Biochemical valorisation of agro-industrial by-products through green extraction techniques, enzymatic digestion and metabolomic approaches with biotechnological and nutraceutical applications.

Agro‑industrial by‑products represent a rapidly growing biomass stream that is still largely underexploited despite its richness in bioactive molecules and functional biopolymers. Within the conceptual framework of the circular economy and green chemistry, this PhD thesis focuses on the biochemical valorisation of these residues through environmentally sustainable extraction, structural and functional characterisation with biochemical, material science and cell biology applications. The global aim is to demonstrate how Citrus and ornamental plant by‑products can be transformed into sources of high‑value compounds with biotechnological and nutraceutical relevance, thereby contributing to a circular bioeconomy model. The first experimental section investigates the effects of the flavone vicenin‑2, present in the by-products obtained from global citrus transformation industry, as a bioprotective agent against thermal‑ and oxidative stress‑induced protein damage. Using human serum albumin (HSA) as a protein-model, the study evaluates the ability of vicenin‑2 to prevent fibril formation and oxidative modifications, combining UV‑visible and fluorescence spectroscopy, microscopy, electrophoretic separation and circular dichroism. Binding equilibria, thermodynamic parameters and displacement assays are integrated with molecular docking to clarify the interaction sites, the predominant non‑covalent forces and the resulting conformational rearrangements in the protein. These data support the potential role of vicenin‑2, with implications for the stabilization of protein structure under stress conditions. The second experimental pillar addresses the development of active food‑packaging systems by functionalising bioplastics based on polyhydroxyalkanoates (PHA), polyvinyl alcohol (PVA) and chitosan with phytochemicals present in citrus “pastazzo” and Callistemon citrinus flowers. Anthocyanin‑enriched fractions and polyphenolic matrices are obtained using green extraction approaches and subsequently incorporated into polymeric films. The resulting materials are characterised by FTIR‑ATR, Raman spectroscopy, SEM and XRD to elucidate structural features and interactions between the polymer matrices and the bioactive components. Optical, mechanical, barrier, swelling and water‑contact properties are systematically evaluated, together with antioxidant and antimicrobial activities, migration and release behaviour and food‑preservation performance. The results show that the incorporation of phytochemicals improves the functional profile of the films, enabling antioxidant and antimicrobial protection while maintaining suitable mechanical integrity and contributing to the extension of food shelf‑life. The third experimental section focuses on the biological activity of an anthocyanin‑enriched fraction obtained from the by-products of Callistemon citrinus in TM3 mouse Leydig cells, with particular attention to mitochondrial function, redox homeostasis and steroidogenesis. Several assays are used to assess metabolic activity, cytotoxicity, intracellular reactive oxygen species, mitochondrial membrane potential, mitochondrial biogenesis, lipid peroxidation, OXPHOS protein levels and intracellular antioxidant capacity. Gene and protein expression analyses are combined with androstenedione quantification to dissect the impact on steroidogenic pathways. In parallel, exometabolomic profiling by 1H-NMR is employed to monitor changes in the extracellular metabolite landscape. The findings reveal that, at specific concentrations, the anthocyanin‑enriched fraction exerts a pro‑oxidant effect, impairs mitochondrial quality and suppresses androgen synthesis through transcriptional repression of CyP17a1 mediated by the nuclear transcriptional factors Nr0b2. Overall, this thesis provides an integrated demonstration that agro‑industrial by‑products can be upgraded into functional ingredients and materials that simultaneously address environmental, technological and biochemical challenges. By combining green extraction technologies, advanced material characterisation and cellular and metabolomic approaches, the work outlines concrete strategies for converting waste streams into added‑value products for active packaging and the modulation of redox‑endocrine pathways, enhancement the link between circular bioeconomy and human health.