A symbiotic preparation containing animal bifidobacterium and galacto-oligosaccharide and its application in preparing products for relieving ulcerative colitis

Abstract

The present application relates to the technical field of biological medicine, in particular to a symbiotic preparation containing animal bifidobacterium and galacto-oligosaccharide and application thereof in preparation of products for relieving ulcerative colitis. The symbiotic preparation is composed of animal bifidobacterium and galacto-oligosaccharide. The results of the present application show that GOS promotes the colonization of animal bifidobacterium and cooperatively amplifies the protective effect of the animal bifidobacterium on DSS-induced colitis, and the effect of combined medication is better than that of any single drug. The synergistic effect is attributed to the coordinated regulation of intestinal flora, which is manifested in the enrichment of beneficial bacteria such as bifidobacterium and lactobacillus and the inhibition of opportunistic pathogens. At the same time, it is accompanied by the enhancement of SCFAs production and the activation of Trp-AhR-IL-22 pathway, which together strengthen the intestinal barrier integrity and inhibit the inflammatory response. The present application provides a theoretical basis for developing the symbiotic preparation of GOS and animal bifidobacterium as an effective dietary intervention for UC management.

Description

Technical Field

[0001] This invention relates to the field of biomedical technology, specifically to a synergistic preparation containing Bifidobacterium animalis and galactooligosaccharides and its application in the preparation of products for relieving ulcerative colitis. Background Technology

[0002] Ulcerative colitis (UC) is a chronic, nonspecific inflammatory bowel disease. Currently, the main medications for treating UC include 5-aminosalicylic acid, glucocorticoids, and immunosuppressants. However, long-term use has serious side effects and does not cure the disease. Therefore, finding safe and effective alternative treatments is of great importance. In recent years, the application of probiotics and prebiotics in the prevention and treatment of UC has received widespread attention. Gut microbiota dysbiosis is a crucial factor in the pathogenesis of UC, characterized by a decrease in beneficial bacteria (such as Bifidobacteria and Lactobacilli) and an increase in pathogenic bacteria, leading to impaired intestinal barrier function and exacerbated inflammatory responses.

[0003] Several patents have been published in the prior art regarding the use of Bifidobacteria or synbiotics for gut health. For example, Chinese patent application CN118044616A discloses a synbiotic composed of Bifidobacterium bifidum and one or more prebiotics, with the prebiotics selected from isomaltooligosaccharides, xylooligosaccharides, soybean oligosaccharides, galactooligosaccharides, polydextrose, pullulan, lactulose, fructooligosaccharides, inulin, and resistant starch. However, the technical solution of this patent is aimed at bacterial infectious diseases (EHEC infection), not inflammatory bowel diseases such as ulcerative colitis; and although galactooligosaccharides are listed as one of the optional prebiotics, its examples and verification data focus entirely on inulin and isomaltooligosaccharides, without providing any technical teaching on the synergistic relief of colitis by galactooligosaccharides and Bifidobacteria. For example, Chinese patent application CN119307425A discloses a strain of Bifidobacterium lactis BLa79, which has broad-spectrum antibacterial properties and the function of regulating intestinal flora. It can alleviate ulcerative colitis by increasing body weight, reducing DAI scores, reducing spleen index, inhibiting colonic atrophy, reducing the levels of pro-inflammatory factors (TNF-α, IL-6, MPO), and promoting SIgA secretion. However, the technical solution of this patent only involves the application of a single probiotic strain, without involving any combination with prebiotics, let alone the synergistic effect of synbiotics.

[0004] Therefore, there is an urgent need in this field for a synergistic formulation for ulcerative colitis that uses a rational combination of specific probiotics and specific prebiotics, has been experimentally verified to have a significant synergistic effect, and has a clearly defined mechanism of action. Summary of the Invention

[0005] In view of the above-mentioned shortcomings in the prior art, the purpose of this invention is to provide a synergistic preparation containing Bifidobacterium animalis and galactooligosaccharide, which utilizes the synergistic effect between the two to effectively alleviate ulcerative colitis, and provides a safe and effective solution for the prevention and treatment of ulcerative colitis.

[0006] To achieve the above-mentioned objectives, the technical solution adopted by this invention is as follows: A synbiotic preparation is provided, which is composed of Bifidobacterium animalis and galactooligosaccharides; wherein the Bifidobacterium animalis is a live bacterium, and its concentration in the synbiotic preparation is 10. 8 ~10¹ 0 CFU / mL; the content of the galactooligosaccharide in the synthetic preparation is 30~90 mg / mL.

[0007] Preferably, the animal bifidobacteria are selected from animal subspecies of Bifidobacterium animalis or lactobacterium animalis.

[0008] Preferably, the animal bifidobacterium is animal subspecies of Bifidobacterium animalis JCM1190.

[0009] Preferably, the concentration of the animal Bifidobacterium is 10. 9 The concentration of CFU / mL and the content of the galactooligosaccharide is 60 mg / mL.

[0010] Provided for the use of the above-mentioned synthetic preparations in the preparation of products for the prevention and / or treatment of ulcerative colitis.

