Novel intestinal probiotic butyric acid monad and application thereof

Abstract

The invention discloses a novel intestinal probiotic butyric acid monad and application thereof, the novel intestinal probiotic butyric acid monad is preserved in Guangdong Microbiological Culture Collection Center, and the preservation number is GDMCC No.67648. A mouse model proves that the butyric acid monad can promote synthesis of intestinal TMP (Trimethoprim) by remodeling intestinal steady state, and then the TMP is converted into thiamine. Through intestinal liver circulation, thiamine accumulates in the liver and is further converted into TPP. The process promotes the degradation of BCAs, further regulates the lipid metabolism of the liver, and obviously alleviates the progress of metabolic dysfunction related fatty liver diseases induced by high fat diet.

Description

Technical Field

[0001] This invention belongs to the field of microbial medicine technology, specifically relating to a novel intestinal probiotic, Butymonas butyricum, and its application in the treatment of obesity and fatty liver disease related to metabolic dysfunction. Background Technology

[0002] The information disclosed in this background section is intended only to enhance some understanding of the overall background of the invention and is not necessarily to be construed as an admission or in any way implying that the information constitutes prior art known to those skilled in the art.

[0003] Metabolic dysfunction-associated fatty liver disease (MASLD) is the most common chronic liver disease worldwide, coexisting with metabolic syndromes such as obesity and insulin resistance. The disease spectrum can progress from simple fatty liver to steatohepatitis, liver fibrosis, and even cirrhosis. Currently, no specific drugs have been approved for direct treatment of the core pathological changes in MASLD. First-line treatment still relies on lifestyle interventions that are difficult to maintain long-term, indicating a significant unmet clinical need.

[0004] In recent years, the "gut-liver axis" theory has provided a new perspective for the treatment of MASLD. Gut microbiota dysbiosis can promote hepatic lipid accumulation and inflammation through multiple pathways. Although some probiotics (such as Lactobacillus and Bifidobacterium strains) have been reported to improve hepatic steatosis in animal models, their mechanisms of action are mostly attributed to relatively indirect and nonspecific pathways such as regulating intestinal barrier function and reducing systemic low-grade inflammation. These studies have a common limitation: they have failed to directly link specific gut microbiota species to specific, verifiable metabolic regulatory pathways within hepatocytes. Therefore, those skilled in the art cannot predict whether there are any gut microbiota strains, and which strains can precisely target and correct intrinsic hepatic metabolic disorders.

[0005] Of particular note is that branched-chain amino acid (BCAA) metabolism disorders have been identified as a key molecular bridge connecting obesity, insulin resistance, and MASLD. Impaired BCAA catabolism in the liver leads to their abnormal accumulation, directly interfering with lipid metabolism and promoting steatosis. Thiamine (vitamin B1) in its active form—thiamine pyrophosphate (TPP)—is a key coenzyme catalyzing the rate-limiting step in BCAA catabolism. However, the scientific question of whether and how the gut microbiota can regulate hepatic BCAA catabolism and lipid homeostasis by influencing host thiamine metabolism remains completely unanswered in the current technology. In other words, the field lacks a microbial preparation that can treat MASLD through this clear and direct "gut microbiota-thiamine-liver BCAA axis" and its well-defined mechanism of action.

[0006] Therefore, developing a novel probiotic strain with a clear mechanism of action that can directly intervene in the core metabolic pathways of the liver has become an urgent technical challenge to overcome the bottleneck in the treatment of MASLD. Summary of the Invention

[0007] To overcome the shortcomings of existing technologies that lack MASLD microbial therapeutic agents that can precisely target liver metabolic disorders and have a clear mechanism of action, this invention aims to provide a novel strain of butyric acid monocytogenes, its composition and other products, and their specific uses.

[0008] The technical solution adopted in this invention is as follows: In a first aspect, the present invention provides a strain of *Butymonas butyricum* (… Butyricimonas virosa AM16-14 was deposited on January 14, 2026 at the Guangdong Provincial Center for Microbial Culture Collection (GDMCC No. 67648), located at 5th Floor, Building 59, No. 100 Xianlie Middle Road, Guangzhou.

[0009] The butyric acid bacillus strain AM16-14 described in this invention is hereinafter referred to as butyric acid bacillus. B. virosa.

[0010] In a second aspect, the present invention provides a composition comprising the butyric acid monocytogenes described in the first aspect. B. virosa .

[0011] Furthermore, the butyric acid bacteria contained in the above composition B. virosa The viable count is not less than 1×10 6 cfu / g.

[0012] Furthermore, the composition is a microbial agent, wherein the *Butymonas butyricum* is present. B. virosa After fermentation and cultivation, the bacterial cells are collected and mixed with a protectant to prepare a microecological solution or freeze-dried bacterial powder.

[0013] Thirdly, the present invention provides the butyric acid monocytogenes described in the first aspect. B. virosa The use of the composition described in the second aspect in the preparation of products for the prevention and / or treatment of obesity and metabolic dysfunction-associated fatty liver disease (MASLD).

[0014] Furthermore, in the application, the strain works by promoting the production of thiamine and related metabolites in the intestine, thereby increasing the level of thiamine pyrophosphate (TPP) in the liver; the elevated TPP can activate the branched-chain α-keto acid dehydrogenase complex (BCKDH) in the liver, thereby accelerating the degradation of branched-chain amino acids (BCAAs) and ultimately improving liver lipid metabolism disorders.

[0015] Furthermore, in the aforementioned application, the butyric acid mononucleosis... B. virosa Its fermentation products or compositions containing it can be used as the sole active ingredient to alleviate fatty liver disease associated with metabolic dysfunction, or they can be used in combination with one or more other active ingredients with similar functions to constitute the active portion of the product.

