Purification And Structural Characterization Ofmetabolic Products From A Potential Probioticescherichia Coli GutM4 In Human Gastrointestinal Tract

Probiotics are defined as live microorganisms which when administered in adequate confer health benefits on the host.The most prevalent probiotics are Lactobacilli,Bifidobacteria,as well as Saccharomyces and Streptococcus.Of particular interest is the Escherichia coli due to its well-knowing as pathogens,but in fact being a very massive commensal nonpathogenic strains in human gastrointestinal（GI）tract indeed.Several E.coli probiotics have been reported as potential therapeutic agents,E.coli Nissle 1917 was known to produce microcin B17,E.coli H22 was shown to against some enteric pathogens,E.coli G3/10 was reported to produce microcin S,E.coli M17 had an inhibitory effect on intestinal inflammation,and E.coli K-12JM105 and E.coli EMO protected germfree mice against infection of Salmonella typhimurium C5.It is such the belief that there are considerably untapped E.coli probiotics because of the nonpathogenic character,commesal properties and the massiveness of the E.coli strains in human gut.In this research,we used test of acid resistance,antioxidative activity,antibacterial activity test and cholesterol-reducing activity for screening potential probiotics from a healthy Chinese female.And then,we took a series of physiological and biochemical test and 16S rDNA test for the identification.Strain of GutM4 was selected primarily as a potential probiotic.MR,VP and O/F tests revealed that this strain can ferment glucose and produce acids.Catalase test indicated that strain had antioxidant activity.Negative results were observed in H₂S production,starch hydrolysis and urease test.Combining with 16S rDNA sequencing,GutM4 was identified being belonged to E.coli.Next,we researched the probiotic bioactivity of E.coli GutM4.The survival percentage of E.coli GutM4 after simulated passage through the GI tract was 92.90%,similar to that of L.bulgaricus.The crude metabolites of GutM4 exhibited significant inhibitory activity against indicator pathogens（P<0.05）,being equal to the capacity of streptomycin sulfate on the concentration of 0.5mg/ml（P>0.05）.E.coli GutM4 was susceptible to majority of the tested antibiotics,expressed antioxidative ability and cholesterol-lowering ability too.Regarding with the action mechanism of this E.coli probiotic,15 compounds were isolated and purified from ethyl acetate extract of E.coli GutM4 by chromatography methods such as HPLC on silica gel,ODS C-18 and Sephadex LH-20.Theses compounds were identified by ¹H-NMR,¹³C-NMR,¹H-¹H COSY,HMQC,HMBC,NOESY and MS as2,5-Diketopeperazine（DKP）,cyclic dipeptide,which belong to nonribosomal peptide（NRP）.The compounds were as follows:1 Cyclo（Val-Ala）,2 Cyclo（Gly-Leu）,3 Cyclo（Pro-Tyr）,4Cyclo（Phe-Gly）,5 Cyclo（4-OH-Pro-Leu）,6 Cyclo（Phe-Ala）,7,8 Cyclo（4-OH-Pro-Phe）,9Cyclo（Tyr-Phe）,10 Cyclo（Trp-Tyr）,11 Cyclo（L-Pro-L-Trp）,12 Cyclo（Pro-Phe）,13Cyclo（D-Pro-L-Trp）,14 Cyclo（Trp-Val）,15 Cyclo（Trp-Leu）,the NRPS biosynthesis pathway assembling the DKPs was confirmed by the positive cloning of NRPS gene in E.coli GutM4.To conclude,the results suggested the potentiality for probiotic candidate of E.coli GutM4which produced 14 diketopiperazines with biosynthesis pathway of nonribosomal peptide synthetase（NRPS）.The present study firstly purified 14 nonribosomal peptides in a human GI E.coli strain which was identified as a potential probiotic.