Protein Quality in Perspective: A Review of Protein Quality Metrics and Their Applications

Abstract

For design of healthy and sustainable diets and food systems, it is important to consider not only the quantity but also the quality of nutrients. This is particularly important for proteins, given the large variability in amino acid composition and digestibility between dietary proteins. This article reviews measurements and metrics in relation to protein quality, but also their application. Protein quality methods based on concentrations and digestibility of individual amino acids are preferred, because they do not only allow ranking of proteins, but also assessment of complementarity of protein sources, although this should be considered only at a meal level and not a diet level. Measurements based on ileal digestibility are preferred over those on faecal digestibility to overcome the risk of overestimation of protein quality. Integration of protein quality on a dietary level should also be done based on measurements on an individual amino acid basis. Effects of processing, which is applied to all foods, should be considered as it can also affect protein quality through effects on digestibility and amino acid modification. Overall, protein quality data are crucial for integration into healthy and sustainable diets, but care is needed in data selection, interpretation and integration.

Full text



Citation: Adhikari, S.; Schop, M.; de
Boer, I.J.M.; Huppertz, T. Protein
Quality in Perspective: A Review of
Protein Quality Metrics and Their
Applications. Nutrients 2022,14, 947.
https://doi.org/10.3390/
nu14050947
Academic Editor: Matteo Tosato
Received: 20 January 2022
Accepted: 21 February 2022
Published: 23 February 2022
Publisher’s Note: MDPI stays neutral
with regard to jurisdictional claims in
published maps and institutional affil-
iations.
Copyright: © 2022 by the authors.
Licensee MDPI, Basel, Switzerland.
This article is an open access article
distributed under the terms and
conditions of the Creative Commons
Attribution (CC BY) license (https://
creativecommons.org/licenses/by/
4.0/).
nutrients
Review
Protein Quality in Perspective: A Review of Protein Quality
Metrics and Their Applications
Shiksha Adhikari 1, Marijke Schop 2, Imke J. M. de Boer 2and Thom Huppertz 1, 3, *
1
Food Quality & Design Group, Wageningen University & Research, 6708 WG Wageningen, The Netherlands;
shiksha.adhikari@wur.nl
2Animal Production Systems Group, Wageningen University & Research,
6708 WD Wageningen, The Netherlands; marijke.schop@wur.nl (M.S.); imke.deboer@wur.nl (I.J.M.d.B.)
3Friesland Campina, Research and Development, 3818 LE Amersfoort, The Netherlands
*Correspondence: thom.huppertz@wur.nl
Abstract:
For design of healthy and sustainable diets and food systems, it is important to consider not
only the quantity but also the quality of nutrients. This is particularly important for proteins, given
the large variability in amino acid composition and digestibility between dietary proteins. This article
reviews measurements and metrics in relation to protein quality, but also their application. Protein
quality methods based on concentrations and digestibility of individual amino acids are preferred,
because they do not only allow ranking of proteins, but also assessment of complementarity of protein
sources, although this should be considered only at a meal level and not a diet level. Measurements
based on ileal digestibility are preferred over those on faecal digestibility to overcome the risk of
overestimation of protein quality. Integration of protein quality on a dietary level should also be done
based on measurements on an individual amino acid basis. Effects of processing, which is applied to
all foods, should be considered as it can also affect protein quality through effects on digestibility and
amino acid modification. Overall, protein quality data are crucial for integration into healthy and
sustainable diets, but care is needed in data selection, interpretation and integration.
Keywords:
protein quality; indispensable amino acids; digestibility; food processing; complementarity
1. Introduction
Food and nutrition play a crucial role in the maintenance of human health and the
prevention of non-communicable diseases. It has been estimated that in 2017, 11 million
deaths and 255 million disability-adjusted life years (DALYs) were attributable to dietary
risk factors [
1
]. The main risk factors identified are diets high in sodium, diets low in
whole grains, diets low in fruit, diets low in vegetables and diets low in nuts and seeds.
Other factors include diets low in milk, diets high in sugar-sweetened beverages and diets
high in processed meat [
1
]. Management of these dietary risk factors requires a balanced
composition of the human diet. Composing an optimal diet from a nutritional perspective
should focus on ensuring that all essential nutrients are provided at the required levels,
while at the same time also on ensuring that this is done through a combination of food
products which does not result in excess intake of nutrient or non-nutrient components
which can lead to dietary risk factors [
2
]. In other words, ensuring sufficient intake of an
essential nutrient like vitamin C should not be in the form of sugar-sweetened beverages
considering that diets high in these products increase risks of cardiovascular diseases and
type 2 diabetes.
To assist the public, but also policy-makers and health professionals, in improving
eating patterns and select healthy diets, dietary guidelines are published in many countries.
These dietary guidelines are often based on food groups, e.g., fruits and vegetables, dairy,
meat, fish, grains, etc. and recommend a minimum or maximum intake of food products
from each group [
3
,
4
]. In the past two decades, however, it has become abundantly
Nutrients 2022,14, 947. https://doi.org/10.3390/nu14050947 https://www.mdpi.com/journal/nutrients

Nutrients 2022,14, 947 2 of 31
clear that diets good for human health are not necessarily good for planetary health and
that in addition to human nutrition, the impact of food production on planetary health
should also be considered [
5
,
6
]. Based hereon, the EAT-Lancet commission on healthy
diets from sustainable food systems introduces a global planetary health diet that was
designed to be optimal for both human health and planetary health [
7
]. This proposed
diet called from a shift from animal-based to plant-based foods [
7
], a recommendation
voiced by others as well, primarily based on the environmental impact of animal-based
food production [5,8–11].
However, it has also been argued extensively that exclusively plant-based diets entail
risks of nutrient deficiencies because some essential nutrients are not present, or only
present at low amounts, in plant-based foods, e.g., vitamin B12 or iodine [
12
–
15
]. For other
essential nutrients, e.g., calcium (Ca) or zinc (Zn), plant-based foods may be able to supply
reasonable levels of intake, but low bioavailability of these minerals in many plant-based
foods, due to the presence of phytates or oxalates, leads to risks of deficiencies [
15
–
19
]. In
addition to micronutrients, concerns have also been raised over one of the macronutrients
in relation shifts to more plant-based diets, i.e., protein [
8
,
20
,
21
]. The class of dietary
proteins is extremely diverse, with notable variations both in amino acid composition and
in digestibility of the protein between different sources of dietary protein. As both the
right amino acid composition and high digestibility are required for proteins to meet the
requirements of the human body, the ability of dietary proteins to meet these requirements
varies widely [
22
,
23
]. Such abilities are often quantitatively expressed in so-called protein
quality metrics, which include amino acid composition and digestibility [
22
,
23
]. It has
been argued that such protein quality metrics should be considered in the assessment of
environmental impact of food proteins. Recent publications in this field have highlighted
that the consideration of protein quality notably affects consideration of the environmental
impact of dietary proteins [24,25].
While consideration of protein quality in the dietary context and in view of dietary
shifts aimed at both human and planetary health is thus important, the implementation of
this concept is not easy. Reported protein quality metrics can be used to rank proteins, the
inclusion of such metrics in a dietary perspective is more complex because protein, unlikely,
e.g., vitamin C or Ca, is not a single nutrient, but the carrier of many essential nutrients,
i.e., 9 indispensable amino acids, plus dietary nitrogen. Integration of protein quality in
a dietary perspective thus requires consideration of multiple aspects. In this paper, we
review the concept of protein quality, with particular emphasis on the inclusion of this
concept in dietary considerations. For this purpose, the importance of protein for human
nutrition and health, as well as protein digestion and amino acid absorption are covered
in Section 2. Subsequently, Section 3covers the different methodologies that have been
applied to determine protein quality. Section 4considers the interpretation and application
of data derived from protein quality measurements. Finally, Sections 5and 6cover two
important aspects for consideration of protein quality on a dietary basis. The effect of
processing and preparation of food products is the focus of Section 5. Section 6covers the
complementarity of different proteins on a dietary basis, but with that also outlines the
crucial importance of the time scales at which complementarity is considered, i.e., on a
meal level rather than on a diet level. Finally, in Section 7, we provide some conclusions
and perspectives on future steps for including the concept of protein quality in dietary
recommendations.
2. Protein Quality
2.1. The Importance of Proteins and Amino Acids for Human Nutrition and Health
Next to carbohydrate and fat, protein is one of the macronutrients. Digestible carbohy-
drates are only a source of energy to the human body, whereas dietary fat is a source of
energy as well as a source of the essential fatty acids linoleic acid (LA) and
α
-linolenic acid
(ALA), which cannot be synthesized by the human body. Adequate intake (AI) levels for
LA and ALA for adults were defined by EFSA as 4% and 0.5% of total energy intake (EI),

