| Ellagic acid（EA）is a kind of natural polyphenols widely distributed in fruits and nuts.It has many physiological activities,such as anti-tumor and prevention of atherosclerosis.Studies have shown that the absorption rate of EA is very low,and the intestinal flora metabolites Uro-S may be the material basis of the physiological activity of EA,and the difference in intestinal flora composition may lead to the difference in the conversion of Urolitin.In this study,UPLC-TOF-MS/MS was used to analyze EA and Uro-S in the feces of volunteers,and three types of EA metabolites were identified.The intestinal flora was co-cultured with EA in vitro to analyze the metabolism of EA to Uro-S.Antioxidant activities,short-chain fatty acids and other indexes were analyzed.Metagenomic sequencing was used to analyze the microfacies characteristics and metabolic pathway differences of the three metabolic types of human intestinal bacteria.Metabonomics was used to analyze the differential metabolites of intestinal flora of different EA metabolic types and association analysis with KEGG differential metabolic pathway of bacterial flora to explore the role of intestinal flora in the process of EA metabolism to produce different types of Uro-S.The main results are as follows:（1）EA metabolism of intestinal flora with different metabolic typesVolunteers were recruited to take EA,and the types and contents of Uro-S in their feces were determined by UPLC-TOF-MS/MS.Different metabolic types of EA were screened,among which 60.00%（21 persons）were type A,22.86%（8 persons）were type B,and17.14%（6 persons）were type 0.Three metabolic types of intestinal flora were co-cultured with EA in vitro.The results showed that when EA was metabolized in vitro for 8,12 and24 h,the degradation of EA by type A intestinal flora was 34.69%,43.21%and 45.92%.Type B degraded to 48.55%,62.68%and 64.29%.Type 0 degraded to 22.38%,51.24%and56.81%.The total metabolic degree of ellagic acid by different intestinal flora was B>0>A.After 8,12 and 24 h of metabolism,the amount of Uro-A in type A was 43.46±1.70 ng/m L,45.36±3.64 ng/m L and 43.91±1.64 ng/m L,respectively,and no Uro-B was detected.The contents of Uro-A and Uro-B in type B were 126.31±8.27 ng/m L,127.48±6.67 ng/m L,130.42±8.61 ng/m L and 12.25±0.01 ng/m L,11.74±0.16 ng/m L,12.02±0.45 ng/m L,respectively.No Uro-S was detected in type 0.After 4 to 48 h of co-culture in vitro,the DPPH·and ABTS+·scavenging abilities of EA-intestinal microbiota metabolites of type A and type 0 were significantly higher than those of type 0.The FRAP values of all types at 4h were significantly higher than those at other time periods.During the metabolism of EA for 4 to 48 h,NH3-N of different intestinal flora ranged from 16.96 to 45.05 mg/L.At 12 h and 24 h,acetic acid and palmitic acid in type A metabolites increased from zero h to 140.99mg/L,135.16 mg/L,15.01 mg/L,15.71 mg/L,respectively.SCFAs in type B change little.The contents of acetic acid,butyric acid,caproic acid,4-methyloctanoic acid and palmitic acid in type 0 increased with the prolonging of metabolism time,and the contents at 24 h were 104.7 mg/L,1.48 mg/L,0.67 mg/L,1.48 mg/L and 21.82 mg/L,respectively.After metabolism for 24 h,the total content of SCFAs in type A was 153.01 mg/L（5 kinds）,type B was 5.51 mg/L（2 kinds）,and type 0 was 132.11 mg/L（8 kinds）.（2）Changes of intestinal flora during EA metabolismThe results of metagenomic sequencing showed that the metabolites changed significantly during 0-12 h,and the changes were relatively small during 12-24 h.The species diversity of intestinal flora of type A was higher than that of type B and type 0 at the level of family,genus and species,and the diversity of intestinal flora of type B was the lowest.In the metabolic process of EA,Bifidobacterium and Streptococcus are the main dominant bacteria genus of type A,Enterobacterium and Enterococcus are the dominant bacteria genus of type B,and Eschella,Enterococcus and Enterobacterium are the absolute dominant bacteria species of type 0.At the species level,the characteristic dominant strains of type A were Bifidobacterium adolescentis,Bifidobacterium longum and Streptococcus alactolyticus,etc.The dominant strains of type B were Enterococcus faecalis,unclassified bacillus and Enterococcus faecalis（the total abundance of both was 88.99%at 12 h and91.05%at 24 h）.Escherichia coli and Enterococcus avium were the characteristic dominant bacteria of type 0 intestinal flora（the 12 h abundance was 38.33%and 9.75%,and the 24 h abundance was 34.37%and 12.29%,respectively）.The results of correlation between different flora and key metabolic pathways showed that the changes of metabolic pathways of type 0 bacteria were significantly different from those of type A and B bacteria.Type 0was significantly upregulated in Metabolic pathways,which may be the main factor contributing to the non-Uro-S metabolism of EA.The Ribosome pathway and Biosynthesis of amino acid pathway were only positively correlated with bifidobacterium longum and streptococcus in the process of EA metabolism of type A flora.The dominant flora of type B was significantly positively correlated with the PTS pathway,while the dominant flora of type A was significantly negatively correlated with the PTS pathway,while the dominant flora of type A was negatively correlated with the amino acid biosynthesis pathway.It is speculated that the amino acid biosynthesis mediated by intestinal flora and the differences in PTS pathway may be related to the production of different types of Uro-S.（3）Metabolic characteristics of intestinal flora of different types of EAThe results of UPLC-Q-TOF-MS/MS analysis showed that the specific metabolites of type A were creatinine,γ-aminobutyric acid,3-phosphoglyceric acid and cinnamic acid.There were 46 species of type B carotenoids,2-methyl-3-propylpyrazine,3-methyladenine and reduced riboflavin,among which amino acids accounted for the highest proportion（32.61%,15 species）.Type 0 included 67 species of 2-hydroxybutyric acid,isobutyric acid,galactos-1-phosphate and phenylpyruvate.Only the metabolites common to type A and B were dipepsinin,L-galactose,ginkolactone and mannitol.The specific metabolic pathways of type A intestinal flora are phosphoinositol metabolism,arachidonic acid metabolism,biosynthesis and degradation of valine,leucine and isoleucine,and propionic acid metabolism.Riboflavin metabolism is a unique metabolic pathway of type B intestinal flora.Biotin metabolism,vitamin B6 metabolism,biosynthesis of ubiquinone and other terpenoid quinones are unique metabolic pathways of type 0 bacteria.The key difference metabolic pathways between type A and type B were 13 pathways,including pyrimidine metabolism,phenylalanine metabolism,D-arginine metabolism and D-ornithine metabolism.The key metabolic pathways of difference between type A and type 0 were 20 pathways such as purine metabolism and phenylalanine metabolism.The key metabolic pathways of difference between type B and type 0 were purine metabolism,pyrimidine metabolism,D-arginine metabolism and D-ornithine metabolism.In combination with the correlation analysis of the differences of intestinal flora of different types and their key metabolic pathways,the differences of metabolic types of EA were mainly related to nucleotide metabolic pathways and amino acid biosynthesis pathways,and the differences of these metabolic pathways and their metabolites may be different from those of intestinal flora metabolizing EA to produce different Uro-S（to produce Uro-A or Uro-B,or Uro-A/Uro-B are not generated）. |
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