Use of ruminococcus flavefaciens in modulating intestinal bile acid metabolic disorders

Abstract

This invention discloses the application of *Gastrococcus xanthophylloides* in regulating intestinal bile acid metabolism disorders, including *Gastrococcus xanthophylloides* (… Ruminococcus flavefaciens The application of this invention in the preparation of compositions for regulating intestinal bile acid metabolism disorders, wherein the compositions promote the conversion of primary bile acids to secondary bile acids, wherein the primary bile acids include cholic acid and chenodeoxycholic acid, and the secondary bile acids include deoxycholic acid and lithocholic acid; wherein the intestinal bile acid metabolism disorders include metabolic disorders caused by antibiotics, wherein the antibiotics include one or more of moxifloxacin and metronidazole; wherein the compositions can be health products, pharmaceuticals, or feed; and wherein the active ingredient of the compositions includes xanthospirococcus flavus.

Description

Technical Field

[0001] This invention belongs to the field of microbial technology, specifically relating to the application of *Gastrococcus xanthospira* in regulating intestinal bile acid metabolism disorders. Background Technology

[0002] Bile acids (BAs) are among the most important metabolic products of the gut microbiota. They dissolve dietary lipids by forming micelles in the small intestine, promoting lipid absorption and excretion. In addition, bile acids can act as hormones, regulating lipid and glucose homeostasis and immune signaling. Bile acids are synthesized in the liver and excreted into the intestine via bile. The gut microbiota can convert conjugated primary bile acids into unconjugated primary bile acids and hydrophobic secondary bile acids. Conjugated bile acids are converted into unconjugated bile acids by bile salt hydrolase (BSH), and further converted into secondary bile acids, namely deoxycholic acid (DCA) and lithocholic acid (LCA), by hydroxysteroid dehydrogenase. The composition of the gut microbiota and the regulation of host bile acid transport and biosynthesis jointly determine the structure of the bile acid pool.

[0003] However, there are few reports on the application of xanthospirococcus in mediating intestinal bile acid metabolism. Summary of the Invention

[0004] The purpose of this section is to outline some aspects of embodiments of the present invention and to briefly describe some preferred embodiments. Simplifications or omissions may be made in this section, as well as in the abstract and title of the invention, to avoid obscuring the purpose of these documents; however, such simplifications or omissions should not be construed as limiting the scope of the invention.

[0005] In view of the problems existing in the above and / or prior art, the present invention is proposed.

[0006] Therefore, the object of this invention is to overcome the shortcomings of the prior art and provide *Gastrococcus xanthophyllus* (… Ruminococcus flavefaciens Application in the preparation of compositions that regulate intestinal bile acid metabolism disorders.

[0007] To solve the above-mentioned technical problems, the present invention provides the following technical solution: the composition is used to promote the conversion of primary bile acids to secondary bile acids, wherein the primary bile acids include cholic acid and chenodeoxycholic acid, and the secondary bile acids include deoxycholic acid and lithocholic acid.

[0008] In a preferred embodiment of the application described in this invention, the intestinal bile acid metabolism disorder includes metabolic disorders caused by antibiotics, wherein the antibiotics include one or more of moxifloxacin and metronidazole.

[0009] As a preferred embodiment of the application described in this invention, the composition comprises one of health products, pharmaceuticals, and feed.

[0010] Another object of the present invention is to overcome the shortcomings of the prior art and provide a composition for regulating intestinal bile acid metabolism disorders.

[0011] To solve the above-mentioned technical problems, the present invention provides the following technical solution: the active ingredient of the composition includes *Gastrococcus xanthophyllosis* (…). Ruminococcus flavefaciens ).

[0012] Beneficial effects of this invention: (1) This invention analyzed the correlation between differential bile acid metabolism and differences in gut microbiota levels. Real-time quantitative PCR (qPCR) results further confirmed that *Gastrococcus xanthophyte* (… Ruminococcus flavefaciens The changes in secondary bile acids are most closely related to these changes.

