Selective removal of green pigments and associated proteins from clover-grass protein concentrates: molecular insights 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 proteins as leaf proteins often possess good nutritional and functional properties. However, the current methods for producing protein isolates from leaves involve partially or fully denaturing conditions, diminishing the 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 grass devoid of attributes such as green color and grassy smell/taste. By sampling at each step along the process, we obtain insights into the fate of the proteins and pigments over each unit operation. This 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, it is found that this can particularly be ascribed to the 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 enriched in proteins with known antioxidant properties while depleted in known food allergens. This work presents an in-depth understanding of the protein-level selectivity in a membrane-based green biorefinery and can help guiding in the process optimization towards improved yields and quality.