- Preprint Article
1- 10.22541/au.174291332.23735717/v1
A collaborative study to validate in vitro assays for protein digestibility assessment using pH-drop and pH-stat methods.
- Mar 25, 2025
- Erin Goldberg + 15 more +15
Protein digestibility is a crucial determinant of nutritional quality in foods, influencing how well the body can utilize amino acids derived from dietary protein. Historically, protein digestibility has been measured using in vivo rodent bioassays, particularly in calculating the PDCAAS. However, with growing public and scientific concerns over the ethical implications of animal testing, there is an urgent need for validated in vitro methods that are scientifically reliable and ethically sound. This report presents findings from an international collaborative study aimed at generating necessary data to position two in vitro methods—the pH-drop and pH-stat assays—for determining protein digestibility as candidates for method approval by an accrediting body. Nine laboratories participated in the study, analyzing 12 diverse protein ingredients from both plant and animal sources having previously published true fecal protein digestibility values. Mean relative standard deviations for repeatability ranged from 0.8-2.1% and 0.5-4.8% and mean relative standard deviation for reproducibility ranged from 1.2-3.6% and 1.1-4.9% for the pH-drop and pH-stat methods, respectively. The strong repeatability and reproducibility of both in vitro methods and the achievement of an official accreditation represent a critical advancement in the process toward acceptance by regulatory adoption as alternatives to in vivo testing to determine protein quality by PDCAAS. The validated methods presented here provide a reliable, affordable, and non-animal-based way to account for protein digestion in food formulation. While regulatory approval is required for use on food labels, these tools are immediately available to product developers to guide ingredient choices and processing decisions.
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