Abstract
In order for probiotics to reach their target site in a viable state they must cope with specific stress challenges throughout the gastrointestinal track among other the presence of bile in the small intestine. In-depth genomics, transcriptomics, and proteomics characterisation of the bile response of lactobacilli has suggested bile response mechanisms such as efflux of bile acids/salts, bile salt hydrolysis, modified sugar metabolism, change in a number of transport systems, and cell membrane or cell wall composition modification. A number of these effects are expected to cause alterations in the metabolome, and an increased understanding of bile response mechanisms could be obtained by analysis of the response by tools within metabolomics. Therefore, the aim of this PhD thesis was to develop a platform for metabolic foot- and fingerprinting of L. paracasei subsp. paracasei strain (L. casei CRL-431®) defined as the semi-quantitative analysis of extra- and intracellular metabolites, respectively.
A number of key steps in the metabolomics protocol was addressed and discussed, from the initial quenching of metabolic activity to the final data analysis, to ensure that the protocol captures a reliable picture of changes in the metabolome. It was shown that the L. paracasei cell membrane was significantly damaged by quenching in buffered methanol. In contrast minimal damage of the cell membrane and better recovery of intracellular metabolites were observed with glycerol saline quenching. Furthermore, it was demonstrated that the subsequent method used to extract intracellular metabolites from the L. paracasei cells altered the metabolic fingerprint.
A comparative study was performed to characterise the effect of the genetic alterations in a set of mutants with enhanced bile tolerance from the parental strain of L. paracasei on the transcriptome and metabolome. The results show that even small genetic alterations between the parental strain and the mutants caused significant physiological changes; both in respect to up-regulation and down-regulation of genes as well as on the metabolic level. Furthermore, the bile response of L. paracasei and the mutants was characterised by transcriptomics and metabolomics. The bile exposure induced changes at the transcript level in genes as well as changes on the metabolic levels; especially in the carbohydrate metabolism.
A number of key steps in the metabolomics protocol was addressed and discussed, from the initial quenching of metabolic activity to the final data analysis, to ensure that the protocol captures a reliable picture of changes in the metabolome. It was shown that the L. paracasei cell membrane was significantly damaged by quenching in buffered methanol. In contrast minimal damage of the cell membrane and better recovery of intracellular metabolites were observed with glycerol saline quenching. Furthermore, it was demonstrated that the subsequent method used to extract intracellular metabolites from the L. paracasei cells altered the metabolic fingerprint.
A comparative study was performed to characterise the effect of the genetic alterations in a set of mutants with enhanced bile tolerance from the parental strain of L. paracasei on the transcriptome and metabolome. The results show that even small genetic alterations between the parental strain and the mutants caused significant physiological changes; both in respect to up-regulation and down-regulation of genes as well as on the metabolic level. Furthermore, the bile response of L. paracasei and the mutants was characterised by transcriptomics and metabolomics. The bile exposure induced changes at the transcript level in genes as well as changes on the metabolic levels; especially in the carbohydrate metabolism.
| Original language | English |
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| Publisher | Department of Plant and Environmental Sciences, Faculty of Science, University of Copenhagen |
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| Number of pages | 185 |
| Publication status | Published - 2015 |