Nine-lab study backs animal-free protein digestibility tests for food labeling

A coordinated study involving nine laboratories across the USA and Canada has supported the use of two in vitro testing methods as alternatives to animal-based protein digestibility assessments used in food labeling.

The research, published with support from the Institute for the Advancement of Food and Nutrition Sciences (IAFNS), evaluated whether the pH-drop and pH-stat laboratory methods could reliably measure protein digestibility across multiple laboratories. The results indicated both approaches demonstrated consistent repeatability and reproducibility, suggesting they could serve as standardized screening tools for evaluating protein quality.

• Nine laboratories in the US and Canada tested 12 plant and animal protein ingredients using pH-drop and pH-stat in vitro methods to evaluate protein digestibility across multiple labs.

• The cross-laboratory study found low variability and strong reproducibility, supporting the potential use of the methods as standardized tools for screening protein digestibility.

• Researchers said the approaches could reduce reliance on animal-based testing and help support innovation in new and alternative protein ingredients.

Protein digestibility remains a key factor in determining protein quality for food labeling in North America. Current regulations rely on digestibility values generated from animal studies. For companies developing new foods or novel ingredients without existing data, this requirement can present cost, logistical and ethical challenges.

Researchers involved in the new work set out to test whether simpler laboratory-based methods could deliver consistent results across different facilities. All nine participating labs analyzed the same set of 12 protein ingredients derived from both plant and animal sources.

The study focused on two in vitro approaches widely discussed in research circles. The pH-drop method measures the decline in pH as proteins are digested by enzymes under controlled conditions, while the pH-stat method tracks the amount of base required to maintain a constant pH during enzymatic digestion.

Both tests are designed to simulate aspects of protein digestion without relying on live animals. According to the study, the results across participating laboratories showed “low interlaboratory variability across samples and confirming stable reproducibility.”

The findings come as interest in plant-based, fermentation-derived and other novel protein sources continues to grow. As developers experiment with new ingredients, accurately assessing protein quality has become increasingly important for formulation, regulatory compliance and labeling claims.

North American food labeling rules currently require protein digestibility correction factors derived from animal testing when calculating overall protein quality. While published values exist for many established ingredients, new or unconventional protein sources may not have these data available.

The study’s authors described this situation as a 'paradox' for companies developing alternative proteins, particularly those targeting consumers who prefer products developed without animal testing.

In such cases, manufacturers seeking to make protein claims on product labels must either rely on existing data that may not reflect their specific ingredients or conduct animal digestibility studies to generate new values.

Researchers suggested validated in vitro methods could help address this challenge by offering a practical early-stage screening approach for ingredient development and processing decisions.

The paper concluded that both standardized pH-drop and pH-stat tests showed acceptable levels of repeatability and reproducibility. According to the authors, the methods are suitable as screening tools for evaluating ingredient selection and understanding how processing conditions affect protein quality.

The researchers added that further work is still needed, including assessing how well the methods apply to hydrolyzed proteins and other specialized ingredients. Nevertheless, they said the techniques could help reduce reliance on animal testing during the early stages of product development.

Chris Marinangeli from Protein Industries Canada said the research could play an important role as food systems evolve. “This work is critical for the ability for industry and consumers to adapt to changing food systems and dietary patterns by, hopefully, expediting protein quality assessments,” he said. “Also, there are always opportunities for regulatory agencies to adopt new and validated methods that reduce regulatory burden.”

If accepted by regulators in the future, in vitro methods such as pH-drop and pH-stat could enable food companies to communicate protein quality on product labels while addressing ethical concerns associated with animal testing.

IAFNS said the work was conducted according to Uniform Methods Committee standards of analysis and supported by its Protein Committee. The nonprofit organization brings together researchers from government, academia and industry to advance science across the food and beverage sector.

As the range of protein ingredients expands, from plant and microbial sources to novel formulations, the ability to evaluate digestibility in a consistent and efficient way is becoming increasingly important for both developers and regulators.