Breast-milk Lacticaseibacillus gasseri FN136 alleviates DSS-induced ulcerative colitis through microbiota-independent metabolite–host interactions

Breast-milk Lacticaseibacillus gasseri FN136 alleviates DSS-induced ulcerative colitis through microbiota-independent metabolite–host interactions | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Research Article Breast-milk Lacticaseibacillus gasseri FN136 alleviates DSS-induced ulcerative colitis through microbiota-independent metabolite–host interactions Qingcui Li, Wenhao Wang, Fan Zhou, Xin Geng, Ce Qi, Chengwen Li, and 1 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-7525483/v1 This work is licensed under a CC BY 4.0 License Status: Published Journal Publication published 28 Nov, 2025 Read the published version in Probiotics and Antimicrobial Proteins → Version 1 posted 11 You are reading this latest preprint version Abstract Chronic, relapsing colonic inflammation defines ulcerative colitis (UC) and heightens long-term colorectal cancer risk. Probiotics, proven beneficial, have emerged as a promising adjunctive strategy for UC management. We conducted a two-tiered evaluation of the breast-milk-derived strain Lacticaseibacillus gasseri FN136. In vitro, FN136 secreted anti-inflammatory amino acids and indoles, displayed pronounced acid- and bile-salt tolerance, and exhibited significant antimicrobial activity. In vivo, daily oral administration of 1 × 10⁹ CFU FN136 markedly attenuated DSS-induced UC: serum l-arginine was restored, splenic Treg frequency increased by 42.09% ( P < 0.05), and nitric oxide production was elevated, while disease activity index, body-weight loss, and colonic shortening were all significantly reduced ( P < 0.05). Histopathology revealed intact crypt architecture and diminished inflammatory infiltration; systemic inflammation was suppressed as evidenced by decreased interleukin-6, myeloperoxidase, lipopolysaccharide, and FITC-dextran levels ( P < 0.05). Critically, 16S rRNA sequencing revealed no significant alterations in global microbial composition, indicating that FN136 exerts protection via a microbiota-independent postbiotic–host axis. Collectively, FN136 mitigates DSS-induced UC by restoring serum arginine and orchestrating immune–barrier homeostasis through a postbiotic pathway, offering a novel framework for precision UC intervention. Lacticaseibacillus gasseri FN136 Ulcerative colitis arginine inflammatory responses regulatory T cells Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 Figure 7 Figure 8 Figure 9 1. Introduction Ulcerative colitis (UC), a prototypical inflammatory bowel disease, is characterized by chronic, relapsing inflammation of the colonic mucosa [ 1 ]. Although its aetiology remains incompletely understood, converging evidence implicates a multifactorial interplay among genetic predisposition [ 2 ], epithelial-barrier dysfunction [ 3 ], dysregulated immune responses [ 4 ] and environmental triggers [ 5 ]. Clinically, patients typically present with abdominal pain, diarrhoea, and haematochezia [ 6 ]. Current pharmacotherapies—including thiopurines, aminosalicylates, and corticosteroids—are frequently associated with adverse events such as headache, abdominal pain, and nausea [ 7 ]. While dietary interventions may offer benefit in select individuals, their efficacy remains inconsistent and is supported by limited high-quality evidence [ 8 ]. Consequently, safe and low-toxicity adjuncts capable of providing sustained symptom relief are urgently required. Probiotics—live microorganisms that confer health benefits when administered in adequate amounts [ 9 ]—represent a promising adjunctive strategy. Breast milk, beyond its nutritional function, constitutes a natural reservoir of early-life microbiota [ 10 ]. Healthy human milk contains approximately 10³–10⁴ CFU mL⁻¹ of bacteria [ 11 ], which are dominated by members of the phyla Proteobacteria and Firmicutes , together with the genera Staphylococcus , Pseudomonas , Enterobacteriaceae , Streptococcus , and lactic acid bacteria [ 12 ]. Among these taxa, Lactobacillus strains isolated from human milk have attracted considerable interest owing to their probiotic potential; they contribute to the establishment and maintenance of a healthy gut microbiota, enhance immune function, and prevent various diseases [ 13 ]. Accumulating evidence demonstrates that milk-derived probiotics can prevent or ameliorate UC. The breast-milk isolate Lacticaseibacillus rhamnosus SHA113 has been shown to mitigate colitis by modulating the gut microbiota, whereas other milk-borne lactobacilli attenuate 2,4,6-trinitrobenzenesulfonic acid -induced colitis via suppression of nuclear factor κ-light-chain-enhancer of activated B cells [ 14 ]. These strains exhibit robust gastrointestinal survival and colonization, thereby ensuring sustained anti-inflammatory activity [ 15 ]. Collectively, breast-milk-derived probiotics provide a safe and practical strategy for UC prevention and adjunctive therapy. UC remains incurable, and relapses are driven by a complex interplay of genetic, immune, epithelial, and environmental factors. Therapeutic strategies that seek to globally remodel the gut microbiota have yielded heterogeneous clinical outcomes, indicating that factors beyond community structure may be decisive. Consequently, research focus is shifting from bacterial colonization per se to the functional metabolome—bioactive metabolites secreted or induced by specific strains that directly interact with host tissues—thereby circumventing the traditional “reshape-first, treat-second” paradigm. The validity of this approach has been preliminarily demonstrated in mice fed a high-fat diet and depleted of their indigenous microbiota by broad-spectrum antibiotics; these animals exhibited reduced inflammatory cytokine levels and improved intestinal barrier integrity after supplementation with Parabacteroides johnsonii [ 16 ]. Importantly, these effects occurred in the absence of detectable microbial engraftment, indicating direct mediation by bacterial metabolites. Inspired by this finding, the selection of breast milk-derived probiotics that possess an inherently efficient metabolic repertoire, coupled with elucidation of their microbiota-independent bacterium–metabolite–host axis, is anticipated to provide a gentle yet precise therapeutic avenue for UC. Lacticaseibacillus gasseri FN136 was isolated from the breast milk of well-nourished mothers in southern Gansu, China. Its metabolic signature was first delineated in a controlled in vitro fermentation model; subsequently, its protective efficacy and underlying mechanisms were systematically evaluated in DSS-induced UC by integrating in vitro fermentation metabolomics, gut microbiota profiling, immunological analyses and serum metabolomics, with the overarching goal of establishing a rigorous scientific foundation for UC prevention and treatment. 2. Materials and Methods 2.1 Probiotic activation Frozen stocks (− 80°C) were thawed, inoculated into de Man, Rogosa and Sharpe (MRS) broth, and incubated at 37°C under static, anaerobic conditions until the late logarithmic phase. The cultures were then transferred at 10% (v/v) into fresh MRS broth and incubated under identical conditions for 24 h. 2.2 Metabolomic profiling of FN136 fermentation broth The activated FN136 culture was washed three times with sterile phosphate-buffered saline (PBS) and resuspended to 1 × 10⁹ CFU mL⁻¹. This suspension was inoculated into modified CDM1 medium [ 17 ] and incubated statically at 37°C. At 0, 8, 12, 16, 20 and 24 h, 1 mL aliquots were withdrawn, centrifuged (15 000 × g, 5 min, 4°C), and the supernatants were collected. Each supernatant was acidified to pH 2–3 with 1 mol L⁻¹ HCl, vortexed for 1 min, held for 10 min, and re-centrifuged under identical conditions. The resulting supernatant was mixed with an equal volume of ice-cold acetonitrile, followed by sequential addition of MgSO₄ (0.2 g mL⁻¹) and NaOAc (0.05 g mL⁻¹). After gentle rotation for 10 min, the mixture was centrifuged (15 000 × g, 5 min, 4°C) and the organic phase was recovered. The extract was filtered through a 0.22 µm membrane and transferred to LC vials. Metabolite profiling was performed on an Agilent 1290 Infinity–6530B UPLC-QTOF-MS system operated in positive electrospray ionisation (ESI⁺) mode. 2.3 Probiotic potential assessment of L. gasseri FN136 2.3.1 Acid and bile-salt tolerance assays The acid (pH 2.0) and bile-salt (0.3%) tolerance of L. gasseri FN136 was assessed according to the protocol described by previous protocol. [ 18 ]. Activated cultures of FN136, Limosilactobacillus reuteri FN041 (positive control) and Lactaseibacillus rhamnosus GG (LGG, positive control) were harvested, washed three times with phosphate-buffered saline adjusted to pH 3.0 or 7.0, and resuspended to approximately 1 × 10⁹ CFU mL⁻¹. A 100 µL aliquot of each suspension was inoculated into fresh medium and incubated at 37°C for 4 h. After incubation, cultures were homogenised, serially diluted, and 50 µL of each dilution was plated on MRS agar. Following 24 h of incubation at 37°C, colonies were enumerated to determine survival. Bile-salt tolerance was evaluated using an identical protocol. Gastric acid or bile-salt tolerance (%) was calculated as (CFU₁/CFU₀) × 100, where CFU₁ and CFU₀ are the viable counts in the experimental and control groups, respectively. 2.3.2 Relative adhesion assay Adhesion of L. gasseri FN136 was quantified according a previous protocol [ 19 ], with minor modifications. Activated cells were harvested, washed three times with sterile PBS, and resuspended to an OD₆₀₀ of 1.0. A 96-well microplate was coated with 200 µL mucin solution (1 mg mL⁻¹ in PBS) and incubated at 4°C for 12 h. After removal of excess mucin, wells were blocked with 0.1% (w/v) bovine serum albumin at 37°C for 1 h and gently rinsed with PBS. Subsequently, 100 µL of the bacterial suspension was added to each well and incubated at 37°C for 2 h. Non-adherent cells were removed by three gentle washes with sterile PBS, the plates were air-dried at room temperature, and absorbance was measured at 450 nm. Relative adhesion was expressed as the percentage of the OD₄₅₀ obtained for LGG, which was set as 100%. 2.3.3 Determination of antimicrobial susceptibility of L. gasseri FN136 Following the micro-broth dilution protocol of Elshikh et al. with minor modifications [ 20 ], the minimum inhibitory concentrations (MICs) of strain FN136 were determined against eleven antibiotics: tetracycline, penicillin, vancomycin, gentamicin, ciprofloxacin, chloramphenicol, ampicillin, streptomycin, erythromycin, kanamycin, and cephalexin. Each antibiotic was dissolved in PBS to obtain a 32768 µg mL⁻¹ stock solution. Activated FN136 cells were harvested, washed three times with sterile PBS, and adjusted to the 0.5 McFarland standard (≈ 1 × 10⁸ CFU mL⁻¹). A 96-well microplate was pre-filled with 100 µL MRS broth per well; the first column received 100 µL of each antibiotic stock, followed by two-fold serial dilutions across the plate. Subsequently, 100 µL of the bacterial suspension was added to each well, and the plates were incubated at 37°C for 24 h. The MIC was defined as the lowest antibiotic concentration that completely inhibited visible growth at 600 nm. All assays were performed in triplicate 2.3.4 Hemolytic activity assay of L. gasseri FN136 To assess hemolytic activity, activated L. gasseri FN136 was streaked onto 5% (w/v) sheep-blood agar and incubated at 37°C for 48 h; Staphylococcus aureus cultured in Luria-Bertani medium served as the positive control. The presence or absence of a clear hemolytic zone around colonies was evaluated visually to determine hemolytic activity [ 21 ]. 2.3.5 Assessment of antimicrobial activity of FN136 To evaluate antimicrobial activity, the activated culture of strain FN136 was centrifuged (15 000 × g, 5 min, 4°C) and the cell-free supernatant was collected. Indicator strains— Escherichia coli , Staphylococcus aureus and Clostridium perfringens —were adjusted to 1 × 10⁶ CFU and evenly spread on Luria–Bertani agar plates. Three sterile Oxford cups were placed on each plate, and 15 µL of supernatant was dispensed into each cup. After complete diffusion, the cups were removed and the plates were incubated at 37°C for 24 h, followed by measurement of inhibition zone diameters. 2.4 Animals All experimental procedures were conducted in strict accordance with the National Guidelines for the Care and Use of Laboratory Animals and were approved by the Animal Ethics Committee of Qingdao University (approval No. QDU-AEC-2024064). Forty male, 5-week-old, specific-pathogen-free C57BL/6J mice were obtained from Beijing Vital River Laboratory Animal Technology Co., Ltd. and were housed five per cage under a 12-hour light/dark cycle at 22 ± 3°C and 40–70% relative humidity. 2.5 Diet and experimental design Five-week-old, specific-pathogen-free male C57BL/6J mice (n = 40) were acclimated for 7 days and then randomly assigned to five groups (n = 8 per group) by means of a random-number table: (1) healthy control (CON); (2) DSS-induced colitis (DSS); (3) Lacticaseibacillus gasseri FN136 intervention (FN136 + DSS); (4) breast-milk-derived Lacticaseibacillus reuteri FN041 positive control (FN041 + DSS); and (5) commercial Lacticaseibacillus rhamnosus GG positive control (LGG + DSS). Throughout the study, all animals had ad libitum access to an AIN-93G purified diet containing 64% carbohydrate, 20% protein, and 7% fat. The CON group received autoclaved drinking water for the entire experimental period. The remaining four groups were subjected to two cycles of 2% (w/v) DSS in the drinking water (days 1–4 and 15–18), interspersed with a 10-day recovery phase during which distilled water was provided. From day 1 to day 32, mice in the CON and DSS groups received 0.9% sterile saline via daily oral gavage, whereas animals in the probiotic-treated groups were administered 1 × 10⁹ CFU of the respective strain suspended in saline. Body weight, stool consistency, and fecal occult blood were recorded daily to calculate the DAI as a measure of colitis severity. On day 32, after a 12-h fast with free access to water, blood was collected by retro-orbital puncture. Mice were then euthanized by cervical dislocation, and colon tissue, cecal contents, and spleen were immediately harvested for subsequent analyses. 2.6 Epithelial paracellular permeability On day 32, mice received an oral gavage of fluorescein isothiocyanate-dextran (molecular weight 4 kDa, FD4) at a dose of 60 mg per 100 g body weight. Four hours later, blood was collected and plasma FD4 fluorescence was quantified using excitation and emission wavelengths of 490 nm and 520 nm, respectively. 2.7 Flow cytometric analyses of the proportion of regulatory T cells in the spleen The spleen was rapidly excised and immersed in ice-cold PBS. After the addition of 5 mL red-blood-cell lysis buffer, the tissue was mechanically dissociated to generate a single-cell suspension. This suspension was transferred to a 50 mL conical tube, diluted to 100 mL with RPMI-1640, and gently inverted 2–3 times. Cells were pelleted by centrifugation at 300 × g for 5 min at 4°C, resuspended in 3 mL fresh medium, and adjusted to 1 × 10⁷ cells mL⁻¹ following enumeration. For flow cytometric analysis, 100 µL of the cell suspension was incubated with FITC-conjugated anti-CD4 and APC-conjugated anti-CD25 antibodies for 30 min at 4°C in the dark. Red-blood-cell lysis buffer and flow cytometry staining buffer were added, and the mixture was centrifuged at 400 × g for 5 min at room temperature. The supernatant was discarded; cells were fixed for 60 min at room temperature in the dark. After a second centrifugation (400 × g, 5 min), the pellet was resuspended and intracellularly stained with PE-conjugated anti-Foxp3 antibody, using an isotype-matched IgG1 control to define background. Samples were acquired on a BD FACSVerse flow cytometer and analyzed using FlowJo v10.0.7. 2.8 Serum inflammatory cytokines, NO and lipopolysaccharide (LPS) Serum concentrations of IL-6, nitric oxide (NO), Interleukin-1 beta (IL-1β), tumor necrosis factor alpha (TNF-α), and LPS were quantified using commercial enzyme-linked immunosorbent assay kits (Huikia Biotechnology Co., Ltd., Xiamen, China) according to the manufacturer’s instructions. 2.9 Serum metabolomics Serum (50 µL) was deproteinized with 250 µL ice-cold acetonitrile, vortex-mixed, and centrifuged at 15 000 rpm (4°C, 15 min). The supernatant was passed through a 0.22-µm membrane filter and transferred to an LC vial for subsequent analysis. Chromatographic separation was conducted on an Agilent 1290 Infinity-6530B UPLC-QTOF-MS system operated in positive electrospray ionization (ESI⁺) mode. Mobile phase A was acetonitrile containing 0.1% formic acid; mobile phase B was 0.1% formic acid in water. The gradient elution program and MS parameters were identical to those reported by Qi et al.[ 22 ]. 2.10 16s rRNA gene amplicon sequencing analysis of colonic microbiota Total genomic DNA was extracted from colonic contents using the QIAamp DNA Stool Mini Kit (Qiagen, Hilden, Germany) and quantified with the Quant-iT™ dsDNA HS Assay Kit (Thermo Fisher Scientific). PCR targeting the 16S rRNA V3–V4 region employed universal primers F (5'-ACTCCTACGGGAGGCAGCA-3') and R (5'-GGACTACHVGGGTWTCTAAT-3'). Cycling parameters were 94°C for 3 min; 30 cycles of 94°C for 45 s, 56°C for 60 s, and 72°C for 60 s; and a final extension at 72°C for 10 min. Indexed adapters were ligated to the purified amplicons for next-generation sequencing. Library concentration was adjusted to 10 nmol L⁻¹ (enzymatic quantification). Paired-end sequencing (2 × 250/300 bp) was performed on an Illumina MiSeq platform (Illumina, San Diego, CA, USA). Sequences were processed with EasyAmplicon [ 23 ] to generate amplicon sequence variants (ASVs), which were taxonomically assigned to the species level using the NCBI 16S Microbial database via BLCA [ 24 ]. Functional prediction was predicted with PICRUSt2 (v2.2.0-b). Raw sequencing data are deposited in the NCBI Sequence Read Archive under BioProject PRJNA1304250. 2.11 Statistical analysis and ‌bioinformatics analysis Statistical analyses were conducted using SPSS 20, and all data are presented as mean ± standard error of the mean (SEM) or median with interquartile range (IQR), as appropriate. GraphPad Prism 8.0 was used for visualization. Homogeneous variances were evaluated by one-way analysis of variance (ANOVA) followed by Tukey’s post-hoc test; heterogeneous variances were assessed with the Kruskal–Wallis test. Alpha-diversity indices for microbiota data were calculated with the micro4all package ( https://nuriamw.github.io/micro4all/tutorial/package_workflow . html). For untargeted metabolomics, raw data were processed with MS-DIAL v4.70 for spectral deconvolution, peak alignment, gap filling, and peak identification. The resulting .mgf files (MS1 and MS2) were further analyzed with the in-house tool MS2Compound to extract metabolite information at both MS levels [ 25 ]. Metabolites were tentatively annotated against the KEGG database after excluding food constituents, environmental pollutants, and pharmaceuticals. In-silico fragmentation of these metabolites was performed from their SMILES identifiers using Competitive Fragmentation Modeling-ID [ 26 ], generating a theoretical spectral library for plasma metabolite matching. Matching criteria were a precursor-ion tolerance of 0.05 Da, a fragment-ion tolerance of 0.5 Da, and a minimum of two matched fragments; the compound with the highest MS score (rank = 1) was retained. Features lacking MS2 evidence were annotated at the MS1 level only. Peak-table preprocessing and orthogonal partial least squares discriminant analysis (OPLS-DA) were performed with the Integrated Mass Spectrometry-based Untargeted Metabolomics (IP4M) platform. Metabolites were considered differential when VIP ≥ 1 in the OPLS-DA model. Correlations were calculated via Spearman’s test and plotted using pheatmap (R); significance was defined as P < 0.05. 