Nutrients 2022,14, 947 3 of 31
whereas total fat intake was recommended to be between 20% and 35% of EI [
26
]. Hence,
while dietary fat is not only a source energy for the human body, 78–87% of fat is actually
used as a source of energy at the recommended intake levels. Like carbohydrates and fat,
protein can also be a source of energy. However, far more importantly, proteins are the
main dietary source of nitrogen and indispensable amino acids (IAAs), which are required
by the body for protein synthesis to enable e.g., tissue growth and maintenance [27].
Proteins play a crucial role in the growth, maintenance and physiological functions of
the human body [
28
]. All amino acids are important in the synthesis and functioning of
muscles and organs, as well as in enzymes, hormones and the immune system [
29
]. Amino
acids are classified as dispensable amino acids (DAAs) and IAAs, based on whether or not
the body can synthesize the particular amino acid. DAAs can be synthesized de novo by the
human body [
29
], whereas IAAs cannot by synthesized by the human body and the only
source of IAAs is dietary protein; hence, it is important to assure adequate dietary intake of
IAAs [
30
]. In addition, some DAAs, such as arginine, cysteine, glutamine, glycine, proline
and tyrosine, can become conditionally indispensable, e.g., for premature neonates [
27
]. In
these cases, the body cannot produce sufficient levels of these amino acids and these amino
acids thus become conditionally IAAs, and need to be supplied through dietary protein to
compensate for insufficient synthesis in these stages of life [
27
,
31
,
32
]. To meet the metabolic
demand and to assure proper functioning of the human body, consumption of adequate
amounts of protein is thus essential to meet both total nitrogen and IAA requirements [
33
].
The general dietary requirement for protein is defined as an estimated average require-
ment (EAR) and recommended dietary allowance (RDA). The EAR is the daily intake level
for a nutrient that is estimated to meet the requirement for 50% of the target population,
whereas the RDA, which is calculated as the EAR plus two times the standard deviation,
meets the requirements for 97–98% of the population [
34
]. For all adults above 18 years
of age, the EAR for protein is 0.66 g protein per kg body weight per day and the RDA
is 0.83 g protein per kg body weight per day [
27
]. EAR and RDA values for children
less than 18 years and for pregnant and lactating women are higher than for the general
adult population [
27
]. Studies have also suggested that protein requirements for elderly
adults could be higher, as summarized by [
27
], and that amino acid requirements can be
amended to minimize aging-related health outcomes [
35
], but these findings have not yet
been translated into clear recommendations by authorities [
27
]. Further details on protein
requirements throughout life cycle are described in further detail elsewhere [
36
]. No upper
limits for protein intake, or the intake of specific amino acids, have been defined to date.
However, findings in the novel area of dietary protein restriction, as recently reviewed [
35
],
warrant further consideration in future.
In addition to total protein intake, requirements for each IAA have also been de-
fined [
30
,
32
]. The requirements for IAAs as defined by FAO [
30
,
32
] and EFSA [
27
] are
presented in Table 1. Similar to RDA values for total protein [
27
], IAA requirements are
highest for the 0.5–1-year-olds, and decrease progressively with increasing age (Table 1).
The decreasing requirements for each IAA with increasing age reflect the fact that in the
early stages of life, the IAAs are required for growth, development and maintenance of
the body, whereas in later stages of life, requirements for growth and development pro-
gressively decrease and requirements for IAAs are mainly based on maintenance [
37
,
38
].
Considering the requirements for IAAs (Table 1) and the fact that protein is the only dietary
source of IAAs, an RDA for protein thus does not only contain a quantitative aspect but
also a qualitative aspect; i.e., the RDA of 0.83 g protein/kg bodyweight/d for adults is only
sufficient to meet the requirements for target population if this intake also provides the
levels of IAAs outlined in the Table 1. For a protein that cannot meet these IAA levels at the
RDA for protein intake, either intake at the RDA level can lead to insufficient intake of one
or more IAAs or higher intake levels than the RDA are needed to achieve recommended
intake of IAA. Such aspects are central to the concept of protein quality.