[0016] This invention relates to bacteria containing the aforementioned butyric acid monoclonal bacteria. B. virosa The concentration of live bacteria in the product is not strictly limited. Those skilled in the art can determine the effective dosage range through routine experiments based on the target use (e.g., treatment or prevention), dosage form, and route of administration. In some preferred embodiments, the product contains *Butymonas butyrate*. B. virosa The concentration of live bacteria can be approximately 1×10⁻⁶. 8 CFU / mL to 1×10 10 Within the range of CFU / mL, for example, approximately 1×10 9 CFU / mL.

[0017] This invention has experimentally confirmed that *Butymonas butyricum* is effective in the application of this invention. B. virosa It can produce the following combined effect: (1) Improve core liver pathology: reduce hepatic steatosis, inflammatory infiltration and hepatocellular damage; (2) Regulates systemic glucose and lipid metabolism: improves insulin resistance and glucose intolerance, and regulates abnormal lipid profile; (3) Remodeling the gut microbiota: Regulating the structure of the gut microbiota and promoting the production of beneficial metabolites; (4) Activate specific protective pathways: promote thiamine metabolism in the intestine, thereby activating the branched-chain amino acid catabolic axis in the liver.

[0018] Fourthly, the present invention provides a health food product comprising the butyric acid monocytogenes described in the first aspect. B. virosa The microbial cells, their fermentation broth, their fermentation products, or the composition described in the second aspect, as well as food-grade excipients.

[0019] Fifthly, the present invention provides a drug comprising the butyric acid monocytogenes described in the first aspect. B. virosa The bacterial cells, their fermentation broth, their fermentation products, or the composition described in the second aspect, and a pharmaceutically acceptable carrier.

[0020] Furthermore, the dosage form of the drug can be a solution, powder, granules, capsules, or tablets.

[0021] In this invention, the 'fermentation broth' refers to the butyric acid monocytogenes. B. virosaThe culture obtained after fermentation may contain bacterial cells and / or supernatant after cell removal. The 'fermentation product' refers to an active substance obtained by separating, extracting, and / or purifying the fermentation broth, including but not limited to metabolites, proteins, and extracts or purified polysaccharides.

[0022] Compared with the related technologies known to the inventors, one of the technical solutions of the present invention has the following beneficial effects: (1) Provides a novel and effective strain resource: This invention is the first to isolate and preserve a new strain of the genus *Butymonas*. B. virosa This study also demonstrated for the first time that this specific strain has a significant effect in alleviating MASLD in an in vivo model, providing a new candidate strain for the development of microbial therapies for liver diseases.

[0023] (2) Pioneeringly elucidating and verifying the precise mechanism of action: The core contribution of this invention lies in the first complete revelation and rigorous verification of the mechanism by which this strain exerts its effects through a clear "gut-hepatic thiamine-BCAAs metabolic axis": the strain B. virosa →Promotes intestinal TMP synthesis →Increases portal vein and hepatic TPP levels →Activates hepatic BCKDH complex →Accelerates BCAAs degradation →Improves hepatic lipid accumulation and inflammation. This elucidation of the complete molecular chain from intestinal microbial function to the regulation of key enzyme activity in host hepatocytes breaks through the limitations of the broad mechanisms of action of probiotics, providing new scientific evidence and clear drug targets for the precision treatment of MASLD.

[0024] (3) A multi-layered and causally strong rigorous evidence system has been constructed: This invention, through an experimental design that combines superficial and in-depth approaches and positive and negative examples, makes the mechanism highly credible. Phenotypic confirmatory layer: Clearly improved core pathological features such as hepatic steatosis and ballooning degeneration, as well as systemic metabolic indicators.

[0025] Pathway analysis layer: Multi-omics was used to pinpoint the thiamine metabolic pathway and the chain reaction of TPP accumulation, BCKDH activation and BCAA reduction was confirmed at the tissue and cellular levels.

[0026] Causal verification layer: via thiamine antagonists ( Pyrithiamine The experiment completely reversed the therapeutic effect, confirming that this pathway is the only essential pathway for the strain to exert its effect; supplemented by a thiamine-deficient diet model and an antibiotic clearance model, the functional independence and synthetic capacity of the strain were further verified. This system of evidence provides a solid scientific foundation and predictability for the technical solution of this invention.

[0027] With clear application prospects and great transformation potential: Based on its clearly defined strain identity and clear target, this invention can be directly used to develop next-generation precision probiotic drugs and functional foods targeting MASLD, or as a benchmark for screening similar functional strains, and has significant industrial transformation value and clinical application prospects. Attached Figure Description

[0028] The accompanying drawings, which form part of this specification, are used to provide a further understanding of the invention. The illustrative embodiments of the invention and their descriptions are used to explain the invention and do not constitute an improper limitation of the invention.

[0029] Figure 1 It is butyric acid mononucleosis. B. virosa A schematic diagram illustrating the mechanism by which MASLD is alleviated through regulation of gut microbiota and TPP metabolism. (The diagram is labeled "...") B. virosa "" refers to the strain of this invention. The "" appearing in the figures below... B. virosa (They all have the same meaning.) Figure 2 butyric acid bacteria B. virosa It can attenuate HFD-induced MASLD and reverse HFD-induced gut microbiota changes. (A) Experimental design, (B) Weight development, (C) Weight gain, (D) Levels of TC and TG in the liver, (E) Liver tissue morphology, H&E staining, and Oil Red O staining, scale bar 50 µm, (F) H&E score of liver tissue, (G) Oil Red O positive area of ​​liver tissue, (H) NCD, HFD, and B. virosa PCoA clustering diagram of the gut microbiota composition of the strain group mice, (I) NCD, HFD and B. virosa Phylogenetic distribution of strain groups, (J) relative abundance of Bacillota in mouse feces, (K) relative abundance of Bacteroidota in mouse feces, (L) changes in the Bacillota / Bacteroidota ratio in mouse feces, (M) relative abundance of Butymonas in mouse intestines, (N) Spearman coefficient analysis of the correlation between Butymonas abundance and MASLD phenotype.

