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

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

doi:10.1038/s41598-025-12561-y

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

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

Research output: Contribution to journal › Article › peer-review

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Abstract

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.

Original language	English
Article number	30985
Journal	Scientific Reports
Volume	15
Issue number	1
DOIs	https://doi.org/10.1038/s41598-025-12561-y
State	Published - Dec 2025

Bibliographical note

Publisher Copyright:
© The Author(s) 2025.

Funding

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). Terri Grassby’s other research work has been funded by McCain Foods Ltd, Mondelez, and the Almond Board of California. All other authors have no competing interests to declare.

Funders	Funder number
Teagasc - Irish Agriculture and Food Development Authority
Irish Department of Agriculture and Marine
University of Surrey, UK
McCain Foods Ltd
Irish Research Council for Science, Engineering and Technology
Ministerie van Landbouw, Natuur en Voedselkwaliteit
Almond Board of California
INFOGEST
Science Foundation Ireland, SFI	21/PATH-S/9418
Horizon 2020 Framework Programme	898013
Fonds Wetenschappelijk Onderzoek	12A0225N
Biotechnology and Biological Sciences Research Council	BBS/E/QU/230001A, BB/X011054/1
Danmarks Frie Forskningsfond	1051-00046B
Department of Agriculture, Food and the Marine, Ireland	696295
Safe Food System	KB-37-001-007, KB-37
Nofima AS	NRC 314599
HORIZON EUROPE Marie Sklodowska-Curie Actions	101107486

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UN SDGs

This output contributes to the following UN Sustainable Development Goals (SDGs)

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Cite this

Freitas, D., Gwala, S., Henry, G., Lazaridou, A., Boesch, C., Duijsens, D., Wheller, F., Lopez-Rodulfo, I. M., Kotsiou, K., Corbin, K. R., Alongi, M., Martinez, M. M., Hafiz, M. S., Tomassen, M. M. M., Perez-Moral, N., Vidal, N. P., Ariëns, R. M. C., Simsek, S., El, S. N., ... Grassby, T. (2025). Interlaboratory validation of an optimized protocol for measuring α-amylase activity by the INFOGEST international research network. Scientific Reports, 15(1), Article 30985. https://doi.org/10.1038/s41598-025-12561-y

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

abstract = "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.",

author = "Daniela Freitas and Shannon Gwala and Gw{\'e}na{\"e}le Henry and Athina Lazaridou and Christine Boesch and Dorine Duijsens and Faye Wheller and Lopez-Rodulfo, \{Ivan M.\} and Kali Kotsiou and Corbin, \{Kendall R.\} and Marilisa Alongi and Martinez, \{Mario M.\} and Hafiz, \{Maryam S.\} and Tomassen, \{Monic M.M.\} and Natalia Perez-Moral and Vidal, \{Natalia P.\} and Ari{\"e}ns, \{Renata M.C.\} and Sebnem Simsek and El, \{Sedef Nehir\} and Sibel Karakaya and \{Le Feunteun\}, Steven and Shanna Bastiaan-Net and Svenja Krause and Bin Zhang and Caroline Orfila and Simon Ballance and Terri Grassby",

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Freitas, D, Gwala, S, Henry, G, Lazaridou, A, Boesch, C, Duijsens, D, Wheller, F, Lopez-Rodulfo, IM, Kotsiou, K, Corbin, KR, Alongi, M, Martinez, MM, Hafiz, MS, Tomassen, MMM, Perez-Moral, N, Vidal, NP, Ariëns, RMC, Simsek, S, El, SN, Karakaya, S, Le Feunteun, S, Bastiaan-Net, S, Krause, S, Zhang, B, Orfila, C, Ballance, S & Grassby, T 2025, 'Interlaboratory validation of an optimized protocol for measuring α-amylase activity by the INFOGEST international research network', Scientific Reports, vol. 15, no. 1, 30985. https://doi.org/10.1038/s41598-025-12561-y

TY - JOUR

T1 - Interlaboratory validation of an optimized protocol for measuring α-amylase activity by the INFOGEST international research network

AU - Freitas, Daniela

AU - Gwala, Shannon

AU - Henry, Gwénaële

AU - Lazaridou, Athina

AU - Boesch, Christine

AU - Duijsens, Dorine

AU - Wheller, Faye

AU - Lopez-Rodulfo, Ivan M.

AU - Kotsiou, Kali

AU - Corbin, Kendall R.

AU - Alongi, Marilisa

AU - Martinez, Mario M.

AU - Hafiz, Maryam S.

AU - Tomassen, Monic M.M.

AU - Perez-Moral, Natalia

AU - Vidal, Natalia P.

AU - Ariëns, Renata M.C.

AU - Simsek, Sebnem

AU - El, Sedef Nehir

AU - Karakaya, Sibel

AU - Le Feunteun, Steven

AU - Bastiaan-Net, Shanna

AU - Krause, Svenja

AU - Zhang, Bin

AU - Orfila, Caroline

AU - Ballance, Simon

AU - Grassby, Terri

PY - 2025/12

Y1 - 2025/12

N2 - 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.

AB - 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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VL - 15

JO - Scientific Reports

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ER -

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

Abstract

Bibliographical note

Funding

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UN SDGs

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