Nutrients 2022,14, 947 4 of 31
Table 1.
Recommended intake of indispensable amino acids (in mg/kg body weight/day) for humans
in different age groups (data from [27,30,32]).
Age (Years) His Ile Leu Lys SAA * AAA ** Thr Trp Val
0.5–1 22 36 73 64 31 59 34 9.5 49
1–2 15 27 54 45 22 40 23 6.4 36
3–10 12 23 44 35 18 30 18 4.8 29
11–14 12 22 44 35 17 30 18 4.8 29
15–18 11 21 42 33 16 28 17 4.5 28
>18 10 20 39 30 15 25 15 4.0 26
* SAA = Sulphur-containing amino acids (Cys + Met); ** AAA = Aromatic amino acids (Phe + Tyr).
2.2. Protein Digestion and Absorption by Humans
As outlined earlier, proteins are the key source of IAAs for the human body. However,
for the IAAs, as well as the DAAs, from a protein to be utilized by the human body, the
protein first needs to be hydrolysed into free amino acids and small (di- and tri-) peptides,
which can subsequently be absorbed in the bloodstream [
39
]. Protein digestion is a complex,
multistage process, as schematically outlined in Figure 1. The protein digestion process
begins with the mechanical disruption of the product matrix containing the protein during
mastication in the mouth. While protein breakdown does not occur at this stage, the oral
phase of digestion can be important for protein digestion, because by disrupting the product
matrix, the surface area increases, which increases exposure of the protein to digestive
proteases and peptidases during the later stages of digestion [
40
,
41
]. Digestion of starch by
salivary amylase can also disrupt the product structure, thereby increasing accessibility of
protein to digestive enzymes in later stages of the digestion process [41].
Nutrients 2022, 14, x FOR PEER REVIEW 5 of 34
Figure 1. Schematic overview of the key steps of protein digestion and absorption in humans.
Following the oral phase of digestion, which typically lasts only for a short time (<1
min), the product passes on to the stomach, where the gastric phase of the digestion pro-
cess takes place. In this phase, the product is mixed with gastric juice, which has a low pH
(1–2 for adults) and contains the protease pepsin [42,43]. The mixing of gastric juice and
product is facilitated through contractions of the stomach. In the stomach, some hydroly-
sis of proteins by pepsin occurs, resulting in (poly)peptide formation [42], but complete
protein digestion to free amino acids and peptides small enough for absorption does not
occur at this stage [44].
Following gastric digestion, the chyme is delivered to the duodenum at a rate deter-
mined by the gastric emptying. In the small intestine, the chyme is mixed with pancreatic
proteases and peptidases, such as trypsin, chymotrypsin, and carboxypeptidase A [40].
Together with intestinal brush border enzymes, these enzymes hydrolyse the proteins and
(poly)peptides into amino acids, di-, tri-, and oligopeptides [45,46]. Pancreatic proteases
and peptidases are considered rigorous compared to pepsin, and most of the protein di-
gestion occurs in the small intestine rather than in the stomach [46]. The amino acids and
di- and tripeptides that are released can be taken up across the small intestinal mucosa
and are generally considered to be almost fully absorbed by the end of small intestine, i.e.,
the terminal ileum [45]. The amino acids and peptides not absorbed at the terminal ileum
pass to the large intestine. The large intestine, especially the caecum, also contains amino
acid transporters, but there is thus far no evidence that the absorption of amino acids in
large intestine occurs in relevant quantities [47,48]. If fully absorbed, the amino acids ab-
sorbed in the large intestine in pigs would increase the level of total amino acids absorbed
by only 0.1% for whey protein and by 3.5% for zein [47]. The quantity of amino acids
passing to large intestine is thus only affected by the absorption of amino acids and small
peptides from the consumed protein to a limited amount. The proportion of unabsorbed
amino acids and peptides, as well as of undigested protein and polypeptides, can also be
digested and fermented by the microbiota [48,49]. Furthermore, colonocytes are capable
Mouth
- Mechanical breakdown of product matrix and hydrolysis of starch
Stomach
- Digestion by pepsin
Small intestine
- Digestion by pancreatic enzymes (chymotrypsin, trypsin, etc.) and brush border enzymes
- Absorption of amino acids and peptides
Large intestine
-Nitrogen absorption and recycling
- Microbial metabolism of protein, peptide, amino acids and metabolites
Figure 1. Schematic overview of the key steps of protein digestion and absorption in humans.

Nutrients 2022,14, 947 5 of 31
Following the oral phase of digestion, which typically lasts only for a short time
(
<1 min
), the product passes on to the stomach, where the gastric phase of the digestion
process takes place. In this phase, the product is mixed with gastric juice, which has a
low pH (1–2 for adults) and contains the protease pepsin [
42
,
43
]. The mixing of gastric
juice and product is facilitated through contractions of the stomach. In the stomach, some
hydrolysis of proteins by pepsin occurs, resulting in (poly)peptide formation [
42
], but
complete protein digestion to free amino acids and peptides small enough for absorption
does not occur at this stage [44].
Following gastric digestion, the chyme is delivered to the duodenum at a rate deter-
mined by the gastric emptying. In the small intestine, the chyme is mixed with pancreatic
proteases and peptidases, such as trypsin, chymotrypsin, and carboxypeptidase A [
40
].
Together with intestinal brush border enzymes, these enzymes hydrolyse the proteins and
(poly)peptides into amino acids, di-, tri-, and oligopeptides [
45
,
46
]. Pancreatic proteases
and peptidases are considered rigorous compared to pepsin, and most of the protein diges-
tion occurs in the small intestine rather than in the stomach [
46
]. The amino acids and di-
and tripeptides that are released can be taken up across the small intestinal mucosa and
are generally considered to be almost fully absorbed by the end of small intestine, i.e., the
terminal ileum [
45
]. The amino acids and peptides not absorbed at the terminal ileum pass
to the large intestine. The large intestine, especially the caecum, also contains amino acid
transporters, but there is thus far no evidence that the absorption of amino acids in large
intestine occurs in relevant quantities [
47
,
48
]. If fully absorbed, the amino acids absorbed
in the large intestine in pigs would increase the level of total amino acids absorbed by only
0.1% for whey protein and by 3.5% for zein [
47
]. The quantity of amino acids passing to
large intestine is thus only affected by the absorption of amino acids and small peptides
from the consumed protein to a limited amount. The proportion of unabsorbed amino acids
and peptides, as well as of undigested protein and polypeptides, can also be digested and
fermented by the microbiota [
48
,
49
]. Furthermore, colonocytes are capable of synthesizing
and metabolizing amino acids which are likely derived through blood circulation rather
than the digesta [
48
]. Given that protein is mainly digested and absorbed in the small
intestine, and microbial protein is formed in the large intestine, digesta samples collected
from either site may differ notably, and cause variation in measurements of protein di-
gestibility [
50
]. This is an important factor in the consideration of the different methods
used for determining protein quality, which are described in Section 3.
3. Protein Quality Measurement
3.1. Defining Protein Quality
As outlined in Section 2, with the existence of RDA values for total protein and require-
ment values for IAAs, protein requirements include both a quantitative and qualitative
aspect. The adequacy of a dietary protein to meet the IAA requirements of humans is often
considered the basis of expression of protein quality. Several commonly used principles of
expressing protein quality are based on the ability of a protein source to supply sufficient
IAA for a specific target group [
51
,
52
], and hence encompasses the three essential elements
outlined in Figure 2: (i) amino acid composition, (ii) digestibility of the IAAs and (iii) IAA
requirements of the target population [
53
], whereby amino acid composition and require-
ments for each IAA are typically expressed per g of protein. The requirements for the IAAs
are combined in a so-called reference protein that, based on 100% digestibility, contains all
IAA at the required level per gram of protein. This reference protein, or scoring pattern,
is developed for different age groups [
54
], e.g., for different age categories, as shown in
Table 2[
27
,
30
]. Digestibility of protein is typically defined as the proportion of ingested
protein that is hydrolysed into amino acids, di- and tripeptides, which are available for
absorption [
55
]. The concentration and digestibility of the IAAs in a protein thus determine
the overall protein quality [
30
], and large variations in protein quality are observed among
food [
30
,
56
]. Some dietary proteins contain all IAAs in digestible form at levels that are