[0030] Figure 3 butyric acid bacteria B. virosa Effects of intervention on glucose and lipid metabolism. (A) Fasting insulin, (B) Fasting blood glucose, (C) HOMA-IR, (D) Plasma glucose profile measured during IPGTT, (E) AUC of glucose tolerance test, (F) Food intake, (G) Energy intake, (H) H&E stained tissue image of epididymal fat, scale bar at 50µm, (I) Adipocyte diameter.

[0031] Figure 4 butyric acid bacteria B. virosa Effects of intervention on MASLD and systemic inflammation. (A) Liver weight, (B) Liver index, (C) Serum ALT level, (D) Serum AST level, (E) Serum inflammatory factor level.

[0032] Figure 5 butyric acid bacteria B. virosa Intervention alters bacterial thiamine synthesis. (A) PCoA clustering of metabolites in fecal samples, (B) Differential effects on metabolite enrichment analysis, (C) Relative content of metabolites related to thiamine metabolism, (D) Metabolic pathway from amino acids to TMP, (E) Glycine level in fecal samples, (F) Tyrosine level in fecal samples, (G) Cysteine ​​level in fecal samples, (H) Expression of thiamine metabolism-related genes, (I) TMP level in fecal samples, (J) TPP level in fecal samples, (K) Thiamine level in fecal samples, (L) Butymonas vaginalis. B. virosa Changes in in vitro synthesized TMP content, (M) TMP level in portal vein, (N) TPP level in portal vein, (O) thiamine level in portal vein.

[0033] Figure 6 butyric acid bacteria B. virosa Effects of TPP on the degradation of BCAAs in the liver. (A) Liver thiamine level, (B) Liver TMP level, (C) Liver TPP level, (D) Representative immunofluorescence images of liver sections stained with phosphorylated BCKDHA (p-BCKDHA), (E) Detection of p-BCKDHA and BCKDHA levels in liver samples by Western blotting, (F) Quantitative ratio analysis of protein expression, (G) p-BCKDHA and BCKDHA levels in HepG2 cells, (H) Quantitative ratio analysis of protein expression, (I) Liver valine level, (J) Liver leucine level, (K) Liver isoleucine level, (L) mRNA expression levels of lipid metabolism-related genes.

[0034] Figure 7 Butymonas hydrophila treated with inhibitors B. virosa Effects on hepatic thiamine levels. (A) Experimental design, (B) TMP levels in fecal samples, (C) TPP levels in fecal samples, (D) Thiamine levels in fecal samples, (E) TMP levels in liver samples, (F) TPP levels in liver samples, (G) Thiamine levels in liver samples, (H) Final weight gain, (I) Liver weight, (J) Epididymal fat weight, (K) Liver tissue H&E staining and Oil Red O staining, (L) Plasma glucose profile measured during IPGTT, (M) Fasting blood glucose, (N) AUC of glucose tolerance test.

[0035] Figure 8butyric acid bacteria B. virosa Effects of thiamine deficiency on hepatic thiamine levels and BCAA metabolism. (A) Experimental design using a thiamine-deficient diet model, (B) Fecal TMP content, (C) Fecal TPP content, (D) Fecal thiamine content, (E) Serum thiamine level, (F) Liver TPP content, (G) Liver thiamine content, (H) Liver TMP level, (I) Experimental design using an ABX treatment model, (J) Relative abundance of *Butymonas butyrate* in fecal samples, (K) Body weight change, (L) Final body weight gain, (M) Liver weight, (N) Liver tissue H&E staining and Oil Red O staining, (O) Liver TMP content, (P) Liver TPP content, (Q) Liver thiamine content, (R) Western blot analysis of p-BCKDHA and BCKDHA content in mouse liver, (S) Quantitative analysis.

[0036] Figure 9 butyric acid bacteria B. virosa The role of gut microbiota in the remission of MASLD. (A) Epididymal fat mass, (B) Quantitative analysis of liver H&E staining, (C) Quantitative analysis of Oil Red O staining. Detailed Implementation

[0037] It should be noted that the following detailed descriptions are exemplary and intended to provide further illustration of the invention. Unless otherwise specified, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention pertains.

[0038] It should be noted that the terminology used herein is for the purpose of describing particular embodiments only and is not intended to limit the exemplary embodiments of the present invention. As used herein, the singular form is intended to include the plural form as well, unless the context clearly indicates otherwise. Furthermore, it should be understood that when the terms "comprising" and / or "including" are used in this specification, they indicate the presence of features, steps, operations, and / or combinations thereof.

[0039] Where specific techniques or conditions are not specified in the examples, they shall be performed in accordance with the techniques or conditions described in the literature in this field or in accordance with the product instructions. Reagents or instruments whose manufacturers are not specified are all commercially available conventional products.

[0040] To enable those skilled in the art to better understand the technical solution of the present invention, the technical solution of the present invention will be described in detail below with reference to specific embodiments.

[0041] The following examples, using a standard MASLD animal model, validated the efficacy of the strain of the present invention and elucidated its mechanism, providing a solid preclinical experimental basis for the further application and development of this strain.

[0042] Example 1: Butyric acid mononucleosis B. virosa Origin, isolation and identification (1) Sample collection Stool samples were collected from healthy adults and transferred to sample tubes under aseptic conditions, then returned to the laboratory for processing within one hour. All participants were informed of the nature of the sampling and testing and gave their consent. Samples were rigorously screened to exclude patients with the following conditions: a history of other types of cancer, a history of other serious systemic diseases or digestive system diseases, chronic viral infections, preoperative bacterial infections, and those who had received immunosuppressive therapy (such as chemotherapy, oral steroids, etc.) or other cancer-related treatments before surgery.