[0013] (2) This invention reveals for the first time that, compared with cefixime and clarithromycin, moxifloxacin and metronidazole inhibit the conversion of primary bile acids to secondary bile acids in the intestine, which is related to their regulation of Ruminococcus, and thus discovers that xanthorumenococci ( Ruminococcus flavefaciens These bacteria are key to the conversion of primary bile acids into secondary bile acids. Attached Figure Description

[0014] To more clearly illustrate the technical solutions of the embodiments of the present invention, the drawings used in the following description of the embodiments will be briefly introduced. Obviously, the drawings described below are only some embodiments of the present invention. For those skilled in the art, other drawings can be obtained based on these drawings without creative effort.

[0015] Figure 1 This is a PCoA analysis diagram of the intestinal flora in each group in Example 1 of the present invention.

[0016] Figure 2 This is a statistical chart of the Chao1 index and Shannon index of each group of intestinal flora in Example 1 of the present invention.

[0017] Figure 3 This is a stacked bar chart showing the percentage of each group of intestinal flora at the phylum level in Example 1 of the present invention.

[0018] Figure 4 This is a box plot showing the relative abundance of gut microbiota at the phylum level in each group in Example 1 of the present invention.

[0019] Figure 5 This is a stacked bar chart showing the percentage of each group of intestinal flora at different genera in Example 1 of the present invention.

[0020] Figure 6 This is a Venn diagram of the intestinal flora of each group in Example 1 of the present invention.

[0021] Figure 7 This is a statistical chart of the number of ASVs in each group of intestinal flora in Example 1 of the present invention.

[0022] Figure 8 This is a graph showing the effect of antibiotic-induced intestinal flora dysbiosis on bile acids in fecal samples in Example 1 of the present invention.

[0023] Figure 9 This is a Spearman correlation heatmap of bile acids and gut microbiota in each group in Example 1 of the present invention.

[0024] Figure 10 This is a scatter plot showing the correlation between rumenococci and various bile acids, as well as the ratios of CDCA / LCA and CA / DCA in each group of Examples 1 of the present invention.

[0025] Figure 11 This is a scatter plot showing the correlation between Clostridium and various bile acids, as well as the CDCA / LCA and CA / DCA ratios in each group of the present invention in Example 1.

[0026] Figure 12 This is a scatter plot showing the correlation between Bacteroides and various bile acids, as well as the CDCA / LCA and CA / DCA ratios in each group of Examples 1 of the present invention.

[0027] Figure 13 This is a scatter plot showing the correlation between coprophytic sterol-producing eubacteria and various bile acids, as well as the ratios of CDCA / LCA and CA / DCA in each group of Example 1 of the present invention.

[0028] Figure 14 This is a scatter plot showing the correlation between lactobacilli and various bile acids, as well as the ratios of CDCA / LCA and CA / DCA in each group of the present invention in Example 1.

[0029] Figure 15 This is a graph showing the changes in the abundance of Bacteroidetes, Lactobacilliaceae, Clostridium, Ruminococciaceae, and Eubacteriaceae families after treatment with four antibiotics in Example 1 of the present invention.

[0030] Figure 16 The images show the activity of bile salt hydrolase and 7α-hydroxysteroid dehydrogenase in intestinal bacteria in Example 1 of this invention, as well as the correlation scatter plots of the activities and relative activities of Ruminococci with 7α-hydroxysteroid dehydrogenase and BSH with BSH.

[0031] Figure 17 The graph shows the correlation between Ruminococcus species and bile acids, as well as a real-time quantitative PCR (qPCR) graph. Detailed Implementation

[0032] To make the above-mentioned objects, features and advantages of the present invention more apparent and understandable, the specific embodiments of the present invention will be described in detail below with reference to the examples in the specification.

[0033] Many specific details are set forth in the following description in order to provide a full understanding of the invention. However, the invention may also be practiced in other ways different from those described herein, and those skilled in the art can make similar extensions without departing from the spirit of the invention. Therefore, the invention is not limited to the specific embodiments disclosed below.

[0034] Secondly, the term "one embodiment" or "embodiment" as used herein refers to a specific feature, structure, or characteristic that may be included in at least one implementation of the present invention. The phrase "in one embodiment" appearing in different places in this specification does not necessarily refer to the same embodiment, nor is it a single or selective embodiment that is mutually exclusive with other embodiments.