[0011] Preferably, the synbiotic alleviates ulcerative colitis through one or more of the following mechanisms: (a) Upregulates the expression levels of messenger ribonucleic acid of tight junction proteins ZO-1, Occludin and mucin MUC2 in colon tissue, thereby enhancing intestinal barrier function; (b) Decreases the levels of TNF-α, IL-1β, and IL-6 in colon tissue and serum, and increases the level of the anti-inflammatory cytokine IL-10; (c) Reduces malondialdehyde content in colon tissue and serum, increases total superoxide dismutase activity, and inhibits myeloperoxidase activity; (d) Regulates the composition of intestinal flora, enriches Bifidobacterium and Lactobacillus, and inhibits opportunistic pathogens; (e) Increased levels of short-chain fatty acids in colonic contents, including acetic acid, propionic acid, butyric acid, isobutyric acid, isovaleric acid, and total short-chain fatty acids; (f) Increased levels of tryptophan metabolites in colonic contents, including indole-3-acetic acid, indole-3-carboxaldehyde, and indole-3-propionic acid; (g) Upregulates the expression of messenger ribonucleic acid of aryl hydrocarbon receptor and its downstream target gene cytochrome P450 family 1 subfamily A member 1 in colon tissue, increases IL-22 level, and activates the aryl hydrocarbon receptor / IL-22 signaling pathway.

[0012] A pharmaceutical composition for relieving ulcerative colitis is provided, comprising the above-mentioned synthetic preparation as the active ingredient, and pharmaceutically acceptable excipients.

[0013] Preferably, the dosage form is selected from powder, tablet, capsule, granule, oral liquid or liquid preparation.

[0014] The beneficial effects of this invention are as follows: This invention is the first to discover that galactooligosaccharides (GOS) can serve as an effective carbon source for Bifidobacterium animalis, promoting its growth more effectively than glucose in the later stages of fermentation. Furthermore, combined administration of GOS is significantly superior to either single drug in alleviating colitis symptoms. This invention also reveals for the first time that GOS and Bifidobacterium animalis synergistically alleviate ulcerative colitis through multiple mechanisms, including regulating intestinal flora, enhancing short-chain fatty acid production, and activating the tryptophan-AhR-IL-22 pathway. Attached Figure Description