Nutrients 2022,14, 947 6 of 31
adequate to meet the requirements in Table 1, whereas in other dietary proteins, one or
more IAAs may not be present at the required level in digestible form [57].
Nutrients 2022, 14, x FOR PEER REVIEW 6 of 34
of synthesizing and metabolizing amino acids which are likely derived through blood cir-
culation rather than the digesta [48]. Given that protein is mainly digested and absorbed
in the small intestine, and microbial protein is formed in the large intestine, digesta sam-
ples collected from either site may differ notably, and cause variation in measurements of
protein digestibility [50]. This is an important factor in the consideration of the different
methods used for determining protein quality, which are described in Section 3.
3. Protein Quality Measurement
3.1. Defining Protein Quality
As outlined in Section 2, with the existence of RDA values for total protein and re-
quirement values for IAAs, protein requirements include both a quantitative and qualita-
tive aspect. The adequacy of a dietary protein to meet the IAA requirements of humans is
often considered the basis of expression of protein quality. Several commonly used prin-
ciples of expressing protein quality are based on the ability of a protein source to supply
sufficient IAA for a specific target group [51,52], and hence encompasses the three essen-
tial elements outlined in Figure 2: (i) amino acid composition, (ii) digestibility of the IAAs
and (iii) IAA requirements of the target population [53], whereby amino acid composition
and requirements for each IAA are typically expressed per g of protein. The requirements
for the IAAs are combined in a so-called reference protein that, based on 100% digestibil-
ity, contains all IAA at the required level per gram of protein. This reference protein, or
scoring pattern, is developed for different age groups [54], e.g., for different age catego-
ries, as shown in Table 2 [27,30]. Digestibility of protein is typically defined as the propor-
tion of ingested protein that is hydrolysed into amino acids, di- and tripeptides, which are
available for absorption [55]. The concentration and digestibility of the IAAs in a protein
thus determine the overall protein quality [30], and large variations in protein quality are
observed among food [30,56]. Some dietary proteins contain all IAAs in digestible form at
levels that are adequate to meet the requirements in Table 1, whereas in other dietary
proteins, one or more IAAs may not be present at the required level in digestible form
[57].
Figure 2. Elements required to quantitively define protein quality.
Protein
Quality
Amino acid
composition
Digestibility of
indispensable
amino acids
Human
requirement of
indispensable
amino acids
Figure 2. Elements required to quantitively define protein quality.
Table 2.
Recommended reference pattern for indispensable amino acids (in mg/g protein) for humans
in different age groups (data from [30,32]).
Age (Years) His Ile Leu Lys SAA * AAA ** Thr Trp Val
0–0.5 21 55 96 69 33 94 44 17 55
0.5–3 20 32 66 57 27 52 31 8.5 43
>3 16 30 61 48 23 41 25 6.6 40
* SAA = Sulphur-containing amino acids (Cys + Met); ** AAA = Aromatic amino acids (Phe + Tyr).
Over the years, many different methods have been developed and implemented for the
determination of protein quality. These methods follow different principles to quantify or
classify protein quality of a protein source. The different principles used in these methods
are outlined in Table 3, which includes the parameters measured and equations used to
calculate the quality of protein. A schematic outline of the different parts of the human
body sampling for measurements for each method is shown in Figure 3. The different
methods used for determining protein quality are covered in detail in Sections 3.2 and 3.3.
These methods vary in several aspects; i.e., they may be
in vivo
or
in vitro
and for
in vivo
methods, they may be carried out in humans or in animals. Furthermore
in vivo
trials
in humans or animals may differ in the point of sampling, with main differences being
sampling for digesta at the terminal ileum or in the faeces, which have a major impact on
the outcomes of protein quality measurements. Section 3.2 will focus on protein quality
methods that are either fully or partially based on the elements outlined in Figure 2[
30
]. In
addition to protein quality methods based on amino acid digestibility, there are also several
methods based on growth studies, whereby growth of animals (typically determined as
weight gain) is determined for a diet containing a test protein and compared to a reference
protein. These are described in Section 3.3. Furthermore,
in vitro
method for protein
digestion and protein quality have been described, which are briefly covered in Section 3.4.

Nutrients 2022,14, 947 7 of 31
Table 3. Overview of in vivo methods used for determining protein quality and protein digestibility.
Method Measurement Principle Calculations Refs.
Protein quality methods
Protein efficiency ratio (PER)
Ratio of weight gain and protein consumed by test
group over control (preferred reference
protein: casein)
(Weight gain (g)TP /Amount o f protein c onsumed (g)TP)
(Weight gain (g)RP /Amo unt o f protein co nsumed (g)RP)[51]
Net protein ratio (or net
protein retention) (NPR)
Difference in weight gain between a test protein
group and protein-free diet group per gram of protein
consumed by the test protein group.
Weight cha nge test grou p (g)−Wei ght change o f protein f ree diet group (g)
Protein consumed (g)[58]
Protein digestibility corrected
amino acid score (PDCAAS)
Ratio of IAAlim in test protein compared to reference
protein corrected for faecal protein digestibility hI AAl im in TP (mg/g TP)
IAAli min RP (mg/g RP)i×Faecal digestibility T P%[32,51,59]
Digestible indispensable
amino acid score (DIAAS)
Ratio of IAAlim in test protein compared to reference
protein corrected for ileal digestibility of IAAlim hIA Alim in TP (mg/g TP)
IAAli min RP (mg/g RP)i×Ileal digestibility IAAlim %[30,60]
Protein digestibility methods
True Digestibility (TD) Percentage of nitrogen observed from protein (food)
consumed in the GI tract
N intake test group (g)−(F aeca l N te st grou p −End eginou s fae cal N) (g)
N intake test group (g)×100 [51]
Biological value (BV) Retained nitrogen over total nitrogen intake, with
corrections for faecal and urinary losses.
N intake TP −(Faecal N −Fec al N on N free diet)−(Urinary N −Urinary N on N f ree diet)
N i ntake of T P −(Fec al N −Fe cal N on N fre e diet )×100 [58]
Net protein utilization (NPU)
Retained nitrogen over total nitrogen intake, with
corrections for faecal and urinary losses.
N intake TP −(Faecal N −Fae cal N o f N −f ree diet)−(U rinar y N −Urin ary N on N −f re e die t)
N i ntake o f TP ×100 [32]
Dual isotope tracer method
Compares AA in circular system from intrinsically
labelled test protein consumed together with a
reference protein with known digestibility
labelled differently
plasma A A (H la bell ed TP)÷meal A A (H la bell ed TP)
plasma A A (C labell ed RP)÷meal A A (C labelled RP)×100 ×Di gesti bilit y o f R P ×
Transa minatio n co rrectio n f actor [61,62]
Abbreviations: TP = Test Protein; RP = Reference Protein; AA = amino acids; N = Nitrogen, IAA = indispensable amino acid IAAlim = first limiting indispensable amino acid.