[0035] The 7-week-old male Wistar rats used in this invention were purchased from Shanghai Slack Laboratory Animal Co., Ltd. (Shanghai, China).

[0036] The Omega PF Mag-Bind fecal DNA extraction kit used in this invention was purchased from Bio-tek, Georgia, USA.

[0037] In this invention, xanthospirococcus ( Ruminococcus flavefaciens The species is *Ruminococcus faecalis* with the international designation DSM 25089. It is available for purchase from Grey Algae Biotechnology Co., Ltd. and can be considered to be publicly available without the requirement for preservation.

[0038] Example 1 This embodiment was used to investigate the effects of four antibiotic-induced gut microbiota dysbiosis on the bile acid profile in fecal samples, specifically: (1) Animal treatment: After rats were acclimatized for one week under a 12-hour light / dark cycle and a temperature of 21±2℃, they were randomly divided into 5 groups: control group (CT), cefixime treatment group (CEF), clarithromycin treatment group (CLR), moxifloxacin treatment group (MFL) and metronidazole treatment group (MDZ). During the period, they had free access to food and water. Rats in the antibiotic treatment group were administered cefixime (20 mg / kg), clarithromycin (50 mg / kg), moxifloxacin (40 mg / kg), and metronidazole (160 mg / kg) by gavage for 3 consecutive days. The control group was given 0.25% sodium carboxymethyl cellulose (CMC-Na) solution as a solvent for 3 consecutive days; After 3 days of treatment, the rats were fasted overnight for 12 hours, and fecal samples were collected and stored at -80°C for subsequent analysis of gut microbiota and bile acids.

[0039] (2) 16S rRNA sequencing analysis: Total genomic DNA was extracted from fecal samples using the Omega PF Mag-Bind fecal DNA extraction kit according to the manufacturer's operating procedures; DNA concentration was determined by 1.0% agarose gel electrophoresis and a NanoDrop® ND-2000 spectrophotometer (Thermo Fisher Scientific, Inc., USA). The hypervariable region V3-V4 of the microbial 16S rRNA gene was amplified using primer pairs 338F (5'-ACTCCTACGGGAGGCAGCAG-3') and 806R (5'-GGACTACHVGGGTWTCTAAT-3') via an ABI GeneAmp® 9700 PCR thermal cycler (ABI, California, USA). The PCR reaction system included 4 μL of 5×Fast Pfu buffer, 2 μL of 2.5 mM dNTPs, 0.8 μL of each primer (5 μM), 0.4 μL of Fast Pfu polymerase, 10 ng of template DNA, and enzyme-free water to a final volume of 20 μL. The PCR amplification cycling conditions were as follows: 95℃ pre-denaturation for 3 min, followed by 23 cycles (95℃ denaturation for 30 s, 55℃ annealing for 30 s, 72℃ extension for 45 s), and a final extension at 72℃ for 10 min, with the reaction terminated at 4℃. All samples underwent three replicate amplifications. The final PCR product library was analyzed by 2% agarose gel electrophoresis to detect the presence of the 600 bp target fragment, and then quantified using a Quantus™ fluorometer (Promega Corporation, USA). The purified amplicons were mixed in equimolar amounts and subjected to paired-end sequencing by Hangzhou Guangke Ande Technology Co., Ltd. (Hangzhou, China) on an Illumina PE250 platform (Illumina Corporation, California, USA) according to the operating instructions.