[0015] Figure 1 In vitro growth and SCFAs yield of *Bifidobacterium animalis* cultured using glucose (GLU) or galactooligosaccharide (GOS) as the sole carbon source; among which, (A) growth curves of *Bifidobacterium animalis* at 0, 12, 24, 36, and 48 h (OD). 600 (B) pH of the culture medium at 0, 12, 24, 36 and 48 h; (C) SCFA concentration at 24 h; (D) SCFA concentration at 48 h; GLU group: Bifidobacterium animalis was cultured in sMRS medium containing 1% (w / v) glucose as the sole carbon source; GOS group: Bifidobacterium animalis was cultured in sMRS medium containing 1% (w / v) GOS as the sole carbon source; Sugar-free control group: Bifidobacterium animalis was cultured in sMRS medium without any added sugar; Data are expressed as mean ± SD (n=6). P<0.05, P*<0.01, P<0.001, compared with the sugar-free control group at the same time point; ^#^P<0.05, ^##^P<0.01, ^###^P<0.001, compared with the GLU group at the same time point; Figure 2Synergistic effects of galactooligosaccharides (GOS) and Bifidobacterium animalis on dextran sulfate sodium (DSS)-induced colitis in mice; (A) Schematic diagram of animal experimental protocol; (B) Body weight change during the 7-day experiment; (C) Disease Activity Index (DAI) score during the 7-day experiment; (D) Colon length on day 7; (E) Representative H&E stained colon sections (scale bar = 400 μm); (F) Histological score; (G) MPO activity in colon tissue; Data are expressed as mean ± SD (n = 8 / group); ^#^P<0.05, ^##^P<0.01, ^###^P<0.001 vs Ctrl group; P<0.05, P*<0.01, P<0.001 vs DSS group; ^&^P<0.05, ^&&^P<0.01, ^&&&^P<0.001 vs BIFGOS group; Ctrl: untreated control group; DSS: DSS-treated group; GOS: DSS + daily gavage 600 mg / kg GOS group; BIF: DSS + daily gavage 10 9 CFU / mL animal Bifidobacterium group; BIFGOS: DSS + daily gavage 600 mg / kg GOS and 10 9 CFU / mL animal Bifidobacteria group; Figure 3 The synergistic effect of galactooligosaccharides (GOS) and Bifidobacterium animalis on intestinal barrier function in DSS-induced colitis mice; the mRNA expression levels of ZO-1, Occludin, and MUC2 in colon tissue were detected by (AC)qRT-PCR; data are expressed as mean ± SD (n=6 / group); different letters indicate significant differences between groups (P<0.05); Ctrl: untreated control group; DSS: DSS-treated group; GOS: DSS + daily gavage 600 mg / kg GOS group; BIF: DSS + daily gavage 10 9 CFU / mL animal Bifidobacterium group; BIFGOS: DSS + daily gavage 600 mg / kg GOS + 10 9 CFU / mL animal Bifidobacteria group; Figure 4The synergistic effects of galactooligosaccharides (GOS) and Bifidobacterium animalis on inflammatory cytokines and oxidative stress in DSS-induced colitis mice; specifically, the levels of T-SOD, MDA, TNF-α, IL-1β, IL-6, and IL-10 in colonic tissue (AF); and the levels of T-SOD, MDA, TNF-α, IL-1β, IL-6, and IL-10 in serum (GL); data are expressed as mean ± SD (n=6 / group); different letters indicate significant differences between groups (P<0.05); Ctrl: untreated control group; DSS: DSS-treated group; GOS: DSS + daily gavage 600 mg / kg GOS group; BIF: DSS + daily gavage 10 9 CFU / mL animal Bifidobacterium group; BIFGOS: DSS + daily gavage 600 mg / kg GOS + 10 9 CFU / mL Bifidobacterium animal group; T-SOD: total superoxide dismutase; MDA: malondialdehyde; TNF-α: tumor necrosis factor-α; IL-1β: interleukin-1β; IL-6: interleukin-6; IL-10: interleukin-10; Figure 5 Synergistic effects of galactooligosaccharides (GOS) and Bifidobacterium animalis on the diversity and composition of gut microbiota in DSS-induced colitis mice; (AD) α-diversity indices: Ace (A), Shannon (B), Chao (C), and Simpson (D); (E) Venn plot showing the unique and shared number of OTUs among the five groups; (F) Principal coordinate analysis (PCoA) based on Bray-Curtis dissimilarity; (G) relative abundance at the phylum level; (H) relative abundance at the family level; (I) heatmap of the top 30 most abundant bacterial genera; data are expressed as mean ± SD (n=6); P<0.05, P*<0.01, P<0.001. Ctrl: untreated control group; DSS: DSS-treated group; GOS: DSS + daily gavage 600 mg / kg GOS group; BIF: DSS + daily gavage 10 9 CFU / mL animal Bifidobacterium group; BIFGOS: DSS + daily gavage 600 mg / kg GOS + 10 9 CFU / mL animal Bifidobacteria group; Figure 6Synergistic effects of galactooligosaccharides (GOS) and Bifidobacterium animalis on differential abundance taxa and key genus colonization in DSS-induced colitis mice; (A) Linear discriminant analysis (LDA) scores of differential abundance taxa among the five groups; (B) Differential abundance taxa between the DSS and BIF groups; (C) Differential abundance taxa between the DSS and GOS groups; (D) Differential abundance taxa between the DSS, BIF, and GOS groups; (E) Relative abundance of Lactobacillus among the five groups based on 16S rRNA sequencing data; (F) Relative abundance of Bifidobacterium among the five groups based on 16S rRNA sequencing data; (G) Absolute quantification of Bifidobacterium by qPCR; Data are expressed as mean ± SD (n=6 / group); P<0.05, P*<0.01, P<0.001; Different letters indicate significant differences between groups (P<0.05); Ctrl: Untreated control group; DSS: DSS-treated group; GOS: DSS + Daily gavage 600 mg / kg GOS group; BIF: DSS + Daily gavage 10 9 CFU / mL animal Bifidobacterium group; BIFGOS: DSS + daily gavage 600 mg / kg GOS + 10 9 CFU / mL animal Bifidobacteria group; Figure 7 The synergistic effects of galactooligosaccharides (GOS) and Bifidobacterium animalis on fatty acid metabolism, short-chain fatty acid (SCFA) production, and their correlation with gut microbiota in DSS-induced colitis mice; (AG) colonic levels of acetic acid (A), propionic acid (B), butyric acid (C), isobutyric acid (D), isovaleric acid (E), valeric acid (F), and total SCFAs (G); (H) Spearman correlation heatmap between gut microbiota and SCFAs; (IJ) PICRUSt2-predicted fatty acid biosynthesis (I) and fatty acid metabolism (J) KEGG pathway abundance; Data are expressed as mean ± SD (n=6 / group); P<0.05, P*<0.01, P<0.001; different letters indicate significant differences between groups (P<0.05); Ctrl: untreated control group; DSS: DSS-treated group; GOS: DSS + daily gavage 600 mg / kg GOS group; BIF: DSS + daily gavage 10 9 CFU / mL animal Bifidobacterium group; BIFGOS: DSS + daily gavage 600 mg / kg GOS + 10 9 CFU / mL animal Bifidobacteria group; Figure 8The synergistic effects of galactooligosaccharides (GOS) and Bifidobacterium animalis on gut microbiota-driven tryptophan (Trp) metabolism and the AhR / IL-22 pathway in DSS-induced colitis mice; including (AD) colonic Trp (A), indole-3-acetic acid (IAA) (B), indole-3-carboxaldehyde (IAId) (C), and indole-3-propionic acid (IPA) (D); (E) colonic tissue IL-22 level; (F) relative AhR mRNA expression in colonic tissue; (G) relative CYP1A1 expression in colonic tissue. mRNA expression; (H) Spearman correlation heatmap of gut microbiota and tryptophan metabolites; (IJ) Tryptophan metabolism predicted by PICRUSt2 (I) and biosynthesis of phenylalanine, tyrosine and tryptophan (J) KEGG pathway abundance; Data are expressed as mean ± SD (n=6 / group); P<0.05, P*<0.01, P<0.001; Different letters indicate significant differences between groups (P<0.05); Ctrl: untreated control group; DSS: DSS-treated group; GOS: DSS + daily gavage 600 mg / kg GOS group; BIF: DSS + daily gavage 10 9 CFU / mL animal Bifidobacterium group; BIFGOS: DSS + daily gavage 600 mg / kg GOS + 10 9 CFU / mL Bifidobacterium animalis group; AhR: aryl hydrocarbon receptor; CYP1A1: member of cytochrome P450 family 1 subfamily A; IL-22: interleukin-22. Detailed Implementation

[0016] The specific embodiments of the present invention are described below to enable those skilled in the art to understand the present invention. However, it should be understood that the present invention is not limited to the scope of the specific embodiments. For those skilled in the art, various changes are obvious as long as they are within the spirit and scope of the present invention as defined and determined by the appended claims. All inventions utilizing the concept of the present invention are protected.

[0017] Example 1. Materials and Methods 1.1. Materials and Strains GOS was purchased from Shanghai Synaura Biotechnology Co., Ltd. DSS was purchased from MP Biomedicals, USA. All other reagents were of analytical grade. Bifidobacterium animalis subspecies JCM1190 was provided by Ningbo Mingzhou Biotechnology Co., Ltd.

