Effect of Extraction Temperature on Protein Recovery and Molecular Weight Distribution of Lemna minor Protein Concentrates

Abstract

Duckweed (Lemna minor) is attracting attention as a sustainable and fast-growing source of plant proteins, particularly due to the abundance of ribulose-1,5-bisphosphate carboxylase/oxygenase (RuBisCO), the major photosynthetic protein in green biomass. Since processing conditions may affect protein recovery and molecular characteristics of obtained concentrates, this study investigated the effect of temperature during ultrasound-assisted alkaline extraction on protein yield, protein content, and molecular weight distribution of Lemna minor protein concentrates. Dried duckweed biomass, previously pretreated with 70% ethanol to reduce phenolic compounds, was subjected to ultrasonic pretreatment to enhance cell disruption and protein accessibility. Alkaline extraction was then performed at pH 11.5 for 2h at different temperatures, followed by isoelectric precipitation at pH 4.5. The concentrates were evaluated for extraction yield and protein content by the Kjeldahl method, while molecular weight distribution was analysed by capillary gel electrophoresis using an Agilent 2100 Bioanalyzer. The temperature treatment during alkaline extraction affected protein recovery and concentrate composition. The control sample showed a protein content of 59.93% and a yield of 0.562 g/100 g biomass. Extraction at 50 °C resulted in 50.88% protein and a yield of 2.533 g/100 g, while treatment at 60 °C yielded 49.80% protein and 2.358 g/100 g. The most favourable result was obtained at 80 °C, where protein content reached 75.34%, with a yield of 4.587 g/100 g biomass, indicating improved protein release and higher extraction selectivity. Capillary electrophoresis revealed changes in molecular weight profile related to temperature treatment. Control, 50 °C and 60 °C protein samples were dominated by low-molecular weight fractions below 50 kDa, accounting for 89.61%, 99.47% and 92.88% of total quantified protein concentration, respectively. These fractions may be associated with RuBisCO subunits commonly reported at 12–15 kDa and 50–60 kDa. In contrast, extraction at 80 °C promoted a broader molecular weight distribution, with higher-molecular weight fractions up to 250 kDa, suggesting protein unfolding, association, and formation of larger complexes or aggregates. Overall, the results indicate that extraction temperature can be used to modulate both recovery efficiency and molecular properties of Lemna minor protein concentrates.