3. Results 3.1 Active metabolites produced by FN136 during fermentation in a chemically defined medium. Untargeted metabolomic profiling of FN136 fermentation broth collected at 8, 12, 16, 20, and 24 h (VIP > 1, P < 0.05) revealed a panel of differential metabolites associated with colitis alleviation (Fig. S1 ). These compounds displayed dynamic temporal patterns, indicating that FN136 orchestrates distinct metabolic pathways to exert anti-inflammatory effects. Typtophan metabolism was markedly up-regulated, leading to the sequential accumulation of anti-inflammatory indole derivatives. Indolepyruvate was significantly elevated as early as 8 h (log₂FC = 1.573) and served as the central precursor. Indole-3-carboxaldehyde accumulated continuously from 12 to 20 h (log₂FC = 1.671, 2.120, and 2.076, respectively), functioning as an aryl hydrocarbon receptor (AhR) ligand that reinforces the intestinal barrier and modulates immunity (Wang et al., 2023). Indole-3-ethanol peaked at 16 h (log₂FC = 1.581), corroborating pathway activation, whereas N-acetyltryptophan rose significantly at 24 h (log₂FC = 1.635), completing the tryptophan metabolic signature. Short-chain fatty acids (SCFAs), the principal microbial metabolites of dietary fiber fermentation, accumulated prominently during the mid-to-late phase. Propionic acid was significantly enriched at 16 and 20 h (log₂FC = 0.867 and 0.363, respectively); in DSS colitis models, propionate markedly suppresses colonic expression of IL-6, IL-1β, and TNF-α [ 27 ]. Isovaleric acid increased sharply at 20 h (log₂FC = 2.393); although its role is pleiotropic, moderate elevations are generally associated with improved gut health. Amino-acid metabolism was extensively reprogrammed. L-arginine was continuously and significantly up-regulated at 20 and 24 h (log₂FC = 1.213 and 1.247, respectively). As a precursor of nitric oxide and polyamines, arginine is essential for mucosal repair and inflammatory resolution [ 28 , 29 ]. Glutamic acid and leucine peaked at 20 h (log₂FC = 1.942 and 1.843, respectively), providing critical energy for enterocytes and maintaining barrier integrity, whereas aspartic acid also rose significantly at 20 h (log₂FC = 1.409), underscoring active amino-acid turnover. Additional bioactive compounds were dynamically altered. Epicatechin was abundant throughout the early phase (8–16 h; maximal log₂FC = 2.503), indicating enhanced polyphenol bioavailability. Hippuric acid accumulated at 24 h (log₂FC = 0.238), reflecting improved microbial diversity and phenylalanine metabolism. Adipic and sebacic acids were significantly elevated at 24 h (log₂FC = 1.327 and 0.910, respectively), potentially contributing to energy metabolism and inflammatory modulation. Collectively, FN136 fermentation sequentially activates tryptophan metabolism, enriches SCFAs, and reprograms an arginine-centric amino-acid network, thereby generating a metabolite milieu conducive to colitis relief and substantiating its probiotic potential. 3.2 Probiotic potential of L. gasseri FN136 In vitro evaluation of probiotic traits is an essential prerequisite for strain selection. Here, the commercial reference strain LGG and Lacticaseibacillus reuteri FN041—a breast-milk-derived probiotic from healthy donors in Gannan—were employed as positive controls to systematically assess survival and adhesion under simulated gastrointestinal stress. Previous work has established that FN041 exhibits robust probiotic properties and confers marked protection in high-fat-diet-induced obese mice [ 30 ]. All tested strains remained viable at pH 3.0 and tolerated 0.3% bile salts. FN136 displayed a survival rate of 56.87% under acidic conditions, comparable to FN041 but significantly lower than LGG ( P < 0.05). In the presence of bile salts, FN136 achieved 53.07% survival and exhibited a relative adhesion of 119.14%, indicating strong adhesive potential. No significant differences were observed between FN136 and FN041 in bile-salt tolerance or adhesion; however, FN136 adhered significantly better than LGG (Fig. 2 A-C). Collectively, these data demonstrate that FN136 satisfies the essential probiotic criteria, showing robust gastrointestinal tolerance and superior intestinal adhesion. 3.3 Antimicrobial activity of L. gasseri FN136 To delineate the in vitro antimicrobial spectrum of FN136, an agar-well diffusion assay was performed against three prevalent enteric pathogens. FN136 produced the largest inhibition zone against the Gram-positive Staphylococcus aureus (20.33 ± 0.58 mm; Fig. 3 H), followed by the Gram-negative Escherichia coli (18.00 ± 0.23 mm; Fig. 3 G), whereas an intermediate but clearly defined zone was observed for Clostridium perfringens (11.67 ± 1.16 mm; Fig. 2 F). Overall, these results demonstrate that FN136 exhibits broad-spectrum antibacterial activity against both Gram-negative and Gram-positive organisms, with the most pronounced efficacy against S. aureus . 3.4 Safety evaluation of L. gasseri FN136 Antibiotic-susceptibility testing of L. gasseri FN136 demonstrated MICs below the EFSA-specified breakpoints for all eleven antibiotics examined (Table 2 ). In hemolysis assays, no zone of clearing was observed on blood agar for FN136, whereas the Staphylococcus aureus control displayed pronounced β-hemolysis (Fig. 2 D). Collectively, these findings confirm that FN136 lacks acquired antibiotic resistance and hemolytic activity, thereby establishing its biosafety. Table 1 Differential Metabolites in FN136 fermentate compared with 0 h medium 8 h 12 h 16 h 20 h 24 h Metabolite VIP Log 2 FC VIP Log 2 FC VIP Log 2 FC VIP Log 2 FC VIP Log 2 FC Epicatechin 2.35 1.36 1.767 2.5 1.43 2.47 Glycine 1.98 2.04 1.11 1.178 1.52 0.02 Indolepyruvate 2.04 1.57 L-Tyrosine 1.76 0.12 1.23 3.5 DHPTP 1.86 0.6 Glycylproline 1.68 3.88 1.48 0.67 1.11 0.726 I3A 1.65 1.67 1.67 2.12 1.11 2.08 1.27 -1.08 m-Cresol 1.59 0.65 Indole-3-ethanol 1.58 1.58 1.01 0.15 Propionic acid 1.57 0.87 1.10 0.36 Glutamic acid 1.11 1.94 Aspartic acid 1.11 1.41 1.24 -1.08 L-Arginine 1.11 1.21 1.69 1.25 Leucine 1.02 1.84 Isovaleric acid 1.17 2.39 1.08 -2.42 L-Asparagine 1.71 0.50 Hippuric acid 1.56 0.24 Sebacic acid 1.56 0.91 Adipic acid 1.80 1.33 Glutarylcarnitine 1.78 0.12 N-Acetyltryptophan 1.54 1.64 VIP were obtained from the OPLS-DA model (n = 3 per group). DHPTP, 1-(3,4- Dihydroxyphenyl)-3-(2,4,6-trihydroxyphenyl)propan-2-ol; I3A, Indole-3-carboxaldehyde; Log 2 FC, log₂ fold change; VIP, variable importance in the projection. Table 2 Minimum inhibitory concentration (µg mL − 1 ) against 11 antibiotics. antibiotic LGG FN041 FN136 Tetracycline 0.25–0.5 0.5–1 0.5–1 Penicillin 0.25–0.5 0.25–0.5 0.25–0.5 Vancomycin 1024–2048 2048–4096 2048–4096 Gentamicin 16–32 16–32 16–32 Ciprofloxacin 256–512 32–64 128–256 Chloramphenicol 0.5–1 45659.00 45659.00 Ampicillin 0.25–0.5 0.50 0.125–0.5 Streptomycin 16–32 18 − 16 32–64 Erythromycin 0.00 0.00 0.00 Kanamycin 16–32 16–32 16–32 Cephalexin 16–32 16–32 45885.00 FN136, Lacticaseibacillus gasseri FN136; FN041, Limosilactobacillus reuteri FN041; LGG, Lacticaseibacillus rhamnosus GG. 3.5 L. gasseri FN136 supplementation markedly ameliorated DSS-induced histopathological injury. Relative to the CON group, DSS mice exhibited significant weight loss ( P < 0.01), DAI ( P < 0.01), pronounced colon shortening ( P < 0.01) and overt rectal bleeding, confirming successful induction of acute colitis. FN136 intervention restored body weight, reduced DAI scores and normalized colon length (Fig. 3 A–F); no gross blood was detected in this group. Histological assessment revealed severe mucosal damage in DSS mice, characterized by crypt distortion, goblet-cell depletion and extensive inflammatory infiltrates. FN136 and FN041 markedly attenuated these lesions, whereas LGG produced only partial improvement with residual inflammation (Fig. 3 G). Peyer’s patch counts were slightly reduced in DSS mice, but differences did not reach statistical significance (Fig. 3 D). Collectively, L. gasseri FN136 significantly mitigated both clinical manifestations and histopathological damage in DSS colitis, underscoring its robust therapeutic potential. 3.6 L. gasseri FN136 supplementation significantly attenuated serum inflammation and restored intestinal barrier integrity in DSS-induced UC mice. Relative to the CON group, DSS-induced UC mice exhibited pronounced increases in the pro-inflammatory mediators IL-6 (29.4%) and MPO (49.9%) ( P < 0.05; Fig. 4 A – C). FN136 intervention reversed these elevations ( P < 0.05). Serum NO, markedly reduced in DSS mice, was restored to near-normal levels by FN136 ( P < 0.05), whereas no comparable recovery was observed in the FN041 + DSS or LGG + DSS groups. TNF-α concentrations did not differ significantly among groups (Fig. 4 E). Serum FD4 and LPS—established markers of intestinal permeability—were elevated in DSS mice, confirming barrier disruption. Co-administration of FN136 significantly decreased FD4 and LPS concentrations by 88.19% and 15.77%, respectively, relative to DSS alone (Fig. 4 E-F). Collectively, FN136 potently attenuated colonic inflammation and restored barrier integrity in the DSS-induced UC model, underscoring its translational potential as a therapeutic probiotic for UC. 3.7 Impact of L. gasseri FN136 on splenic regulatory T-cell frequency in DSS-induced UC mice Tregs are indispensable for immune homeostasis and for restraining excessive intestinal inflammation. Flow-cytometric assessment revealed a pronounced reduction in splenic Tregs in DSS-treated mice compared with CON mice (Fig. 5 A, B). FN136 supplementation fully restored this deficit, increasing the Treg fraction by 42.09% relative to the DSS group ( P < 0.05), whereas FN041 elicited only a modest, non-significant increment (Fig. 5 ). Thus, L. gasseri FN136 expands the Treg compartment and reconfigures Treg-mediated immunoregulatory networks, offering a promising probiotic strategy for alleviating intestinal inflammation. 3.8 Impact of L. gasseri FN136 on the gut microbiota in DSS-induced UC mice To assess the effect of L. gasseri FN136 on the colonic microbiota of DSS-treated mice, 16S rDNA amplicon sequencing of colonic contents was performed. The Shannon index was significantly lower in the DSS group than in the CON group ( P < 0.05), indicating reduced microbial diversity; however, intervention with FN136, FN041, or LGG failed to restore the Chao1, ACE, or Shannon indices (Fig. 6 A). β-diversity analyses (NMDS and PCoA) further demonstrated a clear separation between the DSS and CON groups ( P < 0.05), reflecting substantial compositional shifts. None of the probiotic-treated groups differed significantly from the DSS group (Fig. 6 B). To determine whether FN136 modulated specific taxa, the OTU table was analyzed at the genus and species levels. At the genus level, the DSS group exhibited an increased relative abundance of Escherichia and decreased abundances of Prevotellamassilia , Acetatifactor , Alistipes , Duncaniella , and Lacrimispora compared with the CON group (Fig. S2). These differences were not significant between the DSS and FN136 + DSS groups. Functional pathway prediction also revealed that FN136 intervention did not reverse the differentially enriched pathways identified between the CON and DSS groups (Fig. S3). Collectively, these data indicate that L. gasseri FN136 does not ameliorate DSS-induced colitis through pronounced restructuring of the global gut microbiota. 3.9 Impact of L. gasseri FN136 on the serum metabolome OPLS-DA was used to compare serum metabolite profiles across the five experimental groups. The resulting models exhibited robust predictive capacity (Q²Y > 0.5), and pairwise comparisons revealed clear separation between the DSS group and each of the other four groups. Differentially abundant metabolites (VIP > 1, P < 0.05) were selected for further investigation (Fig. 7 ). Compared with the DSS group, a total of 14 differential metabolites were identified across the experimental cohorts (Table 3 ), ten of which were significantly down-regulated in DSS mice. Notably, FN136 intervention restored several key metabolites to near-control levels. L-arginine was markedly elevated in the FN136 group (log₂FC = 3.69), consistent with our in vitro observation that FN136 synthesizes and accumulates arginine, suggesting that the strain replenishes systemic arginine either directly or via metabolic modulation. Arginine exerts anti-inflammatory effects by suppressing pro-inflammatory cytokine production, limiting NO synthesis, and reinforcing intestinal barrier integrity[ 31 ]. Table 3 Differential serum metabolites relative to DSS-induced colitis control mice. Metabolite CON FN136 FN041 LGG VIP Log 2 FC VIP Log 2 FC VIP Log 2 FC VIP Log 2 FC D-Tryptophan 1.64 - 1.14 0.42 0.27 1.22 1.33 1.01 1.54 Guanidoacetic acid 1.63 -1.04 0.70 0.41 1.27 1.24 1.03 1.40 Guanine 1.61 -2.06 0.94 1.13 1.30 2.40 1.28 2.56 Dihydrouracil 1.59 -2.05 1.19 1.01 0.46 0.68 0.45 0.76 5-Hydroxyindole- 3-acetic acid 1.54 -2.19 0.81 1.44 1.33 2.29 1.18 2.26 Pantothenic Acid 1.49 2.70 1.00 -0.57 0.48 -0.42 0.49 0.46 Niacinamide 1.43 -1.31 1.33 0.94 1.11 1.19 1.04 1.13 Cytosine 1.38 -1.32 1.54 2.17 1.28 1.84 1.13 1.28 L-Arginine 1.27 -2.11 1.27 3.69 1.10 2.16 1.42 4.30 Uracil 1.23 -2.16 1.00 1.01 1.20 2.11 1.09 1.77 Taurocholic acid 0.65 5.94 1.26 -4.88 1.28 -6.01 1.23 -5.25 Glycerophosphocholine 0.73 -0.92 1.23 4.04 1.22 3.91 1.36 3.87 Creatinine 0.35 -0.05 0.60 0.23 1.37 0.83 1.18 1.27 2,6-diaminohexanoic acid 0.15 -0.21 0.40 -0.44 1.07 1.67 1.02 2.29 VIP scores were obtained from the OPLS-DA model (n = 5 per group). CON, healthy control; FN136, L. gasseri FN136-treated DSS UC; FN041, L. reuteri FN041-treated DSS UC; LGG, L. rhamnosus GG-treated DSS UC; DSS, dextran sulfate sodium-induced UC. VIP, variable importance in the projection. Values of log₂FC > 0 indicate up-regulation relative to the DSS group, whereas log₂FC < 0 indicate down-regulation relative to the DSS group. Cyosine and uracil were also significantly increased in the FN136 group (log 2 FC = 2.17 and 1.01, respectively). As nucleic-acid bases, cytosine and uracil participate in DNA and RNA formation and are critical for DNA methylation—an epigenetic process whose dysregulation is implicated in UC pathogenesis [ 32 ]. Additionally, FN136 substantially reduced taurocholic acid (log 2 FC = − 4.88) while elevating glycerophosphocholine (log 2 FC = 4.04). Taurocholic acid, a primary bile acid, aggravates colitis in IL-10-deficient mice [ 33 ], whereas glycerophosphocholine, a choline derivative and acetylcholine precursor, possesses immunomodulatory and anti-inflammatory properties [ 34 ]. Collectively, DSS-induced colitis disrupts amino-acid, bile-acid and energy metabolism. FN136 leverages its intrinsic metabolic capacity—particularly arginine biosynthesis—to restore serum arginine levels, thereby modulating the arginine–NO axis and downstream immune and barrier functions. Concomitant rebalancing of cytosine, bile acids and glycerophosphocholine further amplifies its protective effects. 3.10 Correlation Analysis Spearman correlation analysis identified l-arginine as a central metabolic node that integrates immune, barrier and metabolic homeostasis. L-Arginine was positively correlated with splenic Treg frequency (R² = 0.42, P < 0.01), indicating that it reinforces immunoregulatory circuits to dampen inflammation. As the sole substrate for nitric oxide synthesis, l-arginine was also positively correlated with serum NO ( R² = 0.23, P < 0.05) and negatively correlated with serum LPS( R²= 0.18, P < 0.05) and FD4( R²= 0.21, P < 0.05).—validated indicators of intestinal barrier leakage. Uracil, a nucleic-acid precursor, exhibited concordant patterns: its abundance was negatively associated with IL-6( R²=0.41, P < 0.05), LPS( R²=0.38, P < 0.05) and FD4( R²=0.18, P < 0.05), and positively associated with splenic Treg levels ( R² =0.21, P < 0.05), suggesting that uracil likewise exerts anti-inflammatory effects and strengthens mucosal barrier function(Fig. 8 B). Collectively, l-arginine drives the arginine–NO metabolic axis and, in concert with metabolites such as uracil, sustains metabolic–immune–barrier homeostasis. 4. Discussion In vitro assays revealed that FN136 tolerates acid and bile salts robustly and adheres strongly, confirming its probiotic credentials. Oral administration of FN136 markedly alleviated DSS-induced colitis, as evidenced by reduced disease activity index, decreased serum pro-inflammatory cytokines, restored intestinal barrier integrity, and elevated splenic Treg proportions. Metabolomic profiling of in vitro fermentation showed that FN136 drives the accumulation of l-arginine, propionate, isovalerate, and indole derivatives such as indole-3-carboxaldehyde and indole-3-ethanol, demonstrating its capacity to generate anti-inflammatory metabolites. Although some of these compounds were not detected in serum, FN136 intervention significantly restored serum l-arginine levels (log₂FC = 3.69), mirroring in vitro data and indicating systemic regulation via its metabolites. Serum metabolomics further demonstrated pronounced enrichment of the arginine metabolic pathway and modulation of inflammation- and barrier-associated metabolites, including taurocholic acid and glycerophosphocholine. UC is a chronic, relapsing inflammatory disorder of the gastrointestinal tract characterized by abdominal pain, diarrhea, hematochezia, and weight loss [ 35 ]. Disease progression is driven by LPS-mediated activation of inflammatory signaling pathways, leading to elevated release of pro-inflammatory cytokines such as IL-6, IL-1β, and TNF-α and subsequent disruption of intestinal barrier integrity [ 36 , 37 ]. FD4 is a widely accepted marker of intestinal permeability, with increased serum FD4 indicating compromised barrier function [ 38 ]. In the present study, the FD4 assay demonstrated that FN136 administration significantly reduced serum LPS, IL-6, IL-1β, and FD4 levels, indicating effective attenuation of intestinal barrier damage and inflammation. Multiple studies have demonstrated that gut dysbiosis, aberrant immune activation, and intestinal barrier disruption are closely linked to inflammatory bowel disease and other gastrointestinal disorders[ 39 ]. Amino acids are essential for maintaining mucosal integrity and barrier function[ 40 ]. In vitro fermentation metabolomics revealed that FN136 efficiently utilizes precursor substrates to markedly accumulate l-arginine (log₂FC increased during mid-to-late fermentation) and to activate the tryptophan metabolic pathway, yielding indole derivatives such as indole-3-carboxaldehyde and indole-3-ethanol. These findings indicate that FN136 possesses robust amino-acid metabolic capacity—particularly arginine biosynthesis—that may underlie its immunomodulatory and barrier-protective effects in the host. L-Arginine, a semi-essential amino acid, is indispensable for immune homeostasis [ 41 ], inflammatory regulation and tissue barrier repair [ 29 , 37 ]. It serves as the obligate precursor for nitric oxide, creatine, glutamate, proline and urea [ 28 ]. Although healthy adults can synthesize