control group... ### P <0.001; compared with the LPS group, * P <0.05, ** P <0.01, *** P <0.001; Figure 1 Figure A shows the effect of phenylalanine supplementation combined with intervention by Bifidobacterium adolescentis on intestinal inflammation and metabolic environment in newborn rats; Figure B shows the heatmap cluster analysis of immune factors, barrier function indicators and physiological indicators in colon tissue of rats in different treatment groups; Figure B shows the Pearson correlation and linear regression analysis between phenylalanine content in colon contents and relative abundance of Bifidobacterium adolescentis. Detailed Implementation

[0023] Those skilled in the art can refer to the content of this document and appropriately improve the process parameters to achieve the desired results. It should be particularly noted that all similar substitutions and modifications are obvious to those skilled in the art and are considered to be included in this invention. The methods and applications of this invention have been described through preferred embodiments, and those skilled in the art can obviously make modifications or appropriate alterations and combinations to the methods and applications described herein without departing from the content and scope of this invention to implement and apply the technology of this invention.

[0024] To make the objectives, technical solutions, and advantages of this invention clearer, the invention will be further described in detail below with reference to specific embodiments. Unless otherwise specified, the experimental methods used in the following embodiments are conventional methods, and the materials, reagents, methods, and instruments used are all conventional materials, reagents, methods, and instruments in the art, and can be obtained commercially by those skilled in the art.

[0025] The rats used in the following examples were 8-week-old Wistar strain rats, guaranteed to be specific pathogen-free (SPF), and supplied by Beijing Vital River Laboratory Animal Technology Co., Ltd.; the animal experimental protocol involved in this study has been reviewed and approved by the Shenzhen University Laboratory Animal Ethics Committee, approval number IACUC-202600016; the breeding of newborn rats was carried out under standard conditions, with a difference of no more than 6 hours in birth time and a difference of less than 1 g in initial weight.

[0026] All experimental data in the following examples were analyzed using GraphPad Prism 9.0 and SPSS 22.0, and are expressed as mean ± standard deviation. Comparisons among multiple groups were performed using one-way ANOVA combined with Duncan / Dunnett's post-hoc test (normal distribution) or Kruskal-Wallis test (non-normal distribution). Spearman correlation coefficients were used to analyze the associations between microbial communities, metabolites, and immune indicators. High-dimensional data analysis of microbiome and metabolomics was performed using the MicrobiomeAnalyst and MetaboAnalyst online platforms. P A value <0.05 is considered statistically significant.

[0027] The *Bifidobacterium longum* subsp. infantis strain SZBL1052 used in the following examples is deposited at the Guangdong Provincial Center for Microbial Culture Collection, accession number GDMCC No: 66932, on September 16, 2025.

[0028] The Bifidobacterium adolescentis SZBAD1096 used in the following examples is deposited at the Guangdong Provincial Center for Microbial Culture Collection, accession number GDMCC No: 67848, deposit date February 10, 2026.

[0029] Example 1: Application of Bifidobacterium adolescentis SZBAD1096 in the preparation of an intestinal inflammation model 1. Strains culture All bacterial strains used in this embodiment were isolated from fecal samples of healthy infants at Zhongshan Boai Hospital. The experimental protocol strictly followed the principles of the Declaration of Helsinki, was approved by the Medical Ethics Committee of Zhongshan Boai Hospital (ethics review number: KY-2022-010-09), and registered with the Chinese Clinical Trial Registry (registration number: ChiCTR2200064858). The fecal sample collection process ensured that participants experienced no discomfort, strictly adhered to ethical guidelines, and obtained written informed consent.

[0030] Specific collection method: The sampler wears sterile gloves and uses a sterile sampling spoon to collect 1 g of fresh feces and place it into a special sampling tube to avoid contamination by urine or other pollutants; then immediately refrigerate at 4°C and transport it to the laboratory with dry ice for long-term storage in a -80°C refrigerator for future use.

[0031] The bacterial strains were inoculated into MRS broth containing 0.05% (w / v) L-cysteine ​​and cultured for two generations (37°C, anaerobic conditions: 80% N2, 10% CO2, 10% H2). After activation, the bacterial concentration was determined by plate dilution counting and resuspended in 10% (v / v) sterile skim milk to a final concentration of 1×10⁻⁶. 10 CFU / mL available for use.