Nutrients 2022,14, 947 8 of 31
Nutrients 2022, 14, x FOR PEER REVIEW 7 of 34
Table 2. Recommended reference pattern for indispensable amino acids (in mg/g protein) for hu-
mans in different age groups (data from [30,32]).
Age (Years) His Ile Leu Lys SAA * AAA ** Thr Trp Val
0–0.5 21 55 96 69 33 94 44 17 55
0.5–3 20 32 66 57 27 52 31 8.5 43
>3 16 30 61 48 23 41 25 6.6 40
* SAA = Sulphur-containing amino acids (Cys + Met); ** AAA = Aromatic amino acids (Phe + Tyr).
Over the years, many different methods have been developed and implemented for
the determination of protein quality. These methods follow different principles to quan-
tify or classify protein quality of a protein source. The different principles used in these
methods are outlined in Table 3, which includes the parameters measured and equations
used to calculate the quality of protein. A schematic outline of the different parts of the
human body sampling for measurements for each method is shown in Figure 3. The dif-
ferent methods used for determining protein quality are covered in detail in Sections 3.2
and 3.3. These methods vary in several aspects; i.e., they may be in vivo or in vitro and for
in vivo methods, they may be carried out in humans or in animals. Furthermore in vivo
trials in humans or animals may differ in the point of sampling, with main differences
being sampling for digesta at the terminal ileum or in the faeces, which have a major im-
pact on the outcomes of protein quality measurements. Section 3.2 will focus on protein
quality methods that are either fully or partially based on the elements outlined in Figure
2 [30]. In addition to protein quality methods based on amino acid digestibility, there are
also several methods based on growth studies, whereby growth of animals (typically de-
termined as weight gain) is determined for a diet containing a test protein and compared
to a reference protein. These are described in Section 3.3. Furthermore, in vitro method for
protein digestion and protein quality have been described, which are briefly covered in
Section 3.4.
Figure 3. Overview of site of measurement for different in vivo protein quality measurement meth-
ods.
Figure 3.
Overview of site of measurement for different
in vivo
protein quality measurement methods.
3.2. Methods for Determining Protein Quality Based on Amino Acid Digestibility
From Table 3and Figure 3, it clear that there is a wide variety of methods available
for determining protein quality. Considering, however, that, as outlined in Section 3.1,
the expression of protein quality should reflect the adequacy of a protein to meet the
IAA requirements of humans, protein quality should be ideally measured
in vivo
on an
amino acid basis, whereby digestibility is considered and the amount of digestible IAAs
is compared to a reference pattern of IAA requirements [
54
]. This allows placing protein
quality in a broader context, i.e., not only considering it as a metric for comparing or ranking
individual protein sources, but also in a broader dietary perspective, where multiple protein
sources are included and complementarity among protein sources is also an important
consideration, which is further discussed in Section 6. These protein quality measurements
required the use of digesta, which may be ileal or faecal and may be from humans or
animals.
The preference ranking of the different digesta based on the representativeness to
human digestibility for protein quality measurements as suggested by FAO [
63
] is presented
in Figure 4. This ranking indicates that ileal digesta are preferred over faecal digesta and
that humans are preferred over pigs, which are preferred over rats. Overall, ileal digesta
from humans are thus most preferred for assessment of protein quality. Some studies have
indeed been conducted in humans to determine the ileal digestibility of proteins [
64
–
66
].
However, ileal digesta collection in human is complicated [
67
], and most data available
to date on ileal digestibility of dietary proteins has come from animal models [
68
]. Based
on Figure 4, ileal digesta from pigs and rats are preferred after human ileal digesta, but
over human faecal digesta [
63
]. The endorsed use of pigs and rats as animal models
for studying protein digestibility is based on their physiological resemblance and close
correlation of protein digestibility with that of humans [
69
]. Overall, pigs are preferred
over rats because the anatomy and physiology of digestive tract of growing pigs is more
similar to that of adult humans [
22
,
69
]. Indeed, true ileal digestibility of dietary protein in
humans and pigs have shown a high degree of correlation across different foods [
66
,
69
].
Studies in rats have also shown a good general agreement to humans for true ileal amino
acid digestibility, although it has been reported that rats can potentially better digest some
proteins compared with humans [
69
]. These findings make the available data on pig and
rat models valuable to calculate protein quality in relation to human requirements. In the
absence of ileal digestibility data, faecal digestibility can be considered (Figure 4). However,

Nutrients 2022,14, 947 9 of 31
faecal digestibility of total nitrogen is acceptable to be considered, it is not the case when it
comes to AA digestibility [70,71].
Nutrients 2022, 14, x FOR PEER REVIEW 4 of 34
Figure 4. Ranking preference of digesta used for determination of protein quality. Redrawn from
[63].
Of the different methods for protein quality measurement outlined in Table 3, two
methods stand out for the purpose of determining protein quality at an amino acid level,
i.e., the digestible indispensable amino acid score (DIAAS) method [30] and the protein
digestibility-corrected amino acid score (PDCAAS) method [51]. The DIAAS method cal-
culates a score for each IAA based on the concentration of each digestible IAA, calculated
from the concentration of each IAA (per g protein) and its ileal digestibility [30,50,60]. For
each IAA, this value is then compared with a refence pattern for each age group (see Table
2) and the IAA with lowest score relative to the reference pattern is considered to be the
first limiting IAA (IAAlim); the DIAAS value for the protein is taken as the score for the
IAAlim [60]. For calculation of the DIAAS value, FAO recommends using scoring patterns
for 0–6-month-old babies based on the amino acid composition of human milk, whereas
scoring patterns for 0.5–3-year-old children are based on values for 0.5–1-year-old cate-
gory and values for the older child (>3 years old), adolescents and adults are based on the
3–10-year-old age group (Table 2; [30]). The DIAAS method has been extensively used in
the past decade to study protein quality in food products and food protein ingredients.
Outcomes of these studies are discussed in Section 4.
Due to its longer history of use, the PDCAAS method has been applied more broadly
than the DIAAS method [28]. Like the DIAAS method, the PDCAAS method calculates a
value for protein quality based on the first limiting amino acid in relation to a reference
pattern [59]. However, there are notable differences between the DIAAS method and the
PDCAAS method [72]:
• digestibility in the PDCAAS method is not determined at the ileal but at the faecal
level in test species, and
• digestibility in the PDCAAS method is determined at a protein level, and not at the
individual amino acid level, and the protein digestibility factor is subsequently ap-
plied to every individual IAA.
Like for the DIAAS method, the calculated digestible amino acid level in the
PDCAAS method is subsequently compared to the reference pattern and the score for the
first limiting amino acid is indicated as the PDCAAS value for the protein, with values
truncated at 1 or at 100% [30]. With data available for a large range of foods and the com-
parative ease of measurement, the PDCAAS method has been widely used in the past,
Rat - Faecal
Pig - Faecal
Human - Faecal
Rat - Ileal
Pig - Ileal
Human - Ileal
Figure 4.
Ranking preference of digesta used for determination of protein quality. Redrawn from [
63
].
Of the different methods for protein quality measurement outlined in Table 3, two
methods stand out for the purpose of determining protein quality at an amino acid level,
i.e., the digestible indispensable amino acid score (DIAAS) method [
30
] and the protein
digestibility-corrected amino acid score (PDCAAS) method [
51
]. The DIAAS method
calculates a score for each IAA based on the concentration of each digestible IAA, calculated
from the concentration of each IAA (per g protein) and its ileal digestibility [
30
,
50
,
60
]. For
each IAA, this value is then compared with a refence pattern for each age group (see
Table 2) and the IAA with lowest score relative to the reference pattern is considered to
be the first limiting IAA (IAA
lim
); the DIAAS value for the protein is taken as the score
for the IAA
lim
[
60
]. For calculation of the DIAAS value, FAO recommends using scoring
patterns for 0–6-month-old babies based on the amino acid composition of human milk,
whereas scoring patterns for 0.5–3-year-old children are based on values for 0.5–1-year-old
category and values for the older child (>3 years old), adolescents and adults are based on
the 3–10-year-old age group (Table 2; [
30
]). The DIAAS method has been extensively used
in the past decade to study protein quality in food products and food protein ingredients.
Outcomes of these studies are discussed in Section 4.
Due to its longer history of use, the PDCAAS method has been applied more broadly
than the DIAAS method [
28
]. Like the DIAAS method, the PDCAAS method calculates a
value for protein quality based on the first limiting amino acid in relation to a reference
pattern [
59
]. However, there are notable differences between the DIAAS method and the
PDCAAS method [72]:
•
digestibility in the PDCAAS method is not determined at the ileal but at the faecal
level in test species, and
•
digestibility in the PDCAAS method is determined at a protein level, and not at the
individual amino acid level, and the protein digestibility factor is subsequently applied
to every individual IAA.
Like for the DIAAS method, the calculated digestible amino acid level in the PDCAAS
method is subsequently compared to the reference pattern and the score for the first limiting
amino acid is indicated as the PDCAAS value for the protein, with values truncated at 1 or