[0018] 1.2. Bacterial growth on GOS Bifidobacterium animalis was cultured in sMRS broth containing 1% (w / v) glucose and supplemented with 0.05% (w / v) L-cysteine ​​to support its growth. Stationary cells were collected by centrifugation (3,220 × g, 10 min), washed twice, and resuspended in glucose-free sMRS. The inoculum (1%, v / v) was transferred to fresh sMRS containing 1% (w / v) glucose, 1% (w / v) GOS, or carbohydrate-free sMRS and anaerobically cultured at 37°C for 48 h. Oddi concentrations (OD) were measured at 0, 12, 24, 36, and 48 h in equal aliquots. 600 pH. Culture supernatants were collected by centrifugation (10,000 × g, 10 min, 4°C) at 24 h and 48 h and stored at -80°C for SCFA analysis. Each condition was performed in triplicate.

[0019] 1.3. Animal Experiment Design All animal housing and euthanasia procedures were approved by the Animal Welfare and Animal Experimentation Ethics Committee of China Agricultural University. Six-week-old male C57BL / 6J mice were purchased from Beijing Vital River Laboratory Animal Technology Co., Ltd. Mice were housed at the Experimental Animal Center of China Agricultural University under SPF conditions (22 ± 1 °C, 50 ± 5%, 12 h light / dark cycle) with free access to standard feed and pure water. After one week of acclimatization, 40 mice were randomly divided into five groups (n = 8 / group): untreated control group (Ctrl), DSS treatment group (DSS), DSS + daily gavage with 600 mg / kg GOS group (GOS), DSS + daily gavage with 10 mg / kg GOS group (GOS), and DSS + daily gavage with 10 mg / kg GOS group (GOS). 9 CFU / mL Bifidobacterium animalis group (BIF), DSS + daily gavage 600 mg / kg GOS and 10 9 The CFU / mL Bifidobacterium animalis group (BIFGOS) was used. Except for the Ctrl group, acute colitis was induced in mice by continuous administration of 3% (w / v) DSS in drinking water for 7 days. GOS or Bifidobacterium animalis (0.2 mL per 20 g body weight) was administered by gavage for 7 days, while the Ctrl and DSS groups were administered phosphate-buffered saline (PBS) by gavage. Mouse body weight and activity status were recorded daily. On day 8, blood was collected from the orbital plexus, and serum was obtained by centrifugation (3,000 × g, 15 min, 4°C) and stored at -80°C. The entire colon was removed, and the colonic contents were collected, flash-frozen in liquid nitrogen, and stored at -80°C. After removing the contents, the colonic tissue was segmented; a portion was fixed in 4% paraformaldehyde for histological analysis, and the remaining tissue was flash-frozen at -80°C.

[0020] 1.4. Disease Activity Index (DAI) and Colon Length Mice were monitored daily for body weight, fecal consistency, and fecal occult blood. DAI scores were assessed using a pre-defined scoring system based on weight loss, fecal consistency, and rectal bleeding. At the end of the experiment, the entire colon was removed from the cecum to the anus, and its length was measured using a ruler.

[0021] 1.5. Histopathological evaluation Distal colon tissue fixed in 4% paraformaldehyde was embedded in paraffin, sectioned at 4 μm, and stained with hematoxylin and eosin (H&E). Histological changes were observed under a light microscope. Histological scoring was performed based on parameters such as goblet cell loss, mucosal structure destruction, muscular layer thickening, inflammatory cell infiltration, and crypt abscess formation.

[0022] 1.6. Measurement of inflammatory cytokines and oxidative stress markers Colon tissue was homogenized in pre-cooled PBS (pH 7.4, 1:9 w / v), centrifuged (3,000 × g, 10 min, 4°C), and the supernatant was collected. Inflammatory cytokines TNF-α, IL-1β, IL-6, IL-10, and IL-22 were detected using a commercial ELISA kit. Oxidative stress markers T-SOD activity, MDA content, and MPO activity were determined using a commercial kit (Nanjing Jiancheng Biotechnology Institute, Nanjing, Jiangsu, China) according to the manufacturer's instructions. All samples were analyzed in triplicate.

[0023] 1.7. Quantitative Real-Time Polymerase Chain Reaction (qRT-PCR) Total RNA was extracted from colon tissue using TRIzol reagent, following the manufacturer's instructions. RNA concentration and purity were assessed using a NanoDrop spectrophotometer. 1 μg of RNA was reverse transcribed into cDNA using a PrimeScript RT Reagent Kit. qPCR was performed on a real-time PCR system using TBGreen Premix Ex Taq II under the following conditions: 95°C for 30 s, 40 cycles of 95°C for 5 s and 60°C for 30 s. Two... ^-ΔΔCt^ Relative gene expression was calculated using a method. Primer sequences are shown in Table 1.

