Interlaboratory validation of an optimized protocol for measuring α-amylase activity by the INFOGEST international research network

Daniela Freitas; Terri Grassby; Shannon Gwala; Gwénaële Henry; Athina Lazaridou; Christine Boesch; Dorine Duijsens; Faye Wheller; Ivan Lopez-Rodulfo; Kali Kotsiou; Kendall Corbin; Marilisa Alongi; Mario Martinez; Maryam Hafiz; Monica M. M. Tomassen; Natalia Perez-Moral; Natalia Vidal; Renata M. C. Ariëns; Sebnem Simsek; Sedef El; Steven Le Feunteun; Sibel Karakaya; Shanna Bastiaan-Net; Svenja Krause; Bin Zhang; Caroline Orfila; Simon Ballance

doi:10.1038/s41598-025-12561-y

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Interlaboratory validation of an optimized protocol for measuring α-amylase activity by the INFOGEST international research network

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Interlaboratory validation of an optimized protocol for measuring α-amylase activity by the INFOGEST international research network

Daniela Freitas, Terri Grassby, Shannon Gwala, Gwénaële Henry, Athina Lazaridou, Christine Boesch, Dorine Duijsens, Faye Wheller, Ivan Lopez-Rodulfo, Kali Kotsiou, …

Scientific Reports, Vol.15(1), p.19

23/08/2025

DOI: https://doi.org/10.1038/s41598-025-12561-y

PMID: 40846714

Abstract

Life Sciences

The activity of α-amylases is frequently determined using a single-point assay at 20 °C. Previous work within INFOGEST "Working Group 5 -Starch digestion and amylases" identified significant interlaboratory variation with this protocol. The current study aimed to evaluate the repeatability (intralaboratory precision) and reproducibility (interlaboratory precision), measured as coefficients of variation (CVs), of a newly optimized protocol version based on four time-point measurements at 37 °C. Human saliva (a pool from ten healthy adults) and three porcine enzyme preparations (two pancreatic α-amylases and pancreatin) were tested in 13 laboratories across 12 countries and 3 continents. Assay repeatability for each lab remained below 20% for all test products and the overall repeatability was below 15%, ranging between 8 and 13% for all products. Reproducibility was greatly improved with interlaboratory CVs ranging from 16 to 21%, i.e. up to four times lower than with the original method. Five laboratories repeated the same assay at 20 °C, and the amylolytic activity of each product increased by 3.3-fold (± 0.3) from 20 to 37 °C. The newly optimized protocol is henceforth recommended to ensure precise determinations of α-amylase activity levels and to facilitate comparisons across different studies.

