Bifidobacterium lactis TG11, isolated from the intestines of healthy infants, is now maintained by Jinhua Galaxy Biotechnology Co., Ltd., located in China. The preservation is cataloged under the accession number 20230421003. The bacterial activity has been adjusted to 200 billion CFU/g.

The research protocol for the animal studies received ethical approval from the Animal Welfare Ethics Review Committee of Heilongjiang University of Chinese Medicine (2024032915, Harbin, Heilongjiang, China). Male ICR mice, with a body mass range of 18–22 grams, were accommodated in a controlled laboratory environment and provided with standard care. The experimental process is detailed in Figure 1. The investigation commenced seven days following the adaptation period. Randomly assigned into five cohorts, the mice included a control cohort (NC), a constipation-induced model cohort (CM), and three treatment cohorts (TG11) with varying dosages: low (LG), medium (MG), and high (HG), each consisting of 20 animals. The trial was bifurcated into two phases: the TG11 treatment phase and the constipation induction phase, guided by the “Functional Assessment and Evaluation Protocols for Health Foods (2023 Edition)” for model design and loperamide dosing.

During the TG11 treatment phase (days 1–14), the NC and CM cohorts received saline solution, while the LG, MG, and HG cohorts were dosed with 10^6, 10^7, and 10^8 CFU/mouse of the bacterial preparation, respectively. On day 14, loperamide was orally administered to the CM cohort and all TG11 treatment cohorts at a rate of 4 mg/kg BW, with the NC cohort continuing to receive saline. The animals in all cohorts were subjected to a 16-h fasting period, maintaining access to water. On day 15, a solution of activated charcoal (5% charcoal and 10% arabic gum) at a volume of 10 ml/kg BW was administered. Ten mice from each group were closely monitored to document the time until the first instance of black stool was observed. Concurrently, an additional cohort of ten mice was assessed for gastrointestinal transit rate (GTR). Throughout the duration of the experiment, the mice were afforded unlimited access to both food and water, and their body weights were consistently recorded at regular intervals.

On the 15th day, fecal samples from the groups of mice were collected to measure their fecal water content (FWC). Upon the conclusion of the experiment, blood was drawn and serum was isolated using a refrigerated centrifuge set at 12,000 g for 10 min. This serum was then prepared for subsequent biochemical analyses. Samples from both the small intestine and colon were harvested for histological evaluation and real-time PCR analysis. Before oral administration of a solution of activated charcoal, Feces of mice were collected for short-chain fatty acid and metagenomic analysis, respectively.

Each mouse was housed in a clean cage and monitored immediately after drug administration. The time to first black stool occurrence was documented. Starting from the first black stool emergence (0 h), mice were continuously observed for 5 h, during which the number and weight of black stools were recorded.

The feces of mice in each group were collected for five consecutive hours starting from the first black stool, and wet weight and dry weight are calculated. Immediately after defecation, the feces were weighed while wet. The samples were then placed in an oven at 60°C for 12 h to determine their dry weight. The fecal water content (FWC) was calculated using the following formula:

Twenty-five minutes after administering activated carbon via gavage, the mice were euthanized and their entire intestinal tract was excised to assess the distance traveled by the charcoal. The Gastrointestinal Transit Rate (GTR) was calculated using the following formula:

Then, the thrust is converted to data according to the following formula: X = sinp−1. In the formula, p represents the Gastrointestinal Transit Rate.

Approximately 0.5 cm segments of the small intestine were excised, cleansed with saline, and preserved in 4% paraformaldehyde. These tissue samples were processed for embedding in paraffin and sectioning within 72 h. Following this, they were typically stained using hematoxylin and eosin (H&E). For tissue analysis, an inverted fluorescence microscope (Nikon Eclipse E100, Japan) was employed to evaluate the architectural integrity of intestinal glands, the presence of lymphocyte infiltration, and overall colonic structural changes.

Serum levels of motilin (MTL), substance P (SP), somatostatin (SS), endothelin-1 (ET-1), gastrin (Gas), and vasoactive intestinal peptide (VIP), were assayed using enzyme-linked immunosorbent assay (ELISA) kits provided by Elisa Biotech Co., Ltd. based in Shanghai, China.

Total RNA was extracted from colon tissues using TRIzol reagent (Invitrogen, CA, USA). Subsequently, cDNA was synthesized from 1 μg of RNA as the template, utilizing the RevertAid First Strand cDNA Synthesis Kit (Invitrogen, CA, USA). Real-time PCR was performed on a Bio-Rad system (CA, USA), with GAPDH serving as the housekeeping gene to normalize the data. The primer sequences used in the analysis are summarized in Table 1. The mRNA expression levels were quantified using the 2^–ΔΔCT method, which enables the assessment of fold changes in expression relative to the control group.

