Effects Of Lactobacillus Reuteri LR1 On Ileal And Colonic Luminal Microbial Metabolome In Weaned Piglets

Probiotics have been shown to improve the intestinal microbiota balance and promote the growth and development of animals,and regarded as the most effective substitutes for in-feed antibiotics,but the underlying mechanism remains unclear.The aim of this study was to explore the effects of Lactobacillus reuteri LR1（LR1）on the ileal and colonic luminal microbial metabolites in weaned piglets.A total of 96 crossed weaned piglets（Duroc×Landrace×Yorkshire,21d of age）with average initial body weight of 6.49±0.02 kg,were randomly assigned to 2 dietary treatment groups.Each group had 8replicates（pens）with 6 piglets per replicate.Piglets were fed a basal diet（control group）and a basal diet supplemented with 5×10¹⁰ CFU/kg of LR1（LR1 group）.On the 15th day of the experiment,eight piglets from each group were randomly selected and slaughtered.Samples of ileum and colon contents were collected for metabolomics analysis.The results were shown as follows:1.Metabolic profile analysis of ileal and colonic luminal microbita in piglets.Solvent extraction（methanol:acetonitrile:water=2:2:1）was used to extract the small molecule metabolites in the ileum and colon contents of piglets,and the metabolites were detected by HPLC-QTOFMS.The metabolomics data were analyzed by multivariate pattern recognition analysis which includes unsupervised principal component analysis（PCA）and supervised orthogonal projections to latent structures-discriminant analysis（OPLS-DA）methods.OPLS-DA scores showed that there were obvious clustering phenomena without overfitting between the two groups.According to the OPLS-DA model,differential metabolites were filtrated between different treatment groups in ileal and colon contents by a standard VIP>1,?p（corr）?≥0.5 and all variables are within the 95%confidence interval.Results showed that 2,917 compound signals were detected in the ileal contents of piglets,with 1554 in positive ion mode（ESI+）and 1363 in negative ion mode（ESI-）.Moreover,a total of 2915 compound signals were detected in the colonic contents,with 1551 in positive ion mode（ESI+）and 1364 in negative ion mode（ESI-）.Multidimensional statistical analysis showed that 48 differential metabolites were screened out in the ileal contents of piglets,which were down-regulated in the LR1 group,including 10 known substances such as bile acids,fatty acids etc.A total of 130 differential metabolites were screened out in the colonic contents,wuth 68 up-regulated and 62 down-regulated in the LR1 group,including36 known substances such as lipids,dipeptides etc.2.Verification analysis of ileal and colonic luminal microbial key metabolites in piglets.GC-MS techniques were performed to detect the short-chain fatty acids（SCFAs）in the ileal and colonic contents of piglets.Results showed LR1 significantly increased the content of SCFAs in the ileal and colon contents,especially the content of propionic acid and butyrate in the colonic contents（P<0.05）.The content of bile acids in the ileal and colonic contents of piglets was determined by HPLC-TQMS.LR1 significantly increased the contents of deoxycholic acid（DCA）and lithocholic acid（LCA）in ileal contents（P<0.05）,but significantly reduced the content of LCA in the colonic contents（P<0.05）.Finally,HPLC-UV technology was used to determine the content of three adenosine phosphates（ATP,ADP,AMP）in the colonic contents of piglets.Results showed that LR1significantly increased the contents of ADP and ATP in the colonic contents（P<0.05）.In conclusion,dietary LR1 supplementation exerted a significant effect on the microbial metabolism in the ileal and colonic lumen of piglets.A total of 2,917 compound signals were detected in the ileal contents of the piglets,and 2,915 compound signals were detected in the contents of the colon.Multidimensional statistical analysis showed that 48differential metabolites were screened out in the ileal contents of piglets between two groups,which were down-regulated in the LR1 group,including bile acids and fatty acids.LR1 supplementation induced 130 differential metabolites in the colonic contents of piglets between two groups,with 68 up-regulated and 62 down-regulated,including lipids and dipeptides.LR1 significantly increased the content of DCA and LCA in the ileal contents of piglets,and significantly increased the content of propionic acid,butyric acid,ATP and ADP in the colonic contents of piglets,and significantly reduced the LCA content in the colonic contents.Therefore,dietary supplementation of LR1 could affect the contents of metabolites such as fatty acids and bile acids in intestinal lumen,which might thereby regulate the physiological process of piglets...