[0043] (2) Isolation and purification of Butymonas vaginalis Mix 0.2g of fecal sample with 1ml of sterile PBS, serially dilute and spread on MRS anaerobic broth medium, incubate under anaerobic conditions at 37℃, and select single colonies for isolation.

[0044] (3) Preservation of microbial strains The purified strain was cultured to a concentration of approximately 1×10⁻⁶. 9 The strain was prepared at cfu / ml, with the addition of 20% glycerol, and then stored at -80℃ for long-term preservation. To meet patent procedure requirements, the strain was submitted to the Guangdong Provincial Microbial Culture Collection Center on January 14, 2026, and was approved for preservation with the number GDMCC No. 67648.

[0045] (4) Genomic DNA extraction and 16S rDNA identification Genomic DNA was extracted using a commercial kit and amplified by PCR using universal 16S rDNA primers. Sequencing was performed, and the sequence was compared with the EZbiocloud database. The comparison results showed that the sequence was similar to... Butyricimonas virosa The strains exhibited the highest similarity, and were named based on the identified labeling information. Butyricimonas virosa AM16-14, hereinafter referred to as Butymonas B. virosa .

[0046] Based on subsequent embodiments, this invention, through multi-omics integrated analysis, revealed the presence of *Butyrica butyrica*. B. virosa Potential mechanisms of action that alleviate MASLD, such as Figure 1As illustrated in the diagram, after colonizing the gut, this strain primarily affects the vitamin B1 (thiamine) metabolic pathway by regulating the gut microbiota and host metabolism, increasing the level of its active form, TPP. This, in turn, promotes the activation of the hepatic branched-chain α-keto acid dehydrogenase complex (BCKDH) (manifested as a decrease in p-BCKDHA), accelerating the degradation of branched-chain amino acids (BCAAs). Ultimately, it improves hepatic lipid metabolism and systemic glucose metabolism disorders, thereby alleviating MASLD. This mechanism was systematically validated in the following animal experiments (see Examples 2 and 3).

[0047] Example 2 Butyric acid mononucleosis B. virosa Treatment studies on mouse models of fatty liver disease associated with metabolic dysfunction (1) Establishment of animal models In this example, 24 healthy, 6-8 week old SPF C57BL / 6 mice with an average weight of 18-22g were used for the animal experiment. The mice were acclimatized in an SPF-grade laboratory environment for one week and then randomly assigned to four different experimental groups. The groupings were as follows: normal control group, high-fat diet treatment group, and *Butymonas vaginalis* group. B. virosa The processing groups are detailed in Table 1.

[0048] From week 1 to week 12, the normal control group received a normal maintenance diet throughout the entire period, while the high-fat diet treatment group and Butymonas vaginalis group received treatment. B. virosa The treatment group was given a high-fat diet throughout the treatment to induce a metabolic dysfunction-related fatty liver disease model. Weeks 5-12: *Butymonas vaginalis* B. virosa The mice in the treatment group received 1×10 every two days. 9 CFU was administered via gavage. Simultaneously, mice in the high-fat diet treatment group and the normal control group received an equal volume of phosphate-buffered saline (PBS) via gavage. By week 12, the high-fat diet treatment group mice exhibited significant weight gain, elevated fasting blood glucose, and dyslipidemia, marking the successful establishment of an obesity-induced metabolic dysfunction-associated fatty liver disease (MASLD) model.

[0049] Table 1. Mouse Experimental Design Table

[0050] (2) Observation and detection indicators and processing: ① During modeling and gavage, the rate of change in body weight and energy intake of mice were continuously monitored. In week 11 of the experiment, insulin sensitivity was assessed by intraperitoneal glucose tolerance test (IPGTT), the area under the curve (AUC) was calculated, and fasting blood glucose (FBG), serum insulin levels and fecal samples were collected.

[0051] ② In week 12 of the experiment, after fasting overnight, the mice were weighed, anesthetized, and blood was collected from their eyeballs. The mice were then euthanized by cervical dislocation. The collected blood samples were centrifuged to separate the serum, which was then stored at -80°C for subsequent biochemical analysis. The liver and epididymal fat of the mice were weighed, and samples were taken for histological sectioning. Some liver samples were used to prepare pathological sections for microscopic histological analysis. The remaining liver tissue was cryopreserved at -80°C for future experimental research.

[0052] ③ Liver tissue sections were obtained, fixed in 4% paraformaldehyde, routinely embedded in paraffin, and sectioned. Hematoxylin and eosin (H&E) staining and Oil Red O staining were performed to assess hepatic steatosis, lobular inflammation, and ballooning degeneration, and MASLD pathological scoring was conducted. Simultaneously, transaminase (ALT, AST) and hepatic lipid levels were measured in the liver tissue. The criteria are as follows: Pathological scoring includes steatosis, lobular inflammation, and hepatocyte ballooning degeneration; Steatosis (scored according to the proportion of hepatocyte steatosis): 0-5% = 0 points; 5-33% = 1 point; 34-66% = 2 points; over 66% = 3 points; Lobular inflammation (scored according to the degree of inflammatory cell aggregation): No inflammation = 0 points; Mild = 1 point; Moderate = 2 points; Significant = 3 points; Ballooning degeneration (scored according to the number of hepatocyte ballooning degenerations): None = 0 points; Occasionally = 1 point; Frequently = 2 points. Specific pathological histological scoring criteria are shown in Table 2.

