Mass spectrometry techniques and chemometric approaches for the study of grape metabolome

Grape vine is one of the most important fruit crop in the world. Numerous grape varieties are grown worldwide for production of wine or food. Grape berries contain a large and diverse range of primary and secondary metabolites, that are important for wine quality and sensory attributes but also for their positive impact on human health. This thesis is dedicated to the study of grape metabolomes through LC-MS, GC-MS and advanced multivariate data analysis. This thesis is divided into three main chapters, covering different aspects of grape metabolomics. Chapter 1 is a review on grape metabolomics. The first part is dedicated to the importance of grape and its metabolites, with particular regards on the effect on wine sensory properties. The second part covers the different analytical platforms used in grape metabolomics and the importance of multivariate data analysis, particularly with newly developed OPLS/O2PLS. In the third part, some recent trends in grape metabolomics are described. Chapter 2 is dedicated to the development of new methodological approaches to study terroir, or the effect of environmental and viticultural features on grape and wine quality. Secondary non-volatile and volatile metabolites were extracted from berries of cv. Corvina harvested in three vintages (2006, 2007 and 2008) in eleven vineyards located in the area surrounding Verona. Two different approaches were proposed, based on multivariate data analysis. First, a preliminary unsupervised analysis allowed highlighting the strong effect of climate on cv. Corvina metabolome. The most correct approach resulted to be the analysis of the three vintages together. Several multivariate models were built using vineyard features and validated to avoid over fitting. Few models, beside the one of vintages, gave information in terms of metabolites. Particularly, two vineyards located in Lake Garda area and with by loam soils were characterized by high contents of benzene derivative volatiles compared to other vineyards. A second and completely unsupervised approach was used to reduce the variability in the metabolome that was due to climate, so that to study terroir as the set of metabolites that did not show a year-dependent variation. The analysis of LC-MS dataset revealed that each single vineyard was characterized by specific levels of secondary metabolites. When analysing the GC-MS matrix, two clusters of producers where obtained but no single feature of terroir considered in this survey could explain the grouping. Specific terroir features were suggested to affect the metabolome composition, but such features were mainly correlated to very subtle variation of large sets of metabolites rather than to sharp variation of few metabolites. Chapter 3 is dedicated to the study of the metabolic responses of six different grape varieties (Cabernet Sauvignon, Corvina, Merlot, Oseleta, Sangiovese and Shiraz) during postharvest withering process. A first approach was based on the analysis of raw data and allowed the discrimination of the six varieties by their metabolomes. The second approach was based on the analysis of data matrix normalized for the weight loss, to compensate the concentration effect due to water loss. Modulation of anthocyanin, flavonoid, flavan-3-ol and procyanidin, hydroxybenzoic and hydroxycinnamic acid classes were mainly due to concentration effect and degradation. Compounds belonging to class of stilbenes and viniferins were highly induced during postharvest withering in all the varieties but Cabernet Sauvignon and might act as defence mechanism during stress conditions. The last part of Chapter 3 is focused on the different modulation of volatile compounds in Corvina and Shiraz varieties. Sesquiterpenes, a class of terpene molecules, were highly induced in Corvina during withering and may have a role in stress response and signalling.