[0040] (3) Intestinal bile acid test analysis: Weigh the fecal sample, add ultrapure water at a mass-to-volume ratio of 1:5 (g / mL) to homogenize, centrifuge at 3000×g for 5 min, take 200 μL of supernatant, add 10 μL of internal standard, then add 200 μL of acetonitrile and 10 μL of 14% ammonia, vortex mix for 3 min, centrifuge at 4℃ and 13000×g for 10 min, evaporate to dryness at 65℃, redissolve with 100 μL of 50% methanol, vortex mix for 1 min, centrifuge again at 4℃ and 13000×g for 10 min, take the supernatant to detect the bile acid concentration; The separation of target bile acids was performed on a Waters Xterra RP18 column (4.6 mm × 150 mm inner diameter, 5 μm, Waters Corporation, USA) at 40 °C, equipped with a Phenomenex Security Guard™ C18 pre-column (4 mm × 3.0 mm). An Agilent 1200 high-performance liquid chromatography system was used. The mobile phase consisted of 10 mM ammonium acetate (phase A) and acetonitrile (phase B). The flow rate was 0.8 mL / min, the injection volume was 20 μL, and the elution gradient was: initial 30% phase B, maintenance for 16 min, 16–18 min, and so on. Within 24 minutes, the B phase reached 90% and was maintained for 5 minutes. At 23.5 minutes, the B phase decreased to 30%, and then equilibrated for 6.5 minutes under 30% B phase conditions. Mass spectrometry was performed using an API4000 triple quadrupole tandem mass spectrometer (Applied Biosystems, Sciex, USA) in negative ion mode. The limit of quantitation (LLOQ) for all bile acids was 1 ng / mL, and the calibration curves were linear within the quantitation range. Endogenous substances in the blank matrix did not interfere with the detection of bile acid components and internal standards. The relative standard deviations of intra-day and inter-day precision were both less than 15%. The average extraction recoveries of control samples in fecal homogenate ranged from 83.16% to 109.3%, and the matrix effect ranged from 94.64% to 112.86%. All bile acids were stable at room temperature for 24 hours, at 4°C for 72 hours, at -80°C for 3 months, and after repeated freeze-thaw cycles.

[0041] (4) Analysis of bile salt hydrolase (BSH) and 7α-hydroxysteroid dehydrogenase activities: Sterile physiological saline was added to rat feces at a ratio of 5 mL / g, and a fecal suspension was prepared by homogenization. The homogenate was then vortexed until thoroughly mixed and filtered sequentially through disposable cell sieves of 100 μm and 40 μm. Lysozyme was added to the bacterial suspension, and the mixture was sonicated for 10 min, allowed to stand for 10 min, and then centrifuged at 13000 r / min for 10 min at 4 °C. The supernatant was collected and filtered through a 0.22 μm sterile syringe filter. The enzyme solution was then concentrated to 500 μL in an ultrafiltration tube to obtain the enzyme solution. Assay for bile salt hydrolase and 7α-hydroxysteroid dehydrogenase activities: The reaction system consisted of 50 μL enzyme solution, 50 μL pH 7.4 phosphate buffer (PBS), 100 μL 500 μg / mL taurocholic acid (TCA) or 400 μg / mL cholic acid (CA), and 200 μL distilled water. The mixture was gently and thoroughly mixed and incubated at 37°C for 30 min. After the reaction, the enzyme was inactivated by high temperature and centrifuged at 13000 r / min for 10 min. The bile salt hydrolase activity was calculated by detecting the change in taurocholic acid in the solution after the reaction, and the 7α-hydroxysteroid dehydrogenase activity was calculated by detecting the change in cholic acid in the solution. Enzyme activity unit is defined as the amount of enzyme required to catalyze the conversion of 1 μmol of substrate into product per minute at 37℃ and pH=7.4 (1U=1μmol / min). The relative enzyme activity is calculated with the enzyme activity of the control group as 100%. Dilute the above sample 100 times, take 200 μL, add 200 μL of acetonitrile and 10 μL of 14% ammonia solution; vortex mix for 3 min, centrifuge at 4℃ and 13000 r / min for 10 min, evaporate to dryness at 65℃, redissolve with 100 μL of 50% methanol, vortex mix for 1 min, centrifuge again at 4℃ and 13000 r / min for 10 min, take the supernatant to detect the concentrations of CA and TCA.

