The scientific activities of the LUPINSAFEFOOD project were organized into five work packages (WP), covering the extraction, characterization, and functional evaluation of lupin proteins, as well as their enzymatic hydrolysis, and allergenicity assessment. WP1 focused on the extraction of non-polar (e.g. lipids) and polar (e.g. phenolics) compounds from lupin seeds to concentrate proteins and facilitate their recovery, since the presence of lipids and phenolics can hinder extraction. A green biorefinery strategy was successfully implemented, enabling the sequential removal of non-polar and polar fractions prior to protein isolation. This approach effectively concentrated proteins in the solid matrix, resulting in an 11% increase in protein content post-extraction, while allowing comprehensive characterization of the extracted compounds. These results demonstrate the sustainable valorization of lupin through environmentally friendly extraction technologies. WP2 addressed the extraction and processing of lupin protein extracts and isolates using microwave-assisted extraction (MAE) and ultrasound-assisted extraction (UAE). Both technologies improved protein yield compared to conventional methods, while inducing physical and chemical modifications to the proteins, including changes in proteomic profiles, secondary structure, and techno-functional properties, as further evaluated in WP3. MAE under mild-to-moderate conditions (50–150 W) increased protein solubility up to 95% at pH 7 and 74% at pH 9, while high-power, long-duration treatments (250 W, 8 min) reduced solubility by ~55% due to aggregation. Foaming capacity reached 104.7%, with foam stability highest under low-power conditions (~99%). AE similarly improved protein solubility and foaming properties in a treatment- and pH-dependent manner, with optimal conditions enhancing foam stability and gel formation. The least gelation concentration was reduced from 20% in controls to nearly half in optimized treatments. Emulsions prepared from MAE- and UAE-treated proteins exhibited small droplet sizes and ζ-potential values indicative of good electrostatic stability. WP4 evaluated the antioxidant potential of lupin proteins in fish oil-in-water emulsions. Preliminary screening showed negligible activity in protein isolates and extracts, which led to the production of enzymatic hydrolysates using Alcalase®. Hydrolysates displayed strong iron-chelating activity, with EC₅₀ values below 0.7 mg/mL. When incorporated into emulsions, hydrolysates at 0.1% concentration delayed lipid oxidation, maintaining peroxide values and α-tocopherol levels significantly higher than in untreated controls after eight days, demonstrating their potential as natural antioxidants. WP5 assessed the allergenic potential of the studied samples. Male and female Brown Norway rats were immunized with MAE- and UAE-treated proteins and their hydrolysates. Non-hydrolyzed proteins induced IgE titers of approximately 6–10 log2, whereas hydrolyzed proteins markedly reduced IgE responses near assay cut-off values. Ear swelling tests showed local hypersensitivity responses ranging from 35.3% to 48.4%, with hydrolyzed samples generally eliciting lower reactions. Cross-reactivity studies confirmed partial IgG1 recognition with other legume proteins, while IgE reactivity was minimal for hydrolyzed proteins, indicating a significant reduction in allergenic potential. Overall, the project successfully enhanced the functionality, antioxidant potential, and safety of lupin proteins, providing robust quantitative data on solubility, foaming, emulsifying properties, gelation, enzymatic hydrolysis, oxidative stability, and allergenicity. These results establish a strong scientific foundation for the development of sustainable lupin-based food ingredients with improved techno-functional and health-promoting properties.
