Jorgensen, A. K. | Search bioRxiv

bioRxiv2026

Techno-functional properties of clover grass protein and the effect of different process technologies

This study investigated the effects of different downstream processes for protein isolation on the bulk properties and composition of clover grass protein prototypes (CGPs). A clarified clover grass juice, obtained using membrane filtration (MF), underwent precipitation by acid (AP), heat (HP), or acid+heat (AHP), or underwent ultra- and diafiltration to produce a concentrate (DC) as well as subsequent tryptic hydrolysis of DC (DCH). HP had the highest protein content (p<0.05) and was whiter than other CGPs, although it showed lower aqueous solubility. In contrast, DC showed excellent solubility across a broad pH range. CGPs efficiently decreased oil-water interfacial tension (16-13 mN/m) and displayed viscoelastic and solid-like interfacial behavior. CGPs-stabilized emulsions displayed low physical stability with larger droplets despite high absolute {zeta}-potentials. CGPs were rich in RuBisCO (37-47%) but had varying levels of other proteins. Despite significant protein-level differences, overall protein composition of CGPs was comparable, highlighting that protein state governs bulk functionality more than subtle compositional changes. Graphical abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=108 SRC="FIGDIR/small/701969v1_ufig1.gif" ALT="Figure 1"> View larger version (25K): org.highwire.dtl.DTLVardef@f37756org.highwire.dtl.DTLVardef@1fb5beorg.highwire.dtl.DTLVardef@1d4efe2org.highwire.dtl.DTLVardef@d11ef8_HPS_FORMAT_FIGEXP M_FIG C_FIG Created with BioRender.com HighlightsO_LIThe effect of different processes on functional properties of CGPs was explored. C_LIO_LIHeat treatment increased protein purity and whiteness at the expense of solubility. C_LIO_LICGPs efficiently reduced O/W interfacial tension but produced unstable emulsions. C_LIO_LICGPs were found rich in RuBisCO (34-47%) using quantitative proteomics. C_LIO_LIProtein state had larger influence on functionality than protein-level composition. C_LI

bioengineering↗

bioRxiv2025

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

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

bioengineering↗