[0042] (5) RNA extraction and real-time quantitative PCR of Ruminococcus: Total RNA was extracted from fecal samples using the Trizol reagent via the standard phenol-chloroform method. cDNA was then synthesized from 2 μg of total RNA using a reverse transcription kit (ABM, catalog number: G490). Real-time quantitative PCR was performed on the Applied Biosystems QuantStudio Dx platform (Thermo Fisher Scientific, Inc.) using a universal SYBR Green assay. Green (APE×BIO, USA) was used as the fluorescent dye, and the RNA polymerase β subunit gene (rpoB) was used as the internal reference gene for relative quantification. The forward and reverse primer sequences for real-time PCR are as follows: Ruminococcus luteus (forward primer: 5'-CGAACGGAGATAATTTGAGTTTACTTAGG-3'; reverse primer: 5'-CGGTCTCTGTATGTTATGAGGTATTACC-3'); rpoB (forward primer: 5'-AAGGCGAATCCAGCTTGTTVAGCC-3'; reverse primer: 5'-TGACGTTGCATGTTCGCACCCATCA-3').

[0043] The gut microbiota composition in fecal samples after exposure to four antibiotics was analyzed by 16S rRNA amplicon sequencing. The results are as follows: Figures 1-7 As shown. Among them, Figure 1 For PCoA analysis chart, Figure 2 A statistical chart of the Chao1 index and the Shannon index. Figure 3 A stacked bar chart showing the percentage of doors at different levels. Figure 4 Box plot of relative abundance at the phylum level Figure 5 This is a stacked bar chart representing horizontal percentages. Figure 6 For the petal Venn diagram, Figure 7 This is a statistical chart of ASV numbers.

[0044] from Figure 1As can be seen, the cefixime treatment group (CEF) did not show a significant separation trend from the control group (CT). In contrast, the clarithromycin treatment group (CLR), moxifloxacin treatment group (MFL), and metronidazole treatment group (MDZ) showed significant separation from the control group.

[0045] from Figure 2 As can be seen, there are significant differences in the gut microbiota among the four antibiotic treatment groups. The gut microbiota of the control group rats maintained a high degree of diversity and richness, while the diversity and richness of the gut microbiota decreased after antibiotic intervention. Among them, the decrease was most significant in the moxifloxacin treatment group and the metronidazole treatment group.

[0046] Firmicutes, Bacteroidetes, Proteobacteria, Actinobacteria, and Verrucous are the dominant phyla in the mammalian gut, with Firmicutes and Bacteroidetes being the two major phyla. Generally, the ratio of Firmicutes to Bacteroidetes is correlated with gut microbiota composition, while changes in Proteobacteria are considered microbial markers of gut microbiota dysbiosis. Figure 3 As can be seen, the dominant bacterial phyla at the phylum level in rat fecal samples are Firmicutes, Bacteroidetes, and Proteobacteria.

[0047] contrast Figure 4 It can be seen that, compared with the control group, moxifloxacin and metronidazole have the most significant effects on the intestinal flora at the phylum level, with a significant increase in Proteobacteria and a significant decrease in Firmicutes and Bacteroidetes. Cefixime and clarithromycin did not show significant effects on the composition and abundance of the three major intestinal flora studied at the phylum level.

[0048] At the genus level, the gut microbiota composition varied considerably among all groups. Figure 5 and Figure 6 Furthermore, the number of amplicon sequence variants (ASVs) was significantly reduced after antibiotic treatment. Figure 7 ).

[0049] In short, from Figures 1-7 Studies have shown that antibiotic treatment reduces the bacterial diversity of the gut microbiota, with moxifloxacin and metronidazole having the most significant effects.

[0050] The gut microbiota plays a crucial role in bile acid metabolism through a series of enzymatic transformations. Primary bile acids are synthesized in the liver and released into the intestine after conjugation with taurine or glycine. In the distal small intestine and colon, primary bile acids are converted into secondary bile acids by enzymes produced by the gut microbiota. In Example 1, a total of 19 gut bile acids were detected by liquid chromatography-tandem mass spectrometry (LC-MS / MS) to investigate how antibiotic-induced gut microbiota dysbiosis alters the bile acid profile in fecal samples. The results are as follows: Figure 8As shown, A is the PCA analysis chart, B is the differential bile acid heatmap, C~F are statistical charts of CA, CDCA, DCA, and LCA content, respectively, and G~J are statistical charts of the ratios of CA / DCA, CDCA / LCA, (TCA+GCA) / CA, and (TCDCA+GCDCA) / CDCA, respectively.