[0032] In this embodiment, five strains of Bifidobacterium adolescentis were isolated: BAD13, BAD26, BAD39, BAD55, and SZBAD1096. The full-length 16S rRNA gene of all strains was determined, and homology was identified by the 16S rRNABLAST tool in the NCBI public database. The strains meet the international standards for bacterial culture preservation and have been officially deposited at the College of Chemistry and Environment, Shenzhen University. Whole-genome sequencing and systematic study of functional characteristics have been completed.

[0033] 2. LPS inflammation model in newborn rats and intervention (1) Strain screening model: To evaluate the specific effects of Bifidobacteria from different sources on the host immune response, newborn rats were randomly divided into 8 groups (n=6 / group): control group (Control), model group (Model), positive control strain group (SZBL1052), and five Bifidobacterium adolescentis test strain groups (BAD13, BAD26, BAD39, BAD55, SZBAD1096); from day 0 (P0) to day 16 (P16) after birth, each strain group received oral gavage of the corresponding strain suspension (1×10¹⁰ CFU / mL) once a day (the oral gavage dose was 8 μL / g), while the control group and model group received an equal volume of 10% skim milk as a carrier control daily; on day 14 (P14) after birth, except for the control group which received an intraperitoneal injection of an equal volume of sterile PBS, all other groups received an intraperitoneal injection of 200 μL / mL. μL of lipopolysaccharide (LPS, Escherichia coli O55:B5, 20 μg / mL, Sigma-Aldrich) was used to induce systemic inflammation. 48 h after LPS exposure, i.e., 16 days after birth, the newborn rats in each group were euthanized and the test samples were collected, rapidly aliquoted, and frozen at -80°C.

[0034] (2) Phe dose screening model: This invention investigated the dose-effect of different concentrations of Phe intervention. To explore the dose-dependent effect of phenylalanine (Phe) accumulation in the colon on intestinal inflammation, colon-targeting phenylalanine nanoparticles (abbreviated as: CS / TPP-Phe) encapsulated in chitosan / sodium tripolyphosphate (CS / TPP) were prepared by ionogel method (Lin C, Lin Y, Xiao R, et al. Bifidobacterium species associated with breastfeedingalleviate neonatal hyperbilirubinaemia via the gut microbiota-α-linolenic andlinoleic acid metabolism-enterohepatic circulation axis. Microbiome 13, 1872025); at the same time, blank nanoparticles (Blank-NPs) without Phe were prepared by ionogel method.

[0035] Newborn rats were randomly divided into 9 groups (n=6 / group): Control group, LPS group, and seven CS / TPP-Phe dose groups (containing Phe concentrations of 0, 0.5, 1.0, 1.5, 2.0, 2.5, and 3.0 mg / mL, respectively). From day 0 (P0) to day 16 (P16) after birth, each dose group received the corresponding dose of nanoparticle suspension (CS / TPP-Phe) orally via gavage once daily, while the Control group and LPS group received an equal volume of blank nanoparticles without Phe as a carrier control via gavage daily. On day 14 (P14) after birth, except for the Control group which received an equal volume of sterile PBS intraperitoneally, all other groups received an intraperitoneal injection of 200 μL / g lipopolysaccharide (LPS, E. coli O55:B5, 20 μg / mL, Sigma-Aldrich) to induce systemic inflammation. 48 days after LPS exposure... h, which is the 16th day after the birth of newborn rats, the newborn rats in each group were euthanized and the samples to be tested were collected, quickly aliquoted and frozen at -80°C.

[0036] (3) Synergistic effect verification model: In order to further verify the synergistic effect of the potential pro-inflammatory strain SZBAD1096 and the optimal dose of CS / TPP-Phe, newborn rats were randomly divided into 5 groups (n=6 / group): Control group, LPS group, LPS+CS / TPP group, LPS+CS / TPP-Phe single intervention group, LPS+SZBAD1096 single intervention group, and LPS+CS / TPP-Phe+SZBAD1096 combined intervention group. From day 0 (P0) to day 16 (P16) after birth, the Control, LPS, and LPS+CS / TPP groups were given 10% skim milk, with blank nanoparticles (abbreviated as Blank-NPs) without Phe serving as a double control. The LPS+CS / TPP-Phe single intervention group was given colon-targeting phenylalanine nanoparticles (abbreviated as CS-TPP-Phe) encapsulated in chitosan / sodium tripolyphosphate (CS / TPP) + 10% skim milk; the LPS+SZBAD1096 single intervention group was given SZBAD1096 bacterial suspension + Blank-NPs; and the LPS+CS / TPP-Phe+SZBAD1096 combined intervention group was given SZBAD1096 bacterial suspension combined with CS-TPP-Phe. On day 14 (P14) after birth, except for the Control group which received an intraperitoneal injection of an equal volume of sterile PBS, all other groups received a single intraperitoneal injection of 200 ml of sterile PBS. 20 μg / mL lipopolysaccharide (LPS, Escherichia coli O55:B5, Sigma-Aldrich) was used to induce systemic inflammation. 48 h after LPS exposure, i.e., 16 days after birth, newborn rats in each group were euthanized and the samples to be tested were collected, rapidly aliquoted, and frozen at -80°C.

