Protein quality evaluation twenty years after the introduction of the protein digestibility corrected amino acid score method

Abstract

In 1989 the Joint FAO/WHO Expert Consultation on Protein Quality Evaluation recommended the use of the Protein Digestibility Corrected Amino Acid Score (PDCAAS) method for evaluating protein quality. In calculating PDCAAS, the limiting amino acid score (i.e., ratio of first limiting amino acid in a gram of target food to that in a reference protein or requirement) is multiplied by protein digestibility. The PDCAAS method has now been in use for 20 years. Research emerging during this time has provided useful data on various aspects of protein quality evaluation that has made a review of the current methods used in assessing protein quality necessary. This paper provides an overview of the use of the PDCAAS method as compared to other methods and addresses some of the key challenges that remain in regards to protein quality evaluation. Furthermore, specific factors influencing protein quality including the effects of processing conditions and preparation methods are presented. Protein quality evaluation methods and recommended protein intakes currently used in different countries vis-à-vis the WHO/FAO/UNU standards are further provided. As foods are frequently consumed in complement with other foods, the significance of the PDCAAS of single protein sources may not be evident, thus, protein quality of some key food groups and challenges surrounding the calculation of the amino acid score for dietary protein mixtures are further discussed. As results from new research emerge, recommendations may need to be updated or revised to maintain relevance of methods used in calculating protein quality.

Full text

Protein quality evaluation twenty years after the introduction of the protein
digestibility corrected amino acid score method
Joyce Boye
1,2
*, Ramani Wijesinha-Bettoni
1
and Barbara Burlingame
1
1
Nutrition and Consumer Protection Division, Food and Agriculture Organization of the United Nations,
Viale delle Terme di Caracalla, 00153 Rome, Italy
2
Food Research and Development Centre, Agriculture and Agri-Food Canada, 3600 Casavant Boul West,
Saint Hyacinthe, Quebec, Canada, J2S 8E3
(Submitted 30 August 2011 – Final revision received 17 February 2012 – Accepted 22 February 2012)
Abstract
In 1989 the Joint FAO/WHO Expert Consultation on Protein Quality Evaluation recommended the use of the Protein Digestibility Corrected
Amino Acid Score (PDCAAS) method for evaluating protein quality. In calculating PDCAAS, the limiting amino acid score (i.e., ratio of first
limiting amino acid in a gram of target food to that in a reference protein or requirement) is multiplied by protein digestibility. The PDCAAS
method has now been in use for 20 years. Research emerging during this time has provided useful data on various aspects of protein qual-
ity evaluation that has made a review of the current methods used in assessing protein quality necessary. This paper provides an overview
of the use of the PDCAAS method as compared to other methods and addresses some of the key challenges that remain in regards to
protein quality evaluation. Furthermore, specific factors influencing protein quality including the effects of processing conditions and prep-
aration methods are presented. Protein quality evaluation methods and recommended protein intakes currently used in different countries
vis-a
`-vis the WHO/FAO/UNU standards are further provided. As foods are frequently consumed in complement with other foods, the
significance of the PDCAAS of single protein sources may not be evident, thus, protein quality of some key food groups and challenges
surrounding the calculation of the amino acid score for dietary protein mixtures are further discussed. As results from new research emerge,
recommendations may need to be updated or revised to maintain relevance of methods used in calculating protein quality.
Key words: Protein Quality: Amino Acid: Protein Digestibility Corrected Amino Acid Score: PDCAAS
Introduction
Proteins are important components of the human diet and play
an essential role as structural and functional components of
living systems. Food proteins provide amino acids (AA) which
serve as building blocks of all vital organs, muscles (including
heart muscles), hormones and biological fluids such as blood.
As the human body is incapable of maintaining reserves of pro-
tein, a constant supply of good quality protein is needed to
maintain growth and other physiological functions. Insufficient
intake of protein especially during periods of growth and devel-
opment can affect all organs in the body including the brain,
heart, immune system, and other vital organs. Protein quality
of foods is, therefore, an important criterion for the provision
of adequate nutrition and maintenance of good health.
A significant effort has been made in the last several dec-
ades to establish methods for evaluating protein quality. The
1957 FAO Committee report on Protein Requirements
(1)
rec-
ommended the use of a reference protein with an “ideal” AA
composition to define the pattern of human AA requirements.