Pretreatment: Approximately 50 mg of fecal sample was weighed and mixed with 300 μL of acetonitrile solution (pre-cooled to 4°C) containing internal standards [²H₉]-Pentanoic acid and [²H₁₁]-Hexanoic Acid. The mixture was ground for 3 min at −20°C, followed by ultrasonic extraction in an ice-water bath for 10 min. The extract was centrifuged at 12,000 rpm for 10 min at 4°C, and the supernatant was collected. The supernatant was diluted 5-fold with acetonitrile solution containing the same internal standards.

Derivatization: An 80 μL aliquot of the diluted supernatant was sequentially mixed with 40 μL of 200 mM 3-NPH solution (prepared in 50% acetonitrile-water, v/v) and 40 μL of 120 mM EDC-6% pyridine solution (prepared in 50% acetonitrile-water, v/v). The reaction was carried out at 40°C for 30 min and subsequently cooled on ice for 1 min. The reaction mixture was filtered through a 0.22 μm microporous membrane to obtain the final derivative solution. The standard solutions were processed in the same manner as the samples.

UPLC-ESI-MS/MS Analysis: Chromatographic Conditions: The injection volume was 1 μL. The mobile phase consisted of solvent A (0.1% formic acid aqueous solution) and solvent B (acetonitrile/methanol, 2:1, v/v), with a flow rate of 0.4 mL/min. The gradient elution program was as follows: 0–2 min, 75% A; 2–11 min, 75% A → 45% A; 12–13 min, 25% A. Mass Spectrometry Conditions: Detection was performed in negative ion mode with a spray voltage of −4,500 V and an ion source temperature of 450°C. The column temperature was maintained at 40°C. Curtain gas pressure was set at 35 psi, while both nebulizing gas (Gas1) and auxiliary gas (Gas2) pressures were set at 50 psi. The collision-induced dissociation (CAD) parameter was set to medium.

In the context of this study, a suite of 30 mouse samples was analyzed, yielding a robust dataset with a genomic range spanning 11.19 to 12.43 gigabases. The assemblage of these samples resulted in N50 contig lengths varying between 3,710 and 14,035 base pairs. A comprehensive gene catalog, devoid of redundancy, was constructed from these assemblies, comprising a total of 1,623,375 open reading frames (ORFs). The sample libraries were subjected to sequencing on the state-of-the-art Illumina Novaseq 6000 platform, generating 150-base-pair paired-end reads. Quality control and trimming were meticulously performed using Trimmomatic (version 0.36). Host genome alignment and read filtering were conducted with bowtie2 (version 2.2.9), preceding metagenome assembly via MEGAHIT (version 1.1.2), which led to the reported N50 contig statistics. ORF prediction was executed on the assembled contigs using prodigal (version 2.6.3), and the resulting amino acid sequences were derived. Redundancy was minimized through the application of CDHIT (version 4.5.7), establishing a non-redundant gene set. This set was then aligned to the NR database for taxonomic classification and functionally annotated against various databases using DIAMOND (version 0.9.7). Carbohydrate-active enzymes were identified by comparing the gene sets to the CAZy database with hmmscan (version 3.1), and their activities were approximated by summing gene abundances. Non-metric multidimensional scaling (NMDS) analysis, facilitated by R software (version 3.2.0), was employed to scrutinize the taxonomic and functional abundance profiles across the 30 samples.

Statistical analysis was performed using SPSS software (version 25.0). For normally distributed and homogeneous variance data, group differences were analyzed using one-way analysis of variance (ANOVA) followed by Tukey’s post-hoc test. For other non-normally distributed data, the Kruskal-Wallis test was used to assess differences between groups. Except for differences in species-level taxa within the gut microbiota, which were analyzed using the Kruskal-Wallis test due to their non-normal distribution, all other parameters, including constipation-related phenotypic parameters, gut peptides, cytokine levels, mRNA expression levels, and short-chain fatty acid concentrations, were analyzed using one-way analysis of variance (ANOVA) followed by Tukey’s post-hoc test. Graphs were generated using GraphPad Prism (version 9.5). Statistical significance was set at P < 0.05.

After administering TG11 for 14 days and a single dose of loperamide, the study assessed TG11’s effect on constipation. The findings revealed a significant decrease in the time to the first black stool (F = 22.59, P < 0.05), and increase in both fecal water content (F = 89.78, P < 0.05) and fecal quantity (F = 5.39, P < 0.05) in constipated mice, as depicted in Figures 2A–C. Additionally, the gastrointestinal transit rate (GTR) and 5-h Fecal Weight were markedly lower in the constipation model group (CM) compared to the normal control (NC). TG11 treatment raised GTR (F = 14.35, P < 0.05) and 5-h Fecal Weight (F = 34.17, P < 0.05) significantly (Figures 2D, E). These results confirm that TG11 effectively alleviates constipation in mice.

