Valorisation of olive oil industry by-products and metabolomic insights into olive-Xylella fastidiosa interactions

This doctoral thesis is developed within an interdisciplinary framework that integrates metabolomics, nutraceutical applications, agronomic objectives, and sustainable bioprocesses, with analytical chemistry serving as the central methodological approach. The work is based on two main pillars: the valorisation of olive oil industry by-products to develop bioactive systems and the investigation of olive metabolic responses to the pathogen Xylella fastidiosa. The first phase of the research focused on the recovery, stabilization, and characterization of bioactive compounds from olive mill wastewater (OMWW) and olive leaves. Using technologies such as spray-drying and freeze-drying, stable bioactive-rich matrices were obtained and subsequently applied for the functionalization of biopolymers, using pectin and chitosan as commercial biopolymers, via free radical grafting. Characterization by LC-MS/MS confirmed the efficiency of the grafting process and highlighted the crucial role of lipophenols, amphiphilic molecules with high bioaccessibility, in enhancing bioactive incorporation and the antioxidant properties of the resulting hybrid materials. In parallel, a sustainable extraction strategy for oleuropein from olive leaves was optimized using Natural Deep Eutectic Solvents (NADES) combined with ultrasound-assisted extraction (UAE). This approach demonstrated an extraction efficiency more than four times higher than water and comparable to organic solvents, while significantly reducing environmental impact. The subsequent microencapsulation via spray-drying yielded stable functional powders with preserved biological activity over time. The biological potential of the developed matrices was evaluated through in vitro assays, revealing significant anti-inflammatory (reduction of NO production in RAW 264.7 macrophages) and antiproliferative activities against breast cancer cell lines (MCF-7, MDA-MB-231). In the agronomic field, selected OMWW formulations and functionalized biopolymers (P-LALE) showed inhibitory activity against Xylella fastidiosa subsp. pauca (ST53), suggesting their potential as bio-control agents that may interfere with bacterial quorum sensing or act as competitive substrates against cell wall-degrading enzymes. Finally, a targeted metabolomic study was conducted on the xylem tissues of olive cultivars with varying susceptibility to Xylella fastidiosa (Leccino, Cellina di Nardò, and Ogliarola salentina). Multivariate statistical analysis identified verbascoside as the primary biomarker of infection, followed by quinic acid and oleuropein. The results evidenced a cultivar-dependent metabolic reprogramming: tolerant genotypes (Leccino) exhibited a more regulated and coordinated response compared to susceptible ones, which showed more intense but likely less effective metabolic alterations. In conclusion, this thesis demonstrates that olive by-products are valuable renewable sources for the development of targeted bioactive materials. The integration of advanced analytical methodologies and sustainable biotechnological strategies contributes to the understanding of plant defence mechanisms and supports innovative solutions for the nutraceutical and agri-food sectors within a circular economy framework.