In 1963, a Joint FAO/WHO Expert Group
(2)
further discussed
the need to take into consideration the rate of obligatory nitro-
gen loss from the body on a protein-free diet when determin-
ing human protein requirements. Discrepancies between the
obligatory nitrogen loss and the minimum nitrogen intake
needed to ensure balance, were considered by an Ad Hoc
Expert Committee convened in 1971
(3)
. The concern was
that even for proteins with a high biological value, higher
amounts of protein were required than that indicated by the
obligatory nitrogen loss. The committee also recommended
a safe level of intake for a population, which was defined as
the average protein requirement of the individuals in the
population plus 1·96 times the standard deviation (SD).
A follow-up Joint FAO/WHO/UNU Expert Consultation on
Energy and Protein Requirements in 1981
(4)
emphasised the
need to consider digestibility in the evaluation of protein quality.
Eight years later, the Joint FAO/WHO Expert Consultation on
Protein Quality Evaluation
(5)
recommended the use of the
Protein Digestibility Corrected Amino Acid Score (PDCAAS)
method for protein quality evaluation. The PDCAAS method
has now been in use for 20 years. The 2007 report of the
*Corresponding author: J. Boye, fax (1) 450 773 8461, email joyce.boye@agr.gc.ca
British Journal of Nutrition (2012), 108, S183–S211 doi:10.1017/S0007114512002309
qThe Authors 2012
British Journal of Nutrition

FAO/WHO/UNU Expert Consultation on protein and AA
requirements in human nutrition
(6)
highlighted several aspects
of protein quality that still required consideration. The report
also provided new reference patterns based on AA require-
ments for different age groups.
The current paper provides a review of the use of the
PDCAAS method compared to other methods for evaluating
protein quality and addresses some of the key challenges
that remain in regards to protein quality evaluation. A brief
overview of human AA and protein requirements and factors
influencing dietary protein quality is provided, as well as a
summary of the progress made by the international science
community to identify acceptable methods for evaluating
protein quality. Current recommendations for protein intake
are also reviewed and specific examples of standards and
requirements used in a few selected countries vis-a
`-vis the
WHO/FAO/UNU standards are presented.
Human amino acid and protein requirements
Proteins are made up of amino acids linked by peptide bonds
and are the main supply of nitrogen in the diet. To sustain
bodily functions and growth, humans require certain minimal
levels of protein intake as well as adequate supplies of dietary
essential amino acids that are not synthesized by the body.
The aim of protein quality evaluation is to determine the abil-
ity of a protein to meet maintenance needs plus special needs
for growth, pregnancy, or lactation
(7)
. For most adults, protein
intake should be in equilibrium with protein loss. Positive pro-
tein balance is, however, required for growing infants and
children and during pregnancy and lactation, and require-
ments may also increase during times of illness and recovery.
Nitrogen balance in humans, which is a measure of daily
nitrogen intake minus nitrogen excreted, is a reflection of
both protein and energy intake from the diet. Protein nutri-
tional requirement is, therefore, defined as the lowest level
of dietary protein intake that will balance the losses of nitrogen
from the body, and thus maintain the body protein mass (i.e.,
in persons at energy balance with modest levels of physical
activity)
(6,7)
. In children or pregnant/lactating women, this
further includes needs associated with the deposition of tissues
or the secretion of milk at rates consistent with good health.
Factors influencing dietary protein quality
Protein quality may be defined as the ability of a food protein
to meet the body’s metabolic demand for amino acids and N
and is determined by the AA composition and digestibility
of the protein as well as the bioavailability of the individual
AA. The term is relative allowing a comparison of the ade-
quacy of different sources of food proteins to meet human
protein requirements. Both digestibility and bioavailability
are affected by the food matrix (e.g., levels and types of fat,
carbohydrate and antinutritional compounds). Other factors
influencing protein quality are demands that are specific to
the individual consuming the food, such as age, health
status, physiological status and energy balance
(7)
.
Amino acid composition
Amino acids are classified into those which cannot be syn-
thesized by the body and therefore must be obtained by the
diet, “indispensable” (IAA) or “essential” (EAA) (His, Ile, Leu,
Lys, Met, Phe, Thr, Trp and Val), and those which the body
can produce, “dispensable” or “nonessential” (Asp, Asn, Glu,
Ala, Ser, Cys, Tyr, Gly, Arg, Gln, Pro). However, these two cat-
egories are not absolute or mutually exclusive: the endogen-
ous capacity for the formation of some dispensable AA may
not always meet demand and, potentially, some de novo syn-
thesis of IAA may occur following urea salvage in the lower
gut
(6)
. Some AA, such as Cys, Tyr, Gly, Arg, Gln, Pro and taur-
ine, are termed “conditionally indispensable”, as they become
dietary essential only under specific pathological or physio-
logical conditions.