[0051] Figure 8 Principal component analysis (PCA) showed that the five experimental groups were well separated, indicating that the intestinal bile acid profile after antibiotic treatment was significantly different from that of the control group.

[0052] Analyze the differences in bile acids between each antibiotic treatment group separately, such as Figure 8 B and Figure 8 As shown in Figures C-F, it can be seen that in the intestines of rats treated with moxifloxacin and metronidazole, the levels of primary bile acids cholic acid (CA) and chenodeoxycholic acid (CDCA) were significantly increased, while the levels of secondary bile acids deoxycholic acid (DCA) and lithocholic acid (LCA) were significantly decreased. Therefore, in Figure 8 The ratios of primary bile acids to secondary bile acids (CDCA / LCA and CA / DCA) in GH were significantly increased, indicating that moxifloxacin and metronidazole inhibited the production of secondary bile acids in the intestine. While cefixime and clarithromycin interventions resulted in a decrease in lithocholic acids and an increase in bile acids in the intestine, their effects on intestinal bile acids were far less significant than those of moxifloxacin and metronidazole.

[0053] Furthermore, to explore the relationship between bile acids and gut microbiota, a correlation analysis was performed on altered bile acids and changes in gut microbiota at the anatomical level. The results are as follows: Figures 9-14 As shown. Figure 9 This is a Spearman correlation heatmap. Figures 10-14 The scatter plots show the correlations between Ruminococcus, Clostridium, Bacteroides, coprophytic Eubacterium, Lactobacillus and various bile acids, as well as the CDCA / LCA and CA / DCA ratios.

[0054] The gut microbiota converts conjugated primary bile acids into free primary bile acids through the action of bile salt hydrolases. These free primary bile acids are further converted into secondary bile acids by 7α-hydroxysteroid dehydrogenases. 7α-hydroxysteroid dehydrogenases are key metabolic enzymes determining the production of secondary bile acids in the gut. Bacteroidetes, Lactobacilli, Clostridium, Ruminococci, and Eubacteria are key gut bacterial groups that produce 7α-hydroxysteroid dehydrogenases, mediating the conversion of primary bile acids to secondary bile acids. Figure 9 Spearman analysis showed that the abundance of Bacteroidetes, Clostridium, Ruminococci, and Eubacteriales was negatively correlated with CA and CDCA, and positively correlated with DCA and LCA. According to... Figures 10-14It was also found that the abundance of Bacteroidetes, Clostridium, Ruminococci, and Eubacteriales was negatively correlated with the primary bile acid / secondary bile acid ratios (CDCA / LCA and CA / DCA). Among them, the Ruminococci showed the most significant correlation with CDCA / LCA (r=-0.89, FDR<0.001) and CA / DCA (r=-0.708, FDR<0.01).

[0055] Further comparisons were made of the changes in abundance of Bacteroidetes, Lactobacilliaceae, Clostridium, Ruminococciaceae, and Eubacteriaceae after treatment with four antibiotics. The results are as follows: Figure 15 As shown in the diagram, the abundance of key bacterial groups at the family level reveals that moxifloxacin and metronidazole significantly inhibited the abundance of Lactobacillus, Clostridium, and Ruminococci, while cefixime and clarithromycin did not show a significant effect on Bacteroidetes, Lactobacillus, Clostridium, Ruminococci, or Eubacteria. Therefore, Ruminococci are considered key bacteria in the inhibition of primary bile acid to secondary bile acid conversion caused by moxifloxacin and metronidazole.

