Proof-of-concept: Ultrafiltration as a pre-treatment for the anion exchange chromatography of RuBisCO from alfalfa

Hartono Tanambell; Marianne Danielsen; Anders Hauer Møller; Trine Kastrup Dalsgaard

doi:10.1016/j.fbp.2025.10.011

Proof-of-concept: Ultrafiltration as a pre-treatment for the anion exchange chromatography of RuBisCO from alfalfa

Hartono Tanambell
, Marianne Danielsen
, Anders Hauer Møller
, Trine Kastrup Dalsgaard^*

^*Corresponding author for this work

Research output: Contribution to journal/Conference contribution in journal/Contribution to newspaper › Journal article › Research › peer-review

Abstract

Alfalfa is gaining interest as an alternative protein source. The European Food Safety Authority (EFSA) approval of alfalfa protein concentrate as novel food states presence of metabolites as a barrier for higher amount of consumption. A promising method for protein isolation is anion exchange chromatography, which is scalable through expanded bed chromatography. In this study, ultrafiltration-buffer exchange (UBE) and ultrafiltration-diafiltration was applied as an upstream and downstream processing to anion exchange chromatography, respectively. The performance of these techniques was compared to that of lab scale dilution and centrifugal spin filters. The result showed that UBE shortened the chromatography duration by 50 % and resulted in a cleaner column. However, downstream desalination through ultrafiltration-diafiltration resulted in concentrate with lower protein content (60.41 ± 0.84 %), compared to that obtained from centrifugal spin filters (90.49 ± 1.34 %), suggesting the need to carry out extra diafiltration steps. Untargeted metabolomics revealed that high absorption at 214 nm in the anion exchange flowthrough fraction could be attributed to di- and tripeptides also present in the ultrafiltration permeates. Polyphenols present in ultrafiltration permeate were absent from both flowthrough and 225 mM fraction, suggesting that they might bind strongly to the column. Various carboxylic acids were present in the ultrafiltration permeate, chromatography flowthrough, and 225 mM NaCl fraction. Contaminants eluting at 225 mM NaCl with high absorbance at 214 nm were not successfully identified although their occurrence was lower in UBE samples. These results suggest that UBE is promising as pre-treatment for purification of RuBisCO from alfalfa using anion exchange chromatography.

Original language	English
Journal	Food and Bioproducts Processing
Volume	154
Pages (from-to)	440-448
Number of pages	9
ISSN	0960-3085
DOIs	https://doi.org/10.1016/j.fbp.2025.10.011
Publication status	Published - Dec 2025

Keywords

Alfalfa
Anion exchange chromatography
Metabolomics
RuBisCO
Ultrafiltration

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@article{de33fc794c044a67bb24b177530278d6,

title = "Proof-of-concept: Ultrafiltration as a pre-treatment for the anion exchange chromatography of RuBisCO from alfalfa",

abstract = "Alfalfa is gaining interest as an alternative protein source. The European Food Safety Authority (EFSA) approval of alfalfa protein concentrate as novel food states presence of metabolites as a barrier for higher amount of consumption. A promising method for protein isolation is anion exchange chromatography, which is scalable through expanded bed chromatography. In this study, ultrafiltration-buffer exchange (UBE) and ultrafiltration-diafiltration was applied as an upstream and downstream processing to anion exchange chromatography, respectively. The performance of these techniques was compared to that of lab scale dilution and centrifugal spin filters. The result showed that UBE shortened the chromatography duration by 50 \% and resulted in a cleaner column. However, downstream desalination through ultrafiltration-diafiltration resulted in concentrate with lower protein content (60.41 ± 0.84 \%), compared to that obtained from centrifugal spin filters (90.49 ± 1.34 \%), suggesting the need to carry out extra diafiltration steps. Untargeted metabolomics revealed that high absorption at 214 nm in the anion exchange flowthrough fraction could be attributed to di- and tripeptides also present in the ultrafiltration permeates. Polyphenols present in ultrafiltration permeate were absent from both flowthrough and 225 mM fraction, suggesting that they might bind strongly to the column. Various carboxylic acids were present in the ultrafiltration permeate, chromatography flowthrough, and 225 mM NaCl fraction. Contaminants eluting at 225 mM NaCl with high absorbance at 214 nm were not successfully identified although their occurrence was lower in UBE samples. These results suggest that UBE is promising as pre-treatment for purification of RuBisCO from alfalfa using anion exchange chromatography.",

keywords = "Alfalfa, Anion exchange chromatography, Metabolomics, RuBisCO, Ultrafiltration",

author = "Hartono Tanambell and Marianne Danielsen and M{\o}ller, \{Anders Hauer\} and Dalsgaard, \{Trine Kastrup\}",

note = "Publisher Copyright: {\textcopyright} 2025 The Authors",

year = "2025",

month = dec,

doi = "10.1016/j.fbp.2025.10.011",

language = "English",

volume = "154",

pages = "440--448",

journal = "Food and Bioproducts Processing",

issn = "0960-3085",

publisher = "Institution of Chemical Engineers",

}

Proof-of-concept: Ultrafiltration as a pre-treatment for the anion exchange chromatography of RuBisCO from alfalfa. / Tanambell, Hartono ; Danielsen, Marianne; Møller, Anders Hauer et al.
In: Food and Bioproducts Processing, Vol. 154, 12.2025, p. 440-448.