The 2007 WHO/FAO/UN Expert Consultation on proteins
(6)
stressed the need to interpret these classifications with care, as
there appears to be an absolute metabolic need for both dis-
pensable and indispensable AA. The efficiency of utilization
of IAA is dependent on the total N and the form of N in the
diet. The higher the dietary total N, the lower the amount of
IAA needed to achieve N-balance
(6)
. The report further
states that when all or any of the IAA are present in excess
of demand, the absorbed mixture is unbalanced and limited
by dispensable AA, which would need to be supplied from
oxidation of surplus IAA. However, the biological value of a
protein is defined in terms of how well the profile of IAA in
a protein matches that of the pattern required by body.
Some authors have argued that IAA are important at higher
intakes than those in the requirement pattern, especially in
the case of high quality proteins (e.g. egg, milk, fish and
meat protein products) that are used to supplement other
low quality proteins
(8)
. Another argument supporting higher
intakes of IAA is that their role extends beyond that of sup-
porting growth or N balance (i.e., implications in such diverse
functions as lean body mass retention, cell signalling, bone
health, glucose homeostasis and satiety induction)
(7)
.
Protein digestibility
In order for constituent AA to be released, proteins must first
be digested. A possible exception is in neonates where some
uptake of intact proteins or peptides from the intestinal lumen
into the systemic circulation can occur. Digestibility is usually
defined in terms of the balance of AA across the small intestine
(mouth to terminal ileum: ileal digestibility), or across the
entire gut (mouth to anus: faecal digestibility), based on the
principle that the difference between intake and losses pro-
vides a measure of the extent of digestion and absorption of
food protein as AA by the gastrointestinal tract for use by
the body
(6)
. This net balance across the digestive tract is a
complex process that involves considerable exchange of nitro-
gen in terms of protein, AA and urea between systemic pools
and the gut lumen
(6)
. Large differences can occur between the
digestibility of a protein and specific AA, especially where
antinutritional factors are present, such as in uncooked cereals
and legumes
(9)
.
J. Boye et al.S184
British Journal of Nutrition

The digestive process starts with hydration and solubil-
ization in the mouth. Most food proteins remain intact until
reaching the stomach. A range of different proteolytic
enzymes (some specific to certain AA) are necessary to
break the peptide bonds within the protein chain. HCl in
the stomach denatures some proteins, making the peptide
bonds more accessible for proteolysis by digestive enzymes.
The HCl also converts the pepsinogen secreted by the
stomach to the active protease pepsin, which cleaves the pro-
tein into peptide fragments. The pancreas secretes enzymes
such as trypsin, chymotrypsin, carboxypeptidases, collagenase
and elastase into the duodenum, and these enzymes break up
the peptides into smaller peptide fragments. The final diges-
tion occurs in the small intestine by enzymes such as amino-
peptidases and tripeptidases, which split the remaining short
peptide fragments into single AA or di- and tri-peptides,
which can be absorbed by the intestinal mucosal cells. Undi-
gested and unabsorbed nitrogenous residues are then trans-
ported to the large intestine (colon) where further microbial
modifications of this material are possible within the large
intestine, prior to faecal excretion.
Bioavailability of amino acids
The bioavailability of an amino acid is the proportion of
ingested dietary AA that is absorbed in a chemical form
suitable for it to be utilized for protein synthesis or
metabolism
(6,10)
. There is no direct method for measuring
bioavailability and it is, therefore, generally estimated using
measures of in-vivo digestibility or determined using slope-
ratio assays, which give the bioavailability relative to a refer-
ence protein
(10)
.