and obtain adequate arginine from dietary sources, patients with inflammatory bowel disease frequently exhibit arginine deficiency due to diminished endogenous synthesis, impaired intestinal absorption and heightened inflammatory demand [ 42 ]. Consistent with these observations, DSS-induced UC mice displayed markedly reduced serum arginine concentrations. FN136 intervention restored arginine levels to a degree that mirrored in vitro fermentation profiles, indicating that strain-derived metabolites may directly or indirectly re-establish host arginine homeostasis. Previous investigations have demonstrated that exogenous l-arginine supplementation in DSS colitis models reduces pro-inflammatory cytokine release, accelerates barrier recovery, and attenuates disease severity [ 43 ]. Arginine catabolism ultimately generates NO and polyamines, both of which exert critical regulatory functions [ 29 ]. NO is particularly essential for intestinal homeostasis; patients with UC exhibit diminished arginine uptake and elevated arginase-1 expression, leading to inadequate NO synthesis [ 44 ]. In the present study, DSS-treated mice displayed markedly reduced serum arginine and NO concentrations, and FN136 intervention restored both parameters. Spearman correlation analysis revealed negative associations between arginine and pro-inflammatory mediators and positive associations between arginine and NO levels, further supporting the hypothesis that FN136 exerts anti-inflammatory effects by modulating the arginine–NO metabolic axis. Additionally, the indole derivatives identified in in vitro fermentation are recognized aryl hydrocarbon receptor ligands with established anti-inflammatory and barrier-protective properties, suggesting that FN136 may confer benefits via multi-metabolite synergism. Development and maintenance of immune homeostasis are intimately linked to gut microbiota composition, a relationship that is particularly evident in the pathogenesis of UC [ 45 ].Tregs are pivotal in sustaining intestinal immune balance [ 46 ] and suppress aberrant activation and proliferation of pro-inflammatory Th17 cells in DSS-induced colitis by secreting anti-inflammatory mediators such as TGF-β and IL-10 [ 47 ]. In this study, oral administration of L. gasseri FN136 significantly elevated splenic Treg frequencies, and Spearman correlation analysis revealed a strong positive association between serum arginine levels and Treg numbers. In vitro fermentation metabolomics further confirmed that FN136 efficiently synthesizes and accumulates l-arginine while simultaneously activating the tryptophan metabolic pathway to generate AhR-agonistic indole derivatives, including indole-3-carboxaldehyde and indole-3-ethanol. Collectively, these findings suggest that FN136 enhances Treg function via two complementary mechanisms: (1) direct elevation of host arginine availability, which is known to promote IL-10 secretion and augment Treg immunomodulatory capacity; and (2) synergistic promotion of Treg differentiation or functional maturation through AhR signaling activation by its metabolites, ultimately conferring robust protection against colitis. Conclusion Lacticaseibacillus gasseri FN136 markedly attenuated DSS-induced UC in mice. The protective effect derives from dual-level regulation of metabolism and immunity: the strain augments arginine biosynthesis, elevates systemic arginine availability, and thereby reinforces anti-inflammatory responses and intestinal barrier integrity; concurrently, accumulated arginine promotes the differentiation and functional maturation of regulatory T cells, amplifying their immunoregulatory capacity and jointly restoring gut immune homeostasis. These integrated mechanisms collectively mitigate colitis pathogenesis. Abbreviations aryl hydrocarbon receptor AhR control CON disease activity index DAI sodium dextran sulfate DSS fluorescein isothiocyanate dextran FD4 Lacticaseibacillus reuteri FN041 FN041 Lacticaseibacillus gasseri FN136 FN136 Interleukin-1 beta IL-1β interleukin-6 IL-6 interleukin 10 IL-10 Lacticaseibacillus rhamnosus GG LGG lipopolysaccharide LPS myeloperoxidase MPO nitric oxide NO orthogonal partial least squares-discriminant analysis OPLS-DA partial least squares discriminant analysis phosphate-buffered saline,PBS PLS-DA Staphylococcus aureus S. aureus. tumor necrosis factor alpha TNF-α regulatory T cells Tregs short-chain fatty acids SCFAs TGF-β Transforming Growth Factor-beta ulcerative colitis UC. Declarations Conflict of interest The authors have declared no conflict of interest. Author Contribution Q.L. contributed to the study by acquiring, analyzing, and interpreting the data, as well as drafting the manuscript. F.Z. and C.L. participated in data visualization and revised the manuscript. W.W. and X.G. contributed by acquiring, analyzing, and interpreting the data. C.Q. was responsible for conceiving and designing the study, analyzing and interpreting the data, and drafting and revising the manuscript. J.S.drafted and revised the manuscript and participated in the experimental design. All authors have reviewed and approved the submitted version of the manuscript and agree to be personally accountable for their respective contributions. Acknowledgement This research was supported by the National Natural Science Foundation of China (No. 82574094). References Chen L, Ruan G, Cheng Y et al (2022) The role of Th17 cells in inflammatory bowel disease and the research progress. 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CON, healthy control; DSS, dextran sulfate sodium-induced UC; \u003cem\u003eL. gasseri\u003c/em\u003e FN136-treated DSS UC; FN041+DSS, \u003cem\u003eL. reuteri\u003c/em\u003e FN041-treated DSS UC; LGG+DSS, \u003cem\u003eL. rhamnosus\u003c/em\u003e GG-treated DSS UC.\u003c/p\u003e","description":"","filename":"Figure71.png","url":"https://assets-eu.researchsquare.com/files/rs-7525483/v1/146cc6a2ceeba8b39f3c380d.png"},{"id":91837917,"identity":"d28dc54f-7e74-44b9-a40a-8642edaec745","added_by":"auto","created_at":"2025-09-22 09:41:48","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":666656,"visible":true,"origin":"","legend":"\u003cp\u003eProbiotic potential assessment of \u003cem\u003eL. gasseri\u003c/em\u003e FN136 (n=3 per group). (A) Acid tolerance (B)Bile-salt resistance (C)Relative adhesion of the tested probiotics versus LGG. (D) Hemolytic activity assessment of \u003cem\u003eL. gasseri\u003c/em\u003e FN136. (E) Hemolytic activity of \u003cem\u003eStaphylococcus aureus\u003c/em\u003e. (F) Inhibition of \u003cem\u003eClostridium perfringens\u003c/em\u003e by FN136. (G) Inhibition of\u003cem\u003e Escherichia coli\u003c/em\u003e by FN136. (H) Inhibition of \u003cem\u003eStaphylococcus aureus\u003c/em\u003e by FN136. Data satisfying homogeneity of variance were analysed by one-way ANOVA followed by Tukey’s post-hoc test; otherwise, the Kruskal–Wallis test was applied. All values are presented as mean ± SEM. ** \u003cem\u003eP\u003c/em\u003e \u0026lt; 0.01 vs. LGG. FN136, \u003cem\u003eLacticaseibacillus gasseri\u003c/em\u003e FN136; FN041, \u003cem\u003eLimosilactobacillus reuteri \u003c/em\u003eFN041; LGG, \u003cem\u003eLacticaseibacillus rhamnosus\u003c/em\u003e GG.\u003c/p\u003e","description":"","filename":"Figure72.png","url":"https://assets-eu.researchsquare.com/files/rs-7525483/v1/fc98ce98f29c9590ac308714.png"},{"id":91837910,"identity":"43045274-79f7-4e98-9e68-76d4a9553839","added_by":"auto","created_at":"2025-09-22 09:41:48","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":1097839,"visible":true,"origin":"","legend":"\u003cp\u003eEffects of \u003cem\u003eL. gasseri\u003c/em\u003e FN136 on body weight, disease activity index (DAI), colonic morphology and Peyer’s patch number in mice with DSS-induced UC (n = 8 per group). (A) Terminal body weight; (B) DAI score; (C) Colon length; (D) Representative H\u0026amp;E-stained colonic sections; (E) Fecal bleeding; (F) Number of Peyer’s patches. Data satisfying homogeneity of variance were analysed by one-way ANOVA followed by Tukey’s post-hoc test; otherwise, the Kruskal–Wallis test was applied. All values are presented as mean ± SEM. * \u003cem\u003eP\u003c/em\u003e \u0026lt; 0.05, ** \u003cem\u003eP\u003c/em\u003e\u0026lt; 0.01 vs. CON group; # \u003cem\u003eP\u003c/em\u003e \u0026lt; 0.05, ## p \u0026lt; 0.01 vs. DSS group. CON, healthy control; DSS, dextran sulfate sodium-induced UC; \u003cem\u003eL. gasseri\u003c/em\u003eFN136-treated DSS UC; FN041+DSS, \u003cem\u003eL. reuteri\u003c/em\u003e FN041-treated DSS UC; LGG+DSS, \u003cem\u003eL. rhamnosus\u003c/em\u003e GG-treated DSS UC.\u003c/p\u003e","description":"","filename":"Figure73.png","url":"https://assets-eu.researchsquare.com/files/rs-7525483/v1/746a816774b5924b47503cc5.png"},{"id":91837920,"identity":"a5fad8f4-be76-4891-8c17-f65f3a8dfeaa","added_by":"auto","created_at":"2025-09-22 09:41:48","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":375482,"visible":true,"origin":"","legend":"\u003cp\u003eEffects of \u003cem\u003eL. gasseri\u003c/em\u003e FN136 on serum inflammatory indices in mice with DSS-induced UC (n = 8 per group). (A) Serum IL-6 levels. (B) Serum MPO levels. (C) Serum NO levels. (D) Serum TNF-α levels. (E) Serum LPS levels. (F) Serum FD4 levels. Data satisfying homogeneity of variance were analysed by one-way ANOVA followed by Tukey’s post-hoc test; otherwise, the Kruskal–Wallis test was applied. All values are presented as mean ± SEM. * \u003cem\u003eP\u003c/em\u003e \u0026lt; 0.05, ** \u003cem\u003eP\u003c/em\u003e \u0026lt; 0.01 versus CON; # \u003cem\u003eP\u003c/em\u003e \u0026lt; 0.05, ## \u003cem\u003eP\u003c/em\u003e \u0026lt; 0.01 versus DSS. CON, healthy control; DSS, dextran sulfate sodium-induced UC; FN136+DSS, \u003cem\u003eL. gasseri\u003c/em\u003eFN136-treated DSS UC; FN041+DSS, \u003cem\u003eL. reuteri\u003c/em\u003e FN041-treated DSS UC; LGG+DSS, \u003cem\u003eL. rhamnosus\u003c/em\u003e GG-treated DSS UC.\u003c/p\u003e","description":"","filename":"Figure74.png","url":"https://assets-eu.researchsquare.com/files/rs-7525483/v1/bdf6b58a7b964a19fbcd52c2.png"},{"id":91838871,"identity":"c23af0f2-8d4e-4cb3-bf1c-4392a4abb500","added_by":"auto","created_at":"2025-09-22 09:49:48","extension":"png","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":740086,"visible":true,"origin":"","legend":"\u003cp\u003eImpact of \u003cem\u003eL. gasseri\u003c/em\u003e FN136 on splenic Tregs in DSS-induced UC mice (n = 4 per group). (A) Splenocytes were isolated from the indicated experimental groups. (B) The percentage of CD4⁺CD25⁺Foxp3⁺ Tregs within the splenocyte population was determined. Inter-group comparisons were performed using the Kruskal–Wallis test. # \u003cem\u003eP\u003c/em\u003e \u0026lt; 0.05, ## \u003cem\u003eP\u003c/em\u003e \u0026lt; 0.01 versus DSS. CON, healthy control; DSS, dextran sulfate sodium-induced UC; FN136+DSS, \u003cem\u003eL. gasseri\u003c/em\u003e FN136-treated DSS UC; FN041+DSS, \u003cem\u003eL. reuteri\u003c/em\u003eFN041-treated DSS UC; LGG+DSS, \u003cem\u003eL. rhamnosus\u003c/em\u003e GG-treated DSS UC.\u003c/p\u003e","description":"","filename":"Figure75.png","url":"https://assets-eu.researchsquare.com/files/rs-7525483/v1/02f11aa4fe4581abaa6c42be.png"},{"id":91838875,"identity":"bfaff0b4-5cde-49e9-929b-35c3d1803ecd","added_by":"auto","created_at":"2025-09-22 09:49:49","extension":"png","order_by":6,"title":"Figure 6","display":"","copyAsset":false,"role":"figure","size":516376,"visible":true,"origin":"","legend":"\u003cp\u003eEffects of \u003cem\u003eL. gasseri\u003c/em\u003e FN136 on the gut microbiota of mice with DSS-induced UC (n = 8 per group). (A) α-diversity indices. (B) β-diversity. Data satisfying homogeneity of variance were analysed by one-way ANOVA followed by Tukey’s post-hoc test; otherwise, the Kruskal–Wallis test was applied. All values are presented as mean ± SEM. CON, healthy control; DSS, dextran sulfate sodium-induced UC; FN136+DSS, \u003cem\u003eL. gasseri\u003c/em\u003e FN136-treated DSS UC; FN041+DSS, \u003cem\u003eL. reuteri\u003c/em\u003e FN041-treated DSS UC; LGG+DSS, \u003cem\u003eL. rhamnosus\u003c/em\u003e GG-treated DSS UC.\u003c/p\u003e","description":"","filename":"Figure76.png","url":"https://assets-eu.researchsquare.com/files/rs-7525483/v1/8ba163e2b3b60a8ee8d95cb2.png"},{"id":91837923,"identity":"3f192a0f-f498-4d14-aa44-2c333c27671e","added_by":"auto","created_at":"2025-09-22 09:41:48","extension":"png","order_by":7,"title":"Figure 7","display":"","copyAsset":false,"role":"figure","size":420026,"visible":true,"origin":"","legend":"\u003cp\u003eNon-targeted metabolomic profiling of mouse serum (n = 5 per group). (A) PLS-DA and OPLS-DA score plots comparing two groups. (B) Overfitting test results between two groups. Untargeted metabolomic analysis was performed with UPLC-QTOF MS/MS. CON, healthy control; DSS, dextran sulfate sodium-induced UC; FN136+DSS, \u003cem\u003eL. gasseri\u003c/em\u003e FN136-treated DSS UC; FN041+DSS, \u003cem\u003eL. reuteri\u003c/em\u003e FN041-treated DSS UC; LGG+DSS, \u003cem\u003eL. rhamnosus\u003c/em\u003eGG-treated DSS UC.\u003c/p\u003e","description":"","filename":"Figure77.png","url":"https://assets-eu.researchsquare.com/files/rs-7525483/v1/bd217303bc564012daf1ac32.png"},{"id":91842065,"identity":"554a27bf-24ed-451b-8902-1ef34066b2f7","added_by":"auto","created_at":"2025-09-22 09:57:49","extension":"png","order_by":8,"title":"Figure 8","display":"","copyAsset":false,"role":"figure","size":507024,"visible":true,"origin":"","legend":"\u003cp\u003eRelative abundance and correlation analyses of differential metabolites. (A) Relative abundance of serum arginine. (B) Relative abundance of serum urea. (C) Correlation between serum differential metabolites and inflammatory cytokines. Untargeted metabolomic analysis was performed with UPLC-QTOF MS/MS. All values are presented as mean ± SEM. * \u003cem\u003eP\u003c/em\u003e \u0026lt; 0.01 versus CON; # \u003cem\u003eP\u003c/em\u003e \u0026lt; 0.05, ## \u003cem\u003eP\u003c/em\u003e \u0026lt; 0.01 versus DSS. CON, healthy control; DSS, dextran sulfate sodium-induced UC; FN136+DSS, \u003cem\u003eL. gasseri\u003c/em\u003e FN136-treated DSS UC; FN041+DSS, \u003cem\u003eL. reuteri\u003c/em\u003e FN041-treated DSS UC; LGG+DSS, \u003cem\u003eL. rhamnosus\u003c/em\u003e GG-treated DSS UC.\u003c/p\u003e","description":"","filename":"Figure78.png","url":"https://assets-eu.researchsquare.com/files/rs-7525483/v1/d043dbd9a99b4098e99c4730.png"},{"id":91837959,"identity":"0e32c9a5-668c-4a87-900e-bd94068c9b45","added_by":"auto","created_at":"2025-09-22 09:41:49","extension":"png","order_by":9,"title":"Figure 9","display":"","copyAsset":false,"role":"figure","size":367419,"visible":true,"origin":"","legend":"\u003cp\u003eSchematic representation of the mechanism by which \u003cem\u003eL. gasseri\u003c/em\u003e FN136 attenuates DSS-induced ulcerative colitis in mice. DSS,sodium dextran sulfate; FD4, fluorescein isothiocyanate dextran; IL-1β, Interleukin-1 beta; IL-6, interleukin-6; \u0026nbsp;LPS, lipopolysaccharide; NO, nitric oxide; regulatory Tregs,T cells.\u003c/p\u003e","description":"","filename":"Figure79.png","url":"https://assets-eu.researchsquare.com/files/rs-7525483/v1/4336fe50dfd7a3c95649b7a5.png"},{"id":97179373,"identity":"961715ad-bb7f-4b35-86c8-edc57c669d43","added_by":"auto","created_at":"2025-12-01 16:15:10","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":6205062,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-7525483/v1/5f916bac-89ec-4671-9f08-bfa8ed4476ac.pdf"},{"id":91838869,"identity":"af82c114-f714-4583-a36c-01687826b71c","added_by":"auto","created_at":"2025-09-22 09:49:48","extension":"docx","order_by":0,"title":"","display":"","copyAsset":false,"role":"supplement","size":4720321,"visible":true,"origin":"","legend":"","description":"","filename":"Supplementarymaterials.docx","url":"https://assets-eu.researchsquare.com/files/rs-7525483/v1/c77aac786ca2ddc9ef6f655d.docx"}],"financialInterests":"No competing interests reported.","formattedTitle":"Breast-milk Lacticaseibacillus gasseri FN136 alleviates DSS-induced ulcerative colitis through microbiota-independent metabolite–host interactions","fulltext":[{"header":"1. Introduction","content":"\u003cp\u003eUlcerative colitis (UC), a prototypical inflammatory bowel disease, is characterized by chronic, relapsing inflammation of the colonic mucosa [\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e]. Although its aetiology remains incompletely understood, converging evidence implicates a multifactorial interplay among genetic predisposition [\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e], epithelial-barrier dysfunction [\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e], dysregulated immune responses [\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e] and environmental triggers [\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e]. Clinically, patients typically present with abdominal pain, diarrhoea, and haematochezia [\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e]. Current pharmacotherapies\u0026mdash;including thiopurines, aminosalicylates, and corticosteroids\u0026mdash;are frequently associated with adverse events such as headache, abdominal pain, and nausea [\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e]. While dietary interventions may offer benefit in select individuals, their efficacy remains inconsistent and is supported by limited high-quality evidence [\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e]. Consequently, safe and low-toxicity adjuncts capable of providing sustained symptom relief are urgently required.\u003c/p\u003e\u003cp\u003eProbiotics\u0026mdash;live microorganisms that confer health benefits when administered in adequate amounts [\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e]\u0026mdash;represent a promising adjunctive strategy. Breast milk, beyond its nutritional function, constitutes a natural reservoir of early-life microbiota [\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e]. Healthy human milk contains approximately 10\u0026sup3;\u0026ndash;10⁴ CFU mL⁻\u0026sup1; of bacteria [\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e], which are dominated by members of the phyla \u003cem\u003eProteobacteria\u003c/em\u003e and \u003cem\u003eFirmicutes\u003c/em\u003e, together with the genera \u003cem\u003eStaphylococcus\u003c/em\u003e, \u003cem\u003ePseudomonas\u003c/em\u003e, \u003cem\u003eEnterobacteriaceae\u003c/em\u003e, \u003cem\u003eStreptococcus\u003c/em\u003e, and lactic acid bacteria [\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e]. Among these taxa, \u003cem\u003eLactobacillus\u003c/em\u003e strains isolated from human milk have attracted considerable interest owing to their probiotic potential; they contribute to the establishment and maintenance of a healthy gut microbiota, enhance immune function, and prevent various diseases [\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e].\u003c/p\u003e\u003cp\u003eAccumulating evidence demonstrates that milk-derived probiotics can prevent or ameliorate UC. The breast-milk isolate \u003cem\u003eLacticaseibacillus rhamnosus\u003c/em\u003e SHA113 has been shown to mitigate colitis by modulating the gut microbiota, whereas other milk-borne lactobacilli attenuate 2,4,6-trinitrobenzenesulfonic acid -induced colitis via suppression of nuclear factor κ-light-chain-enhancer of activated B cells [\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e]. These strains exhibit robust gastrointestinal survival and colonization, thereby ensuring sustained anti-inflammatory activity [\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e]. Collectively, breast-milk-derived probiotics provide a safe and practical strategy for UC prevention and adjunctive therapy.