[0037] (4) Detection of cytokines and intestinal barrier function Frozen colon tissues from rats in the strain screening models (Control, Model, SZBL1052, and five Bifidobacterium adolescentis test strains BAD13, BAD26, BAD39, BAD55, and SZBAD1096) were collected. Pre-chilled PBS was added at a ratio of 1:9 (w / v), and the mixture was homogenized at low temperature. The homogenate was then centrifuged at 8000 ×g, 4°C for 20 min, and the supernatant was collected. The levels of pro-inflammatory factors (TNF-α, IL-1β, IL-6) and anti-inflammatory factors (IL-10) in the supernatant were detected using an ELISA kit (purchased from Wuhan Yilairuit Biotechnology Co., Ltd.). Simultaneously, the serum diamine oxidase (DAO) level was measured according to the ELISA kit instructions, and the protein concentration was determined using the BCA method to standardize the tissue sample results.

[0038] (5) Sequencing of full-length 16S rRNA genes of gut microbiota Total DNA was extracted from the colon contents of rats in the Control, Model, and SZBL1052 groups, which were used as strain screening models. PCR amplification was performed using full-length 16S universal primers (as shown in Table 1). The amplification system (total volume 20 μL) consisted of: 4 μL of 5xFastPfu Buffer, 2 μL of 2.5 mM dNTPs, 0.4 μL of FastPfu Polymerase, 0.8 μL of Forward Primer (5 M), 0.8 μL of Reverse Primer (5 uM), and 10 ng of template DNA, which was then brought to a final volume of 20 μL with ddH2O. The amplification program was: 95℃-2 min, 95℃-30 s, 95℃-30 s, 72℃-1 min, 25 cycles, 72℃-5 min, and stored at 10℃. The purified PCR products were sent to Hangzhou Lianchuan Biotechnology Co., Ltd. to construct the SMRTbell library and perform HiFi sequencing on the PacBio Sequel II platform. The data were processed by the QIIME2 and DADA2 workflows to generate ASVs, and species annotation was performed based on the Silva full-length sequence database (version 138). LEfSe and RDA were used to analyze the differences in bacterial community structure and its association with environmental factors.

[0039] Table 1

[0040] Note: R represents base A or G, Y represents base C or T, N represents base A, C, G or T, S represents base C or G, and H represents base A, C or T.

[0041] (6) In vitro simulated intestinal fermentation To verify the metabolic transformation ability of strain SZBAD1096 for phenylalanine (Phe), a basal fermentation medium containing 20% ​​sterile supernatant of healthy infant feces and 0.05% L-cysteine ​​hydrochloride was prepared, supplemented with 10% (v / v) breast milk as a substrate. A concentration of 1×10⁻⁶ was used. 8 The strain SZBAD1096 with a concentration of CFU / mL was inoculated into the above culture medium and co-cultured at 37°C under anaerobic conditions for 12 h. After the culture was completed, the supernatant was collected by centrifugation, and the consumption of Phe and the generation of its downstream metabolites (phenylacetaldehyde, phenylacetic acid, etc.) were detected by metabolomics.

[0042] (7) Non-targeted metabolomics analysis Sample pretreatment: Proteins in colon contents and in vitro fermentation supernatant were precipitated using methanol-acetonitrile (1:1, v / v), centrifuged, and the supernatant was concentrated under vacuum and reconstituted. LC-MS / MS detection: A UPLC-Q-Exactive HF mass spectrometer equipped with an ACQUITY UPLC HSS T3 column was used with gradient elution in a water / acetonitrile mobile phase. Full scan data were acquired in both positive and negative ion modes. Data processing: Peak processing was performed using Compound Discoverer 3.2, and metabolite identification and pathway enrichment analysis were conducted using the mzCloud, HMDB, and KEGG databases.