[0053] Table 2. Histopathological Scoring Criteria

[0054] like Figure 2 As shown, Butyric acid bacteria B. virosa The intervention significantly reduced weight gain ( Figure 2 BC), and downregulated the levels of TC and TG in the liver ( Figure 2 D). Based on histological evaluation, *Butymonas vaginalis* was significantly higher in mice compared to those fed with HFD. B. virosa After intervention, hepatic lipid accumulation, inflammatory cell infiltration, and fibrosis were significantly reduced. Figure 2 E and Figure 2 F). Furthermore, Oil Red O staining indicated that a HFD diet significantly increased fat accumulation in the liver, while Butymonas vaginalis... B. virosa Intervention reduces fat accumulation. Figure 2 E and Figure 2 G). The above experimental data show that Butyric acid mononucleosis B. virosa The intervention had a positive impact on body weight, liver lipids, and histopathological parameters in MASLD model mice. Metagenomics and metabolomics analyses were used to further explore the potential anti-MASLD mechanism of AM16-14. (Butymonas butyrate) B. virosa Intervention modulates the composition of the gut microbiota ( Figure 2 H), and reversed the difference caused by HFD at the gate level ( Figure 2 I). At the phylum level, *Butymonas vaginalis* B. virosa Intervention can slightly inhibit Bacillus phylum ( Figure 2 J), and can significantly counteract HFD-induced Bacteroidetes ( Figure 2 K). The decrease in the Bacillota / Bacteroidota ratio further indicates that Butymonas vaginalis... B. virosa Intervention improved the balance of gut microbiota ( Figure 2 L). These data indicate that Butyric acid mononucleosis B. virosa The changes in gut microbiota induced by the intervention were associated with its ameliorative effect on systemic metabolism. Metagenomic analysis also showed that the abundance of *Butymonas butyrate* in the feces of mice fed AM16-14 was significantly increased compared with mice fed HFD alone. Figure 2 M). In the MASLD model, the abundance of *Butymonas vaginalis* was negatively correlated with liver total cholesterol (TC), serum triglycerides (TG), body weight, serum IL-6, fasting blood glucose, adipocyte diameter, and liver weight. Figure 2 N). In summary, Butyric acid bacteria B. virosa By modulating gut microbiota composition and gut metabolites, metabolic disorders associated with HFD-induced MASLD can be improved.

[0055] like Figure 3 As shown, Butyric acid bacteria B. virosa Administration significantly reduced fasting insulin ( Figure 3 A) Fasting blood glucose ( Figure 3 B) and HOMA-IR ( Figure 3 C). Compared with the HFD group, *Butymonas vaginalis* B. virosa The increase in blood glucose levels in the treated mice was significantly reduced after glucose loading. Figure 3 DE), but energy intake remained unchanged ( Figure 3 FG). The results showed that continuous administration of AM16-14 improved glucose tolerance and insulin sensitivity. Furthermore, we observed a significant decrease in the size of adipocytes in the epididymal fat (FG). Figure 3 The results (HI) indicate that the high-fat diet-induced MASLD-related adipose tissue dysfunction was effectively alleviated.

[0056] like Figure 4 As shown, compared with the HFD group, mice supplemented with Butymonas vaginalis... B. virosa Significantly reduced liver weight ( Figure 4 A) Liver index ( Figure 4 B). In addition, *Butyricum* B. virosaTreatment reduced serum AST and ALT levels in mice ( Figure 4 CD) also reduced the levels of serum inflammatory factors TNF-α, IL-1β, and IL-6, and increased the level of anti-inflammatory cytokine IL-10 (CCD). Figure 4 E). In summary, these findings suggest that supplementing with Butymonas vaginalis... B. virosa It can be used as a potential adjunctive treatment to relieve MASLD symptoms and improve metabolic abnormalities.

[0057] like Figure 5 As shown, in order to further study Butymonas vaginalis B. virosa To investigate the mechanisms by which MASLD is alleviated, we conducted both non-targeted and targeted metabolomics analyses. For example... Figure 5 As shown in A, Butyric acid bacteria B. virosa Treatment significantly altered the composition of metabolites in fecal samples. Furthermore, enrichment analysis indicated that supplementation with *Butymonas butyrate* significantly improved the metabolite composition. B. virosa Subsequently, the pathways involved in thiamine metabolism underwent the most significant changes. Figure 5 B). Further examination of thiamine-related metabolites revealed that in *Butymonas*... B. virosa After treatment, thiamine and L-tyrosine levels increased significantly, while thiol levels decreased significantly. Figure 5 C). In addition to L-tyrosine, we also observed significant changes in metabolic pathways associated with multiple amino acids, including arginine, proline, alanine, and glycine. Figure 5 B). Amino acids are key precursor molecules in the synthesis of thiamine. Among them, glycine, tyrosine, and cysteine ​​are the most important amino acids involved in this process. Figure 5 D). Subsequently, we performed targeted detection of the content of these three amino acids. The results showed that *Butymonas vaginalis*... B. virosa Treatment significantly increased the levels of glycine and cysteine. Figure 5 EG). Based on the thiamine biosynthesis pathway in prokaryotes, the metabolic process of converting amino acids into thiamine requires the prior synthesis of TMP (…). Figure 5 D). Based on the results of RT-qPCR, *Butymonas vaginalis* B. virosa Treatment significantly upregulated the mRNA levels of bacterial thiamine metabolism-related genes in the gut microbiota. Figure 5 H). Butyric acid mononuclear bacteria B. virosa The expression of the genes ThiG, ThiE, and ALPI in the treated bacteria was significantly upregulated. These genes are involved in the biosynthesis of TMP and thiamine. Conversely, the expression of ThiL, a key enzyme in the conversion of TMP to TPP, was significantly downregulated. To further quantify the levels of thiamine-related metabolites, we performed targeted metabolomics analysis, measuring the levels of TMP, TPP, and thiamine in fecal samples. (In *Butymonas butyricum*) B. virosa After treatment, we observed an increase in the levels of TMP, TPP, and thiamine in the feces. Figure 5 IK). In addition, it is *Butyricum*. B. virosa Providing the precursors required for the synthesis of thiamine significantly increased the TMP content in the culture medium after culture. Figure 5 L). To identify Butyrica... B. virosa To determine whether TMP from this source enters the liver directly via enterohepatic circulation, we measured the levels of TMP, TPP, and thiamine in the portal vein. Figure 5 The results showed that *Butyricum* B. virosa The TMP level in the portal vein of the treatment group was significantly higher than that of the high-fat diet group, confirming the pathway of TMP transport from the intestine to the liver. These data indicate that *Butymonas vaginalis*... B. virosa The intervention improved the utilization rate of bacterial-derived thiamine. Subsequent validation experiments (see Example 3) Figure 7 Further research will be conducted to explore the relationship between this pathway and the remission of hepatic steatosis.

