A collaborative study to validate in vitro assays for protein digestibility assessment using pH-drop and pH-stat methods

The diversification of protein sources used in food formulation has increased the need to assess protein quality beyond traditional food forms. Protein digestibility is a key component of the Protein Digestibility Corrected Amino Acid Score (PDCAAS), the regulatory metric derived from rodent bioassays. Ethical concerns and the high cost of animal testing limit the ability of food formulators to screen protein ingredients and evaluate processing effects on digestibility. Findings from an international collaborative study are presented to position two in vitro methods, the pH-drop and pH-stat assays, as accredited approaches for determining protein digestibility. Nine laboratories participated in the study and analyzed 12 protein ingredients. Relative standard deviations for repeatability ranged from 0.8–2.1% and 0.5–4.8%, while reproducibility ranged from 1.2–3.6% and 1.1–4.9% for the pH-drop and pH-stat methods, respectively. Comparison between the two assays demonstrated strong correlation and moderate agreement, with the pH-stat assay yielding slightly lower digestibility values. In vitro protein digestibility coefficients aligned well with literature reported true faecal protein digestibility values for comparable, non-identical protein ingredients. Both methods received official AOCS Uniform Methods Committee approval, offering simple, affordable, reliable, and ethical tools to support informed decisions on protein digestibility during food formulation. ● 9 laboratories successfully evaluated protein digestibility of 12 diverse proteins ● Both pH assays showed strong repeatability and reproducibility across laboratories ● pH-stat assay yielded slightly lower digestibility values than the pH-drop method ● Validated in vitro assays offer ethical, affordable tools for protein screening ● American Oil Chemists' Society approved pH-drop and pH-stat methods in 2025