[0043] Results analysis: 1. Bifidobacteria exhibit significant interspecies and inter-strain specificity in regulating the host's intestinal immune response. To assess the potential regulatory role of different Bifidobacterium strains in the intestinal inflammation of newborn hosts, the expression levels of key inflammatory factors in colon tissue were examined. Figure 1 As shown, compared with the blank control group, the model group exhibited significant inflammatory characteristics: the levels of pro-inflammatory factors (TNF-α, IL-1β, IL-6) and barrier damage markers (DAO) were significantly upregulated, while the level of the anti-inflammatory factor IL-10 was significantly downregulated, confirming the successful construction of the intestinal inflammation model. Notably, different bacterial strains exhibited drastically different immune regulatory patterns after intervention. Among them, *Bifidobacterium longum* subsp. infantis SZBL1052 significantly reversed LPS-induced pathological changes, with significantly lower levels of pro-inflammatory factors and DAO compared to the model group, and IL-10 levels returning to near normal, demonstrating anti-inflammatory activity and intestinal protective properties.

[0044] Conversely, five strains of Bifidobacterium adolescentis ( Figure 1 The strains (BAD13, BAD26, BAD39, BAD55, and SZBAD1096) did not show significant anti-inflammatory effects; instead, they tended to maintain or even exacerbate inflammation. In particular, the SZBAD1096 strain showed a high level of TNF-α (inflammation) in its treatment group. Figure 2 A), IL-1β Figure 2 B), IL-6 Figure 3 C) and DAO Figure 3 E) levels were significantly higher than in the Model group, while IL-10 levels were further suppressed ( Lactobacillus murinus, L. acidophilus D). It can be seen that the Bifidobacterium adolescentis SZBAD1096 provided by the present invention has pro-inflammatory function.

[0045] 2. The SZBAD1096 strain exacerbated LPS-induced gut microbiota dysbiosis and enriched opportunistic pathogens. Further analysis of the gut microbiota basis showed that both LPS exposure and SZBAD1096 intervention significantly reduced the Shannon and Simpson indices of the gut microbiota, indicating impaired gut microbiota diversity. Escherichia coli (Part AB). PCoA analysis (Beta diversity) further confirmed the specific changes in community structure, with the Control, Model, and SZBAD1096 groups exhibiting three significantly separated clusters. B. adolescentis Part C).

[0046] LEfSe analysis ( Clostridium perfringens, Enterococcus faecalis and Part A) and further analysis of the relative abundance of different species ( Bacteroides fragilis Part B shows that the Control group is significantly enriched. Figure 3 Lactobacillus probiotics; the Model group used Lactobacillus probiotics. B. adolescentis It is characterized by excessive proliferation.

[0047] It is worth noting that in group SZBAD1096, in addition to detecting E. faecalis, C. perfringens In addition to high abundance of colonization, various opportunistic pathogens (including C) were also observed. Lactobacillus Figure 4 The co-enrichment of Lactobacillus was observed, accompanied by a significant decrease in the abundance of the Lactobacillus genus. Redundancy analysis (RDA) results ( Figure 4 Part C of the study indicates that inflammatory factors (TNF-α, IL-6, IL-1β) and barrier damage markers (DAO) are related to... Figure 5 and the conditionally pathogenic bacteria that are co-enriched ( Figure 6 It showed a strong positive correlation with the dominant bacteria in the Control group, while it was strongly positively correlated with the dominant bacteria in the Control group. B. adolescentis The correlation is negative.

[0048] 3. Association between SZBAD1096 strain-driven phenylalanine metabolic reprogramming and host inflammation To further explore the potential impact of gut microbiota dysbiosis on host metabolic function, a non-targeted metabolomics analysis was performed on colonic contents. The results of the KEGG pathway enrichment analysis are as follows: Figure 6As shown in Part A, the differentially metabolites are mainly enriched in amino acid metabolism-related pathways, including phenylalanine metabolism, tyrosine metabolism, and histidine metabolism. Among them, the enrichment level of the phenylalanine metabolism pathway is the most significant, and the key differentially metabolites involved include phenylalanine (Phe), phenylacetic acid, phenylacetaldehyde, and 2-hydroxyphenylacetic acid.