\u003c/p\u003e\u003cp\u003eUC remains incurable, and relapses are driven by a complex interplay of genetic, immune, epithelial, and environmental factors. Therapeutic strategies that seek to globally remodel the gut microbiota have yielded heterogeneous clinical outcomes, indicating that factors beyond community structure may be decisive. Consequently, research focus is shifting from bacterial colonization per se to the functional metabolome\u0026mdash;bioactive metabolites secreted or induced by specific strains that directly interact with host tissues\u0026mdash;thereby circumventing the traditional \u0026ldquo;reshape-first, treat-second\u0026rdquo; paradigm. The validity of this approach has been preliminarily demonstrated in mice fed a high-fat diet and depleted of their indigenous microbiota by broad-spectrum antibiotics; these animals exhibited reduced inflammatory cytokine levels and improved intestinal barrier integrity after supplementation with \u003cem\u003eParabacteroides johnsonii\u003c/em\u003e [\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e]. Importantly, these effects occurred in the absence of detectable microbial engraftment, indicating direct mediation by bacterial metabolites. Inspired by this finding, the selection of breast milk-derived probiotics that possess an inherently efficient metabolic repertoire, coupled with elucidation of their microbiota-independent bacterium\u0026ndash;metabolite\u0026ndash;host axis, is anticipated to provide a gentle yet precise therapeutic avenue for UC.\u003c/p\u003e\u003cp\u003e\u003cem\u003eLacticaseibacillus gasseri\u003c/em\u003e FN136 was isolated from the breast milk of well-nourished mothers in southern Gansu, China. Its metabolic signature was first delineated in a controlled in vitro fermentation model; subsequently, its protective efficacy and underlying mechanisms were systematically evaluated in DSS-induced UC by integrating in vitro fermentation metabolomics, gut microbiota profiling, immunological analyses and serum metabolomics, with the overarching goal of establishing a rigorous scientific foundation for UC prevention and treatment.\u003c/p\u003e"},{"header":"2. Materials and Methods","content":"\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e\u003ch2\u003e2.1 Probiotic activation\u003c/b\u003e\u003c/h2\u003e\u003cp\u003eFrozen stocks (\u0026minus;\u0026thinsp;80\u0026deg;C) were thawed, inoculated into de Man, Rogosa and Sharpe (MRS) broth, and incubated at 37\u0026deg;C under static, anaerobic conditions until the late logarithmic phase. The cultures were then transferred at 10% (v/v) into fresh MRS broth and incubated under identical conditions for 24 h.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec4\" class=\"Section2\"\u003e\u003ch2\u003e2.2 Metabolomic profiling of FN136 fermentation broth\u003c/h2\u003e\u003cp\u003eThe activated FN136 culture was washed three times with sterile phosphate-buffered saline (PBS) and resuspended to 1 \u0026times; 10⁹ CFU mL⁻\u0026sup1;. This suspension was inoculated into modified CDM1 medium [\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e] and incubated statically at 37\u0026deg;C. At 0, 8, 12, 16, 20 and 24 h, 1 mL aliquots were withdrawn, centrifuged (15 000 \u0026times; g, 5 min, 4\u0026deg;C), and the supernatants were collected. Each supernatant was acidified to pH 2\u0026ndash;3 with 1 mol L⁻\u0026sup1; HCl, vortexed for 1 min, held for 10 min, and re-centrifuged under identical conditions. The resulting supernatant was mixed with an equal volume of ice-cold acetonitrile, followed by sequential addition of MgSO₄ (0.2 g mL⁻\u0026sup1;) and NaOAc (0.05 g mL⁻\u0026sup1;). After gentle rotation for 10 min, the mixture was centrifuged (15 000 \u0026times; g, 5 min, 4\u0026deg;C) and the organic phase was recovered. The extract was filtered through a 0.22 \u0026micro;m membrane and transferred to LC vials. Metabolite profiling was performed on an Agilent 1290 Infinity\u0026ndash;6530B UPLC-QTOF-MS system operated in positive electrospray ionisation (ESI⁺) mode.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec5\" class=\"Section2\"\u003e\u003ch2\u003e2.3 Probiotic potential assessment of \u003cem\u003eL. gasseri\u003c/em\u003e FN136\u003c/h2\u003e\u003cdiv id=\"Sec6\" class=\"Section3\"\u003e\u003ch2\u003e2.3.1 Acid and bile-salt tolerance assays\u003c/h2\u003e\u003cp\u003eThe acid (pH 2.0) and bile-salt (0.3%) tolerance of \u003cem\u003eL. gasseri\u003c/em\u003e FN136 was assessed according to the protocol described by previous protocol. [\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e]. Activated cultures of FN136, \u003cem\u003eLimosilactobacillus reuteri\u003c/em\u003e FN041 (positive control) and \u003cem\u003eLactaseibacillus rhamnosus\u003c/em\u003e GG (LGG, positive control) were harvested, washed three times with phosphate-buffered saline adjusted to pH 3.0 or 7.0, and resuspended to approximately 1 \u0026times; 10⁹ CFU mL⁻\u0026sup1;. A 100 \u0026micro;L aliquot of each suspension was inoculated into fresh medium and incubated at 37\u0026deg;C for 4 h. After incubation, cultures were homogenised, serially diluted, and 50 \u0026micro;L of each dilution was plated on MRS agar. Following 24 h of incubation at 37\u0026deg;C, colonies were enumerated to determine survival. Bile-salt tolerance was evaluated using an identical protocol. Gastric acid or bile-salt tolerance (%) was calculated as (CFU₁/CFU₀) \u0026times; 100, where CFU₁ and CFU₀ are the viable counts in the experimental and control groups, respectively.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec7\" class=\"Section3\"\u003e\u003ch2\u003e2.3.2 Relative adhesion assay\u003c/h2\u003e\u003cp\u003eAdhesion of \u003cem\u003eL. gasseri\u003c/em\u003e FN136 was quantified according a previous protocol [\u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e], with minor modifications. Activated cells were harvested, washed three times with sterile PBS, and resuspended to an OD₆₀₀ of 1.0. A 96-well microplate was coated with 200 \u0026micro;L mucin solution (1 mg mL⁻\u0026sup1; in PBS) and incubated at 4\u0026deg;C for 12 h. After removal of excess mucin, wells were blocked with 0.1% (w/v) bovine serum albumin at 37\u0026deg;C for 1 h and gently rinsed with PBS. Subsequently, 100 \u0026micro;L of the bacterial suspension was added to each well and incubated at 37\u0026deg;C for 2 h. Non-adherent cells were removed by three gentle washes with sterile PBS, the plates were air-dried at room temperature, and absorbance was measured at 450 nm. Relative adhesion was expressed as the percentage of the OD₄₅₀ obtained for LGG, which was set as 100%.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec8\" class=\"Section3\"\u003e\u003ch2\u003e2.3.3 Determination of antimicrobial susceptibility of \u003cem\u003eL. gasseri\u003c/em\u003e FN136\u003c/h2\u003e\u003cp\u003eFollowing the micro-broth dilution protocol of Elshikh et al. with minor modifications [\u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e], the minimum inhibitory concentrations (MICs) of strain FN136 were determined against eleven antibiotics: tetracycline, penicillin, vancomycin, gentamicin, ciprofloxacin, chloramphenicol, ampicillin, streptomycin, erythromycin, kanamycin, and cephalexin. Each antibiotic was dissolved in PBS to obtain a 32768 \u0026micro;g mL⁻\u0026sup1; stock solution. Activated FN136 cells were harvested, washed three times with sterile PBS, and adjusted to the 0.5 McFarland standard (\u0026asymp;\u0026thinsp;1 \u0026times; 10⁸ CFU mL⁻\u0026sup1;). A 96-well microplate was pre-filled with 100 \u0026micro;L MRS broth per well; the first column received 100 \u0026micro;L of each antibiotic stock, followed by two-fold serial dilutions across the plate. Subsequently, 100 \u0026micro;L of the bacterial suspension was added to each well, and the plates were incubated at 37\u0026deg;C for 24 h. The MIC was defined as the lowest antibiotic concentration that completely inhibited visible growth at 600 nm. All assays were performed in triplicate\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec9\" class=\"Section3\"\u003e\u003ch2\u003e2.3.4 Hemolytic activity assay of \u003cem\u003eL. gasseri\u003c/em\u003e FN136\u003c/h2\u003e\u003cp\u003eTo assess hemolytic activity, activated \u003cem\u003eL. gasseri\u003c/em\u003e FN136 was streaked onto 5% (w/v) sheep-blood agar and incubated at 37\u0026deg;C for 48 h; \u003cem\u003eStaphylococcus aureus\u003c/em\u003e cultured in Luria-Bertani medium served as the positive control. The presence or absence of a clear hemolytic zone around colonies was evaluated visually to determine hemolytic activity [\u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e].\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec10\" class=\"Section3\"\u003e\u003ch2\u003e2.3.5 Assessment of antimicrobial activity of FN136\u003c/h2\u003e\u003cp\u003eTo evaluate antimicrobial activity, the activated culture of strain FN136 was centrifuged (15 000 \u0026times; g, 5 min, 4\u0026deg;C) and the cell-free supernatant was collected. Indicator strains\u0026mdash;\u003cem\u003eEscherichia coli\u003c/em\u003e, \u003cem\u003eStaphylococcus aureus\u003c/em\u003e and \u003cem\u003eClostridium perfringens\u003c/em\u003e\u0026mdash;were adjusted to 1 \u0026times; 10⁶ CFU and evenly spread on Luria\u0026ndash;Bertani agar plates. Three sterile Oxford cups were placed on each plate, and 15 \u0026micro;L of supernatant was dispensed into each cup. After complete diffusion, the cups were removed and the plates were incubated at 37\u0026deg;C for 24 h, followed by measurement of inhibition zone diameters.\u003c/p\u003e\u003c/div\u003e\u003c/div\u003e\u003cdiv id=\"Sec11\" class=\"Section2\"\u003e\u003ch2\u003e2.4 Animals\u003c/h2\u003e\u003cp\u003e All experimental procedures were conducted in strict accordance with the National Guidelines for the Care and Use of Laboratory Animals and were approved by the Animal Ethics Committee of Qingdao University (approval No. QDU-AEC-2024064). Forty male, 5-week-old, specific-pathogen-free C57BL/6J mice were obtained from Beijing Vital River Laboratory Animal Technology Co., Ltd. and were housed five per cage under a 12-hour light/dark cycle at 22\u0026thinsp;\u0026plusmn;\u0026thinsp;3\u0026deg;C and 40\u0026ndash;70% relative humidity.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec12\" class=\"Section2\"\u003e\u003ch2\u003e2.5 Diet and experimental design\u003c/h2\u003e\u003cp\u003eFive-week-old, specific-pathogen-free male C57BL/6J mice (n\u0026thinsp;=\u0026thinsp;40) were acclimated for 7 days and then randomly assigned to five groups (n\u0026thinsp;=\u0026thinsp;8 per group) by means of a random-number table: (1) healthy control (CON); (2) DSS-induced colitis (DSS); (3) \u003cem\u003eLacticaseibacillus gasseri\u003c/em\u003e FN136 intervention (FN136\u0026thinsp;+\u0026thinsp;DSS); (4) breast-milk-derived \u003cem\u003eLacticaseibacillus reuteri\u003c/em\u003e FN041 positive control (FN041\u0026thinsp;+\u0026thinsp;DSS); and (5) commercial \u003cem\u003eLacticaseibacillus rhamnosus\u003c/em\u003e GG positive control (LGG\u0026thinsp;+\u0026thinsp;DSS). Throughout the study, all animals had \u003cem\u003ead libitum\u003c/em\u003e access to an AIN-93G purified diet containing 64% carbohydrate, 20% protein, and 7% fat. The CON group received autoclaved drinking water for the entire experimental period. The remaining four groups were subjected to two cycles of 2% (w/v) DSS in the drinking water (days 1\u0026ndash;4 and 15\u0026ndash;18), interspersed with a 10-day recovery phase during which distilled water was provided. From day 1 to day 32, mice in the CON and DSS groups received 0.9% sterile saline via daily oral gavage, whereas animals in the probiotic-treated groups were administered 1 \u0026times; 10⁹ CFU of the respective strain suspended in saline. Body weight, stool consistency, and fecal occult blood were recorded daily to calculate the DAI as a measure of colitis severity. On day 32, after a 12-h fast with free access to water, blood was collected by retro-orbital puncture. Mice were then euthanized by cervical dislocation, and colon tissue, cecal contents, and spleen were immediately harvested for subsequent analyses.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec13\" class=\"Section2\"\u003e\u003ch2\u003e2.6 Epithelial paracellular permeability\u003c/h2\u003e\u003cp\u003eOn day 32, mice received an oral gavage of fluorescein isothiocyanate-dextran (molecular weight 4 kDa, FD4) at a dose of 60 mg per 100 g body weight. Four hours later, blood was collected and plasma FD4 fluorescence was quantified using excitation and emission wavelengths of 490 nm and 520 nm, respectively.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec14\" class=\"Section2\"\u003e\u003ch2\u003e2.7 Flow cytometric analyses of the proportion of regulatory T cells in the spleen\u003c/h2\u003e\u003cp\u003eThe spleen was rapidly excised and immersed in ice-cold PBS. After the addition of 5 mL red-blood-cell lysis buffer, the tissue was mechanically dissociated to generate a single-cell suspension. This suspension was transferred to a 50 mL conical tube, diluted to 100 mL with RPMI-1640, and gently inverted 2\u0026ndash;3 times. Cells were pelleted by centrifugation at 300 \u0026times; g for 5 min at 4\u0026deg;C, resuspended in 3 mL fresh medium, and adjusted to 1 \u0026times; 10⁷ cells mL⁻\u0026sup1; following enumeration.\u003c/p\u003e\u003cp\u003eFor flow cytometric analysis, 100 \u0026micro;L of the cell suspension was incubated with FITC-conjugated anti-CD4 and APC-conjugated anti-CD25 antibodies for 30 min at 4\u0026deg;C in the dark. Red-blood-cell lysis buffer and flow cytometry staining buffer were added, and the mixture was centrifuged at 400 \u0026times; g for 5 min at room temperature. The supernatant was discarded; cells were fixed for 60 min at room temperature in the dark. After a second centrifugation (400 \u0026times; g, 5 min), the pellet was resuspended and intracellularly stained with PE-conjugated anti-Foxp3 antibody, using an isotype-matched IgG1 control to define background. Samples were acquired on a BD FACSVerse flow cytometer and analyzed using FlowJo v10.0.7.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec15\" class=\"Section2\"\u003e\u003ch2\u003e2.8 Serum inflammatory cytokines, NO and lipopolysaccharide (LPS)\u003c/h2\u003e\u003cp\u003e Serum concentrations of IL-6, nitric oxide (NO), Interleukin-1 beta (IL-1β), tumor necrosis factor alpha (TNF-α), and LPS were quantified using commercial enzyme-linked immunosorbent assay kits (Huikia Biotechnology Co., Ltd., Xiamen, China) according to the manufacturer\u0026rsquo;s instructions.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec16\" class=\"Section2\"\u003e\u003ch2\u003e2.9 Serum metabolomics\u003c/h2\u003e\u003cp\u003eSerum (50 \u0026micro;L) was deproteinized with 250 \u0026micro;L ice-cold acetonitrile, vortex-mixed, and centrifuged at 15 000 rpm (4\u0026deg;C, 15 min). The supernatant was passed through a 0.22-\u0026micro;m membrane filter and transferred to an LC vial for subsequent analysis.\u003c/p\u003e\u003cp\u003eChromatographic separation was conducted on an Agilent 1290 Infinity-6530B UPLC-QTOF-MS system operated in positive electrospray ionization (ESI⁺) mode. Mobile phase A was acetonitrile containing 0.1% formic acid; mobile phase B was 0.1% formic acid in water. The gradient elution program and MS parameters were identical to those reported by Qi et al.[\u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e].\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec17\" class=\"Section2\"\u003e\u003ch2\u003e2.10 16s rRNA gene amplicon sequencing analysis of colonic microbiota\u003c/h2\u003e\u003cp\u003eTotal genomic DNA was extracted from colonic contents using the QIAamp DNA Stool Mini Kit (Qiagen, Hilden, Germany) and quantified with the Quant-iT\u0026trade; dsDNA HS Assay Kit (Thermo Fisher Scientific). PCR targeting the 16S rRNA V3\u0026ndash;V4 region employed universal primers F (5'-ACTCCTACGGGAGGCAGCA-3') and R (5'-GGACTACHVGGGTWTCTAAT-3'). Cycling parameters were 94\u0026deg;C for 3 min; 30 cycles of 94\u0026deg;C for 45 s, 56\u0026deg;C for 60 s, and 72\u0026deg;C for 60 s; and a final extension at 72\u0026deg;C for 10 min. Indexed adapters were ligated to the purified amplicons for next-generation sequencing. Library concentration was adjusted to 10 nmol L⁻\u0026sup1; (enzymatic quantification). Paired-end sequencing (2 \u0026times; 250/300 bp) was performed on an Illumina MiSeq platform (Illumina, San Diego, CA, USA). Sequences were processed with EasyAmplicon [\u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e] to generate amplicon sequence variants (ASVs), which were taxonomically assigned to the species level using the NCBI 16S Microbial database via BLCA [\u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e]. Functional prediction was predicted with PICRUSt2 (v2.2.0-b). Raw sequencing data are deposited in the NCBI Sequence Read Archive under BioProject PRJNA1304250.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec18\" class=\"Section2\"\u003e\u003ch2\u003e2.11 Statistical analysis and \u0026zwnj;bioinformatics analysis\u003c/h2\u003e\u003cp\u003eStatistical analyses were conducted using SPSS 20, and all data are presented as mean\u0026thinsp;\u0026plusmn;\u0026thinsp;standard error of the mean (SEM) or median with interquartile range (IQR), as appropriate. GraphPad Prism 8.0 was used for visualization. Homogeneous variances were evaluated by one-way analysis of variance (ANOVA) followed by Tukey\u0026rsquo;s post-hoc test; heterogeneous variances were assessed with the Kruskal\u0026ndash;Wallis test. Alpha-diversity indices for microbiota data were calculated with the micro4all package (\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://nuriamw.github.io/micro4all/tutorial/package_workflow\u003c/span\u003e\u003cspan address=\"https://nuriamw.github.io/micro4all/tutorial/package_workflow\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e. html).\u003c/p\u003e\u003cp\u003eFor untargeted metabolomics, raw data were processed with MS-DIAL v4.70 for spectral deconvolution, peak alignment, gap filling, and peak identification. The resulting .mgf files (MS1 and MS2) were further analyzed with the in-house tool MS2Compound to extract metabolite information at both MS levels [\u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e]. Metabolites were tentatively annotated against the KEGG database after excluding food constituents, environmental pollutants, and pharmaceuticals. In-silico fragmentation of these metabolites was performed from their SMILES identifiers using Competitive Fragmentation Modeling-ID [\u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e], generating a theoretical spectral library for plasma metabolite matching. Matching criteria were a precursor-ion tolerance of 0.05 Da, a fragment-ion tolerance of 0.5 Da, and a minimum of two matched fragments; the compound with the highest MS score (rank\u0026thinsp;=\u0026thinsp;1) was retained. Features lacking MS2 evidence were annotated at the MS1 level only.\u003c/p\u003e\u003cp\u003ePeak-table preprocessing and orthogonal partial least squares discriminant analysis (OPLS-DA) were performed with the Integrated Mass Spectrometry-based Untargeted Metabolomics (IP4M) platform. Metabolites were considered differential when VIP\u0026thinsp;\u0026ge;\u0026thinsp;1 in the OPLS-DA model. Correlations were calculated via Spearman\u0026rsquo;s test and plotted using pheatmap (R); significance was defined as \u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.05.