The utilization of an amino acid may be influenced by ingre-
dient characteristics, genetics, physiological state and dietary
factors
(11)
. Food processing can sometimes reduce the bio-
availability of AA. One common example is Lys which has
undergone Maillard reactions with reducing sugars or other
aldehyde compounds during heat processing such as in
heated skim milk powder
(9)
: although it can be digested and
partially absorbed by the body, it cannot be utilized for protein
synthesis. Other changes associated with alkaline and/or heat
processing include racemization of L-amino acids, and the for-
mation of crosslinked peptide chains such as lysinoalanine,
which result in a loss of Lys, Cys and Thr, together with reduced
protein digestibility
(8,9)
. Antinutritional factors can also cause a
reduction in protein bioavailability. Some workers, therefore,
suggest that digestibility as determined traditionally may not
be a good approximation of bioavailability in products contain-
ing antinutritional factors present naturally or formed during
processing or storage
(9)
.
Overview of methods used for evaluating protein quality
A list of some of the principal methods used in evaluating pro-
tein quality is provided in Table 1, along with some consider-
ations on their appropriateness and effectiveness. Methods
frequently used include amino acid score (AAS), nitrogen
balance (NB), in vivo protein digestibility (apparent, corrected
or true), in vitro protein digestibility, protein efficiency ratio
(PER), estimated PER, maximum PER, net protein ratio
(or retention) (NPR), protein rating (PR), net protein utilization
(NPU), biological value (BV) (apparent, true, relative) and the
PDCAAS. Details on these different methodologies and their
use have been extensively reported in the literature.
AAS is the ratio of the amino acid content in 1 g of a target
protein to that of a reference protein or requirement. AA anal-
ysis, which is typically undertaken using ion exchange chro-
matography (IEC), gas chromatography (GC) or reverse
phase high performance liquid chromatography (HPLC), is
needed for calculating the amino acid content. A standard pro-
cedure for AA analysis was recommended by the 1989 Joint
FAO/WHO Expert Consultation. This is because data can
vary markedly depending on the method and conditions
used. The WHO/FAO/UNU Expert Committee
(6)
suggested
that since in practice dietary proteins are likely to be limited
only by Lys (most cereal proteins), the sulphur AA (legume
proteins), Trp (some cereals such as maize) or Thr (some cer-
eals), in calculating scores it is usually only necessary to use a
pattern based on these four AA. In addition to variations in
results if not properly calculated, major issues regarding the
use of amino acid score include lack of information on AA
bioavailability. Furthermore, processing can modify IAA
which can influence digestibility. Additionally, the degree of
amino acid absorption may depend on the length of peptide
released after hydrolysis (limit peptides).
Nitrogen balance provides a measure of body nitrogen
retention based on directly measuring daily nitrogen intake
minus nitrogen excreted. Digestibility measurements estimate
the extent to which proteins are hydrolysed by gastrointestinal
enzymes into AA, and the extent to which these AA are
absorbed, and provide a measure of the dietary protein
intake which is made available to the organism after digestion
and absorption. Protein efficiency ratio measures the ability of
a protein to support the growth of young growing rats and is
reported as the gain in weight per gram of protein consumed.
As has been discussed extensively in the literature, rats have a
higher need for sulphur containing IAA, thus, the PER method
overestimates the requirements for humans and likely under-
estimates the quality of some proteins, especially plant pro-
teins. NPR is similar to PER except that an additional factor
(average weight loss of rats fed a non-protein diet) is taken
into consideration. Protein rating is the product of the PER
of a protein multiplied by the amount of protein in a reason-
able daily intake. NPU provides a measure of overall protein
utilization and reflects the proportion of ingested protein
retained. Biological value, on the other hand, provides a
measure of how well the absorbed amino acid profile matches
that of the requirement. Challenges associated with BV include
the following: results for the same food vary significantly
depending on N intake; results for different foods may be
similar at low N intake and very different at higher intake
levels; proteins which are completely devoid of one IAA
can still have a BV of up to 40%; the method ignores the
importance of factors which influence digestion of the protein
Protein Quality Evaluation S185
British Journal of Nutrition

Table 1. List of some of the principal methods used in evaluating dietary protein quality
Protein Quality Evaluation
Method Abbrev Method Summary and/or Equation Unit Issues Reference
Amino Acid Score AAS AAS ¼mg of limiting amino acid in 1 g test protein / mg of limiting
amino acid in a reference protein or requirement pattern
dmnl
d
†Does not provide information on bioavailability of IAA. (5)
†Processing can modify structure of AA.
†Processing can impact digestibility.
†Amino acid absorption will potentially depend on length of peptide
released after hydrolysis which is not accounted for.
Apparent Protein Digestibility APD APD ¼100 [(Protein intake 2Faecal protein)/Protein intake] % †Does not take into consideration concentration and availability of IAA. (9)
In vivo method †Does not account for metabolic faecal protein.
