Selective removal of green pigments and associated proteins from clover-grass protein concentrates: Molecular insight into a non-destructive, two-stage membrane-based biorefinery concept for high-quality food protein production

Abstract

Green leaves are gaining traction as an emerging protein source and a sustainable alternative to animal-based protein as leaf proteins often possess good nutritional and functional properties. Current methods for producing protein isolates from leaves include partially or fully denaturing conditions, diminishing protein solubility and functionality. Here, we characterize the performance of a multi-stage, membrane-based green biorefinery concept capable of producing a native protein product from clover grasses devoid of attributes such as green color and grassy smell/taste. By sampling at each step along the process, we obtain insights on the fate of proteins and pigments over each unit operation. The process efficiently removes green pigments (>99.9%), when comparing the product stream with the initial feed stream, based on UV/Vis analysis. Using mass spectrometry-based proteomics, two complementary quantification strategies, and subsequent bioinformatic analysis, this can particularly be ascribed to a very selective retention (>99%) of unwanted membrane-associated and pigment-binding proteins in the first-stage filtration. In the second-stage filtration and subsequent diafiltration stage, residual unwanted proteins, fragments, and pigments are efficiently washed out while retaining the overall protein composition. The product maintains the high RuBisCO content of green juice and is furthermore enriched in proteins with known antioxidant properties while depleted in known food allergens. This work presents an in-depth understanding of protein-level selectivity in membrane-based green biorefinery and can help guide process optimization towards improved yields and quality. HighlightsO_LIStreams from a two-stage membrane-based green biorefinery were characterized C_LIO_LI>99.9% pigment-binding proteins were removed primarily in first stage filtration C_LIO_LIThe 2nd filtration and diafiltration removed residual pigment and retained protein C_LIO_LIThe full process efficiently removed >99.9% of chlorophylls and carotenoids C_LIO_LIThe soluble product is rich in RuBisCO and enriched in known antioxidant proteins C_LI