\u003c/p\u003e\u003c/div\u003e"},{"header":"3. Results","content":"\u003cdiv id=\"Sec20\" class=\"Section2\"\u003e\u003ch2\u003e3.1 Active metabolites produced by FN136 during fermentation in a chemically defined medium.\u003c/h2\u003e\u003cp\u003eUntargeted metabolomic profiling of FN136 fermentation broth collected at 8, 12, 16, 20, and 24 h (VIP\u0026thinsp;\u0026gt;\u0026thinsp;1, \u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.05) revealed a panel of differential metabolites associated with colitis alleviation (Fig. \u003cspan refid=\"MOESM1\" class=\"InternalRef\"\u003eS1\u003c/span\u003e). These compounds displayed dynamic temporal patterns, indicating that FN136 orchestrates distinct metabolic pathways to exert anti-inflammatory effects.\u003c/p\u003e\u003cp\u003eTyptophan metabolism was markedly up-regulated, leading to the sequential accumulation of anti-inflammatory indole derivatives. Indolepyruvate was significantly elevated as early as 8 h (log₂FC\u0026thinsp;=\u0026thinsp;1.573) and served as the central precursor. Indole-3-carboxaldehyde accumulated continuously from 12 to 20 h (log₂FC\u0026thinsp;=\u0026thinsp;1.671, 2.120, and 2.076, respectively), functioning as an aryl hydrocarbon receptor (AhR) ligand that reinforces the intestinal barrier and modulates immunity (Wang et al., 2023). Indole-3-ethanol peaked at 16 h (log₂FC\u0026thinsp;=\u0026thinsp;1.581), corroborating pathway activation, whereas N-acetyltryptophan rose significantly at 24 h (log₂FC\u0026thinsp;=\u0026thinsp;1.635), completing the tryptophan metabolic signature.\u003c/p\u003e\u003cp\u003eShort-chain fatty acids (SCFAs), the principal microbial metabolites of dietary fiber fermentation, accumulated prominently during the mid-to-late phase. Propionic acid was significantly enriched at 16 and 20 h (log₂FC\u0026thinsp;=\u0026thinsp;0.867 and 0.363, respectively); in DSS colitis models, propionate markedly suppresses colonic expression of IL-6, IL-1β, and TNF-α [\u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e]. Isovaleric acid increased sharply at 20 h (log₂FC\u0026thinsp;=\u0026thinsp;2.393); although its role is pleiotropic, moderate elevations are generally associated with improved gut health.\u003c/p\u003e\u003cp\u003eAmino-acid metabolism was extensively reprogrammed. L-arginine was continuously and significantly up-regulated at 20 and 24 h (log₂FC\u0026thinsp;=\u0026thinsp;1.213 and 1.247, respectively). As a precursor of nitric oxide and polyamines, arginine is essential for mucosal repair and inflammatory resolution [\u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e, \u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e]. Glutamic acid and leucine peaked at 20 h (log₂FC\u0026thinsp;=\u0026thinsp;1.942 and 1.843, respectively), providing critical energy for enterocytes and maintaining barrier integrity, whereas aspartic acid also rose significantly at 20 h (log₂FC\u0026thinsp;=\u0026thinsp;1.409), underscoring active amino-acid turnover.\u003c/p\u003e\u003cp\u003eAdditional bioactive compounds were dynamically altered. Epicatechin was abundant throughout the early phase (8\u0026ndash;16 h; maximal log₂FC\u0026thinsp;=\u0026thinsp;2.503), indicating enhanced polyphenol bioavailability. Hippuric acid accumulated at 24 h (log₂FC\u0026thinsp;=\u0026thinsp;0.238), reflecting improved microbial diversity and phenylalanine metabolism. Adipic and sebacic acids were significantly elevated at 24 h (log₂FC\u0026thinsp;=\u0026thinsp;1.327 and 0.910, respectively), potentially contributing to energy metabolism and inflammatory modulation.\u003c/p\u003e\u003cp\u003eCollectively, FN136 fermentation sequentially activates tryptophan metabolism, enriches SCFAs, and reprograms an arginine-centric amino-acid network, thereby generating a metabolite milieu conducive to colitis relief and substantiating its probiotic potential.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec21\" class=\"Section2\"\u003e\u003ch2\u003e3.2 Probiotic potential of \u003cem\u003eL. gasseri\u003c/em\u003e FN136\u003c/h2\u003e\u003cp\u003eIn vitro evaluation of probiotic traits is an essential prerequisite for strain selection. Here, the commercial reference strain LGG and \u003cem\u003eLacticaseibacillus reuteri\u003c/em\u003e FN041\u0026mdash;a breast-milk-derived probiotic from healthy donors in Gannan\u0026mdash;were employed as positive controls to systematically assess survival and adhesion under simulated gastrointestinal stress. Previous work has established that FN041 exhibits robust probiotic properties and confers marked protection in high-fat-diet-induced obese mice [\u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e30\u003c/span\u003e]. All tested strains remained viable at pH 3.0 and tolerated 0.3% bile salts. FN136 displayed a survival rate of 56.87% under acidic conditions, comparable to FN041 but significantly lower than LGG (\u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.05). In the presence of bile salts, FN136 achieved 53.07% survival and exhibited a relative adhesion of 119.14%, indicating strong adhesive potential. No significant differences were observed between FN136 and FN041 in bile-salt tolerance or adhesion; however, FN136 adhered significantly better than LGG (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e2\u003c/span\u003eA-C). Collectively, these data demonstrate that FN136 satisfies the essential probiotic criteria, showing robust gastrointestinal tolerance and superior intestinal adhesion.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec22\" class=\"Section2\"\u003e\u003ch2\u003e3.3 Antimicrobial activity of \u003cem\u003eL. gasseri\u003c/em\u003e FN136\u003c/h2\u003e\u003cp\u003eTo delineate the in vitro antimicrobial spectrum of FN136, an agar-well diffusion assay was performed against three prevalent enteric pathogens. FN136 produced the largest inhibition zone against the Gram-positive \u003cem\u003eStaphylococcus aureus\u003c/em\u003e (20.33\u0026thinsp;\u0026plusmn;\u0026thinsp;0.58 mm; Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e3\u003c/span\u003eH), followed by the Gram-negative \u003cem\u003eEscherichia coli\u003c/em\u003e (18.00\u0026thinsp;\u0026plusmn;\u0026thinsp;0.23 mm; Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e3\u003c/span\u003eG), whereas an intermediate but clearly defined zone was observed for \u003cem\u003eClostridium perfringens\u003c/em\u003e (11.67\u0026thinsp;\u0026plusmn;\u0026thinsp;1.16 mm; Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e2\u003c/span\u003eF). Overall, these results demonstrate that FN136 exhibits broad-spectrum antibacterial activity against both Gram-negative and Gram-positive organisms, with the most pronounced efficacy against \u003cem\u003eS. aureus\u003c/em\u003e.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec23\" class=\"Section2\"\u003e\u003ch2\u003e3.4 Safety evaluation of\u003c/b\u003e \u003cb\u003eL. gasseri\u003c/b\u003e \u003cb\u003eFN136\u003c/b\u003e\u003c/h2\u003e\u003cp\u003eAntibiotic-susceptibility testing of \u003cem\u003eL. gasseri\u003c/em\u003e FN136 demonstrated MICs below the EFSA-specified breakpoints for all eleven antibiotics examined (Table\u0026nbsp;\u003cspan refid=\"Tab2\" class=\"InternalRef\"\u003e2\u003c/span\u003e). In hemolysis assays, no zone of clearing was observed on blood agar for FN136, whereas the Staphylococcus aureus control displayed pronounced β-hemolysis (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e2\u003c/span\u003eD). Collectively, these findings confirm that FN136 lacks acquired antibiotic resistance and hemolytic activity, thereby establishing its biosafety.\u003c/p\u003e\u003cp\u003e\u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab1\" border=\"1\"\u003e\u003ccaption language=\"En\"\u003e\u003cdiv class=\"CaptionNumber\"\u003eTable 1\u003c/div\u003e\u003cdiv class=\"CaptionContent\"\u003e\u003cp\u003eDifferential Metabolites in FN136 fermentate compared with 0 h medium\u003c/p\u003e\u003c/div\u003e\u003c/caption\u003e\u003ccolgroup cols=\"11\"\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c4\" colnum=\"4\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c5\" colnum=\"5\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c6\" colnum=\"6\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c7\" colnum=\"7\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c8\" colnum=\"8\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c9\" colnum=\"9\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c10\" colnum=\"10\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c11\" colnum=\"11\"\u003e\u003c/div\u003e\u003cthead\u003e\u003ctr\u003e\u003cth align=\"left\" colname=\"c1\"\u003e\u0026nbsp;\u003c/th\u003e\u003cth align=\"left\" colspan=\"2\" nameend=\"c3\" namest=\"c2\"\u003e\u003cp\u003e8 h\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colspan=\"2\" nameend=\"c5\" namest=\"c4\"\u003e\u003cp\u003e12 h\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colspan=\"2\" nameend=\"c7\" namest=\"c6\"\u003e\u003cp\u003e16 h\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colspan=\"2\" nameend=\"c9\" namest=\"c8\"\u003e\u003cp\u003e20 h\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colspan=\"2\" nameend=\"c11\" namest=\"c10\"\u003e\u003cp\u003e24 h\u003c/p\u003e\u003c/th\u003e\u003c/tr\u003e\u003c/thead\u003e\u003ctbody\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eMetabolite\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003eVIP\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003eLog\u003csub\u003e2\u003c/sub\u003eFC\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003eVIP\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003eLog\u003csub\u003e2\u003c/sub\u003eFC\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u003cp\u003eVIP\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c7\"\u003e\u003cp\u003eLog\u003csub\u003e2\u003c/sub\u003eFC\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c8\"\u003e\u003cp\u003eVIP\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c9\"\u003e\u003cp\u003eLog\u003csub\u003e2\u003c/sub\u003eFC\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c10\"\u003e\u003cp\u003eVIP\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c11\"\u003e\u003cp\u003eLog\u003csub\u003e2\u003c/sub\u003eFC\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eEpicatechin\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e2.35\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e1.36\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e1.767\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003e2.5\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u003cp\u003e1.43\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c7\"\u003e\u003cp\u003e2.47\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c8\"\u003e\u0026nbsp;\u003c/td\u003e\u003ctd align=\"left\" colname=\"c9\"\u003e\u0026nbsp;\u003c/td\u003e\u003ctd align=\"left\" colname=\"c10\"\u003e\u0026nbsp;\u003c/td\u003e\u003ctd align=\"left\" colname=\"c11\"\u003e\u0026nbsp;\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eGlycine\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e1.98\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e2.04\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u0026nbsp;\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u0026nbsp;\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u0026nbsp;\u003c/td\u003e\u003ctd align=\"left\" colname=\"c7\"\u003e\u0026nbsp;\u003c/td\u003e\u003ctd align=\"left\" colname=\"c8\"\u003e\u003cp\u003e1.11\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c9\"\u003e\u003cp\u003e1.178\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c10\"\u003e\u003cp\u003e1.52\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c11\"\u003e\u003cp\u003e0.02\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eIndolepyruvate\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e2.04\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e1.57\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u0026nbsp;\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u0026nbsp;\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u0026nbsp;\u003c/td\u003e\u003ctd align=\"left\" colname=\"c7\"\u003e\u0026nbsp;\u003c/td\u003e\u003ctd align=\"left\" colname=\"c8\"\u003e\u0026nbsp;\u003c/td\u003e\u003ctd align=\"left\" colname=\"c9\"\u003e\u0026nbsp;\u003c/td\u003e\u003ctd align=\"left\" colname=\"c10\"\u003e\u0026nbsp;\u003c/td\u003e\u003ctd align=\"left\" colname=\"c11\"\u003e\u0026nbsp;\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eL-Tyrosine\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u0026nbsp;\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u0026nbsp;\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e1.76\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003e0.12\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u0026nbsp;\u003c/td\u003e\u003ctd align=\"left\" colname=\"c7\"\u003e\u0026nbsp;\u003c/td\u003e\u003ctd align=\"left\" colname=\"c8\"\u003e\u0026nbsp;\u003c/td\u003e\u003ctd align=\"left\" colname=\"c9\"\u003e\u0026nbsp;\u003c/td\u003e\u003ctd align=\"left\" colname=\"c10\"\u003e\u003cp\u003e1.23\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c11\"\u003e\u003cp\u003e3.5\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eDHPTP\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u0026nbsp;\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u0026nbsp;\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e1.86\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003e0.6\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u0026nbsp;\u003c/td\u003e\u003ctd align=\"left\" colname=\"c7\"\u003e\u0026nbsp;\u003c/td\u003e\u003ctd align=\"left\" colname=\"c8\"\u003e\u0026nbsp;\u003c/td\u003e\u003ctd align=\"left\" colname=\"c9\"\u003e\u0026nbsp;\u003c/td\u003e\u003ctd align=\"left\" colname=\"c10\"\u003e\u0026nbsp;\u003c/td\u003e\u003ctd align=\"left\" colname=\"c11\"\u003e\u0026nbsp;\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eGlycylproline\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u0026nbsp;\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u0026nbsp;\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e1.68\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003e3.88\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u003cp\u003e1.48\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c7\"\u003e\u003cp\u003e0.67\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c8\"\u003e\u003cp\u003e1.11\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c9\"\u003e\u003cp\u003e0.726\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c10\"\u003e\u0026nbsp;\u003c/td\u003e\u003ctd align=\"left\" colname=\"c11\"\u003e\u0026nbsp;\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eI3A\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u0026nbsp;\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u0026nbsp;\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e1.65\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003e1.67\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u003cp\u003e1.67\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c7\"\u003e\u003cp\u003e2.12\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c8\"\u003e\u003cp\u003e1.11\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c9\"\u003e\u003cp\u003e2.08\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c10\"\u003e\u003cp\u003e1.27\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c11\"\u003e\u003cp\u003e-1.08\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003em-Cresol\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u0026nbsp;\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u0026nbsp;\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u0026nbsp;\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u0026nbsp;\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u003cp\u003e1.59\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c7\"\u003e\u003cp\u003e0.65\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c8\"\u003e\u0026nbsp;\u003c/td\u003e\u003ctd align=\"left\" colname=\"c9\"\u003e\u0026nbsp;\u003c/td\u003e\u003ctd align=\"left\" colname=\"c10\"\u003e\u0026nbsp;\u003c/td\u003e\u003ctd align=\"left\" colname=\"c11\"\u003e\u0026nbsp;\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eIndole-3-ethanol\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u0026nbsp;\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u0026nbsp;\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u0026nbsp;\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u0026nbsp;\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u003cp\u003e1.58\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c7\"\u003e\u003cp\u003e1.58\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c8\"\u003e\u003cp\u003e1.01\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c9\"\u003e\u003cp\u003e0.15\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c10\"\u003e\u0026nbsp;\u003c/td\u003e\u003ctd align=\"left\" colname=\"c11\"\u003e\u0026nbsp;\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003ePropionic acid\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u0026nbsp;\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u0026nbsp;\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u0026nbsp;\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u0026nbsp;\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u003cp\u003e1.57\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c7\"\u003e\u003cp\u003e0.87\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c8\"\u003e\u003cp\u003e1.10\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c9\"\u003e\u003cp\u003e0.36\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c10\"\u003e\u0026nbsp;\u003c/td\u003e\u003ctd align=\"left\" colname=\"c11\"\u003e\u0026nbsp;\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eGlutamic acid\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u0026nbsp;\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u0026nbsp;\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u0026nbsp;\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u0026nbsp;\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u0026nbsp;\u003c/td\u003e\u003ctd align=\"left\" colname=\"c7\"\u003e\u0026nbsp;\u003c/td\u003e\u003ctd align=\"left\" colname=\"c8\"\u003e\u003cp\u003e1.11\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c9\"\u003e\u003cp\u003e1.94\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c10\"\u003e\u0026nbsp;\u003c/td\u003e\u003ctd align=\"left\" colname=\"c11\"\u003e\u0026nbsp;\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eAspartic acid\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u0026nbsp;\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u0026nbsp;\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u0026nbsp;\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u0026nbsp;\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u0026nbsp;\u003c/td\u003e\u003ctd align=\"left\" colname=\"c7\"\u003e\u0026nbsp;\u003c/td\u003e\u003ctd align=\"left\" colname=\"c8\"\u003e\u003cp\u003e1.11\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c9\"\u003e\u003cp\u003e1.41\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c10\"\u003e\u003cp\u003e1.24\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c11\"\u003e\u003cp\u003e-1.08\u003c/p\u003e \u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eL-Arginine\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u0026nbsp;\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u0026nbsp;\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u0026nbsp;\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u0026nbsp;\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u0026nbsp;\u003c/td\u003e\u003ctd align=\"left\" colname=\"c7\"\u003e\u0026nbsp;\u003c/td\u003e\u003ctd align=\"left\" colname=\"c8\"\u003e\u003cp\u003e1.11\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c9\"\u003e\u003cp\u003e1.21\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c10\"\u003e\u003cp\u003e1.69\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c11\"\u003e\u003cp\u003e1.25\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eLeucine\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u0026nbsp;\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u0026nbsp;\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u0026nbsp;\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u0026nbsp;\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u0026nbsp;\u003c/td\u003e\u003ctd align=\"left\" colname=\"c7\"\u003e\u0026nbsp;\u003c/td\u003e\u003ctd align=\"left\" colname=\"c8\"\u003e\u003cp\u003e1.02\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c9\"\u003e\u003cp\u003e1.84\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c10\"\u003e\u0026nbsp;\u003c/td\u003e\u003ctd align=\"left\" colname=\"c11\"\u003e\u0026nbsp;\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eIsovaleric acid\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u0026nbsp;\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u0026nbsp;\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u0026nbsp;\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u0026nbsp;\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u0026nbsp;\u003c/td\u003e\u003ctd align=\"left\" colname=\"c7\"\u003e\u0026nbsp;\u003c/td\u003e\u003ctd align=\"left\" colname=\"c8\"\u003e\u003cp\u003e1.17\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c9\"\u003e\u003cp\u003e2.39\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c10\"\u003e\u003cp\u003e1.08\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c11\"\u003e\u003cp\u003e-2.42\u003c/p\u003e \u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eL-Asparagine\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u0026nbsp;\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u0026nbsp;\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u0026nbsp;\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u0026nbsp;\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u0026nbsp;\u003c/td\u003e\u003ctd align=\"left\" colname=\"c7\"\u003e\u0026nbsp;\u003c/td\u003e\u003ctd align=\"left\" colname=\"c8\"\u003e\u0026nbsp;\u003c/td\u003e\u003ctd align=\"left\" colname=\"c9\"\u003e\u0026nbsp;\u003c/td\u003e\u003ctd align=\"left\" colname=\"c10\"\u003e\u003cp\u003e1.71\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c11\"\u003e\u003cp\u003e0.50\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eHippuric acid\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u0026nbsp;\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u0026nbsp;\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u0026nbsp;\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u0026nbsp;\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u0026nbsp;\u003c/td\u003e\u003ctd align=\"left\" colname=\"c7\"\u003e\u0026nbsp;\u003c/td\u003e\u003ctd align=\"left\" colname=\"c8\"\u003e\u0026nbsp;\u003c/td\u003e\u003ctd align=\"left\" colname=\"c9\"\u003e\u0026nbsp;\u003c/td\u003e\u003ctd align=\"left\" colname=\"c10\"\u003e\u003cp\u003e1.56\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c11\"\u003e\u003cp\u003e0.24\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eSebacic acid\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u0026nbsp;\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u0026nbsp;\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u0026nbsp;\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u0026nbsp;\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u0026nbsp;\u003c/td\u003e\u003ctd align=\"left\" colname=\"c7\"\u003e\u0026nbsp;\u003c/td\u003e\u003ctd align=\"left\" colname=\"c8\"\u003e\u0026nbsp;\u003c/td\u003e\u003ctd align=\"left\" colname=\"c9\"\u003e\u0026nbsp;\u003c/td\u003e\u003ctd align=\"left\" colname=\"c10\"\u003e\u003cp\u003e1.56\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c11\"\u003e\u003cp\u003e0.91\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eAdipic acid\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u0026nbsp;\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u0026nbsp;\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u0026nbsp;\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u0026nbsp;\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u0026nbsp;\u003c/td\u003e\u003ctd align=\"left\" colname=\"c7\"\u003e\u0026nbsp;\u003c/td\u003e\u003ctd align=\"left\" colname=\"c8\"\u003e\u0026nbsp;\u003c/td\u003e\u003ctd align=\"left\" colname=\"c9\"\u003e\u0026nbsp;\u003c/td\u003e\u003ctd align=\"left\" colname=\"c10\"\u003e\u003cp\u003e1.80\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c11\"\u003e\u003cp\u003e1.33\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eGlutarylcarnitine\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u0026nbsp;\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u0026nbsp;\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u0026nbsp;\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u0026nbsp;\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u0026nbsp;\u003c/td\u003e\u003ctd align=\"left\" colname=\"c7\"\u003e\u0026nbsp;\u003c/td\u003e\u003ctd align=\"left\" colname=\"c8\"\u003e\u0026nbsp;\u003c/td\u003e\u003ctd align=\"left\" colname=\"c9\"\u003e\u0026nbsp;\u003c/td\u003e\u003ctd align=\"left\" colname=\"c10\"\u003e\u003cp\u003e1.78\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c11\"\u003e\u003cp\u003e0.12\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eN-Acetyltryptophan\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u0026nbsp;\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u0026nbsp;\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u0026nbsp;\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u0026nbsp;\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u0026nbsp;\u003c/td\u003e\u003ctd align=\"left\" colname=\"c7\"\u003e\u0026nbsp;\u003c/td\u003e\u003ctd align=\"left\" colname=\"c8\"\u003e\u0026nbsp;\u003c/td\u003e\u003ctd align=\"left\" colname=\"c9\"\u003e\u0026nbsp;\u003c/td\u003e\u003ctd align=\"left\" colname=\"c10\"\u003e\u003cp\u003e1.54\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c11\"\u003e\u003cp\u003e1.64\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003c/tbody\u003e\u003c/colgroup\u003e\u003ctfoot\u003e\u003ctr\u003e\u003ctd colspan=\"11\"\u003eVIP were obtained from the OPLS-DA model (n\u0026thinsp;=\u0026thinsp;3 per group). DHPTP, 1-(3,4- Dihydroxyphenyl)-3-(2,4,6-trihydroxyphenyl)propan-2-ol; I3A, Indole-3-carboxaldehyde; Log\u003csub\u003e2\u003c/sub\u003eFC, log₂ fold change; VIP, variable importance in the projection.\u003c/td\u003e\u003c/tr\u003e\u003c/tfoot\u003e\u003c/table\u003e\u003c/div\u003e\u003c/p\u003e\u003cp\u003e\u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab2\" border=\"1\"\u003e\u003ccaption language=\"En\"\u003e\u003cdiv class=\"CaptionNumber\"\u003eTable 2\u003c/div\u003e\u003cdiv class=\"CaptionContent\"\u003e\u003cp\u003eMinimum inhibitory concentration (\u0026micro;g mL\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e) against 11 antibiotics.\u003c/p\u003e\u003c/div\u003e\u003c/caption\u003e\u003ccolgroup cols=\"4\"\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c4\" colnum=\"4\"\u003e\u003c/div\u003e\u003cthead\u003e\u003ctr\u003e\u003cth align=\"left\" colname=\"c1\"\u003e\u003cp\u003eantibiotic\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c2\"\u003e\u003cp\u003eLGG\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c3\"\u003e\u003cp\u003eFN041\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c4\"\u003e\u003cp\u003eFN136\u003c/p\u003e\u003c/th\u003e\u003c/tr\u003e\u003c/thead\u003e\u003ctbody\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eTetracycline\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e0.25\u0026ndash;0.5\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e0.5\u0026ndash;1\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e0.5\u0026ndash;1\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003ePenicillin\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e0.25\u0026ndash;0.5\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e0.25\u0026ndash;0.5\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e0.25\u0026ndash;0.5\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eVancomycin\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e1024\u0026ndash;2048\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e2048\u0026ndash;4096\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e2048\u0026ndash;4096\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eGentamicin\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e16\u0026ndash;32\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e16\u0026ndash;32\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e16\u0026ndash;32\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eCiprofloxacin\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e256\u0026ndash;512\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e32\u0026ndash;64\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e128\u0026ndash;256\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eChloramphenicol\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e0.5\u0026ndash;1\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e45659.00\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e45659.00\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eAmpicillin\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e0.25\u0026ndash;0.5\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e0.50\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e0.125\u0026ndash;0.5\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eStreptomycin\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e16\u0026ndash;32\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e18\u0026thinsp;\u0026minus;\u0026thinsp;16\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e32\u0026ndash;64\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eErythromycin\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e0.00\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e0.00\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e0.00\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eKanamycin\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e16\u0026ndash;32\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e16\u0026ndash;32\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e16\u0026ndash;32\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eCephalexin\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e16\u0026ndash;32\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e16\u0026ndash;32\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e45885.00\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003c/tbody\u003e\u003c/colgroup\u003e\u003ctfoot\u003e\u003ctr\u003e\u003ctd colspan=\"4\"\u003eFN136, \u003cem\u003eLacticaseibacillus gasseri\u003c/em\u003e FN136; FN041, \u003cem\u003eLimosilactobacillus reuteri\u003c/em\u003e FN041; LGG, \u003cem\u003eLacticaseibacillus rhamnosus\u003c/em\u003e GG.\u003c/td\u003e\u003c/tr\u003e\u003c/tfoot\u003e\u003c/table\u003e\u003c/div\u003e\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec24\" class=\"Section2\"\u003e\u003ch2\u003e3.5 \u003cem\u003eL. gasseri\u003c/em\u003e FN136 supplementation markedly ameliorated DSS-induced histopathological injury.\u003c/h2\u003e\u003cp\u003eRelative to the CON group, DSS mice exhibited significant weight loss (\u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.01), DAI (\u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.01), pronounced colon shortening (\u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.01) and overt rectal bleeding, confirming successful induction of acute colitis. FN136 intervention restored body weight, reduced DAI scores and normalized colon length (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e3\u003c/span\u003eA\u0026ndash;F); no gross blood was detected in this group.\u003c/p\u003e\u003cp\u003eHistological assessment revealed severe mucosal damage in DSS mice, characterized by crypt distortion, goblet-cell depletion and extensive inflammatory infiltrates. FN136 and FN041 markedly attenuated these lesions, whereas LGG produced only partial improvement with residual inflammation (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e3\u003c/span\u003eG). Peyer\u0026rsquo;s patch counts were slightly reduced in DSS mice, but differences did not reach statistical significance (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e3\u003c/span\u003eD).\u003c/p\u003e\u003cp\u003eCollectively, \u003cem\u003eL. gasseri\u003c/em\u003e FN136 significantly mitigated both clinical manifestations and histopathological damage in DSS colitis, underscoring its robust therapeutic potential.\u003c/p\u003e\u003cp\u003e\u003cb\u003e3.6\u003c/b\u003e \u003cb\u003eL. gasseri\u003c/b\u003e \u003cb\u003eFN136 supplementation significantly attenuated serum inflammation and restored intestinal barrier integrity in DSS-induced UC mice.\u003c/b\u003e\u003c/p\u003e\u003cp\u003eRelative to the CON group, DSS-induced UC mice exhibited pronounced increases in the pro-inflammatory mediators IL-6 (29.4%) and MPO (49.9%) (\u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.05; Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e4\u003c/span\u003eA \u0026ndash; C). FN136 intervention reversed these elevations (\u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.05). Serum NO, markedly reduced in DSS mice, was restored to near-normal levels by FN136 (\u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.05), whereas no comparable recovery was observed in the FN041\u0026thinsp;+\u0026thinsp;DSS or LGG\u0026thinsp;+\u0026thinsp;DSS groups. TNF-α concentrations did not differ significantly among groups (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e4\u003c/span\u003eE).\u003c/p\u003e\u003cp\u003eSerum FD4 and LPS\u0026mdash;established markers of intestinal permeability\u0026mdash;were elevated in DSS mice, confirming barrier disruption. Co-administration of FN136 significantly decreased FD4 and LPS concentrations by 88.19% and 15.77%, respectively, relative to DSS alone (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e4\u003c/span\u003eE-F).\u003c/p\u003e\u003cp\u003eCollectively, FN136 potently attenuated colonic inflammation and restored barrier integrity in the DSS-induced UC model, underscoring its translational potential as a therapeutic probiotic for UC.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec25\" class=\"Section2\"\u003e\u003ch2\u003e3.7 Impact of \u003cem\u003eL. gasseri\u003c/em\u003e FN136 on splenic regulatory T-cell frequency in DSS-induced UC mice\u003c/h2\u003e\u003cp\u003eTregs are indispensable for immune homeostasis and for restraining excessive intestinal inflammation. Flow-cytometric assessment revealed a pronounced reduction in splenic Tregs in DSS-treated mice compared with CON mice (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e5\u003c/span\u003eA, B). FN136 supplementation fully restored this deficit, increasing the Treg fraction by 42.09% relative to the DSS group (\u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.05), whereas FN041 elicited only a modest, non-significant increment (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e5\u003c/span\u003e). Thus, \u003cem\u003eL. gasseri\u003c/em\u003e FN136 expands the Treg compartment and reconfigures Treg-mediated immunoregulatory networks, offering a promising probiotic strategy for alleviating intestinal inflammation.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec26\" class=\"Section2\"\u003e\u003ch2\u003e3.8 Impact of \u003cem\u003eL. gasseri\u003c/em\u003e FN136 on the gut microbiota in DSS-induced UC mice\u003c/h2\u003e\u003cp\u003eTo assess the effect of \u003cem\u003eL. gasseri\u003c/em\u003e FN136 on the colonic microbiota of DSS-treated mice, 16S rDNA amplicon sequencing of colonic contents was performed. The Shannon index was significantly lower in the DSS group than in the CON group (\u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.05), indicating reduced microbial diversity; however, intervention with FN136, FN041, or LGG failed to restore the Chao1, ACE, or Shannon indices (Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e6\u003c/span\u003eA).\u003c/p\u003e\u003cp\u003eβ-diversity analyses (NMDS and PCoA) further demonstrated a clear separation between the DSS and CON groups (\u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.05), reflecting substantial compositional shifts. None of the probiotic-treated groups differed significantly from the DSS group (Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e6\u003c/span\u003eB).\u003c/p\u003e\u003cp\u003eTo determine whether FN136 modulated specific taxa, the OTU table was analyzed at the genus and species levels. At the genus level, the DSS group exhibited an increased relative abundance of \u003cem\u003eEscherichia\u003c/em\u003e and decreased abundances of \u003cem\u003ePrevotellamassilia\u003c/em\u003e, \u003cem\u003eAcetatifactor\u003c/em\u003e, \u003cem\u003eAlistipes\u003c/em\u003e, \u003cem\u003eDuncaniella\u003c/em\u003e, and \u003cem\u003eLacrimispora\u003c/em\u003e compared with the CON group (Fig. S2). These differences were not significant between the DSS and FN136\u0026thinsp;+\u0026thinsp;DSS groups. Functional pathway prediction also revealed that FN136 intervention did not reverse the differentially enriched pathways identified between the CON and DSS groups (Fig. S3).\u003c/p\u003e\u003cp\u003eCollectively, these data indicate that \u003cem\u003eL. gasseri\u003c/em\u003e FN136 does not ameliorate DSS-induced colitis through pronounced restructuring of the global gut microbiota.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec27\" class=\"Section2\"\u003e\u003ch2\u003e3.9 Impact of \u003cem\u003eL. gasseri\u003c/em\u003e FN136 on the serum metabolome\u003c/h2\u003e\u003cp\u003eOPLS-DA was used to compare serum metabolite profiles across the five experimental groups. The resulting models exhibited robust predictive capacity (Q\u0026sup2;Y\u0026thinsp;\u0026gt;\u0026thinsp;0.5), and pairwise comparisons revealed clear separation between the DSS group and each of the other four groups. Differentially abundant metabolites (VIP\u0026thinsp;\u0026gt;\u0026thinsp;1, \u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.05) were selected for further investigation (Fig.\u0026nbsp;\u003cspan refid=\"Fig8\" class=\"InternalRef\"\u003e7\u003c/span\u003e).