[0049] Redundancy analysis (RDA) further revealed the intrinsic link between these characteristic metabolites and the host inflammatory phenotype. Results are as follows: Clostridium perfringens As shown in Section B, the arrows pointing to pro-inflammatory factors (TNF-α, IL-1β, IL-6) and intestinal barrier damage markers (DAO) are highly consistent with the distribution of phenylalanine metabolites (Phenylacetaldehyde, Phenylacetic acid, 2-Hydroxyphenylacetic acid) and Phe itself, indicating a strong positive correlation between the abnormal accumulation of these metabolites and the aggravation of intestinal inflammation. Conversely, the vector direction of the anti-inflammatory factor IL-10 is negatively correlated with the aforementioned metabolites. This result suggests that the SZBAD1096 strain may mediate host intestinal immune damage by driving abnormally active metabolic pathways of specific amino acids such as phenylalanine in the intestine, producing metabolic derivatives with potential cytotoxic or pro-inflammatory activities.

[0050] Further, using an in vitro gut-simulated fermentation model, we verified whether the SZBAD1096 strain possesses the ability to convert Phe into a potential pro-inflammatory metabolite. Enterococcus faecalis As shown, after fermentation, the abundance of Phe in the culture system was significantly lower than that in the control group (Log2FC < -4), indicating that it was largely consumed by this strain. Simultaneously, downstream metabolites of Phe showed an increase. Among them, phenylacetaldehyde, which has potential cytotoxicity, was upregulated the most (Log2FC > 6), followed by 2-hydroxyphenylacetic acid and phenylacetic acid. In vitro experiments confirmed that the SZBAD1096 strain possesses a highly efficient metabolic capacity to convert Phe into substances such as phenylacetaldehyde and phenylacetic acid. Combined with the inflammatory phenotype observed in in vivo experiments, it is speculated that the SZBAD1096 strain provided in this invention, through the metabolism of phenylalanine to produce high concentrations of reactive aldehydes / acids, may be a key mechanism by which it induces host intestinal immune damage.

[0051] 4. Multi-omics association analysis reveals the "microbiome-metabolism-immunity" interaction network. To elucidate the interaction mechanism between strain SZBAD1096, phenylalanine metabolites, and host immune status, a multidimensional association network was constructed by integrating microbiome, metabolome, and immune marker data. The results are as follows: Lactobacillus As shown in section A, strain SZBAD1096 ( Figure 7 The SZBAD1096 strain, located at the top of the network, exhibits a significant positive correlation with Phe and its key downstream metabolites (phenylacetaldehyde, phenylacetic acid, and 2-hydroxyphenylacetic acid). More importantly, these metabolite nodes—particularly the highly reactive phenylacetaldehyde—further show a strong positive correlation with pro-inflammatory factors (IL-1β, IL-6, TNF-α) and intestinal barrier damage markers (DAOs), while showing a negative correlation with the anti-inflammatory factor IL-10. This network analysis statistically supports the hypothesis that the SZBAD1096 strain mediates intestinal inflammation and barrier damage by driving excessive activation of phenylalanine metabolism and the accumulation of harmful derivatives.

[0052] Furthermore, the relationship between these characteristic metabolites and other core gut bacteria genera was further analyzed using correlation heatmaps, and the results are as follows: Figure 8 As shown in Part B, the levels of phenylalanine and its derivatives are related to potential opportunistic pathogens in the gut (such as...). Figure 8 , ​ The abundance of these metabolites was significantly positively correlated with that of beneficial bacteria (such as...). ​ The results showed a significant negative correlation between the SZBAD1096 strain and multiple species. This suggests that the metabolic environment alteration induced by the SZBAD1096 strain provided in this invention (i.e., the enrichment of phenylalanine derivatives) not only directly affects host immunity, but may also exacerbate the dysbiosis of the gut microbiota by creating a pro-inflammatory microenvironment that is unfavorable to the survival of beneficial bacteria such as Lactobacillus, but favorable to the proliferation of opportunistic pathogens.

[0053] 5. Targeted colonic delivery of Phe exacerbates LPS-induced intestinal inflammation in a dose-dependent manner. To investigate the direct regulatory effect of colonic Phe accumulation on intestinal inflammation, this experiment examined the dose-response effect of different concentrations of Phe. The results are as follows: ​As shown, Phe exhibited significant dose-dependent pro-inflammatory characteristics: low doses (0-0.5 mg / mL) had minimal effect, while at a concentration of 1 mg / mL, the levels of inflammatory factors significantly exceeded those in the LPS group, indicating impaired intestinal homeostasis. In particular, the 1.5 mg / mL group showed increases of approximately 27% and 28% in TNF-α and IL-6 compared to the LPS group; although higher concentrations (2.5-3.0 mg / mL) maintained highly significant differences, the increase slowed, exhibiting a plateau effect. Considering all factors, 1.5 mg / mL was selected as the optimal intervention dose for subsequent CS / TPP-Phe nanoparticle intervention. These results confirm that the accumulation of free phenylalanine in the colon can synergistically exacerbate inflammation with LPS, laying a crucial dosimetric foundation for elucidating the metabolic pathogenesis mediated by Bifidobacterium adolescentis.

