Optimization of protein extraction from duckweed (Lemna minor L.) and advanced proteomic investigation for allergens unveiling

Plant-based proteins have long been considered a sustainable alternative to animal proteins although the large-scale cultivation of some crops may still lead to environmental concerns such as deforestation or biodiversity loss. In this context, aquatic plants — particularly floating species — offer an even more sustainable protein source, requiring minimal land and freshwater resources, with the possibility to be cultivated in vertical farms. Duckweed (Lemna minor L.) is a small floating aquatic plant that has recently gained increasing interest for potential use in animal and human food systems due to its peculiar properties such as high biomass production rate (up to 10–20 g dry matter per m2/day), protein content (20 to 35% based on dry weight), high levels of nutrients and resilience to several climates. Moreover, since 2024, the European Food Safety Authority (EFSA) authorized the marketing and commercialization of a protein concentrate from L. gibba and L. minor as a novel food (EFSA, https://eur-lex.europa.eu/eli/reg_impl/2024/1048/oj). In line with the increasing interest in duckweed proteins, and in the perspective of safety consumers’ protection, this study aims to give insights into the complete protein profile of L. minor also focusing attention into the potential allergenicity of this plant. Firstly, a protocol optimized for a comprehensive extraction of proteins from L. minor was developed, while resulting proteins were explored via SDS-PAGE and the relative bands identified via High Resolution Mass Spectrometry (HRMS) assisted by bioinformatic search. The performance of high denaturant and reducent buffers at different pH (7,9,11) and matrix:solvent ratio (1:10, 1:25) were explored. As for cell disruption and protein release, 50°C heat or ultrasound-assisted extraction (UAE) at different time and conditions, were investigated. Protein content estimated by Bradford assay revealed no significative differences among the various test performed although, when analyzed by SDS-PAGE, some protein profiles disclosed different composition and different band intensity. Accordingly, the use of a pH 9 buffer with 1:25 matrix:solvent ratio for resuspending and UAE (10 min) for the extraction, resulted the most effective conditions for L. minor proteins investigation. From a proteomic point of view, SDS-PAGE profiles of L. minor samples highlighted bands referred to a wide range of proteins among which chlorophyll-binding proteins (LHCB1), RuBisCO (ribulose-1,5-bisphosphate carboxylase/oxygenase) subunits, ATP synthase subunits and other structural proteins as confirmed by identification studies accomplished by in-gel digestion experiments and HRMS/ bioinformatic analysis. Full lenght protein sequences identified by HRMS were finally evaluated in silico for their potential allergenicity by using AllerCatPro (v. 2.0) (https://allercatpro.bii.a-star.edu.sg/) and cross-reactivity with multiple food allergens was found.