The histological assessment using Hematoxylin and Eosin staining revealed the impact of TG11 on the intestinal morphology in mice suffering from constipation. Loperamide treatment at low doses was observed to lead to the disappearance of intestinal glandular structures within the colonic lamina propria, coupled with a significant decrease in lymphocyte infiltration. In contrast, therapy with TG11 was able to maintain the architectural integrity of the intestinal glands in the lamina propria. Furthermore, TG11 exhibited an inhibitory effect on lymphocyte infiltration in the intestinal tract (as illustrated in Figure 2F). However, it should be noted that at low doses, the therapeutic effects of TG11 on preserving colonic structural integrity were less pronounced in mice with constipation.

To evaluate the therapeutic potential of TG11 in modulating intestinal function through the regulation of critical neurotransmitters and hormones, we conducted quantification of mouse motilin (MTL), substance P (SP), somatostatin (SS), endothelin-1 (ET-1), gastrin (Gas), and vasoactive intestinal peptide (VIP). The constipation group exhibited significantly diminished levels of MTL, SP, and Gas, along with marked elevations in SS, ET-1, and VIP, as compared to the normal control group (Figures 3A–F). Administration of TG11, however, led to a notable restoration of MTL (F = 10.73, P < 0.05), SP (F = 35.93, P < 0.001), and Gas (F = 42.19, P < 0.001) to near physiological levels, concomitant with a significant reduction in SS (F = 6.23, P < 0.05), ET-1 (F = 114.93, P < 0.001) and VIP (F = 170.81, P < 0.001) concentrations. These data imply that TG11 may represent a promising intervention for the management of slow transit constipation, by re-equilibrating the intestinal neurotransmitter and hormone profile.

Studies have indicated that the mRNA expression levels of SCF and c-kit are associated with intestinal function. Constipation may be related to weakened intestinal motility and abnormal intestinal mucosal function. A decrease in the mRNA expression of SCF or c-kit could lead to insufficient activation of the c-kit receptor, thereby affecting intestinal motility and secretory functions, potentially exacerbating constipation symptoms. In mice with constipation, the mRNA expression levels of SCF and c-kit in the colon were significantly reduced. Compared to the control group (CM), treatment with various doses of TG11 increased the expression of SCF (F = 119.44, P < 0.001) and c-kit (F = 119.44, P < 0.001) by two to threefold, thereby alleviating the symptoms of constipation (Figures 4A, B).

Utilizing Ultra-Performance Liquid Chromatography-Electrospray Ionization Tandem Mass Spectrometry (UPLC-ESI-MS/MS), we investigated the impact of TG11 on the Short-Chain Fatty Acids (SCFAs) profile in fecal samples. The study comprised five cohorts: Control, Constipation Model (CM), and Low-, Medium-, and High-dose TG11 treatment groups. Our findings revealed a significant reduction in various SCFAs, including acetic (F = 77.58, P < 0.001), butyric (F = 86.49, P < 0.001), glutaric (F = 94.85, P < 0.001), isovaleric (F = 59.31, P < 0.001), malonic (F = 67.27, P < 0.001), and propionic acids (F = 14.25, P < 0.001), in the CM group compared to the Control. This observation suggests a substantial alteration in the metabolic functionality of the gut microbiota in the constipation model, resulting in diminished SCFA production. Intriguingly, we observed a dose-dependent restoration of SCFA levels with TG11 treatment. As the TG11 dosage increased, SCFA concentrations progressively recovered, converging toward those observed in the Control group. This trend implies a potential therapeutic effect of TG11 in normalizing gut microbiota metabolism and SCFA production in constipation (Figures 5A–F). In addition to the six SCFAs mentioned above, we also detected six other SCFAs, including pentanoic acid, hexanoic acid, 2,3-dihydroxy-3-methylbutanoic acid, lactic acid, malonic acid, and glutaric acid. However, these SCFAs did not show statistically significant differences between groups (P > 0.05).

The NMDS analysis at the species level indicated that the NC group was distinct from the CM group, whereas the LG group showed a close relationship with the CM group. The MG and HG groups were also similar to the CM group, indicating that medium and high doses of TG11 can maintain a balanced state of the intestinal microbiota in mice (Figure 6A). We observed that higher doses of TG11 were linked to significant alterations in the composition of the gut microbiota, particularly at high doses (Figure 6B). At the species level, loperamide increased the abundance of Muribaculum_sp. (P < 0.05) and uncultured_Duncaniella (P < 0.05), while reducing the abundance of Muribaculum sp._NM65 B17, an effect that was reversed by TG11 treatment (Figures 6D–E).

In our KEGG pathway analysis, we detected substantial disparities in a multitude of biochemical pathways between the TG11 intervention groups at varying doses, the disease model group, and the control. These pathways include, but are not limited to, fatty acid metabolism, drug metabolism, DNA repair, amino acid biosynthesis, and sulfur metabolism (Figure 7A). Notably, the high-dose TG11 intervention group (HG) exhibited pronounced alterations in pathways related to amino acid synthesis, neurogrowth factor signaling, and angiogenesis, implying a regulatory function of TG11 in these physiological processes. The moderate-dose (MG) and low-dose (LG) groups also revealed dose-dependent changes in select KEGG pathways, further highlighting the gradient of TG11’s impact (Figure 7B).