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Title: Interlaboratory validation of an optimized protocol for measuring α-amylase activity by the INFOGEST international research network
Creators: Daniela Freitas (Corresponding Author) - Teagasc Food Research Centre [Fermoy, Ireland]
Terri Grassby (Author) - University of Surrey, School of Biosciences
Shannon Gwala (Author) - Teagasc - The Irish Agriculture and Food Development Authority
Gwénaële Henry (Author) - Laboratoire Science & technologie du Lait & de l'œuf
Athina Lazaridou (Author) - Aristotle University of Thessaloniki
Christine Boesch (Author) - University of Leeds
Dorine Duijsens (Author) - KU Leuven
Faye Wheller (Author) - University of Surrey, School of Biosciences
Ivan Lopez-Rodulfo (Author) - Aarhus University
Kali Kotsiou (Author) - Aristotle University of Thessaloniki
Kendall Corbin (Author) - University of Kentucky
Marilisa Alongi (Author) - University of Udine
Mario Martinez (Author) - Aarhus University
Maryam Hafiz (Author) - King Abdulaziz University
Monica M. M. Tomassen (Author) - Wageningen Food & Biobased Research
Natalia Perez-Moral (Author) - Quadram Institute
Natalia Vidal (Author) - Aarhus University
Renata M. C. Ariëns (Author) - Wageningen Food & Biobased Research
Sebnem Simsek (Author) - Ege University
Sedef El (Author) - Ege University
Steven Le Feunteun (Author) - Laboratoire Science & technologie du Lait & de l'œuf
Sibel Karakaya (Author) - Ege University
Shanna Bastiaan-Net (Author) - Wageningen University & Research
Svenja Krause (Author) - Paris-Saclay Food and Bioproduct Engineering
Bin Zhang (Author) - South China University of Technology
Caroline Orfila (Author) - University of Leeds
Simon Ballance (Author)
Publication Details: Scientific Reports, Vol.15(1), p.19
Publisher: Nature Research
Publication Date: 23/08/2025
Date accepted for publication: 17/07/2025
Grants: ERA-NET Cofund, ERA-HDHL (GA N° 696295), European Union (Belgium, Brussels) - EU
Teagasc - The Irish Agriculture and Food Development Authority (Ireland, Carlow)
898013, European Union (Belgium, Brussels) - EU
SFI-IRC Pathway grant, 21/PATH-S/9418, Taighde Éireann - Research Ireland (Ireland, Dublin)
Department of Agriculture, Food and the Marine
12A0225N, Research Foundation - Flanders (Belgium, Brussels) - FWO
Sapere Aude Starting Grant, 1051-00046B, Danmarks Frie Forskningsfond (Denmark, Odense) - DFF
BBSRC Institute Strategic Programme: Food Microbiome and Health (FMH), BB/X011054/1, Biotechnology and Biological Sciences Research Council (United Kingdom, Swindon) - BBSRC
BBS/E/QU/230001A, Biotechnology and Biological Sciences Research Council (United Kingdom, Swindon) - BBSRC
Healthy and Safe Food System (KB-37), KB-37-001-007, Ministry of Agriculture, Nature and Food Quality (The Netherlands, The Hague) - LNV
SusHealth, NRC 314599, Fund for Research Fees for Agricultural Products (FFL)
Grant note: The authors would like to thank the INFOGEST research network for financially supporting this research. The authors would like to acknowledge Teagasc (the Irish Agriculture and Food Development Authority) for supporting the publication fees of this article. Daniela Freitas received funding from the European Union’s Horizon 2020 research and innovation program under the Marie Sklodowska-Curie grant agreement number 898013 as well as Research Ireland (previously Science Foundation Ireland and the Irish Research Council) under the SFI-IRC Pathway grant number 21/PATH-S/9418. Shannon Gwala received funding from the Irish Department of Agriculture and Marine (DAFM) under the umbrella of the European Joint Programming Initiative “A Healthy Diet for a Healthy Life” and of the ERA-NET co-fund ERA-HDHL (GA N° 696295) as well as the Marie Sklodowska-Curie grant agreement number 101107486, both from the EU Horizon 2020 Research and Innovation Programme. Consumables and salary for Terri Grassby were provided by University of Surrey. Dorine Duijsens is a postdoctoral researcher funded by the Research Foundation Flanders (FWO, grant 12A0225N). Mario M. Martinez received financial support from Independent Fund Denmark—Danmarks Frie Forskningsfond (Sapere Aude Starting Grant, project number 1051-00046B). Natalia Perez-Moral received funding from the BBSRC Institute Strategic Programme Food Microbiome and Health BB/X011054/1 and its constituent project BBS/E/QU/230001A (Theme 1, Micro- and macro-nutrient bioavailability from plant-based foods across the GIT). Shanna Bastiaan-Net acknowledges the financial support by the Dutch Ministry of Agriculture, Nature and Food Quality via the “Healthy and Safe Food System (KB-37)” knowledge base program (grant no. KB-37-001-007). The Norwegian Fund for Research Fees for Agricultural Products (FFL) is thanked for supporting the contribution of NOFIMA through the project SusHealth (NRC 314599).
Identifiers: 991035366402346; WOS:001559637000002
Copyright: Copyright © 2025, The Author(s). This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons licence, and indicate if changes were made. The images or other third party material in this article are included in the article’s Creative Commons licence, unless indicated otherwise in a credit line to the material. If material is not included in the article’s Creative Commons licence and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this licence, visit http://creativecommons.org/licenses/by/4.0/.This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons licence, and indicate if changes were made. The images or other third party material in this article are included in the article’s Creative Commons licence, unless indicated otherwise in a credit line to the material. If material is not included in the article’s Creative Commons licence and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this licence, visit http://creativecommons.org/licenses/by/4.0/. For the purpose of open access, the authors have applied a Creative Commons Attribution (CC BY) licence to any Author Accepted Manuscript version arising.
Academic Unit: School of Biosciences
Language: English
Resource Type: Journal article
Data Access Statement: The data supporting the findings of this study is provided within the manuscript or supplementary information files. Should any raw data files be needed, they are available from the corresponding author upon reasonable request.

Interlaboratory validation of an optimized protocol for measuring α-amylase activity by the INFOGEST international research network

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