In-Vitro DIAAS: A Practical Guide to “Protein Quality” Testing

Introduction

Protein quality is no longer judged solely by how much protein a food contains, but by how efficiently its amino acids are digested and absorbed by the human body. For decades, the Protein Digestibility Corrected Amino Acid Score (PDCAAS) served as the global standard. However, limitations such as reliance on fecal digestibility and score truncation led to the development of a more physiologically relevant metric.

That is where Digestible Indispensable Amino Acid Score (DIAAS) and its lab-friendly cousin IV-DIAAS become important—especially for India’s fast-growing market of plant proteins, fortified foods, and high-protein packaged products.

What is DIAAS?

•   Indispensable (essential) amino acids in the food (histidine, isoleucine, leucine, lysine, methionine, phenylalanine, threonine, tryptophan, and valine)
•   Digestibility of each essential amino acid at the ileum (end of the small intestine)
•   Comparison against a reference amino-acid requirement pattern for a target group (children vs adults)

Unlike PDCAAS, an older approach, DIAAS focuses on amino-acid-by-amino-acid digestibility, which matters a lot for cereals, pulses, millets, and processed protein ingredients.

The general DIAAS equation is:

DIAAS (%) = 100 × (mg of digestible indispensable amino acid in 1 g of test protein / mg of the same amino acid in 1 g of FAO/WHO reference protein)

The lowest score among indispensable amino acids defines the final DIAAS value.

Methodology of In-Vitro DIAAS

In-vitro DIAAS is an estimated DIAAS-style score derived using laboratory-simulated gastrointestinal digestion rather than animal or human ileal digestibility trials. In this approach, essential amino acids are quantified in the digested (bioaccessible) fraction, and a DIAAS-like score is subsequently calculated based on the first limiting indispensable amino acid

This type of simulated gastrointestinal digestion is widely used in protein research to understand what remains stable or becomes available after digestion.

For real-world use cases where IV-DIAAS is useful

1. Product development for Indian protein foods

IV-DIAAS helps companies and researchers compare: Whey/casein vs soy vs pea, Pulse proteins (chana, moong, urad, masoor), Millet + pulse blends (ragi–urad, jowar–soy, bajra–moong), Protein isolates/concentrates used in bars, RTD drinks, powders, and baked foods.

Why it matters: Two products can both claim “20 g protein,” yet one can deliver much less digestible lysine or sulfur amino acids, especially after processing.

2. Improving plant-based proteins (a big Indian need)

Many Indian diets are cereal-heavy, while many plant proteins have one or two limiting amino acids. IV-DIAAS helps to:

•   Identify the limiting amino acid (often lysine in cereals; methionine/cysteine in many pulses)
•   Design better cereal–pulse complementation
•   Evaluate the effect of soaking, germination, fermentation, extrusion, roasting

3. Evidence for nutrition claims (and stronger R&D decisions)

IV-DIAAS can support internal R&D decisions about:

•   Choosing protein sources
•   Deciding processing conditions
•   Deciding whether fortification with specific amino acids (e.g., lysine) is justified
(Regulatory acceptance and claim wording depend on jurisdiction and the exact method/validation—IV-DIAAS is often best viewed as a high-quality R&D tool unless formally standardized for claims.)

Why only a few labs in India can do IV-DIAAS well (today):

High-end infrastructure: Accurate IV-DIAAS requires full essential amino acid profiling, reproducible digestion simulations, and LC/LC–MS–grade analytics with robust QA/QC—capabilities beyond routine protein testing labs.

Rare interdisciplinary expertise: The method spans protein chemistry, digestive enzymology, analytical amino acid methods, and nutrition science, a skill set concentrated mainly in advanced R&D labs.

Validation-driven credibility: Meaningful IV-DIAAS data depend on rigorous method validation, repeatability, and comparability—requiring sustained time, expertise, and investment.

Experimental example of 95% crude plant protein sample:

Calculate In-vitro Ileal AA Digestibility and In-vitro predicted DIAAS:

Conclusion:

For internal R&D comparisons, the FAO reference amino acid pattern for adults (≥18 years) is used to assess whether the essential amino acid requirements are met by the sample protein. The in vitro–predicted DIAAS (IV-DIAAS) reveals substantial amino acid–specific variability in protein quality. Several indispensable amino acids, including leucine, histidine, and phenylalanine + tyrosine, exhibits IV-DIAAS values exceeding 100%. In contrast, lysine, isoleucine, and valine does not meet the reference requirements. Notably, L-threonine is identified as the first limiting amino acid. Additionally, the mean in vitro ileal digestibility of the protein is comparatively low, indicating reduced enzymatic hydrolysis and amino acid release under simulated gastrointestinal conditions. According to DIAAS principles, this protein is classified as an incomplete protein and is not suitable as a sole dietary protein source for adults (≥18 years); therefore, supplementation with complementary protein sources is required to meet essential amino acid requirements.

IV-DIAAS is a powerful, lab-based way to estimate protein quality, especially valuable for India’s plant-protein innovation and nutrition programs. But it remains expensive, method-sensitive, and analytically demanding, which is why only a limited number of Indian labs currently produce high-confidence IV-DIAAS results.

Contributed by : Abu Saleh Musha Islam https://www.linkedin.com/in/abu-saleh-musha-islam-69b49820b/

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