[0024] Table 1 Primer sequences for real-time quantitative PCR experiments 1.8. 16S rRNA gene sequencing Genomic DNA was extracted from colon contents using the EZNA® Stool DNA Kit according to the manufacturer's instructions. The V3-V4 hypervariable regions of the bacterial 16S rRNA gene were amplified using universal primers 338F (5'-ACTCCTACGGGAGGCAGCAG-3') and 806R (5'-GGACTACHVGGGTWTCTAAT-3'). The PCR products were purified, quantified, and sequenced on the Illumina MiSeq platform. Sequencing was performed by Shanghai Meiji Biotechnology Co., Ltd. Raw sequencing data were processed using QIIME2 (version 2022.2). Brief procedure: paired-end reads were quality filtered, denoised, and merged (DADA2 plugin). OTUs were clustered using UCLUST at 97% similarity. Representative sequences for each OTU were aligned to the Silva 16S rRNA database (version 138) with a confidence threshold of 70%. Alpha diversity was assessed using the Chao1 and Shannon indices. β-diversity was assessed using principal coordinate analysis (PCoA) based on Bray-Curtis dissimilarity, with differences evaluated using the PERMANOVA test (999 permutations). LEfSe was used to identify differentially abundant taxa between groups; an LDA score ≥3 and P < 0.05 were considered significant. PICRUSt2 was used to predict gut microbiota function based on the KEGG database. Correlation analysis employed Spearman's rank correlation coefficient to assess the relationship between differentially abundant bacterial genera and host physiological parameters; only P < 0.05 was considered significant.

[0025] 1.9. SCFAs Content Analysis SCFAs in colonic contents were analyzed using gas chromatography-mass spectrometry (GC-MS). In short, the sample was mixed with 30% phosphoric acid and acetone, homogenized, and centrifuged. The supernatant was filtered and analyzed on a DB-FFAP column (30 m × 0.25 mm × 0.25 μm). The injection volume was 1 μL (split ratio 10:1), with high-purity helium as the carrier gas, and a flow rate of 1.0 mL / min. The column temperature was maintained at 70°C for 5 min, then increased to 100°C at a rate of 6°C / min. Data were processed using MassHunter and Quant-My-Way software.

[0026] 1.10. Analysis of tryptophan and its metabolites UHPLC-MS / MS was used to quantify tryptophan metabolites in colonic contents. In short, 20 mg of sample was homogenized in 200 µL of water, then 600 µL of pre-cooled methanol containing the internal standard 1-methyltryptophan was added, vortexed, and centrifuged. The supernatant was dried under vacuum, reconstituted in 100 µL of 10% methanol, and centrifuged again. Separation was performed on an Agilent 1290 UHPLC-6470A QQQ system with a mobile phase of 0.1% formic acid (A) and methanol (B). The flow rate was 0.3 mL / min, and the column temperature was 35°C. Mass spectrometry detection was performed using ESI+, dMRM mode. Key parameters: gas temperature 300°C, gas flow rate 5 L / min, nebulizer 45 psi, sheath gas 300°C / 10 L / min, capillary 4000 V. Data were processed using MassHunter (B.04.00). Metabolites were quantified using a standard curve (purity ≥98%; Shanghai Yuanye Biotechnology Co., Ltd., China).

[0027] 1.11. Statistical Analysis Data are expressed as mean ± standard deviation (SD). Statistical analysis was performed using SPSS software (version 24.0, Chicago, IL, USA). For comparisons among multiple groups, one-way ANOVA was used for normally distributed data, and the Kruskal-Wallis test was used for nonparametric data. A p-value <0.05 was considered statistically significant. All experiments were performed in at least three independent replicates.

[0028] 2. Results 2.1. In vitro utilization of GOS by Bifidobacterium animalis OD within 48 hours 600 Measure and evaluate the growth of Bifidobacterium animals on different carbon sources. For example... Figure 1 As shown in Figure A, *Bifidobacterium animalis* grew significantly in the presence of glucose and GOS, while no significant growth was observed in the glucose-free control group. Notably, the OD values ​​in the GOS group at 36 h and 48 h were significantly higher. 600 The pH values ​​were significantly higher than those in the glucose group, reaching maximum values ​​of 1.87 ± 0.02 and 1.20 ± 0.01 at 48 h, respectively. These results indicate that GOS can serve as an effective carbon source for Bifidobacterium animalis and promotes its growth more effectively than glucose in the later stages of fermentation. The pH of the culture medium was monitored throughout the fermentation process. The pH in the sugar-free control group remained stable within 48 h. When GOS was used as the sole carbon source, the pH of the culture medium decreased significantly after 12 h of fermentation and remained stable from 12 to 48 h (pH 4.95 ± 0.08 at 12 h, pH 4.48 ± 0.09 at 48 h). A similar pH decrease was observed in the glucose group after 12 h (pH 4.81 ± 0.12 at 12 h). However, there was no significant difference in pH between the glucose and GOS groups at any time point. Figure 1 C and Figure 1 As shown in Figure D, SCFA analysis at 24 h and 48 h revealed that Bifidobacterium animalis primarily produced acetic acid and propionic acid. Notably, butyric acid levels in the GOS group were significantly higher than those in the glucose group at both 24 h and 48 h (P<0.05), and acetic acid levels in the GOS group were also significantly higher than those in the glucose group at 24 h (P<0.05). These findings demonstrate that GOS not only supports the growth of Bifidobacterium animalis but also enhances the production of beneficial SCFAs, especially acetic acid and butyric acid.