[0058] like Figure 6 As shown, to investigate whether increased thiamine levels in the gut lead to changes in thiamine content and lipid metabolism in the liver, we measured the levels of thiamine, TMP, and TPP in the liver. Figure 6 As shown in AC, a high-fat diet significantly reduced the levels of hepatic thiamine, TMP, and TPP, while butyric acid mononucleosis... B. virosa Treatment significantly increased hepatic thiamine and TPP concentrations. Branched-chain amino acids have been shown to play diverse metabolic roles and are associated with insulin resistance and other metabolic disorders. The branched-chain α-keto acid dehydrogenase E1 subunit α (BCKDHA) regulates the degradation of BCKDHA, while TPP, as a coenzyme, is crucial for the function of the E1 component in the BCKDH complex, promoting the dephosphorylation and activation of BCKDHA. Figure 6 As shown in the EG, the level of p-BCKDHA in the liver of MASLD mice was significantly increased, while that of Butymonas vaginalis was significantly increased. B. virosa Intervention resulted in decreased BCKDHA phosphorylation levels. In the colon, TMPs are normally taken up directly by intestinal epithelial cells and used for metabolic processes. Thiamine can be further transported to the liver via the portal vein, where it is subsequently converted to TPP. In vitro analysis in HepG2 cells showed that thiamine treatment enhanced BCKDHA dephosphorylation. Conversely, the addition of bacimethrin prevented the conversion of thiamine to TPP, thereby inhibiting this dephosphorylation process. Figure 6 HI). In the AM16-14 treatment group, there was an increase in liver TPP levels ( Figure 6 C) Accompanied by BCKDHA dephosphorylation ( Figure 6Increased EG and decreased liver BCAA levels ( Figure 6 IK). Therefore, we found that HFD increased the level of branched-chain amino acids in the liver (IK). Figure 6 JL), while butyric acid bacteria B. virosa Treatment significantly reduced valine and leucine levels. Therefore, these results indicate that gut microbiota-derived thiamine metabolism enhances branched-chain amino acid metabolism by regulating hepatic TPP levels and BCKDHA activity. Finally, we determined the expression of genes playing important roles in hepatic metabolic regulation. This analysis showed that after consuming a high-fat diet, adiponectin, ACOX1 , Fgfr1c and βklotho The expression of [a substance] was significantly reduced, but using [a strain of] butyric acid bacteria [was effective]. B. virosa After intervention, adiponectin, ACOX1 , Fgfr1c and βklotho The expression of was significantly increased ( Figure 6 L). Therefore, Butyric acid bacteria B. virosa By increasing liver TPP levels, it promotes the degradation of BCAAs, improves various metabolic parameters, and increases the expression of genes related to lipid catabolism and the FGF21-related signaling pathway.

[0059] Example 3 Butyric acid mononucleosis B. virosa Mechanism validation study of a mouse model of fatty liver disease associated with metabolic dysfunction (1) Establishment of animal models Experiment 1: In this animal experiment, 32 healthy 6-8 week old SPF C57BL / 6 mice with an average weight of 18-22g were selected. The mice were acclimatized in an SPF-grade laboratory environment for one week and then randomly assigned to four different experimental groups. The groupings were as follows: normal control group, high-fat diet treatment group, and *Butymonas vaginalis* group. B. virosa The treatment group and the Pyri combined treatment group are detailed in Table 3.

[0060] From week 1 to week 12, the normal control group received a normal maintenance diet throughout the entire period, while the high-fat diet treatment group and Butymonas vaginalis group received treatment. B. virosa The treatment group was given a high-fat diet throughout the treatment to induce a metabolic dysfunction-related fatty liver disease model. Weeks 5-12: *Butymonas vaginalis* B. virosa The mice in the treatment group received 1×10 every two days. 9 Mice in the CFU bacterial solution administered via gavage, and mice in the Pyri combination treatment group received 1×10⁻⁶ CFU twice daily. 9Mice were administered CFU via gavage and pyrithiamine (500 μg / kg) via intraperitoneal injection. Simultaneously, mice in the high-fat diet treatment group and the normal control group received an equal volume of phosphate-buffered saline (PBS) via gavage. By week 12, the high-fat diet treatment group exhibited significant weight gain, elevated fasting blood glucose, and dyslipidemia, marking the successful establishment of an obesity-induced metabolic dysfunction-associated fatty liver disease (MASLD) model.

[0061] Experiment 2: In this animal experiment, 28 healthy SPF C57BL / 6 mice, aged 6–8 weeks with an average weight of 18–22 g, were selected. The mice were acclimatized in an SPF-grade laboratory environment for one week and then randomly assigned to four different experimental groups. The groupings were as follows: high-fat diet treatment group, high-fat diet butyric acid bacteria group, and [other groups]. B. virosa Treatment group, thiamine-deficient high-fat diet group, thiamine-deficient high-fat diet group, butyric acid mononucleosis B. virosa Treatment group, detailed in Table 3.

