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PreprintCited by 5Membrane Separation TechnologiesProteins in Food SystemsProtein purification and stability

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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

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Gregersen Echers, Simon, Abdul-Khalek, Naim, Jensen, Nete Hassing, Jørgensen, Anders Kjær, Mattsson, Tuve, Jørgensen, Mads Koustrup, Lübeck, Peter Stephensen, Lübeck, Mette·2025·Sustainable Food Technology
DOI 10.1101/2025.10.24.684365

Preprint — published as 10.1039/d5fb00771b · Paper page

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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. Highlights: Streams from a two-stage membrane-based green biorefinery were characterized >99.9% pigment-binding proteins were removed primarily in first stage filtration The 2 nd filtration and diafiltration removed residual pigment and retained protein The full process efficiently removed >99.9% of chlorophylls and carotenoids The soluble product is rich in RuBisCO and enriched in known antioxidant proteins

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Metadata

Authors
Gregersen Echers, S., Abdul-Khalek, N., Jensen, N. H., Jørgensen, A. K., Mattsson, T., Jørgensen, M. K., Lübeck, P. S., & Lübeck, M.
Affiliations
Aalborg University (DK)
ORCID
Venue
Sustainable Food Technology
Publisher
openRxiv
Published
2025-10-26
Volume
4
Issue
2
Pages
1689-1713
ISSN
2753-8095 (ISSN-L)
DOI
10.1101/2025.10.24.684365
OpenAlex
W7108069026
Cited by
5
References
1
Citations by year
2026: 5
FWCI
1.27
Type
journal article
Language
en
Open access
Licence
Locations
  • Sustainable Food Technology (publishedVersion, cc-by-nc)
  • bioRxiv (Cold Spring Harbor Laboratory) (acceptedVersion, cc-by-nd)
  • VBN Forskningsportal (Aalborg Universitet) (submittedVersion, cc-by-nc)
  • VBN Forskningsportal (Aalborg Universitet) (submittedVersion, cc-by-nc-nd)
Funders
  • Danish Green Development and Demonstration Program (34009-20-1657)
  • Independent Research Foundation Denmark (1127-00245B)
Topics
  • Physical Sciences › Environmental Science › Water Science and Technology › Membrane Separation Technologies
  • Life Sciences › Agricultural and Biological Sciences › Food Science › Proteins in Food Systems
  • Life Sciences › Biochemistry, Genetics and Molecular Biology › Molecular Biology › Protein purification and stability
Keywords
green pigments, chlorophyll, carotenoids, Rubisco, antioxidant proteins, membrane separation, selective retention, biorefinery

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CoChat. (2026). Citation check: 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. https://app.cochat.ai/papers/10.1101/2025.10.24.684365
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Gregersen Echers, S., Abdul-Khalek, N., Jensen, N. H., Jørgensen, A. K., Mattsson, T., Jørgensen, M. K., Lübeck, P. S., & Lübeck, M. (2025). 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. Sustainable Food Technology. https://doi.org/10.1101/2025.10.24.684365