[0029] 2.2. GOS and Bifidobacterium animalis synergistically alleviate DSS-induced colitis symptoms and histopathological damage in mice. To evaluate the synergistic protective effect of GOS and Bifidobacterium animalis against DSS-induced colitis, according to Figure 2 A colonic dysplasia (UC) model was established. DSS-induced weight loss, elevated DAI score, and shortened colon length are reliable indicators of successful UC model establishment. Figure 2 As shown in the BD diagram, compared with the Ctrl group, mice in the DSS group had significantly lower body weight, significantly higher DAI scores, and significantly shorter colon length (P<0.05). Conversely, compared with the DSS group, mice treated with Bifidobacterium animalis, GOS, or a combination of both experienced less weight loss, lower DAI scores, and longer colon lengths (P<0.05). Among these, the BIFGOS group showed the most significant improvement in weight loss, DAI score, and colon length, superior to the groups treated with GOS or Bifidobacterium animalis alone (P<0.05). Further histopathological analysis of the colon tissue revealed severe mucosal damage, crypt destruction, and extensive inflammatory cell infiltration in the DSS group. Figure 2 E, Figure 2 The mean F; P<0.05). In the Ctrl group, the colonic mucosa was normal and the epithelium intact. In contrast, all intervention groups showed significant improvement in these pathological changes (P<0.05 compared to the DSS group). MPO activity has been reported to be associated with disease severity and can serve as a biomarker for assessing disease status in IBD patients (Plasma Calprotectin and Myeloperoxidase as Biomarkers in Inflammatory Bowel Disease). Compared to the Ctrl group, the DSS group showed significantly increased MPO activity (P<0.05). Figure 2 (Middle G). All intervention groups significantly inhibited MPO activity (P<0.05 compared with the DSS group). Among them, the BIFGOS group showed the most significant reduction in MPO activity, further supporting the synergistic anti-inflammatory effect of the combination therapy.

[0030] 2.3. GOS and Bifidobacterium animalis synergistically improve intestinal barrier function in DSS-induced colitis mice. To assess the effects of GOS and Bifidobacterium animalis on intestinal barrier integrity, the mRNA expression levels of tight junction proteins ZO-1, Occludin, and the mucus layer component MUC2 in colonic tissue were measured. Figure 3 As shown in Figures AC, compared with the Ctrl group, DSS treatment significantly downregulated the mRNA expression of ZO-1, Occludin, and MUC2 (P<0.05). Compared with the DSS group, GOS or Bifidobacterium animalis alone significantly upregulated MUC2 expression (P<0.05). Notably, the combined use of GOS and Bifidobacterium animalis significantly increased the expression of ZO-1, Occludin, and MUC2 (compared to the DSS group), and MUC2 expression was higher than that of GOS or Bifidobacterium animalis alone (P<0.05). These results indicate that GOS and Bifidobacterium animalis synergistically enhance intestinal barrier function by upregulating the production of tight junction proteins and mucins, thereby protecting against DSS-induced epithelial damage.

[0031] 2.4. GOS and Bifidobacterium animalis synergistically reduce inflammation and oxidative stress in DSS-induced colitis mice. like Figure 4 As shown in the AF, compared with the Ctrl group, DSS treatment significantly increased the levels of pro-inflammatory cytokines TNF-α, IL-1β, and IL-6, as well as MDA content in colon tissue (P<0.05), while significantly decreasing the activities of anti-inflammatory cytokines IL-10 and T-SOD (P<0.01). Compared with the DSS group, GOS or Bifidobacterium animalis alone significantly reduced MDA, TNF-α, and IL-1β levels and increased IL-10 levels (P<0.05). Notably, the combined use of GOS and Bifidobacterium animalis was the most effective, further reducing TNF-α, IL-1β, IL-6, and MDA, and increasing IL-10 and SOD to a greater extent (P<0.05 compared with the DSS group). Consistent with the colon tissue results, a similar trend was observed in serum. Figure 4 (GL). GOS or Bifidobacterium animalis alone significantly increased T-SOD levels and decreased MDA, IL-1β, and IL-6 levels (P<0.05). Combined administration showed the most significant improvement, further reducing TNF-α, IL-1β, IL-6, and MDA, and increasing T-SOD to a greater extent (P<0.05 compared to the DSS group). These results indicate that GOS and Bifidobacterium animalis synergistically alleviated the systemic inflammatory response and enhanced antioxidant capacity in DSS-induced colitis mice.

[0032] 2.5. GOS and Bifidobacterium animalis synergistically regulate the gut microbiota composition in mice with DSS-induced colitis. To assess the impact of GOS and Bifidobacterium animalis on gut microbiota composition, 16S rRNA gene sequencing was performed on colonic contents. Alpha diversity was assessed using the Ace, Shannon, Chao, and Simpson indices. Figure 5 (AD). Compared with the Ctrl group, the Ace and Chao indices of the DSS group were significantly increased (P<0.05), indicating that the microbial richness was significantly increased after DSS treatment. PCoA based on Bray-Curtis dissimilarity showed significant separation among the five groups (R=0.2052, P=0.005). Figure 5 The middle F) indicates that DSS treatment significantly altered the overall microbial community structure, and intervention with GOS and Bifidobacterium animalis shifted the microbial composition towards a healthier direction. Venn plot of OTU levels ( Figure 5 The E-group displays a total of 193 OTUs across five groups. The Ctrl, DSS, BIF, GOS, and BIFGOS groups contain 832, 1099, 884, 900, and 1082 unique OTUs respectively. At the gate level ( Figure 5 In the middle G group, Bacteroidetes and Bacillota are the two dominant phyla in all groups. At the family level ( Figure 5 In the middle H group, Muribaculaceae, Lachnospiraceae, Lactobacillaceae, and Erysipelotrichaceae are the main components. At the genus level, heatmaps of the top 30 most abundant bacterial genera further illustrate the similarities and differences in microbial community structure among groups. Figure 5 Middle I).