\u003c/p\u003e\u003cp\u003eCompared with the DSS group, a total of 14 differential metabolites were identified across the experimental cohorts (Table\u0026nbsp;\u003cspan refid=\"Tab3\" class=\"InternalRef\"\u003e3\u003c/span\u003e), ten of which were significantly down-regulated in DSS mice. Notably, FN136 intervention restored several key metabolites to near-control levels. L-arginine was markedly elevated in the FN136 group (log₂FC\u0026thinsp;=\u0026thinsp;3.69), consistent with our in vitro observation that FN136 synthesizes and accumulates arginine, suggesting that the strain replenishes systemic arginine either directly or via metabolic modulation. Arginine exerts anti-inflammatory effects by suppressing pro-inflammatory cytokine production, limiting NO synthesis, and reinforcing intestinal barrier integrity[\u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e31\u003c/span\u003e].\u003c/p\u003e\u003cp\u003e\u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab3\" border=\"1\"\u003e\u003ccaption language=\"En\"\u003e\u003cdiv class=\"CaptionNumber\"\u003eTable 3\u003c/div\u003e\u003cdiv class=\"CaptionContent\"\u003e\u003cp\u003eDifferential serum metabolites relative to DSS-induced colitis control mice.\u003c/p\u003e\u003c/div\u003e\u003c/caption\u003e\u003ccolgroup cols=\"12\"\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c4\" colnum=\"4\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c5\" colnum=\"5\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c6\" colnum=\"6\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c7\" colnum=\"7\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c8\" colnum=\"8\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c9\" colnum=\"9\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c10\" colnum=\"10\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c11\" colnum=\"11\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c12\" colnum=\"12\"\u003e\u003c/div\u003e\u003cthead\u003e\u003ctr\u003e\u003cth align=\"left\" colname=\"c1\" morerows=\"1\" rowspan=\"2\"\u003e\u003cp\u003eMetabolite\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colspan=\"2\" nameend=\"c3\" namest=\"c2\"\u003e\u003cp\u003eCON\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colspan=\"3\" nameend=\"c6\" namest=\"c4\"\u003e\u003cp\u003eFN136\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colspan=\"3\" nameend=\"c9\" namest=\"c7\"\u003e\u003cp\u003eFN041\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colspan=\"3\" nameend=\"c12\" namest=\"c10\"\u003e\u003cp\u003eLGG\u003c/p\u003e\u003c/th\u003e\u003c/tr\u003e\u003ctr\u003e\u003cth align=\"left\" colname=\"c2\"\u003e\u003cp\u003eVIP\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colspan=\"2\" nameend=\"c4\" namest=\"c3\"\u003e\u003cp\u003eLog\u003csub\u003e2\u003c/sub\u003eFC\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c5\"\u003e\u003cp\u003eVIP\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colspan=\"2\" nameend=\"c7\" namest=\"c6\"\u003e\u003cp\u003eLog\u003csub\u003e2\u003c/sub\u003eFC\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c8\"\u003e\u003cp\u003eVIP\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colspan=\"2\" nameend=\"c10\" namest=\"c9\"\u003e\u003cp\u003eLog\u003csub\u003e2\u003c/sub\u003eFC\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c11\"\u003e\u003cp\u003eVIP\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c12\"\u003e\u003cp\u003eLog\u003csub\u003e2\u003c/sub\u003eFC\u003c/p\u003e\u003c/th\u003e\u003c/tr\u003e\u003c/thead\u003e\u003ctbody\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eD-Tryptophan\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e1.64\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colspan=\"2\" nameend=\"c4\" namest=\"c3\"\u003e\u003cp\u003e\u003cb\u003e-\u003c/b\u003e1.14\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003e0.42\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colspan=\"2\" nameend=\"c7\" namest=\"c6\"\u003e\u003cp\u003e0.27\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c8\"\u003e\u003cp\u003e1.22\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colspan=\"2\" nameend=\"c10\" namest=\"c9\"\u003e\u003cp\u003e1.33\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c11\"\u003e\u003cp\u003e1.01\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c12\"\u003e\u003cp\u003e1.54\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eGuanidoacetic acid\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e1.63\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colspan=\"2\" nameend=\"c4\" namest=\"c3\"\u003e\u003cp\u003e-1.04\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003e0.70\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colspan=\"2\" nameend=\"c7\" namest=\"c6\"\u003e\u003cp\u003e0.41\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c8\"\u003e\u003cp\u003e1.27\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colspan=\"2\" nameend=\"c10\" namest=\"c9\"\u003e\u003cp\u003e1.24\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c11\"\u003e\u003cp\u003e1.03\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c12\"\u003e\u003cp\u003e1.40\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eGuanine\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e1.61\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colspan=\"2\" nameend=\"c4\" namest=\"c3\"\u003e\u003cp\u003e-2.06\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003e0.94\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colspan=\"2\" nameend=\"c7\" namest=\"c6\"\u003e\u003cp\u003e1.13\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c8\"\u003e\u003cp\u003e1.30\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colspan=\"2\" nameend=\"c10\" namest=\"c9\"\u003e\u003cp\u003e2.40\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c11\"\u003e\u003cp\u003e1.28\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c12\"\u003e\u003cp\u003e2.56\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eDihydrouracil\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e1.59\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colspan=\"2\" nameend=\"c4\" namest=\"c3\"\u003e\u003cp\u003e-2.05\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003e1.19\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colspan=\"2\" nameend=\"c7\" namest=\"c6\"\u003e\u003cp\u003e1.01\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c8\"\u003e\u003cp\u003e0.46\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colspan=\"2\" nameend=\"c10\" namest=\"c9\"\u003e\u003cp\u003e0.68\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c11\"\u003e\u003cp\u003e0.45\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c12\"\u003e\u003cp\u003e0.76\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e5-Hydroxyindole-\u003c/p\u003e\u003cp\u003e3-acetic acid\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e1.54\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colspan=\"2\" nameend=\"c4\" namest=\"c3\"\u003e\u003cp\u003e-2.19\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003e0.81\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colspan=\"2\" nameend=\"c7\" namest=\"c6\"\u003e\u003cp\u003e1.44\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c8\"\u003e\u003cp\u003e1.33\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colspan=\"2\" nameend=\"c10\" namest=\"c9\"\u003e\u003cp\u003e2.29\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c11\"\u003e\u003cp\u003e1.18\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c12\"\u003e\u003cp\u003e2.26\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003ePantothenic Acid\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e1.49\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colspan=\"2\" nameend=\"c4\" namest=\"c3\"\u003e\u003cp\u003e2.70\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003e1.00\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colspan=\"2\" nameend=\"c7\" namest=\"c6\"\u003e\u003cp\u003e-0.57\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c8\"\u003e\u003cp\u003e0.48\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colspan=\"2\" nameend=\"c10\" namest=\"c9\"\u003e\u003cp\u003e-0.42\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c11\"\u003e\u003cp\u003e0.49\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c12\"\u003e\u003cp\u003e0.46\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eNiacinamide\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e1.43\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colspan=\"2\" nameend=\"c4\" namest=\"c3\"\u003e\u003cp\u003e-1.31\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003e1.33\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colspan=\"2\" nameend=\"c7\" namest=\"c6\"\u003e\u003cp\u003e0.94\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c8\"\u003e\u003cp\u003e1.11\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colspan=\"2\" nameend=\"c10\" namest=\"c9\"\u003e\u003cp\u003e1.19\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c11\"\u003e\u003cp\u003e1.04\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c12\"\u003e\u003cp\u003e1.13\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eCytosine\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e1.38\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colspan=\"2\" nameend=\"c4\" namest=\"c3\"\u003e\u003cp\u003e-1.32\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003e1.54\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colspan=\"2\" nameend=\"c7\" namest=\"c6\"\u003e\u003cp\u003e2.17\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c8\"\u003e\u003cp\u003e1.28\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colspan=\"2\" nameend=\"c10\" namest=\"c9\"\u003e\u003cp\u003e1.84\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c11\"\u003e\u003cp\u003e1.13\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c12\"\u003e\u003cp\u003e1.28\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eL-Arginine\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e1.27\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colspan=\"2\" nameend=\"c4\" namest=\"c3\"\u003e\u003cp\u003e-2.11\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003e1.27\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colspan=\"2\" nameend=\"c7\" namest=\"c6\"\u003e\u003cp\u003e3.69\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c8\"\u003e\u003cp\u003e1.10\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colspan=\"2\" nameend=\"c10\" namest=\"c9\"\u003e\u003cp\u003e2.16\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c11\"\u003e\u003cp\u003e1.42\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c12\"\u003e\u003cp\u003e4.30\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eUracil\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e1.23\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colspan=\"2\" nameend=\"c4\" namest=\"c3\"\u003e\u003cp\u003e-2.16\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003e1.00\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colspan=\"2\" nameend=\"c7\" namest=\"c6\"\u003e\u003cp\u003e1.01\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c8\"\u003e\u003cp\u003e1.20\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colspan=\"2\" nameend=\"c10\" namest=\"c9\"\u003e\u003cp\u003e2.11\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c11\"\u003e\u003cp\u003e1.09\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c12\"\u003e\u003cp\u003e1.77\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eTaurocholic acid\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e0.65\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colspan=\"2\" nameend=\"c4\" namest=\"c3\"\u003e\u003cp\u003e5.94\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003e1.26\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colspan=\"2\" nameend=\"c7\" namest=\"c6\"\u003e\u003cp\u003e-4.88\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c8\"\u003e\u003cp\u003e1.28\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colspan=\"2\" nameend=\"c10\" namest=\"c9\"\u003e\u003cp\u003e-6.01\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c11\"\u003e\u003cp\u003e1.23\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c12\"\u003e\u003cp\u003e-5.25\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eGlycerophosphocholine\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e0.73\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colspan=\"2\" nameend=\"c4\" namest=\"c3\"\u003e\u003cp\u003e-0.92\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003e1.23\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colspan=\"2\" nameend=\"c7\" namest=\"c6\"\u003e\u003cp\u003e4.04\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c8\"\u003e\u003cp\u003e1.22\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colspan=\"2\" nameend=\"c10\" namest=\"c9\"\u003e\u003cp\u003e3.91\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c11\"\u003e\u003cp\u003e1.36\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c12\"\u003e\u003cp\u003e3.87\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eCreatinine\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e0.35\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colspan=\"2\" nameend=\"c4\" namest=\"c3\"\u003e\u003cp\u003e-0.05\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003e0.60\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colspan=\"2\" nameend=\"c7\" namest=\"c6\"\u003e\u003cp\u003e0.23\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c8\"\u003e\u003cp\u003e1.37\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colspan=\"2\" nameend=\"c10\" namest=\"c9\"\u003e\u003cp\u003e0.83\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c11\"\u003e\u003cp\u003e1.18\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c12\"\u003e\u003cp\u003e1.27\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e2,6-diaminohexanoic acid\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e0.15\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colspan=\"2\" nameend=\"c4\" namest=\"c3\"\u003e\u003cp\u003e-0.21\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003e0.40\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colspan=\"2\" nameend=\"c7\" namest=\"c6\"\u003e\u003cp\u003e-0.44\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c8\"\u003e\u003cp\u003e1.07\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colspan=\"2\" nameend=\"c10\" namest=\"c9\"\u003e\u003cp\u003e1.67\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c11\"\u003e\u003cp\u003e1.02\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c12\"\u003e\u003cp\u003e2.29\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003c/tbody\u003e\u003c/colgroup\u003e\u003ctfoot\u003e\u003ctr\u003e\u003ctd colspan=\"12\"\u003eVIP scores were obtained from the OPLS-DA model (n\u0026thinsp;=\u0026thinsp;5 per group). CON, healthy control; FN136, \u003cem\u003eL. gasseri\u003c/em\u003e FN136-treated DSS UC; FN041, \u003cem\u003eL. reuteri\u003c/em\u003e FN041-treated DSS UC; LGG, \u003cem\u003eL. rhamnosus\u003c/em\u003e GG-treated DSS UC; DSS, dextran sulfate sodium-induced UC. VIP, variable importance in the projection. Values of log₂FC\u0026thinsp;\u0026gt;\u0026thinsp;0 indicate up-regulation relative to the DSS group, whereas log₂FC\u0026thinsp;\u0026lt;\u0026thinsp;0 indicate down-regulation relative to the DSS group.\u003c/td\u003e\u003c/tr\u003e\u003c/tfoot\u003e\u003c/table\u003e\u003c/div\u003e\u003c/p\u003e\u003cp\u003eCyosine and uracil were also significantly increased in the FN136 group (log\u003csub\u003e2\u003c/sub\u003eFC\u0026thinsp;=\u0026thinsp;2.17 and 1.01, respectively). As nucleic-acid bases, cytosine and uracil participate in DNA and RNA formation and are critical for DNA methylation\u0026mdash;an epigenetic process whose dysregulation is implicated in UC pathogenesis [\u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e32\u003c/span\u003e]. Additionally, FN136 substantially reduced taurocholic acid (log\u003csub\u003e2\u003c/sub\u003eFC\u0026thinsp;=\u0026thinsp;\u0026minus;\u0026thinsp;4.88) while elevating glycerophosphocholine (log\u003csub\u003e2\u003c/sub\u003eFC\u0026thinsp;=\u0026thinsp;4.04). Taurocholic acid, a primary bile acid, aggravates colitis in IL-10-deficient mice [\u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e33\u003c/span\u003e], whereas glycerophosphocholine, a choline derivative and acetylcholine precursor, possesses immunomodulatory and anti-inflammatory properties [\u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e34\u003c/span\u003e].\u003c/p\u003e\u003cp\u003eCollectively, DSS-induced colitis disrupts amino-acid, bile-acid and energy metabolism. FN136 leverages its intrinsic metabolic capacity\u0026mdash;particularly arginine biosynthesis\u0026mdash;to restore serum arginine levels, thereby modulating the arginine\u0026ndash;NO axis and downstream immune and barrier functions. Concomitant rebalancing of cytosine, bile acids and glycerophosphocholine further amplifies its protective effects.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec28\" class=\"Section2\"\u003e\u003ch2\u003e3.10 Correlation Analysis\u003c/h2\u003e\u003cp\u003eSpearman correlation analysis identified l-arginine as a central metabolic node that integrates immune, barrier and metabolic homeostasis. L-Arginine was positively correlated with splenic Treg frequency (R\u0026sup2; = 0.42, \u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.01), indicating that it reinforces immunoregulatory circuits to dampen inflammation. As the sole substrate for nitric oxide synthesis, l-arginine was also positively correlated with serum NO ( R\u0026sup2; = 0.23, \u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.05) and negatively correlated with serum LPS( R\u0026sup2;= 0.18, \u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.05) and FD4( R\u0026sup2;= 0.21, \u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.05).\u0026mdash;validated indicators of intestinal barrier leakage.\u003c/p\u003e\u003cp\u003eUracil, a nucleic-acid precursor, exhibited concordant patterns: its abundance was negatively associated with IL-6( R\u0026sup2;=0.41, \u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.05), LPS( R\u0026sup2;=0.38, \u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.05) and FD4( R\u0026sup2;=0.18, \u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.05), and positively associated with splenic Treg levels ( R\u0026sup2; =0.21, \u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.05), suggesting that uracil likewise exerts anti-inflammatory effects and strengthens mucosal barrier function(Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e8\u003c/span\u003eB).\u003c/p\u003e\u003cp\u003eCollectively, l-arginine drives the arginine\u0026ndash;NO metabolic axis and, in concert with metabolites such as uracil, sustains metabolic\u0026ndash;immune\u0026ndash;barrier homeostasis.\u003c/p\u003e\u003c/div\u003e"},{"header":"4. Discussion","content":"\u003cp\u003e\u003cem\u003eIn vitro\u003c/em\u003e assays revealed that FN136 tolerates acid and bile salts robustly and adheres strongly, confirming its probiotic credentials. Oral administration of FN136 markedly alleviated DSS-induced colitis, as evidenced by reduced disease activity index, decreased serum pro-inflammatory cytokines, restored intestinal barrier integrity, and elevated splenic Treg proportions.\u003c/p\u003e\u003cp\u003eMetabolomic profiling of \u003cem\u003ein vitro\u003c/em\u003e fermentation showed that FN136 drives the accumulation of l-arginine, propionate, isovalerate, and indole derivatives such as indole-3-carboxaldehyde and indole-3-ethanol, demonstrating its capacity to generate anti-inflammatory metabolites. Although some of these compounds were not detected in serum, FN136 intervention significantly restored serum l-arginine levels (log₂FC\u0026thinsp;=\u0026thinsp;3.69), mirroring in vitro data and indicating systemic regulation via its metabolites. Serum metabolomics further demonstrated pronounced enrichment of the arginine metabolic pathway and modulation of inflammation- and barrier-associated metabolites, including taurocholic acid and glycerophosphocholine.