Nutrients 2022,14, 947 10 of 31
at 100% [
30
]. With data available for a large range of foods and the comparative ease of
measurement, the PDCAAS method has been widely used in the past, and is still being
used, to determine protein quality of food products [
28
]. However, the PDCAAS method
has come under criticism for several aspects:
•
the determination of faecal rather than ileal digestibility in the PDCAAS method,
despite the fact that it is established that amino acids absorbed past the terminal ileum
do not contribute to protein metabolism and that faecal nitrogen levels may be affected
by nitrogen metabolism of gut microbiota [28];
•
the fact that digestibility in the PDCAAS method is determined on a protein basis,
rather than on an individual amino acid basis, despite the fact that it is known that
digestibility values between amino acids in protein sources vary widely [30];
•
the truncation of protein quality scores at 100% in the PDCAAS method not allowing
to consider complementarity of different protein sources on an amino acid basis (for
further explanation see Section 6).
It has been indicated that the PDCAAS method can overestimate the protein quality,
especially of protein sources with poor digestibility, due to the use of faecal rather than
ileal digestibility measurements [
57
,
72
,
73
]. To illustrate this, data were collected from
studies that directly compared ileal and faecal digestibility of dietary proteins in either
pigs or rats. From the results shown in Figure 5, it is clear that for most products faecal
protein digestibility was higher than ileal digestibility. In some cases, the overestimation
of faecal digestibility compared to ileal digestibility exceeds 10% (Figure 5). Considering
that, as outlined earlier, any amino acids absorbed post the terminal ileum are unlikely
to contribute to the metabolic amino acid pool [
47
], measurement of faecal digestibility,
as used in the PDCAAS method, results in the risk of overestimating protein quality,
compared to measurements based on ileal digestibility used in the DIAAS method. One
study in pigs also reported a lower digestibility score for faecal digestibility compared
to ileal digestibility, which may be due to nitrogen secretion into hindgut [
57
]. In case of
dietary protein with low protein digestibility, a lower degree of correlation between the
ileal and faecal digestibility has been reported previously [
71
], which was also observed in
Figure 5.
Nutrients 2022, 14, x FOR PEER REVIEW 5 of 34
and is still being used, to determine protein quality of food products [28]. However, the
PDCAAS method has come under criticism for several aspects:
• the determination of faecal rather than ileal digestibility in the PDCAAS method, de-
spite the fact that it is established that amino acids absorbed past the terminal ileum
do not contribute to protein metabolism and that faecal nitrogen levels may be af-
fected by nitrogen metabolism of gut microbiota [28];
• the fact that digestibility in the PDCAAS method is determined on a protein basis,
rather than on an individual amino acid basis, despite the fact that it is known that
digestibility values between amino acids in protein sources vary widely [30];
• the truncation of protein quality scores at 100% in the PDCAAS method not allowing
to consider complementarity of different protein sources on an amino acid basis (for
further explanation see Section 6).
It has been indicated that the PDCAAS method can overestimate the protein quality,
especially of protein sources with poor digestibility, due to the use of faecal rather than
ileal digestibility measurements [57,72,73]. To illustrate this, data were collected from
studies that directly compared ileal and faecal digestibility of dietary proteins in either
pigs or rats. From the results shown in Figure 5, it is clear that for most products faecal
protein digestibility was higher than ileal digestibility. In some cases, the overestimation
of faecal digestibility compared to ileal digestibility exceeds 10% (Figure 5). Considering
that, as outlined earlier, any amino acids absorbed post the terminal ileum are unlikely to
contribute to the metabolic amino acid pool [47], measurement of faecal digestibility, as
used in the PDCAAS method, results in the risk of overestimating protein quality, com-
pared to measurements based on ileal digestibility used in the DIAAS method. One study
in pigs also reported a lower digestibility score for faecal digestibility compared to ileal
digestibility, which may be due to nitrogen secretion into hindgut [57]. In case of dietary
protein with low protein digestibility, a lower degree of correlation between the ileal and
faecal digestibility has been reported previously [71], which was also observed in Figure
5.
Figure 5. Relation between faecal digestibility and ileal digestibility protein from milk and milk
protein concentrates and isolates (,), roasted nuts (,), cooked cereals (), raw cereals (,),
legume and cereal protein isolates and concentrates (,) and cooked legumes () tested in pigs
y = 0.576x + 41.8
R² = 0.685
70
75
80
85
90
95
100
105
70 75 80 85 90 95 100 105
Faecal digestibility (%)
Ileal digestibility (%)
Figure 5.
Relation between faecal digestibility and ileal digestibility protein from milk and milk protein

Nutrients 2022,14, 947 11 of 31
concentrates and isolates (
•
,

), roasted nuts (
•
,

), cooked cereals (
•
), raw cereals (
•
,

), legume
and cereal protein isolates and concentrates (
•
,

) and cooked legumes (
•
) tested in pigs (squares)
or rats (circles). Data from [
57
,
72
–
74
]; Black dotted line: trend line based on linear regression; grey
dashed line: line of unity.
In addition to differences in the type of digesta being analyses (i.e., ileal or faecal),
differences in outcomes between the DIAAS method and the PDCAAS method for protein
quality measurement are suggested to be due also to the fact that the former determines the
digestibility of each IAA individually, whereas the latter determines protein digestibility
and applies this factor to each IAA [
60
]. Because of differences in the digestibility between
different IAAs [
70
,
75
], it has been suggested that only determining protein digestibility
impacts outcomes [
50
]. To explore this, we studied the relationship between standardized
ileal digestibility (SID) of IAA
lim
with the SID of average amino acid for different foods.
Results are shown in Figure 6and indicate that although some deviations were observed,
particularly in products where the IAA
lim
was lysine, the overall trend showed a high
degree of correlation (R
2
> 0.99) with a slope very close to 1. This indicates that the use of
average amino acid digestibility rather than digestibility of IAA
lim
can lead to deviations,
but is unlikely to lead to a structural over- or underestimation in dietary protein quality
values. As a result, structural differences between (untruncated) PDCAAS values and
DIAAS values for different protein sources appear mainly attributable to the use of faecal
digestibility rather than ileal digestibility values and not from the use of total digestibility
of protein rather than the digestibility of the IAAlim.
Nutrients 2022, 14, x FOR PEER REVIEW 7 of 34
Figure 6. Relation between ileal digestibility of total amino acids (AA) and of the first limiting in-
dispensable amino acid (IAA) histidine (), sulphur-containing amino acids (), lysine (), valine
() or leucine () of different food sources. Data from studies in pigs [57,76–80], and rats [72,81].
3.3. Methods for Determining Protein Quality Based on Growth Studies
In addition to the PDCAAS method and DIAAS method described in Section 3.2,
various other methods to measure protein quality have been used and are included in
Table 3 and Figure 4. These methods, however, use some notably different measurement
principals, e.g., the change in body weight, net nitrogen utilization, rather than the previ-
ously described digestibility of amino acids at faecal or intestinal level [30]. Methods using
change in body weight to evaluate protein quality include the protein efficiency ratio
(PER) and the net protein retention/ratio (NPR) and are commonly determined compared
to a reference protein, often casein [33]. The PER and NPR methods have an advantage of
being relatively easy to conduct [28]. These methods involve data collection from rodent
feeding trials, where growing rodents are fed either the test protein or the reference pro-
tein for a set period of time [51]. At the end of trial, the final weights of the experimental
rodents from each group are determined, and weight gain is expressed relative to amount
of protein consumed. The final protein quality value for the test protein is calculated rel-
ative to that of the reference protein, which is most commonly casein [60]. One drawback
of these methods is that, the digestibility of sulphur-containing amino acids (SAAs) in
rodents is higher than in humans, and the outcomes thus can overestimate the protein
quality for humans [69]. The PER and NPR methods also overlook the role of other nutri-
ents in the experimental diet that possibly contribute to the weight gain of the test subject
[28].
3.4. Methods for Determining Protein Quality and Protein Digestibility In Vitro
In addition to in vivo methods listed in Table 3, in vitro methods for determining
protein digestibility and protein quality have been reported. In vitro methods simulate
the digestion process in a laboratory setting to measure protein quality and these methods
may resemble the digestion process in either a static or (semi-)dynamic manner. Static
models do not replicate actual human digestion processes, including peristalsis and grad-
ual introduction of different enzymes at different time, while dynamic methods can rep-
licate these better [82]. In vitro methods, such as in vitro protein digestibility (IVPD)
method, compare total protein content of the food and total digested protein, while other
y = 0.975x
R² = 0.995
60
65
70
75
80
85
90
95
100
105
110
60 70 80 90 100 110
First limiting IAA digestibility (%)
Total AA digestibility (%)
Figure 6.
Relation between ileal digestibility of total amino acids (AA) and of the first limiting
indispensable amino acid (IAA) histidine (
•
), sulphur-containing amino acids (
•
), lysine (
•
), valine
(•) or leucine (•) of different food sources. Data from studies in pigs [57,76–80], and rats [72,81].
In addition to ileal and faecal digestibility, as applied in DIAAS and PDCAAS method-
ology, respectively, there are also other methods for determining protein digestibility
in vivo
, which are included in Table 3. This includes several methods based on nitrogen
balance, i.e., the biological value (BV) method, net protein utilization (NPU) method and the
true digestibility (TD) method. In these methods based on nitrogen balances, rodents are
fed with experimental diets containing the test protein as sole source of nitrogen and with
a nitrogen-free diet for the control group [
32
]. Endogenous nitrogen losses are measured