[0033] LEfSe analysis was used to identify the differential abundance taxa among the five groups. Figure 6 In the DSS group, *Alistipes* and *norank_f_Anaerovoracaceae* were significantly enriched. In the GOS group, *norank_f_Oscillospiraceae* was significantly increased. In the BIFGOS group, *Bacilli*, *Bifidobacterium*, *Lactobacillus*, and *Dubosiella* were significantly enriched. Further enrichment was observed between the DSS and BIF groups (…). Figure 6 In the DSS group, Anaerovoracaceae, Ruminococcaceae, Marinifilaceae, Anaerorotruncus, Odoribacter, and norank_f Christensenellaceae are enriched in the DSS group, while Lactobacillus and Bifidobacterium are enriched in the BIF group. The DSS group is enriched in the BIF group. (The text abruptly ends here, likely due to an incomplete sentence or missing information.) Figure 6 In the DSS group, Saccharimonadaceae, Atopobiaceae, Candidatus_Saccharimonas, Christensenella, Muribacculum, [Eubacterium]_xylanophilum_group, and norank_f_Ruminococcaceae were enriched, while Cyanobacteriota and Gastanaerophilaceae were enriched in the GOS group. The DSS and BIFGOS groups showed a difference in enrichment. Figure 6 Bifidobacterium (Bifidobacterium), Acholeplasmataceae, Anaerovoracaceae, and Alistipes were enriched in the DSS group, while Bifidobacterium, Faecalibaculum, Lactobacillus, Litchfieldia, and Staphylococcus were enriched in the BIFGOS group. Notably, the relative abundance of Bifidobacterium and Lactobacillus was significantly increased in the BIFGOS group compared to the DSS group, and the abundance of Bifidobacterium was also higher than in other treatment groups (P<0.05). This result is consistent with the qPCR results, confirming the significant enrichment of Bifidobacteria in the BIFGOS group, indicating that animal Bifidobacteria administered by gavage in the presence of GOS can effectively colonize and proliferate, with better efficacy than GOS or animal Bifidobacteria alone.

[0034] 2.6. Co-regulation of SCFA production in DSS-induced colitis mice by GOS and Bifidobacterium animalis and its correlation with gut microbiota. To predict the functional potential of the gut microbiota, PICRUSt2 analysis was performed based on 16S rRNA sequencing data. Figure 7 As shown in Figure IJ, the combined use of GOS and Bifidobacterium animalis significantly upregulated the KEGG pathway, which is associated with fatty acid biosynthesis and fatty acid metabolism (P<0.01 compared to the DSS group), indicating that the synbiotic enhances the functional potential of the gut microbiota in fatty acid production and utilization. To further validate these functional predictions, the levels of SCFAs in colonic contents were measured to assess the effects of GOS and Bifidobacterium animalis on microbial metabolic function. Figure 7(AG). Compared with the Ctrl group, DSS treatment significantly reduced the levels of acetic acid, propionic acid, butyric acid, isobutyric acid, isovaleric acid, and total SCFAs (P<0.05). GOS or Bifidobacterium animalis alone partially restored these SCFA levels, while combined use significantly increased the concentrations of acetic acid, propionic acid, butyric acid, isobutyric acid, isovaleric acid, and total SCFAs (P<0.05). Spearman correlation analysis was used to assess the relationship between gut microbiota and SCFAs (…). Figure 7 The relative abundance of *Bifidobacterium* was significantly positively correlated with the levels of acetic acid, butyric acid, isobutyric acid, valeric acid, isovaleric acid, and total SCFAs (P < 0.05). The relative abundance of *Lactobacillus* was also positively correlated with the levels of isovaleric acid and total SCFAs (P < 0.05). Furthermore, *Akkermansia*, *Faecalibaculum*, *Turicibacter*, *unclassified_c__Bacilli*, *Litchfieldia*, *Blautia*, and *Adlercreutzia* were positively correlated with these SCFAs. Conversely, *Odoribacter*, *Alistipes*, *norank_o__Clostridia_vadinBB60_group*, *Helicobacter*, *unclassified_f__Ruminococcaceae*, *unclassified_f__Erysipelotrichaceae*, *[Eubacterium]_siraeum_group*, *Acutalibacter*, *norank_o__RF39*, ​​*unclassified_f__Oscillospiraceae*, *unclassified_k__norank_d__Bacteria*, *Prevotellaceae_UCG-001*, *Rikenellaceae_RC9_gut_group*, and *norank_f__Muribaculaceae* were negatively correlated with SCFAs. In summary, these correlation results further support the conclusion that combined administration of GOS and *Bifidobacterium animalis* promotes the enrichment of SCFA-producing bacteria, which is closely related to elevated SCFA levels.

