Periodic Reporting for period 1 - LUPINSAFEFOOD (Strategies to promote the lupin proteins as a new and safe functional food ingredient: Assessment of oxidative and physical stability of food emulsions and allergenic potential)
Periodo di rendicontazione: 2024-01-01 al 2025-12-31
The LUPINSAFEFOOD project was developed in response to the increasing demand for sustainable, plant-based protein ingredients to support healthier diets and environmentally responsible food systems. Lupin seeds are a promising protein source due to their high protein content and favorable nutritional profile, yet their broader application in food products is limited by challenges related to extraction efficiency, functionality, oxidative stability and allergenicity.
The main objective of the project was to develop sustainable, green processing strategies to improve the extraction, functionality, and applicability of lupin proteins. This involved implementing a biorefinery approach to concentrate proteins through the sequential removal of non-polar and polar compounds, followed by advanced physical processing technologies—microwave- and ultrasound-assisted extraction—to enhance protein yield, structural properties, and functional performance. The project also addressed the antioxidant potential of lupin proteins through enzymatic hydrolysis, demonstrating the ability of hydrolysates to reduce lipid oxidation in emulsions, and evaluated allergenic potential, showing that enzymatic hydrolysis significantly attenuates IgE reactivity and reduces hypersensitivity responses.
The main objective of the project was to develop sustainable, green processing strategies to improve the extraction, functionality, and applicability of lupin proteins. This involved implementing a biorefinery approach to concentrate proteins through the sequential removal of non-polar and polar compounds, followed by advanced physical processing technologies—microwave- and ultrasound-assisted extraction—to enhance protein yield, structural properties, and functional performance. The project also addressed the antioxidant potential of lupin proteins through enzymatic hydrolysis, demonstrating the ability of hydrolysates to reduce lipid oxidation in emulsions, and evaluated allergenic potential, showing that enzymatic hydrolysis significantly attenuates IgE reactivity and reduces hypersensitivity responses.
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.
The LUPINSAFEFOOD project advanced the current state of the art by providing a comprehensive and integrated assessment of sustainable processing strategies for lupin proteins, combining biorefinery-based concentration, microwave- and ultrasound-assisted extraction, enzymatic hydrolysis, proteomic profiling, functional characterization, antioxidant assessment, and allergenicity evaluation. Novel insights were generated on the impact of physical processing on protein composition, structure–function relationships, and techno-functional properties. Microwave and ultrasound treatments were shown to modulate solubility, foaming, emulsifying, and gelling behavior, providing a level of functional control not previously reported for lupin proteins. Enzymatic hydrolysis unlocked antioxidant activity, enabling hydrolysates to delay lipid oxidation in fish oil-in-water emulsions, demonstrating the practical application of lupin proteins as natural antioxidants in complex food systems. In terms of allergenic potential, the study revealed that enzymatic hydrolysis substantially reduces IgE reactivity and attenuates hypersensitivity responses in vivo, while preserving protein functionality. This provides a novel strategy to enhance the safety of lupin protein ingredients, addressing a key limitation for their widespread use in food products. Overall, the project produced a unique and openly accessible dataset linking processing conditions to functional, proteomic, antioxidant, and immunological outcomes. These results offer critical guidance for future research and industrial development of sustainable, plant-based protein ingredients, and establish a foundation for designing lupin-derived proteins with improved functionality, oxidative stability, and safety. Future studies could further explore the digestibility, bioavailability, and incorporation of these proteins into diverse plant-based food systems, extending their application and supporting the development of nutritious and sustainable alternatives in the food industry.