Nutrients 2022,14, 947 12 of 31
from the control group fed the nitrogen-free diet and, together with total nitrogen con-
sumption and the nitrogen content of the excreta in the experimental group are measured.
These values can further be used to calculate the protein quality [
51
] (Table 3). One of the
fundamental assumptions of the nitrogen balance methods is that the amount of nitrogen
consumed is either used or excreted without any other metabolic consequences [
52
]. These
methods, however, overlook that there can be delay in nitrogen excretion (especially in
case of a large urea pool), the metabolic contributions to the nitrogen excreted and ignore
variation in digestibility of consumed protein [28,51].
3.3. Methods for Determining Protein Quality Based on Growth Studies
In addition to the PDCAAS method and DIAAS method described in Section 3.2,
various other methods to measure protein quality have been used and are included in
Table 3and Figure 4. These methods, however, use some notably different measurement
principals, e.g., the change in body weight, net nitrogen utilization, rather than the previ-
ously described digestibility of amino acids at faecal or intestinal level [
30
]. Methods using
change in body weight to evaluate protein quality include the protein efficiency ratio (PER)
and the net protein retention/ratio (NPR) and are commonly determined compared to a
reference protein, often casein [
33
]. The PER and NPR methods have an advantage of being
relatively easy to conduct [
28
]. These methods involve data collection from rodent feeding
trials, where growing rodents are fed either the test protein or the reference protein for a set
period of time [
51
]. At the end of trial, the final weights of the experimental rodents from
each group are determined, and weight gain is expressed relative to amount of protein
consumed. The final protein quality value for the test protein is calculated relative to that of
the reference protein, which is most commonly casein [
60
]. One drawback of these methods
is that, the digestibility of sulphur-containing amino acids (SAAs) in rodents is higher than
in humans, and the outcomes thus can overestimate the protein quality for humans [
69
].
The PER and NPR methods also overlook the role of other nutrients in the experimental
diet that possibly contribute to the weight gain of the test subject [28].
3.4. Methods for Determining Protein Quality and Protein Digestibility In Vitro
In addition to
in vivo
methods listed in Table 3,
in vitro
methods for determining
protein digestibility and protein quality have been reported.
In vitro
methods simulate the
digestion process in a laboratory setting to measure protein quality and these methods
may resemble the digestion process in either a static or (semi-)dynamic manner. Static
models do not replicate actual human digestion processes, including peristalsis and gradual
introduction of different enzymes at different time, while dynamic methods can replicate
these better [
82
].
In vitro
methods, such as
in vitro
protein digestibility (IVPD) method,
compare total protein content of the food and total digested protein, while other methods,
such as
in vitro
protein digestibility corrected amino acid score (IVPDCAAS) method,
further correct for the first limiting amino acid compared to reference protein [
83
]. Although
in vitro
analysis can be cheaper and easier methods to predict the outcome of
in vivo
digestibility, the complexity of
in vivo
digestibility has not been realized fully in an
in vitro
model [
84
]. However, development of in-vitro analysis has potential to provide insight
into processing of protein during digestion. Analytical methods such as size exclusion
chromatography can estimate the percentage of small peptide available for absorption. A
physiologically relevant protein digestibility is estimated by combining the total dissolved
protein and percentage small peptide [
85
]. Overall, though, data from
in vivo
studies
remain preferred, particularly when based on ileal digestibility. Outcomes from such
studies are described in Section 4.
4. Protein Quality Data from DIAAS Measurements: Interpretation and Application
As outlined in Section 3, protein quality can be determined by various methods, but
estimates based on ileal digestibility of individual amino acids, as done in the DIAAS
method, are preferred. To illustrate the variability and the underlying reasons for this

Nutrients 2022,14, 947 13 of 31
variability, we collected available DIAAS values for dietary proteins from food products or
protein ingredients. The overview of available DIAAS values is shown in Table 4, which
also includes the IAA
lim
for each product and the SID of the IAA
lim
. Furthermore, the
species in which SID measurements were carried out (pigs or rats) and the reference pattern
against which the DIAAS values were calculated are also included in Table 4.
Table 4.
Overview of digestible indispensable amino acid score (DIAAS) values, including the first
limiting indispensable amino acid (IAA
lim
) and its standardized ileal digestibility (SID) as well as the
species in which testing was performed and the protein reference pattern against which DIAAS was
calculated for different food items. Items are ranked from highest to lowest DIAAS value 1.
Food Item Food
Group
DIAAS
Value (%) IAAlim
SID of
IAAlim (%)
Test
Species
Protein
Reference
Pattern
References
Dry milk Dairy 144 SAA 94 Pig >3-year-old [77]
Bacon
(smoked-cooked) Pork 142 Valine 95 Pig >3-year-old [79]
Milk protein
concentrate Dairy 141 SAA 101 Pig >3-year-old [57]
Pork loin (medium) Pork 139 Valine 95 Pig >3-year-old [79]
Whey protein
concentrate Dairy 133 Histidine 97 Pig >3-year-old [57]
Ham
(alternatively-cured) Pork 133 Valine 95 Pig >3-year-old [79]
Ribeye
(roast, medium) Beef 130 Valine 95 Pig >3-year-old [76]
Bologna Pork 128 Leucine 97 Pig >3-year-old [76]
Ham
(
conventionally-cured
)
Pork 126 Valine 96 Pig >3-year-old [79]
Whey protein isolate Dairy 125 Histidine 100 Pig >3-year-old [57]
Ham (non-cured) Pork 124 Valine 93 Pig >3-year-old [79]
Skimmed milk
powder Dairy 123 SAA 99 Pig >3-year-old [57]
Egg Egg 122 SAA 75 Pig >3-year-old [86]
Ground beef (raw) Beef 121 Leucine 99 Pig >3-year-old [76]
Beef jerky Beef 120 SAA 98 Pig >3-year-old [76]
Salami Pork 120 Valine 96 Pig >3-year-old [76]
Pork belly (raw) Pork 119 Valine 97 Pig >3-year-old [79]
Milk protein
concentrate Dairy 118 SAA 94 Rat 0.5–3-year-old [72]
Pork loin
(medium-well done) Pork 118 Valine 95 Pig >3-year-old [79]
Bacon (smoked) Pork 117 Valine 95 Pig >3-year-old [79]
Pork loin (well-done) Pork 117 Valine 95 Pig >3-year-old [79]
Ribeye
(roast, medium-rare) Beef 111 Valine 97 Pig >3-year-old [76]
Whey protein isolate Dairy 109 Histidine 99 Rat 0.5–3-year-old [72]
Ribeye (well-done) Beef 107 Valine 97 Pig >3-year-old [76]
Soy flour Legumes 105 SAA 101 Pig >3-year-old [57]