Research output: Contribution to journal/Conference contribution in journal/Contribution to newspaper › Journal article › Research › peer-review

TY - JOUR

T1 - Proof-of-concept

T2 - Ultrafiltration as a pre-treatment for the anion exchange chromatography of RuBisCO from alfalfa

AU - Tanambell, Hartono

AU - Danielsen, Marianne

AU - Møller, Anders Hauer

AU - Dalsgaard, Trine Kastrup

PY - 2025/12

Y1 - 2025/12

N2 - Alfalfa is gaining interest as an alternative protein source. The European Food Safety Authority (EFSA) approval of alfalfa protein concentrate as novel food states presence of metabolites as a barrier for higher amount of consumption. A promising method for protein isolation is anion exchange chromatography, which is scalable through expanded bed chromatography. In this study, ultrafiltration-buffer exchange (UBE) and ultrafiltration-diafiltration was applied as an upstream and downstream processing to anion exchange chromatography, respectively. The performance of these techniques was compared to that of lab scale dilution and centrifugal spin filters. The result showed that UBE shortened the chromatography duration by 50 % and resulted in a cleaner column. However, downstream desalination through ultrafiltration-diafiltration resulted in concentrate with lower protein content (60.41 ± 0.84 %), compared to that obtained from centrifugal spin filters (90.49 ± 1.34 %), suggesting the need to carry out extra diafiltration steps. Untargeted metabolomics revealed that high absorption at 214 nm in the anion exchange flowthrough fraction could be attributed to di- and tripeptides also present in the ultrafiltration permeates. Polyphenols present in ultrafiltration permeate were absent from both flowthrough and 225 mM fraction, suggesting that they might bind strongly to the column. Various carboxylic acids were present in the ultrafiltration permeate, chromatography flowthrough, and 225 mM NaCl fraction. Contaminants eluting at 225 mM NaCl with high absorbance at 214 nm were not successfully identified although their occurrence was lower in UBE samples. These results suggest that UBE is promising as pre-treatment for purification of RuBisCO from alfalfa using anion exchange chromatography.

AB - Alfalfa is gaining interest as an alternative protein source. The European Food Safety Authority (EFSA) approval of alfalfa protein concentrate as novel food states presence of metabolites as a barrier for higher amount of consumption. A promising method for protein isolation is anion exchange chromatography, which is scalable through expanded bed chromatography. In this study, ultrafiltration-buffer exchange (UBE) and ultrafiltration-diafiltration was applied as an upstream and downstream processing to anion exchange chromatography, respectively. The performance of these techniques was compared to that of lab scale dilution and centrifugal spin filters. The result showed that UBE shortened the chromatography duration by 50 % and resulted in a cleaner column. However, downstream desalination through ultrafiltration-diafiltration resulted in concentrate with lower protein content (60.41 ± 0.84 %), compared to that obtained from centrifugal spin filters (90.49 ± 1.34 %), suggesting the need to carry out extra diafiltration steps. Untargeted metabolomics revealed that high absorption at 214 nm in the anion exchange flowthrough fraction could be attributed to di- and tripeptides also present in the ultrafiltration permeates. Polyphenols present in ultrafiltration permeate were absent from both flowthrough and 225 mM fraction, suggesting that they might bind strongly to the column. Various carboxylic acids were present in the ultrafiltration permeate, chromatography flowthrough, and 225 mM NaCl fraction. Contaminants eluting at 225 mM NaCl with high absorbance at 214 nm were not successfully identified although their occurrence was lower in UBE samples. These results suggest that UBE is promising as pre-treatment for purification of RuBisCO from alfalfa using anion exchange chromatography.

KW - Alfalfa

KW - Anion exchange chromatography

KW - Metabolomics

KW - RuBisCO

KW - Ultrafiltration

UR - https://www.scopus.com/pages/publications/105018673134

U2 - 10.1016/j.fbp.2025.10.011

DO - 10.1016/j.fbp.2025.10.011

M3 - Journal article

AN - SCOPUS:105018673134

SN - 0960-3085

VL - 154

SP - 440

EP - 448

JO - Food and Bioproducts Processing

JF - Food and Bioproducts Processing

ER -

Proof-of-concept: Ultrafiltration as a pre-treatment for the anion exchange chromatography of RuBisCO from alfalfa

Abstract

Keywords

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