Development of analytical methodologies applied to metabolomic studies in graciano wine variety

Resum

Metabolomics is a recent ¿omics¿ field based on biological science devoted to fully identify phenotypes, the connectivity that relates their constituents and the dynamical response to perturbations, as well as the relationships with the rest of the cellular molecular machinery, namely at the regulatory levels such as genomics, transcriptomics and proteomics at the systems biology stage. The interest in the metabolomic study of the Graciano Vitis vinifera wine variety lays on the lack of knowledge of the composition of this type of wine in addition to its high market demand due to its unique and appreciated organoleptic characteristics. The main item of this work is the characterization of the metabolites that constitute the metabolome of red wine elaborated from the grape variety Vitis vinifera Graciano. To achieve this goal, a first study of the volatile fraction was performed. An analytical method based on thermal desorption cold trap on-line with gas chromatography coupled with mass spectrometry (dual-SBSE-ATD/GC-MS) detection was developed. The application of a novel dual mode stir-bar sorptive extraction methodology, allowed the identification of about 205 volatile and semi-volatile metabolites, six of which have been identified in wine for the first time. In a second stage, the metabolome of the nonvolatile fraction was studied. To this end, liquid chromatography coupled to a mass spectrometer hybrid quadrupole time of flight analyzer (LC/Q-TOF) was used. In a comparative study between a classic ESI source and an ESI source provided with Jet Stream technology, it was found that the use of the Jet Stream technology did not provide better results. It rather causes a significant decrease in the number of possible metabolites in the wine matrix. For a correct analysis of the obtained results, the use of various deconvolution and alignment softwares was necessary. In addition, different databases were also required with identification purposes. It has even been created a homemade database (WinMet) with compounds that have been already identified in oenological matrices. This database includes the name of the metabolite, the CAS number, the molecular formula, its exact mass and its family. The whole process LC/Q-TOF (WinMet) enables the identification of 403 metabolites in the non volatile fraction.The last part of this thesis is focused on the development of a method for the untargeted phenol profiling by LLE-ESI-LC-QTOF and chemometric analyses. A comparative study, between Graciano wines from Australia and from Spain, was carried out to propose possible markers in the Graciano Vitis Vinifera variety that enable discriminating wines based upon their origin.