Protein is an essential macronutrient in our diet, source of nitrogen and essential
amino acids, but the biological utilization of dietary protein depends on its
digestibility and the absorption of amino acids and peptides in the gastrointestinal
tract. The methods to define the amount and the quality of protein to meet
human nutritional needs, such as the Digestible Indispensable Amino Acid
Score (DIAAS), require the use of animal models or human studies. These in vivo
methods are the reference in protein quality evaluation, but they are expensive
and long-lasting procedures with significant ethical restrictions. Therefore, the
development of rapid, reproducible and in vitro digestion methods validated
with in vivo data is an old demand. This review describes the challenges of the
in vitro digestion methods in the evaluation of the protein nutritional quality.
In addition to the technical difficulties to simulate the complex and adaptable
processes of digestion and absorption, these methods are affected by similar
limitations as the in vivo procedures, i.e., analytical techniques to accurately
determine bioavailable amino acids and the contribution of the endogenous
nitrogen. The in vitro methods used for the evaluation of protein digestibility,
with special attention on those showing comparative data, are revised,
emphasizing their pros and cons. The internationally harmonized digestion
protocol proposed by the INFOGEST network is being adapted to evaluate
protein and amino acid digestibility. The inter-laboratory reproducibility of this
protocol was demonstrated for dairy products. The in vivo/in vitro comparability
results obtained to date with this protocol for several plant and animal sources
are promising, but it requires an extensive validation with a wider range of foods
and substrates with known in vivo digestibility. These in vitro methods will
probably not be applicable to all foods, and therefore, it is important to identify
their limitations, not to elude their use, but to apply them within the limits, by
using the appropriate standards and references, and always as a complementary
tool to in vivo tests to reduce their number.