In the context of the valorization of agri-food by-products, tomato (Solanum lycopersicum L.) seeds represent a protein-rich matrix containing potential bioactives. The aim of the present work is to develop a biochemical pipeline for (i) achieving high protein recovery from tomato seed, (ii) optimizing the hydrolysis with different proteases, and (iii) characterizing the resulting peptides. This approach was instrumental for obtaining and selecting the most promising peptide mixture to test for antifungal activity. To this purpose, proteins from an alkaline extraction were treated with bromelain, papain, and pancreatin, and the resulting hydrolysates were assessed for their protein/peptide profiles via SDS-PAGE, SEC-HPLC, and RP-HPLC. Bromelain hydrolysate was selected for antifungal tests due to its greater quantity of peptides, in a broader spectrum of molecular weights and polarity/hydrophobicity profiles, and higher DPPH radical scavenging activity, although all hydrolysates exhibited antioxidant properties. In vitro assays demonstrated that the bromelain-digested proteins inhibited the growth of Fusarium graminearum and F. oxysporum f.sp. lycopersici in a dose-dependent manner, with a greater effect at a concentration of 0.1 mg/mL. The findings highlight that the enzymatic hydrolysis of tomato seed protein represents a promising strategy for converting food by-products into bioactive agents with agronomic applications, supporting sustainable biotechnology and circular economy strategies.

β-vignin is the main storage protein in cowpea (Vigna unguiculata, Walp) seeds. Similar to other vicilins, differently glycosylated isoforms organize in trimers whose oligomerization is influenced by salt concentration and pH. It was previously observed that two polypeptides, resistant to further proteolysis, are generated and persist throughout the germination process. This study aimed to characterize the structural features of partially degraded, yet structured, cleaved β-vignin using a combination of biochemical and biophysical techniques. The cleavage site is present only in the non-glycosylated isoform, located between the two cupin domains. Thermal Shift Assay (TSA), Differential Scanning Calorimetry (DSC) and thermodynamic analysis of β-vignin in its native form highlighted the complex stability scenario, in line with the structural evidence that accounts for two isoforms and the monomer-to-trimer equilibrium. Furthermore, DSC data suggest that the protein's tertiary and quaternary structure is resilient to the structural modifications resulting from enzymatic cleavage. The tertiary conformation is "released" from some constraints of the primary structure, producing an even more compact protein. In addition, by exploiting the pH-dependent dissociation equilibrium of β-vignin, we investigated how glycosylated and non-glycosylated β-vignin isoforms combine to form the trimer. This study, revealing that the stable breakdown intermediate maintains a higher-order structure, provides novel insights into the current understanding of seed storage protein dynamics during germination. The molecular characterisation of the stable breakdown products is instrumental in further investigating their functional roles during germination, such as insecticidal or antifungal properties, and/or as mediators of seed adaptation to environmental conditions.