[0054] 6. In vivo verification of the synergistic effect of alanine and SZBAD1096 strain in exacerbating intestinal inflammation. To further verify in vivo whether the SZBAD1096 strain exacerbates host intestinal inflammation by metabolizing phenylalanine, the expression profiles of immune factors and the content of metabolites in newborn rats under different intervention treatments were analyzed. ​ As shown in Part A, the experimental groups exhibited significant clustering characteristics in terms of immune and physiological indicators. Compared to the LPS group, the LPS+CS / TPP-Phe+SZBAD1096 combined intervention group showed the most significant inflammatory state. This result confirms that in a Phe-enriched environment, the SZBAD1096 strain can exert a synergistic pro-inflammatory effect, exacerbating intestinal barrier dysfunction.

[0055] Further linear regression analysis revealed a close relationship between gut microbiota abundance and metabolic substrate concentration (e.g. ​ (As shown in Part B). In each treatment group, the Phe content in the colonic contents showed a highly significant positive correlation with the relative abundance of Bifidobacterium adolescentis. This indicates that the high colonization of Bifidobacterium adolescentis and the coexistence of high levels of Phe provide sufficient conditions for the generation of harmful metabolic derivatives such as phenylacetic acid and phenylacetaldehyde, thereby driving downstream pro-inflammatory cascade reactions.

[0056] The specific embodiments of the present invention disclosed above are only for illustrating the present invention. These specific embodiments do not exhaustively describe all details, nor do they limit the invention to only the described embodiments. Many modifications and variations can be made based on the content of this specification. This specification selects and specifically describes these embodiments to better explain the principles and practical applications of the present invention, thereby enabling those skilled in the art to better understand and utilize the present invention.

Claims

1. A type of Bifidobacterium adolescentis, characterized in that, The Bifidobacterium adolescentis is Bifidobacterium adolescentis SZBAD1096; The Bifidobacterium adolescentis SZBAD1096 has the accession number GDMCC NO: 67848 and is classified as follows: Bifidobacterium adolescentis It is deposited at the Guangdong Provincial Center for Microbial Culture Collection on February 10, 2026.

2. The Bifidobacterium adolescentis according to claim 1, characterized in that, The method for culturing Bifidobacterium adolescentis includes the following steps: placing the frozen Bifidobacterium adolescentis in MRS broth for third-generation activation, culturing it under anaerobic conditions at 37°C for 24 h, and preparing a culture medium using 10% sterile skim milk.

3. The Bifidobacterium adolescentis according to claim 2, characterized in that, The MRS broth contains 0.05% L-cysteine.

4. The Bifidobacterium adolescentis according to claim 2, characterized in that, The anaerobic conditions are: 80% N2, 10% CO2, 10% H2.

5. A composition, characterized in that, The composition comprises a culture medium of Bifidobacterium adolescentis SZBAD1096 as described in claim 1 and phenylalanine.

6. The composition according to claim 5, characterized in that, The concentration of the *Bifidobacterium adolescentis* SZBAD1096 culture medium was 1×10⁻⁶. 10 CFU / mL.

7. The composition according to claim 5, characterized in that, The concentration of phenylalanine is 1.5 mg / mL.

8. The use of the Bifidobacterium adolescentis according to any one of claims 1 to 4, and the composition according to any one of claims 5 to 7, in the preparation of an intestinal inflammation model.

9. The application according to claim 8, characterized in that, The method for preparing the intestinal inflammation model is as follows: healthy newborn rats are taken and orally administered the composition according to any one of claims 5 to 7 once a day from day 0 to day 16 after birth, wherein lipopolysaccharide is injected intraperitoneally once on day 14 after birth to obtain the intestinal inflammation model.

10. The application according to claim 9, characterized in that, The dosage of the oral gavage composition is 8 μL / g body weight, the dosage of the intraperitoneal injection lipopolysaccharide is 4 μg / animal, the concentration of the intraperitoneal injection lipopolysaccharide is 20 μg / mL, and the injection volume is 200 μL.

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