Protein digestibility and absorption quality

The protein transition is in full swing. But what about the nutritional value of all these innovative protein products? How much of it do we actually absorb as humans? And what about individual variation in digestion and absorption? To measure is to know. And methods to assess quality using human participants are available.

Protein content in products is assessed using validated methods (e.g., Kjeldahl or DUMAS). This information appears on the nutritional labels of products. However, it only tells you something about the quantity, and nothing about the ideal balance of amino acids or their digestibility. And that also matters.

Protein quality

Protein quality is determined by the amount of protein in the product, the balance of essential amino acids, and digestibility—meaning amino acid absorption in the small intestine. If essential amino acids are missing, or if proteins are poorly broken down into amino acids in our digestive system, their value to our body diminishes. Proteins perform countless crucial functions in the human body, such as building muscle tissue, providing energy, supporting enzyme activity, regulating hormonal processes, and more. They are composed of 20 different amino acids, some of which the human body cannot produce itself. These are called essential amino acids and must be obtained through our diet. An expert team from the WHO/FAO has determined the ideal balance of these essential amino acids for our diet, while the Netherlands Nutrition Centre provides recommendations on the daily protein intake for individuals. Our health depends, in part, on the proteins we consume.

Protein quality evaluations

Protein quality is currently evaluated primarily using the Protein Digestibility Corrected Amino Acid Score (PDCAAS) or the Digestible Indispensable Amino Acid Score (DIAAS). Both scores are calculated based on unused proteins found in feces or at the end of the small intestine (Mathai et al., 2017). These methods rely on animal testing, a practice that we would like to reduce given the availability of good alternatives. A second limitation is that these methods do not account for differences in absorption among individuals or target groups. In vitro laboratory models are valuable during the product development phase. Methods based on the consensus digestion protocol (INFOGEST) and methanol-based separation into absorbable and non-absorbable fractions are widely accepted by research groups (including WUR), however, further validation is still needed. Hopefully the method will receive an ISO certification soon.

We believe that a good alternative way to evaluate protein quality in products is by testing with human subjects. Several research methods are available for this, such as nasogastric intubation in healthy individuals. However, this approach is burdensome for participants (Guillin et al., 2022). Recruiting human ileostomies offers an alternative (Hodgkinson et al., 2022), though caution is required due to potential physiological abnormalities in digestion.

Intrinsically labeled proteins

Using intrinsically labeled proteins (with stable isotopes like 13C, 15N) is another promising method (Bandyopadhyay et al., 2022), but this methology comes with higher costs. The plants (or other biomass that produce these proteins for research) must be specially cultivated in controlled environments, where the label is supplied through water or air. The labeled plant material can then be used to extract proteins for digestion research with human subjects. The labeled proteins absorbed can be distinguished from those already present in the body. However, the scale of plant cultivation under these conditions is never large enough to extract proteins for use in industrial-scale production of protein-rich products. We are very interested in the effect of large-scale processes on protein quality since these are the products consumers actually eat.

Postprandial amino acid testing

A simpler approach to estimate relative protein quality in humans is the postprandial amino acid absorption test in blood. In this method, a person consumes 20 grams of protein (in any form or food category), and amino acid concentrations in their blood are measured at different intervals afterward. Participants come to the facility after a standard evening meal and an overnight fast, during which blood amino acid levels are low. We begin by measuring these fasting concentrations. After consuming the protein rich product, blood samples are taken 14 times over the next 5 hours. A week later, the same participants consume a different product. We can measure up to six products over six weeks in a cross-over study, with one or two reference products often included. Using standardized software developed at WUR (Wehrens et al., 2024), we compare the postprandial amino acid levels in the blood and evaluate the digestibility and absorption quality of different products. At WUR, we have already validated many products this way, often in collaboration with the food industry (e.g., Mes et al., Ummels et al., Esser et al., 2023). We hope to test many more products in the future.

Globally, more research groups are using this method. These studies have resulted in over 75 publications, from which we have compiled a database of results. This database aids in further standardizing the method and facilitating comparisons between studies and products. A review publication is currently in progress. The postprandial amino acid test also provides deeper insights into individual variations in protein digestion and absorption (Mensink et al., submitted for publication). Even among young, healthy individuals, we observed a two- to threefold difference between individuals in the amount of amino acids absorbed from the same product. People with consistently poor protein digestion and absorption may face protein deficiencies—even if they follow dietary protein recommendations from the Nutrition Centre. The effects of product development and individual variation in digestion and absorption will be further explored in a newly launched TKI research project, PROUD (LVVN 23.106).

Evaluating quality more often

For protein-rich products, the focus should not only be on the amount of protein they contain but also on the balance of amino acids and the digestibility and absorption of these amino acids. We hope the food industry will evaluate the quality of their products more frequently. The research models for this are available, from laboratory tests for initial indications to human testing for more detailed insights.

ReferenCes

Bandyopadhyay S, Kashyap S, Calvez J, Devi S, Azzout-Marniche D, Tomé D, Kurpad AV, Gaudichon C. Evaluation of Protein Quality in Humans and Insights on Stable Isotope Approaches to Measure Digestibility - A Review. Adv Nutr. 2022 Aug 1;13(4):1131-1143.

Esser D, Wehrens R, Lenaerts K, Engel J, van den Dool RTM, Bastiaan-Net S, Mes JJ, Wichers HJ. Evaluating and comparing tolerance, nutritional quality and bio-functional activity of bovine-plasma, corn and whey proteins, outcomes of a randomized double blind controlled trial. Curr Res Food Sci. 2023 Sep 12;7:100588.

Guillin FM, Gaudichon C, Guérin-Deremaux L, Lefranc-Millot C, Airinei G, Khodorova N, Benamouzig R, Pomport PH, Martin J, Calvez J. Real ileal amino acid digestibility of pea protein compared to casein in healthy humans: a randomized trial. Am J Clin Nutr. 2022 Feb 9;115(2):353-363.

Hodgkinson SM, Stroebinger N, van der Wielen N, Mensink M, Montoya C, Hendriks WH, de Vries S, Stein HH, Moughan PJ. Comparison of True Ileal Amino Acid Digestibility between Adult Humans and Growing Pigs. J Nutr. 2022 Jul 6;152(7):1635-1646.

Mathai JK, Liu Y, Stein HH. Values for digestible indispensable amino acid scores (DIAAS) for some dairy and plant proteins may better describe protein quality than values calculated using the concept for protein digestibility-corrected amino acid scores (PDCAAS). Br J Nutr. 2017 Feb;117(4):490-499.

Mes JJ, Esser D, Oosterink E, van den Dool RTM, Engel J, de Jong GAH, Wehrens R, van der Meer IM. A controlled human intervention trial to study protein quality by amino acid uptake kinetics with the novel Lemna protein concentrate as case study. Int J Food Sci Nutr. 2022 Mar;73(2):251-262.

Ummels M, JanssenDuijghuijsen L, Mes JJ, van der Aa C, Wehrens R, Esser D. Evaluating Brewers' Spent Grain Protein Isolate Postprandial Amino Acid Uptake Kinetics: A Randomized, Cross-Over, Double-Blind Controlled Study. Nutrients. 2023 Jul 19;15(14):3196.

Wehrens R, Engel J, Mes J, de Jong A, Esser D. Analysing postprandial amino acid responses in crossover studies with the aaresponse package for R. Amino Acids. 2024 Mar 21;56(1):25.