Nutrients 2022,14, 947 14 of 31
Table 4. Cont.
Food Item Food
Group
DIAAS
Value (%) IAAlim
SID of
IAAlim (%)
Test
Species
Protein
Reference
Pattern
References
Ground beef (cooked) Beef 99 Leucine 97 Pig >3-year-old [76]
Topside steak (boiled) Beef 99 Valine 99 Pig >3-year-old [78]
Topside steak
(pan fried) Beef 98 Valine 98 Pig >3-year-old [78]
Soy protein isolate Legumes 98 SAA 98 Pig >3-year-old [57]
Whey protein
concentrate Dairy 97 Histidine 98 Rat 0.5–3-year-old [72]
Topside steak (raw) Beef 97 Valine 98 Pig >3-year-old [78]
Mung beans (cooked) Legumes 94 2Threonine 77 Pig >3-year-old [87]
Topside steak (roasted)
Beef 91 Valine 98 Pig >3-year-old [78]
Soy protein isolate Legumes 91 SAA 94 Rat 0.5–3-year-old [72]
Soy protein isolate Legumes 90 SAA 92 Rat 0.5–3-year-old [72]
Peas (cooked) Legumes 88 2Valine 87 Pig >3-year-old [87]
Broad beans (cooked) Legumes 87 2Valine 91 Pig >3-year-old [87]
Pistachio (raw) Nuts 86 Lysine 87 Pig >3-year-old [74]
Pistachio (roasted) Nuts 83 Lysine 77 Pig >3-year-old [74]
Pea protein
concentrate Legumes 82 SAA 95 Rat 0.5–3-year-old [72]
Topside steak (grilled) Beef 80 Valine 97 Pig >3-year-old [78]
Adzuki beans
(cooked) Legumes 78 2SAA 87 Pig >3-year-old [87]
Dehulled oats Cereals 77 Lysine 85 Pig >3-year-old [80]
Kidney beans (cooked)
Legumes 74 2SAA 68 Pig >3-year-old [87]
Pea protein
concentrate Legumes 73 SAA 78 Pig >3-year-old [57]
Chickpeas (cooked) Legumes 71 2Valine 83 Pig >3-year-old [87]
Buckwheat (cooked) Cereals 68 SAA 86 Rat 0.5–3-year-old [81]
Quick oats Cereals 67 Lysine 83 Pig >3-year-old [77]
Oat protein
concentrate Cereals 67 Lysine 86 Pig >3-year-old [88]
Polished white rice Cereals 64 Lysine 92 Pig >3-year-old [80]
Rice (cooked) Cereals 60 Lysine 92 Rat 0.5–3-year-old [72]
Kidney beans (cooked)
Legumes 59 SAA 75 Rat 0.5–3-year-old [72]
Peas (cooked) Legumes 58 SAA 89 Rat 0.5–3-year-old [72]
Rolled oats (cooked) Cereals 54 Lysine 84 Rat 0.5–3-year-old [72]
Nutridense maize Cereals 54 Lysine 79 Pig >3-year-old [80]
Dehulled barley Cereals 51 Lysine 74 Pig >3-year-old [80]
Yellow dent maize Cereals 48 Lysine 75 Pig >3-year-old [80]
Rey Cereals 47 Lysine 67 Pig >3-year-old [80]
Tartary buckwheat
(cooked) Cereals 47 SAA 72 Rat 0.5–3-year-old [81]

Nutrients 2022,14, 947 15 of 31
Table 4. Cont.
Food Item Food
Group
DIAAS
Value (%) IAAlim
SID of
IAAlim (%)
Test
Species
Protein
Reference
Pattern
References
Peanuts (roasted) Legumes 43 Lysine 92 Rat 0.5–3-year-old [72]
Wheat Cereals 43 Lysine 73 Pig >3-year-old [80]
Oats (cooked) Cereals 43 Lysine 83 Rat 0.5–3-year-old [81]
Brown rice (cooked) Cereals 42 Lysine 93 Rat 0.5–3-year-old [81]
Wheat bran Cereals 41 Lysine 73 Rat 0.5–3-year-old [72]
Rice protein
concentrate Cereals 37 Lysine 86 Rat 0.5–3-year-old [72]
Polished rice cooked Cereals 37 Lysine 92 Rat 0.5–3-year-old [81]
Sorghum Cereals 29 Lysine 69 Pig >3-year-old [80]
Whole wheat (cooked) Cereals 20 Lysine 96 Rat 0.5–3-year-old [81]
Cornflakes Cereals 19 Lysine 78 Pig >3-year-old [77]
Adlay (cooked) Cereals 13 Lysine 90 Rat 0.5–3-year-old [81]
Foxtail millet (cooked) Cereals 10 Lysine 88 Rat 0.5–3-year-old [81]
Proso millet (cooked) Cereals 7 Lysine 96 Rat 0.5–3-year-old [81]
Corn-based
breakfast cereal Cereals 1 Lysine 13 Rat 0.5–3-year-old [72]
SAA = Sulphur-containing amino acids (cysteine + methionine);
1
Only studies are considered where reported
DIAAS values were calculated based on determination of standardized ileal digestibility and amino acid composi-
tion on the same material. Calculated DIAAS values based on calculations with data from different studies was
not considered.
2
DIAAS values are calculated from data provided and not the reported DIAAS values in the
publication based on discrepancies between published data and reported DIAAS values and communications
with the authors.
Table 4shows a wide variability in both DIAAS values (ranging from 1 to 144%) and
SID of the IAA
lim
(ranging from 13 to 101%) between the different products. The DIAAS
score is essentially the product of the SID and a normalized concentration of the IAA
lim
(expressed relative to the concentration, per g protein, of this IAA in the reference pattern),
with the former, like the DIAAS score, expressed as a percentage and the latter expressed
as a fraction. Hence, a normalized concentration of IAA
lim
= 1 yields DIAAS (%) = SID (%),
whereas for SID = 100%, DIAAS = normalized concentration of IAA
lim ×
100%. Hence, a
comparison of DIAAS values with SID values for IAA
lim
provides useful insights in the
relative contributions of SID and the concentration of IAA
lim
to the DIAAS score. With
only one exception (i.e., eggs) all products with a DIAAS value > 100% show a SID for