[0062] After mice were fed an HFD diet for 4 weeks, the thiamine-deficient high-fat diet group and the thiamine-deficient high-fat diet group were compared with the butyric acid monoclonal antibody group. B. virosa The treatment group received a two-week high-fat diet treatment for thiamine deficiency, and the thiamine-deficient high-fat diet was associated with butyric acid bacteria. B. virosa Treatment group and high-fat diet with butyric acid bacteria B. virosa The treatment group received (1×10) every two days. 9 The patients received CFU (carbohydrated sulfonamide) via gavage, while the thiamine-deficient high-fat diet group and the high-fat diet treatment group received phosphate-buffered saline (PBS) via gavage every two days.

[0063] Experiment 3: In this animal experiment, 12 healthy 6-8 week old SPF C57BL / 6 mice with an average weight of 18-22g were selected. The mice were acclimatized in an SPF-grade laboratory environment for one week and then randomly assigned to two different experimental groups. The groupings were as follows: antibiotic bowel cleansing group and antibiotic bowel cleansing group (containing *Butymonas butyricum*). B. virosa Treatment group, detailed in Table 3.

[0064] Weeks 1-12, antibiotic bowel cleansing group and antibiotic bowel cleansing with butyric acid bacteria B. virosa The treatment group received a high-fat diet throughout the treatment to induce a metabolic dysfunction-related fatty liver disease model. In week four, they underwent bowel cleansing with a quadruple antibiotic regimen (ampicillin 200 mg / kg; metronidazole 200 mg / kg; vancomycin 100 mg / kg; neomycin 200 mg / kg). From weeks 5 to 12, antibiotics were used to cleanse the bowels of *Butyrosporum* bacteria. B. virosa The mice in the treatment group received 1×10 every two days. 9CFU was administered via gavage. Meanwhile, mice in the antibiotic bowel cleansing group and the control group received an equal volume of phosphate-buffered saline (PBS) via gavage.

[0065] Table 3. Mouse Experimental Design Table

[0066] (2) Observation and detection indicators and processing: ① During modeling and gavage, the rate of change in body weight and energy intake of mice were continuously monitored. In week 11 of the experiment, insulin sensitivity was assessed by intraperitoneal glucose tolerance test (IPGTT), the area under the curve (AUC) was calculated, fasting blood glucose (FBG) was measured, and fecal samples were collected.

[0067] ② In week 12 of the experiment, after fasting overnight, the mice were weighed, anesthetized, and blood was collected from their eyeballs. The mice were then euthanized by cervical dislocation. The collected blood samples were centrifuged to separate the serum, which was then stored at -80°C for subsequent biochemical analysis. The liver and epididymal fat of the mice were weighed, and samples were taken for histological sectioning. Some liver samples were used to prepare pathological sections for microscopic histological analysis. The remaining liver tissue was cryopreserved at -80°C for future experimental research.

[0068] ③ After fixing the liver with 4% paraformaldehyde, it was routinely embedded in paraffin and sectioned to obtain liver tissue sections. Hematoxylin-eosin (H&E) staining and Oil Red O staining were performed to assess the degree of liver degeneration.

[0069] like Figure 7 As shown, to further verify this, we used pyrithiamine (Pyri), a structural analog of thiamine, as a thiamine antagonist to competitively inhibit the absorption and utilization of thiamine. Figure 7 A). Supplement with Butyric Acid Monoclonal bacteria B. virosa These levels were partially restored, but co-treatment with Pyri did not reverse this restoration, indicating that Pyri cannot regulate intestinal thiamine excretion. Figure 7 (BD). Furthermore, Pyri combined with other medications eliminated *Butymonas butyricum*. B. virosa The resulting increase in hepatic TPP and thiamine confirms that thiamine has the ability to specifically inhibit the production or retention of hepatic TPP. Figure 7 EG). Compared to NCD, HFD increases body weight ( Figure 7 H) and liver weight ( Figure 7 I), while the effect of the AM16-14 treatment group was weakened. (Compared to Butymonas) B. virosaIn contrast, Pyri combined treatment partially reversed the aforementioned improvements, leading to increased body weight and liver weight in mice. Figure 7 K). Pyri does not affect the weight of white adipose tissue. Figure 7 J). with Butyric acid bacteria B. virosa Compared to the treatment group, mice receiving Pyri combined treatment showed significantly higher blood glucose levels after glucose loading. Figure 7 LN). Adiponectin, through Pyri combination therapy, was used to treat... ACOX1 , Fgfr1c and βklotho Restored to HFD level ( Figure 7 L). These results indicate that Butyric acid mononucleosis B. virosa Supplementing the promoted intestinal thiamine synthesis is a key pathway to increase hepatic TPP accumulation and thereby regulate branched-chain amino acid and lipid metabolism.

[0070] like Figures 8-9 As shown, to investigate the importance of TPP in the liver, we performed Butymonas vaginalis assays. B. virosa Gavage therapy, while simultaneously administering a high-fat thiamine-deficient (HFTD) diet. Figure 8 A). Given that HFTD feeding causes severe neurological damage in mice, a two-week HFTD dietary intervention is typically implemented under normal experimental conditions. Two weeks of HFTD treatment significantly reduced fecal TMP (…). Figure 8 B), TPP ( Figure 8 C) and thiamine ( Figure 8 The level of D). In contrast, Butyrica... B. virosa Treatment increased fecal TMP ( Figure 8 B), TPP ( Figure 8 C) and thiamine levels ( Figure 8 D). We further examined serum thiamine levels and found that HTFD feeding significantly reduced serum thiamine levels. (Butymonas butyricum) B. virosa Treatment did not significantly increase serum thiamine concentration. Figure 8 E). However, Butyric acid bacteria B. virosa Post-treatment liver TPP ( Figure 8 F) and thiamine ( Figure 8 G) levels were significantly elevated. However, liver TMP levels ( Figure 8 H) Supplementing with butyric acid monocytogenes B. virosa No statistically significant changes were observed afterward. Therefore, these data suggest that thiamine derived from gut microbiota plays a crucial role in restoring host thiamine metabolite homeostasis.