[0035] 2.7. GOS and Bifidobacterium animalis synergistically regulate gut microbiota-driven Trp metabolism and AhR / IL-22 pathway in DSS-induced colitis mice. like Figure 8As shown in Figure IJ, the combined use of GOS and Bifidobacterium animalis significantly upregulated the KEGG pathway, which is associated with Trp metabolism and the biosynthesis of phenylalanine, tyrosine, and Trp (P<0.05 compared to the DSS group), indicating that the synbiotic enhances the functional potential of the gut microbiota in Trp metabolism. To further validate these functional predictions, the levels of Trp and its indole pathway metabolites were measured. Figure 8 As shown in the AD diagram, compared with the Ctrl group, DSS treatment significantly reduced the levels of IAA, IAId, and IPA (P<0.05). GOS or Bifidobacterium animalis alone partially restored these metabolites, while combined use significantly increased the levels of IAA, IAId, and IPA (P<0.05). Consistently, qPCR analysis showed that DSS treatment significantly reduced AhR mRNA expression in colonic tissue compared with the Ctrl group (P<0.05), while GOS or Bifidobacterium animalis treatment alleviated this reduction (P<0.05). Figure 8 In addition, the mRNA expression of CYP1A1 (a downstream target gene of AhR) in the BIFGOS group was significantly upregulated compared with that in the DSS group (P<0.05), further confirming the activation of AhR signaling by the synthetic preparation. Figure 8 (G). Studies have shown that AhR activation can stimulate innate lymphoid cells and CD4+. + T cells secrete IL-22, a cytokine crucial for tissue repair and maintaining intestinal barrier function. Figure 8 As shown in Figure E, DSS treatment significantly reduced IL-22 levels in the colon (P<0.05), while the combined use of GOS and Bifidobacterium animalis significantly restored IL-22 levels (P<0.05). Spearman correlation analysis was used to assess the relationship between gut microbiota and Trp metabolites. Figure 8(H). The relative abundance of *Bifidobacterium* was positively correlated with IAA and IAId levels (P<0.05), and *Lactobacillus* was positively correlated with IAA levels (P<0.05). Furthermore, *Litchfieldia* was positively correlated with *Trp*, *Faecalibaculum* with IAA and IAId, *Turicibacter* with IAA, and *norank_Clostridia_UCG-014* with IPA (P<0.05). Conversely, *Odoribacter* was negatively correlated with IAA, IAId, and IPA (P<0.05). Alistipes and norank_o_Clostridia_vadinBB60_group were negatively correlated with Trp, IAA, and IAId; Helicobacter was negatively correlated with IAId and IPA; Bacteroides were negatively correlated with Trp and IPA; Prevotellaceae_UCG_001, unclassified_k__norank_d__Bacteria, and unclassified_f__Oscillospiraceae were negatively correlated with IAA; norank_o__RF39 was negatively correlated with IAId (P<0.05).

[0036] In summary, the results of this invention demonstrate that GOS promotes the colonization of Bifidobacteria in animals and synergistically amplifies its protective effect against DSS-induced colitis, with the combined use being superior to either single agent. This synergistic effect is attributed to the coordinated regulation of the gut microbiota, manifested as the enrichment of beneficial genera such as Bifidobacteria and Lactobacillus, and the inhibition of opportunistic pathogens. Simultaneously, enhanced SCFA production and activation of the Trp-AhR-IL-22 pathway further strengthen intestinal barrier integrity and suppress inflammatory responses. This invention provides a theoretical basis for developing synergistic formulations of GOS and Bifidobacteria in animals as an effective dietary intervention for the management of UC.

[0037] It will be apparent to those skilled in the art that the present invention is not limited to the details of the exemplary embodiments described above, and that the invention can be implemented in other specific forms without departing from the spirit or essential characteristics of the invention. Therefore, the embodiments should be considered in all respects as exemplary and non-limiting, and the scope of the invention is defined by the appended claims rather than the foregoing description. Thus, it is intended that all variations falling within the meaning and scope of equivalents of the claims be included within the present invention.

[0038] Furthermore, it should be understood that although this specification describes embodiments, not every embodiment contains only one independent technical solution. This narrative style is merely for clarity. Those skilled in the art should consider the specification as a whole, and the technical solutions in each embodiment can also be appropriately combined to form other embodiments that can be understood by those skilled in the art.

Claims

1. A synthetic preparation, characterized in that, Composed of Bifidobacterium animalis and galactooligosaccharides; wherein the Bifidobacterium animalis is a live bacterium, and its concentration in the synbiotic preparation is 10. 8 ~10¹ 0 CFU / mL; the content of the galactooligosaccharide in the synthetic preparation is 30~90 mg / mL.

2. The synthetic preparation according to claim 1, characterized in that, The animal bifidobacteria are selected from the animal subspecies of Bifidobacterium animalis.

3. The synthetic preparation according to claim 1, characterized in that, The concentration of the animal Bifidobacterium is 10. 9 The concentration of CFU / mL and the content of the galactooligosaccharide is 60 mg / mL.

4. The use of the synthetic preparation according to any one of claims 1-3 in the preparation of products for the prevention and / or treatment of ulcerative colitis.

5. The application according to claim 4, characterized in that, The synbiotic alleviates ulcerative colitis through one or more of the following mechanisms: (a) Upregulates the expression levels of messenger ribonucleic acid of tight junction proteins ZO-1, Occludin and mucin MUC2 in colon tissue, thereby enhancing intestinal barrier function; (b) Decreases the levels of TNF-α, IL-1β, and IL-6 in colon tissue and serum, and increases the level of the anti-inflammatory cytokine IL-10; (c) Reduces malondialdehyde content in colon tissue and serum, increases total superoxide dismutase activity, and inhibits myeloperoxidase activity; (d) Regulates the composition of intestinal flora, enriches Bifidobacterium and Lactobacillus, and inhibits opportunistic pathogens; (e) Increased levels of short-chain fatty acids in colonic contents, including acetic acid, propionic acid, butyric acid, isobutyric acid, isovaleric acid, and total short-chain fatty acids; (f) Increased levels of tryptophan metabolites in colonic contents, including indole-3-acetic acid, indole-3-carboxaldehyde, and indole-3-propionic acid; (g) Upregulates the expression of messenger ribonucleic acid of aryl hydrocarbon receptor and its downstream target gene cytochrome P450 family 1 subfamily A member 1 in colon tissue, increases IL-22 level, and activates the aryl hydrocarbon receptor / IL-22 signaling pathway.

6. A pharmaceutical composition for relieving ulcerative colitis, characterized in that, The active ingredient is a synthetic preparation according to any one of claims 1-3, and pharmaceutically acceptable excipients.

7. The pharmaceutical composition according to claim 6, characterized in that, The dosage form is selected from powder, tablet, capsule, granule, oral liquid or liquid preparation.

Images