\u003c/p\u003e\u003cp\u003eUC is a chronic, relapsing inflammatory disorder of the gastrointestinal tract characterized by abdominal pain, diarrhea, hematochezia, and weight loss [\u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e35\u003c/span\u003e]. Disease progression is driven by LPS-mediated activation of inflammatory signaling pathways, leading to elevated release of pro-inflammatory cytokines such as IL-6, IL-1β, and TNF-α and subsequent disruption of intestinal barrier integrity [\u003cspan citationid=\"CR36\" class=\"CitationRef\"\u003e36\u003c/span\u003e, \u003cspan citationid=\"CR37\" class=\"CitationRef\"\u003e37\u003c/span\u003e]. FD4 is a widely accepted marker of intestinal permeability, with increased serum FD4 indicating compromised barrier function [\u003cspan citationid=\"CR38\" class=\"CitationRef\"\u003e38\u003c/span\u003e]. In the present study, the FD4 assay demonstrated that FN136 administration significantly reduced serum LPS, IL-6, IL-1β, and FD4 levels, indicating effective attenuation of intestinal barrier damage and inflammation.\u003c/p\u003e\u003cp\u003eMultiple studies have demonstrated that gut dysbiosis, aberrant immune activation, and intestinal barrier disruption are closely linked to inflammatory bowel disease and other gastrointestinal disorders[\u003cspan citationid=\"CR39\" class=\"CitationRef\"\u003e39\u003c/span\u003e]. Amino acids are essential for maintaining mucosal integrity and barrier function[\u003cspan citationid=\"CR40\" class=\"CitationRef\"\u003e40\u003c/span\u003e]. In vitro fermentation metabolomics revealed that FN136 efficiently utilizes precursor substrates to markedly accumulate l-arginine (log₂FC increased during mid-to-late fermentation) and to activate the tryptophan metabolic pathway, yielding indole derivatives such as indole-3-carboxaldehyde and indole-3-ethanol. These findings indicate that FN136 possesses robust amino-acid metabolic capacity\u0026mdash;particularly arginine biosynthesis\u0026mdash;that may underlie its immunomodulatory and barrier-protective effects in the host.\u003c/p\u003e\u003cp\u003eL-Arginine, a semi-essential amino acid, is indispensable for immune homeostasis [\u003cspan citationid=\"CR41\" class=\"CitationRef\"\u003e41\u003c/span\u003e], inflammatory regulation and tissue barrier repair [\u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e, \u003cspan citationid=\"CR37\" class=\"CitationRef\"\u003e37\u003c/span\u003e]. It serves as the obligate precursor for nitric oxide, creatine, glutamate, proline and urea [\u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e]. Although healthy adults can synthesize and obtain adequate arginine from dietary sources, patients with inflammatory bowel disease frequently exhibit arginine deficiency due to diminished endogenous synthesis, impaired intestinal absorption and heightened inflammatory demand [\u003cspan citationid=\"CR42\" class=\"CitationRef\"\u003e42\u003c/span\u003e]. Consistent with these observations, DSS-induced UC mice displayed markedly reduced serum arginine concentrations. FN136 intervention restored arginine levels to a degree that mirrored in vitro fermentation profiles, indicating that strain-derived metabolites may directly or indirectly re-establish host arginine homeostasis.\u003c/p\u003e\u003cp\u003ePrevious investigations have demonstrated that exogenous l-arginine supplementation in DSS colitis models reduces pro-inflammatory cytokine release, accelerates barrier recovery, and attenuates disease severity [\u003cspan citationid=\"CR43\" class=\"CitationRef\"\u003e43\u003c/span\u003e]. Arginine catabolism ultimately generates NO and polyamines, both of which exert critical regulatory functions [\u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e]. NO is particularly essential for intestinal homeostasis; patients with UC exhibit diminished arginine uptake and elevated arginase-1 expression, leading to inadequate NO synthesis [\u003cspan citationid=\"CR44\" class=\"CitationRef\"\u003e44\u003c/span\u003e]. In the present study, DSS-treated mice displayed markedly reduced serum arginine and NO concentrations, and FN136 intervention restored both parameters. Spearman correlation analysis revealed negative associations between arginine and pro-inflammatory mediators and positive associations between arginine and NO levels, further supporting the hypothesis that FN136 exerts anti-inflammatory effects by modulating the arginine\u0026ndash;NO metabolic axis. Additionally, the indole derivatives identified in in vitro fermentation are recognized aryl hydrocarbon receptor ligands with established anti-inflammatory and barrier-protective properties, suggesting that FN136 may confer benefits via multi-metabolite synergism.\u003c/p\u003e\u003cp\u003eDevelopment and maintenance of immune homeostasis are intimately linked to gut microbiota composition, a relationship that is particularly evident in the pathogenesis of UC [\u003cspan citationid=\"CR45\" class=\"CitationRef\"\u003e45\u003c/span\u003e].Tregs are pivotal in sustaining intestinal immune balance [\u003cspan citationid=\"CR46\" class=\"CitationRef\"\u003e46\u003c/span\u003e] and suppress aberrant activation and proliferation of pro-inflammatory Th17 cells in DSS-induced colitis by secreting anti-inflammatory mediators such as TGF-β and IL-10 [\u003cspan citationid=\"CR47\" class=\"CitationRef\"\u003e47\u003c/span\u003e]. In this study, oral administration of \u003cem\u003eL. gasseri\u003c/em\u003e FN136 significantly elevated splenic Treg frequencies, and Spearman correlation analysis revealed a strong positive association between serum arginine levels and Treg numbers. In vitro fermentation metabolomics further confirmed that FN136 efficiently synthesizes and accumulates l-arginine while simultaneously activating the tryptophan metabolic pathway to generate AhR-agonistic indole derivatives, including indole-3-carboxaldehyde and indole-3-ethanol. Collectively, these findings suggest that FN136 enhances Treg function via two complementary mechanisms: (1) direct elevation of host arginine availability, which is known to promote IL-10 secretion and augment Treg immunomodulatory capacity; and (2) synergistic promotion of Treg differentiation or functional maturation through AhR signaling activation by its metabolites, ultimately conferring robust protection against colitis.\u003c/p\u003e"},{"header":"Conclusion","content":"\u003cp\u003e\u003cem\u003eLacticaseibacillus gasseri\u003c/em\u003e FN136 markedly attenuated DSS-induced UC in mice. The protective effect derives from dual-level regulation of metabolism and immunity: the strain augments arginine biosynthesis, elevates systemic arginine availability, and thereby reinforces anti-inflammatory responses and intestinal barrier integrity; concurrently, accumulated arginine promotes the differentiation and functional maturation of regulatory T cells, amplifying their immunoregulatory capacity and jointly restoring gut immune homeostasis. These integrated mechanisms collectively mitigate colitis pathogenesis.\u003c/p\u003e"},{"header":"Abbreviations","content":"\u003cdiv class=\"DefinitionList\"\u003e\u003cdiv class=\"DefinitionListEntry\"\u003e\u003cdiv class=\"Term\"\u003earyl hydrocarbon receptor\u003c/div\u003e\u003cdiv class=\"Description\"\u003e\u003cp\u003eAhR\u003c/p\u003e\u003c/div\u003e\u003c/div\u003e\u003cdiv class=\"DefinitionListEntry\"\u003e\u003cdiv class=\"Term\"\u003econtrol\u003c/div\u003e\u003cdiv class=\"Description\"\u003e\u003cp\u003eCON\u003c/p\u003e\u003c/div\u003e\u003c/div\u003e\u003cdiv class=\"DefinitionListEntry\"\u003e\u003cdiv class=\"Term\"\u003edisease activity index\u003c/div\u003e\u003cdiv class=\"Description\"\u003e\u003cp\u003eDAI\u003c/p\u003e\u003c/div\u003e\u003c/div\u003e\u003cdiv class=\"DefinitionListEntry\"\u003e\u003cdiv class=\"Term\"\u003esodium dextran sulfate\u003c/div\u003e\u003cdiv class=\"Description\"\u003e\u003cp\u003eDSS\u003c/p\u003e\u003c/div\u003e\u003c/div\u003e\u003cdiv class=\"DefinitionListEntry\"\u003e\u003cdiv class=\"Term\"\u003efluorescein isothiocyanate dextran\u003c/div\u003e\u003cdiv class=\"Description\"\u003e\u003cp\u003eFD4\u003c/p\u003e\u003c/div\u003e\u003c/div\u003e\u003cdiv class=\"DefinitionListEntry\"\u003e\u003cdiv class=\"Term\"\u003e\u003cem\u003eLacticaseibacillus reuteri\u003c/em\u003e FN041\u003c/div\u003e\u003cdiv class=\"Description\"\u003e\u003cp\u003eFN041\u003c/p\u003e\u003c/div\u003e\u003c/div\u003e\u003cdiv class=\"DefinitionListEntry\"\u003e\u003cdiv class=\"Term\"\u003e\u003cem\u003eLacticaseibacillus gasseri\u003c/em\u003e FN136\u003c/div\u003e\u003cdiv class=\"Description\"\u003e\u003cp\u003eFN136\u003c/p\u003e\u003c/div\u003e\u003c/div\u003e\u003cdiv class=\"DefinitionListEntry\"\u003e\u003cdiv class=\"Term\"\u003eInterleukin-1 beta\u003c/div\u003e\u003cdiv class=\"Description\"\u003e\u003cp\u003eIL-1β\u003c/p\u003e\u003c/div\u003e\u003c/div\u003e\u003cdiv class=\"DefinitionListEntry\"\u003e\u003cdiv class=\"Term\"\u003einterleukin-6\u003c/div\u003e\u003cdiv class=\"Description\"\u003e\u003cp\u003eIL-6\u003c/p\u003e\u003c/div\u003e\u003c/div\u003e\u003cdiv class=\"DefinitionListEntry\"\u003e\u003cdiv class=\"Term\"\u003einterleukin 10\u003c/div\u003e\u003cdiv class=\"Description\"\u003e\u003cp\u003eIL-10\u003c/p\u003e\u003c/div\u003e\u003c/div\u003e\u003cdiv class=\"DefinitionListEntry\"\u003e\u003cdiv class=\"Term\"\u003e\u003cem\u003eLacticaseibacillus rhamnosus\u003c/em\u003e GG\u003c/div\u003e\u003cdiv class=\"Description\"\u003e\u003cp\u003eLGG\u003c/p\u003e\u003c/div\u003e\u003c/div\u003e\u003cdiv class=\"DefinitionListEntry\"\u003e\u003cdiv class=\"Term\"\u003elipopolysaccharide\u003c/div\u003e\u003cdiv class=\"Description\"\u003e\u003cp\u003eLPS\u003c/p\u003e\u003c/div\u003e\u003c/div\u003e\u003cdiv class=\"DefinitionListEntry\"\u003e\u003cdiv class=\"Term\"\u003emyeloperoxidase\u003c/div\u003e\u003cdiv class=\"Description\"\u003e\u003cp\u003eMPO\u003c/p\u003e\u003c/div\u003e\u003c/div\u003e\u003cdiv class=\"DefinitionListEntry\"\u003e\u003cdiv class=\"Term\"\u003enitric oxide\u003c/div\u003e\u003cdiv class=\"Description\"\u003e\u003cp\u003eNO\u003c/p\u003e\u003c/div\u003e\u003c/div\u003e\u003cdiv class=\"DefinitionListEntry\"\u003e\u003cdiv class=\"Term\"\u003eorthogonal partial least squares-discriminant analysis\u003c/div\u003e\u003cdiv class=\"Description\"\u003e\u003cp\u003eOPLS-DA\u003c/p\u003e\u003c/div\u003e\u003c/div\u003e\u003cdiv class=\"DefinitionListEntry\"\u003e\u003cdiv class=\"Term\"\u003epartial least squares discriminant analysis\u003c/div\u003e\u003cdiv class=\"Description\"\u003e\u003cp\u003ephosphate-buffered saline,PBS\u003c/p\u003e\u003c/div\u003e\u003c/div\u003e\u003cdiv class=\"DefinitionListEntry\"\u003e\u003cdiv class=\"Term\"\u003ePLS-DA\u003c/div\u003e\u003cdiv class=\"Description\"\u003e\u003c/div\u003e\u003c/div\u003e\u003cdiv class=\"DefinitionListEntry\"\u003e\u003cdiv class=\"Term\"\u003e\u003cem\u003eStaphylococcus aureus\u003c/em\u003e\u003c/div\u003e\u003cdiv class=\"Description\"\u003e\u003cp\u003eS. aureus.\u003c/p\u003e\u003c/div\u003e\u003c/div\u003e\u003cdiv class=\"DefinitionListEntry\"\u003e\u003cdiv class=\"Term\"\u003etumor necrosis factor alpha\u003c/div\u003e\u003cdiv class=\"Description\"\u003e\u003cp\u003eTNF-α\u003c/p\u003e\u003c/div\u003e\u003c/div\u003e\u003cdiv class=\"DefinitionListEntry\"\u003e\u003cdiv class=\"Term\"\u003eregulatory T cells\u003c/div\u003e\u003cdiv class=\"Description\"\u003e\u003cp\u003eTregs\u003c/p\u003e\u003c/div\u003e\u003c/div\u003e\u003cdiv class=\"DefinitionListEntry\"\u003e\u003cdiv class=\"Term\"\u003eshort-chain fatty acids\u003c/div\u003e\u003cdiv class=\"Description\"\u003e\u003cp\u003eSCFAs\u003c/p\u003e\u003c/div\u003e\u003c/div\u003e\u003cdiv class=\"DefinitionListEntry\"\u003e\u003cdiv class=\"Term\"\u003eTGF-β\u003c/div\u003e\u003cdiv class=\"Description\"\u003e\u003cp\u003eTransforming Growth Factor-beta\u003c/p\u003e\u003c/div\u003e\u003c/div\u003e\u003cdiv class=\"DefinitionListEntry\"\u003e\u003cdiv class=\"Term\"\u003eulcerative colitis\u003c/div\u003e\u003cdiv class=\"Description\"\u003e\u003cp\u003eUC.\u003c/p\u003e\u003c/div\u003e\u003c/div\u003e\u003c/div\u003e"},{"header":"Declarations","content":"\u003ch2\u003eConflict of interest\u003c/h2\u003e\u003cp\u003eThe authors have declared no conflict of interest.\u003c/p\u003e\u003ch2\u003eAuthor Contribution\u003c/h2\u003e\u003cp\u003eQ.L. contributed to the study by acquiring, analyzing, and interpreting the data, as well as drafting the manuscript. F.Z. and C.L. participated in data visualization and revised the manuscript. W.W. and X.G. contributed by acquiring, analyzing, and interpreting the data. C.Q. was responsible for conceiving and designing the study, analyzing and interpreting the data, and drafting and revising the manuscript. J.S.drafted and revised the manuscript and participated in the experimental design. All authors have reviewed and approved the submitted version of the manuscript and agree to be personally accountable for their respective contributions.\u003c/p\u003e\u003ch2\u003eAcknowledgement\u003c/h2\u003e\u003cp\u003eThis research was supported by the National Natural Science Foundation of China (No. 82574094).\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\u003cli\u003e\u003cspan\u003eChen L, Ruan G, Cheng Y et al (2022) The role of Th17 cells in inflammatory bowel disease and the research progress. 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Cells 8:709. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.3390/cells8070709\u003c/span\u003e\u003cspan address=\"10.3390/cells8070709\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":false,"highlight":"","institution":"","isAcceptedByJournal":true,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"probiotics-and-antimicrobial-proteins","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"paap","sideBox":"Learn more about [Probiotics and Antimicrobial Proteins](http://link.springer.com/journal/12601)","snPcode":"12602","submissionUrl":"https://submission.nature.com/new-submission/12602/3","title":"Probiotics and Antimicrobial Proteins","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"em","reportingPortfolio":"Springer Hybrid","inReviewEnabled":true,"inReviewRevisionsEnabled":false},"keywords":"Lacticaseibacillus gasseri FN136, Ulcerative colitis, arginine, inflammatory responses, regulatory T cells","lastPublishedDoi":"10.21203/rs.3.rs-7525483/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-7525483/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003eChronic, relapsing colonic inflammation defines ulcerative colitis (UC) and heightens long-term colorectal cancer risk. Probiotics, proven beneficial, have emerged as a promising adjunctive strategy for UC management. We conducted a two-tiered evaluation of the breast-milk-derived strain \u003cem\u003eLacticaseibacillus gasseri\u003c/em\u003e FN136. In vitro, FN136 secreted anti-inflammatory amino acids and indoles, displayed pronounced acid- and bile-salt tolerance, and exhibited significant antimicrobial activity. In vivo, daily oral administration of 1 \u0026times; 10⁹ CFU FN136 markedly attenuated DSS-induced UC: serum l-arginine was restored, splenic Treg frequency increased by 42.09% (\u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.05), and nitric oxide production was elevated, while disease activity index, body-weight loss, and colonic shortening were all significantly reduced (\u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.05). Histopathology revealed intact crypt architecture and diminished inflammatory infiltration; systemic inflammation was suppressed as evidenced by decreased interleukin-6, myeloperoxidase, lipopolysaccharide, and FITC-dextran levels (\u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.05). Critically, 16S rRNA sequencing revealed no significant alterations in global microbial composition, indicating that FN136 exerts protection via a microbiota-independent postbiotic\u0026ndash;host axis. Collectively, FN136 mitigates DSS-induced UC by restoring serum arginine and orchestrating immune\u0026ndash;barrier homeostasis through a postbiotic pathway, offering a novel framework for precision UC intervention.\u003c/p\u003e","manuscriptTitle":"Breast-milk Lacticaseibacillus gasseri FN136 alleviates DSS-induced ulcerative colitis through microbiota-independent metabolite–host interactions","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2025-09-22 09:41:43","doi":"10.21203/rs.3.rs-7525483/v1","editorialEvents":[{"type":"communityComments","content":0},{"type":"decision","content":"Revision requested","date":"2025-10-25T17:26:28+00:00","index":"","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2025-10-20T14:28:15+00:00","index":"hide","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2025-10-18T01:04:03+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"286520676270041400945153530426622900046","date":"2025-09-23T06:14:46+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"21173953422931397420753953894906682425","date":"2025-09-18T05:31:55+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"125473350309234407107516826179400921372","date":"2025-09-17T22:38:33+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"85139696974679334203990011793712468930","date":"2025-09-16T18:29:51+00:00","index":"hide","fulltext":""},{"type":"reviewersInvited","content":"","date":"2025-09-14T17:20:59+00:00","index":"","fulltext":""},{"type":"editorAssigned","content":"","date":"2025-09-04T11:03:03+00:00","index":"","fulltext":""},{"type":"checksComplete","content":"","date":"2025-09-04T11:03:00+00:00","index":"","fulltext":""},{"type":"submitted","content":"Probiotics and Antimicrobial Proteins","date":"2025-09-03T09:43:04+00:00","index":"","fulltext":""}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"probiotics-and-antimicrobial-proteins","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"paap","sideBox":"Learn more about [Probiotics and Antimicrobial Proteins](http://link.springer.com/journal/12601)","snPcode":"12602","submissionUrl":"https://submission.nature.com/new-submission/12602/3","title":"Probiotics and Antimicrobial Proteins","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"em","reportingPortfolio":"Springer Hybrid","inReviewEnabled":true,"inReviewRevisionsEnabled":false}}],"origin":"","ownerIdentity":"4bf4e03a-56c3-4030-80d8-4eaed9389645","owner":[],"postedDate":"September 22nd, 2025","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"published-in-journal","subjectAreas":[],"tags":[],"updatedAt":"2025-12-01T16:09:30+00:00","versionOfRecord":{"articleIdentity":"rs-7525483","link":"https://doi.org/10.1007/s12602-025-10858-y","journal":{"identity":"probiotics-and-antimicrobial-proteins","isVorOnly":false,"title":"Probiotics and Antimicrobial Proteins"},"publishedOn":"2025-11-28 15:57:54","publishedOnDateReadable":"November 28th, 2025"},"versionCreatedAt":"2025-09-22 09:41:43","video":"","vorDoi":"10.1007/s12602-025-10858-y","vorDoiUrl":"https://doi.org/10.1007/s12602-025-10858-y","workflowStages":[]},"version":"v1","identity":"rs-7525483","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-7525483","identity":"rs-7525483","version":["v1"]},"buildId":"8U1c8b4HqxoKbykW_rLl7","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}