[0071] To further study Butyrica... B. virosaThe potential role of butyric acid mononucleosis was investigated after treatment with antibiotics (ABX). B. virosa treat( Figure 8 I). Compared with the ABX group, the ABX group mice had more Butymonas citrate in their feces. B. virosa The number has increased significantly ( Figure 8 J). After ABX treatment, Butyric acid mononucleosis B. virosa It can still reduce the weight loss caused by HFD ( Figure 8 K), weight gain ( Figure 8 L), liver quality ( Figure 8 M) and epididymal fat mass ( Figure 9 An increase in A). In addition, *Butymonas vaginalis*. B. virosa The processing reduced the characteristics of MASLD ( Figure 8 N and Figure 9 B) reduces liver fat accumulation ( Figure 9 C). In addition, Butyric acid bacteria B. virosa Significantly improved TMP ( Figure 8 O), TPP ( Figure 8 P) and thiamine ( Figure 8 The level of phosphorylated BCKDHA (p-BCKDHA) in the liver was analyzed. We found that even after ABX treatment, *Butymonas vaginalis*... B. virosa The treatment still enhanced the dephosphorylation of BCKDHA. Figure 8 RS). This indicates that despite antibiotic intervention, *Butymonas vaginalis*... B. virosa Its beneficial effects in regulating liver metabolism and mitigating the effects of HFD remain intact, indicating that *Butymonas vaginalis* has preserved these benefits. B. virosa It plays a crucial role in regulating thiamine levels and influencing the phosphorylation state of key metabolic enzymes during BCAA degradation.

[0072] Example 4: Butyric acid-containing bacteria B. virosa Preparation of microecological solution preparations (1) Preparation of probiotic suspension Butyric acid bacteria B. virosa Single colonies were inoculated into MRS anaerobic broth and cultured anaerobicly at 37°C until the growth plateau phase, and *Butymonas butyricum* were collected. B. virosa The bacterial suspension was centrifuged at 3000×g and 4℃ for 10 minutes, the supernatant was discarded, the bacterial cells were collected and resuspended in physiological saline to a concentration of 1×10⁻⁶. 9 CFU / mL, which yields Butyrica doxorubicin B. virosa A suspension.

[0073] (2) Microecological preparation formulation Butyric acid bacteria B. virosaThe suspension is mixed with a protectant (e.g., skim milk, trehalose, glycerol or more) according to the number of viable bacteria to prepare a microecological solution suitable for oral administration.

[0074] (3) Formulation stability test The prepared probiotic preparations were subjected to stability testing to ensure the activity of probiotics during storage and use.

[0075] Example 5: Butyric acid-containing bacteria B. virosa Preparation of microecological freeze-dried bacterial powder preparation (1) Probiotic culture Butyric acid bacteria B. virosa Single colonies were inoculated into MRS anaerobic broth and cultured anaerobicly at 37°C until the growth plateau phase, and *Butymonas butyricum* were collected. B. virosa The bacterial suspension was centrifuged at 3000×g and 4℃ for 10 minutes, the supernatant was discarded, and the bacterial cells were collected, mixed with a protective agent, and then freeze-dried to obtain *Butymonas butyric acid*. B. virosa Bacterial powder.

[0076] (2) Microecological preparation formulation Butyric acid bacteria B. virosa The bacterial strain powder is mixed with a protectant (e.g., one or more of skim milk, trehalose, glycerol) according to the number of live bacteria to prepare a microecological freeze-dried powder formulation suitable for oral administration.

[0077] (3) Formulation stability test The prepared probiotic preparations were subjected to stability testing to ensure the activity of probiotics during storage and use.

[0078] The above embodiments are preferred embodiments of the present invention, but the embodiments of the present invention are not limited to the above embodiments. Any changes, modifications, substitutions, combinations, or simplifications made without departing from the spirit and principle of the present invention shall be considered equivalent substitutions and shall be included within the protection scope of the present invention.

Claims

1. A strain of *Butyricum* AM16-14, characterized in that, The butyric acid monocytogenes strain AM16-14 was deposited at the Guangdong Provincial Center for Microbial Culture Collection on January 14, 2026, with accession number GDMCC No. 67648.

2. A composition, characterized in that, It includes the butyric acid monoclonal bacteria AM16-14 as described in claim 1.

3. The use of the butyric acid monocytogenes AM16-14 of claim 1 or the composition of claim 2 in the preparation of a medicament for the prevention and / or treatment of obesity and metabolic dysfunction-associated fatty liver disease (MASLD).

4. The application according to claim 3, characterized in that, The drug can promote the biosynthesis of thiamine or its precursor TMP in the intestine.

5. The application according to claim 3, characterized in that, The drug can increase the level of thiamine pyrophosphate (TPP) in liver tissue.

6. The application according to claim 3, characterized in that, The drug can promote the activation of the hepatic branched-chain α-keto acid dehydrogenase complex (BCKDH).

7. The application according to claim 3, characterized in that, The drug can reduce the level of branched-chain amino acids (BCAAs) in liver tissue.

8. A health food product, characterized in that, The mixture comprises the cells of Butymonas butyricum AM16-14 as described in claim 1, its fermentation broth, its fermentation products, or the composition as described in claim 2, and food-grade excipients.

9. A drug, characterized in that, The mixture comprises the cells of Butymonas butyricum AM16-14 as described in claim 1, its fermentation broth, its fermentation products, or the composition as described in claim 2, and a pharmaceutically acceptable carrier.

10. The medicament according to claim 9, characterized in that, The drug is an oral formulation.

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