[0056] Bile salt hydrolases produced by gut microbiota decompose primary bile acids, which are then further metabolized into secondary bile acids via 7α-hydroxylation. Bile salt hydrolases are highly conserved across all major gut microbiota phyla, including Bacteroidetes, Firmicutes, and Actinobacteria. Firmicutes and Bacteroidetes, particularly Clostridium, Lactobacillus, Ruminococcus, Eubacterium, and Bacteroides, are the main producers of 7α-hydroxysteroid dehydrogenases. The results of detecting the activities of bile salt hydrolases and 7α-hydroxysteroid dehydrogenases in gut bacteria are as follows: Figure 16 As shown in Figures A through B, A and B are statistical graphs of bile salt hydrolase and 7α-hydroxysteroid dehydrogenase activities, respectively; C and E are scatter plots showing the correlation between Ruminococcus and 7α-hydroxysteroid dehydrogenase activities and relative activities, respectively; and D and F are scatter plots showing the correlation between Ruminococcus and BSH activities and relative activities.

[0057] from Figure 16 A and Figure 16 In study B, moxifloxacin and metronidazole were found to significantly reduce the activity of bile salt hydrolases and 7α-hydroxysteroid dehydrogenases in the gut microbiota. Figure 16 As can be seen from C to F, the abundance of Ruminococci is most strongly correlated with the activity of 7α-hydroxysteroid dehydrogenase (r=0.701, FDR<0.001), indicating that a significant decrease in the relative abundance of Ruminococci may lead to a reduction in the production of secondary bile acids.

[0058] Further analysis of the correlation between Ruminococci species and bile acids yielded the following results: Figure 17 As shown in A, from Figure 17 A heatmap showing the correlation between *Gastrococcus xanthophyllus* and bile acids revealed that *Gastrococcus xanthophyllus* (… Ruminococcus flavefaciensIt is most closely associated with the conversion of primary bile acids to secondary bile acids (r<-0.68, FDR<0.001).

[0059] Real-time quantitative PCR (qPCR), results as follows Figure 17 As shown in B, Figure 17 B further confirmed that after intervention with moxifloxacin and metronidazole, xanthospirococci ( Ruminococcus flavefaciens The expression of ) was significantly reduced.

[0060] In summary, this invention first analyzed the gut microbiota composition in rat fecal samples after administration of four antibiotics using 16S rRNA amplicon sequencing, and then detected a total of 19 intestinal bile acids using liquid chromatography-tandem mass spectrometry (LC-MS / MS) to explore how the gut microbiota dysbiosis induced by the four antibiotics altered the bile acid profile in fecal samples.

[0061] Secondly, to explore the relationship between gut microbiota and gut bile acid metabolism, we performed a correlation analysis between differential bile acid metabolism and differences at the gut microbiota family level. This was further confirmed by real-time quantitative PCR (qPCR) using *Gastrococcus xanthophyte* (…). Ruminococcus flavefaciens The changes in secondary bile acids are most closely related to these changes.

[0062] This invention reveals for the first time that, compared with cefixime and clarithromycin, moxifloxacin and metronidazole inhibit the conversion of primary bile acids to secondary bile acids in the intestine, which is related to their regulation of Ruminococcus, and thereby discovers that *Xanthomonas xanthomonas* (… Ruminococcus flavefaciens It plays a key role in mediating the conversion of primary bile acids to secondary bile acids.

[0063] It should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention and are not intended to limit it. Although the present invention has been described in detail with reference to preferred embodiments, those skilled in the art should understand that modifications or equivalent substitutions can be made to the technical solutions of the present invention without departing from the spirit and scope of the technical solutions of the present invention, and all such modifications or substitutions should be covered within the scope of the claims of the present invention.

Claims

1. Xanthomonas acinosa ( Ruminococcus flavefaciens The application of this compound in the preparation of compositions for regulating intestinal bile acid metabolism disorders is characterized by: The composition is used to promote the conversion of primary bile acids to secondary bile acids, the primary bile acids including cholic acid and chenodeoxycholic acid, and the secondary bile acids including deoxycholic acid and lithocholic acid.

2. The application as described in claim 1, characterized in that: The intestinal bile acid metabolism disorder includes metabolic disorders caused by antibiotics, including one or more of moxifloxacin and metronidazole.

3. The application as described in claim 1 or 2, characterized in that: The composition includes one of the following: health products, pharmaceuticals, and animal feed.

4. A composition for regulating intestinal bile acid metabolism disorders, characterized in that: The active ingredient of the composition includes xanthospirococcus ( Ruminococcus flavefaciens ).

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