Corrected Protein Digestibility
a
(also sometimes called True
Protein Digestibility)
CPD (TD) CPD or TD ¼100 [(Protein intake 2(Faecal protein 2metabolic
faecal protein) /Protein intake)]
%†Does not take into consideration concentration and availability of IAA. (9)
In vivo method
In vitro Protein Digestibility IVPD IVPD ¼210·46 218·10X % †Does not measure true digestibility (i.e., digestibility is calculated). (55)
where, X ¼pH of sample suspension (during hydrolysis)at
10 min.
†Does not take into consideration concentration and availability of IAA.
Nitrogen Balance NB Measure of daily nitrogen intake minus nitrogen excreted g †Large pool of urea in body water means that there is an extended
delay before any change in nitrogen input or output becomes fully
apparent.
(5), and refer-
ences therein;
(6)
NB ¼Nitrogen intake (NI)2nitrogen losses (NL)†An arbitary adjustment factor
e
sometimes used in calculation.
†Underestimation of actual minimum physiological needs.
Protein Efficiency Ratio PER Gain in weight of rat per gram of protein eaten dmnl †Rats have a higher requirement than humans for some amino acids. (5), and refer-
ences thereinPER ¼Gain in body mass (g)/Protein intake (g)†Does not properly credit protein used for maintenance purposes.
†Gain in body weight does not necessarily correspond to gain in body
protein.
†PER values vary with levels of protein intake.
†Poor precision and reproducibility
†Animal studies are expensive.
Estimated Protein Efficiency
Ratio
ePER Protein efficiency ratio (PER) is estimated using a regression
equation
dmnl †Does not measure true protein efficiency (i.e., PER is calculated). (83)
ePER ¼0·468 þ0·454 (Leu)20·105 (Tyr)
Maximum Protein Efficiency
Ratio
PERmax Defined as the PER value estimated at the optimum dietary protein
level
dmnl †Same issues as for PER except determined at optimal dietary protein
intake.
(84)
PER ¼Gain in body mass (g)/Protein intake (g)
Net Protein Ratio (or retention) NPR NPR ¼(weight gain of test rat þweight loss of non-protein rat)/
(protein consumed by test rat)
dmnl †Same issues as for PER (8)
Net Protein Utilization
b
NPU Proportion of ingested protein retained dmnl †Measured when protein content of diet is below that of requirement
and may not be appropriate when diet is adequate.
(6)
NPU ¼((0·16£(24 hour protein intake in grams)) 2((24 hour
urinary urea nitrogen)þ2)2(0·1 £(ideal body weight in kilo-
grams))) /(0·16£(24 hour protein intake in grams))
Protein Rating PR PR ¼Protein in a reasonable daily intake, g x PER g †Same limitations as for PER.
Relative Nutritive Value RNV Measures feed/food intake (FI) and the efficiency of extraction of
nutrients during digestion (digestibility).
†Similar issues as for NB, PER, etc. (85), and refer-
ences therein†Method becomes limited in samples severely lacking in Lys.
Apparent Biological Value ABV Proportion of the absorbed nitrogen retained % †Does not take into consideration metabolic nitrogen loss.
BV
c
¼[(N
i
2N
e(f)
2N
e(u)
)/(Ni 2N
e(f)
)]£100
True Biological Value TBV Proportion of the absorbed nitrogen retained taking into consider-
ation the metabolic nitrogen loss
%†Ignores variation in digestibility of a food (i.e., ignores the importance
of factors which influence digestion of the protein and interaction of
protein with other dietary factors before absorption).
(4,86,87)
BV
c
¼[(N
i
2(N
e(f)
2N
epf(f)
)2(N
e(u)
-N
epf(u)
))/(Ni 2(N
e(f)
2N
epf(f)
))]£100
†Measured when protein content of diet is below that of requirement
and may not be appropriate when diet is adequate.
†BV results for the same food varies significantly depending on N
intake.
†BV results for different foods may be similar at low N intake and very
different at higher intake levels.
†Proteins which are completely devoid of one IAA can still have a BV
of up to 40.
Relative Biological Value rBV Biological value of a food source relative to egg dmnl †Same limitations as for BV above. Additionally, due to differences in
BV at different N intakes, proportionately between proteins seen at
lower levels will not be maintained at higher levels.
(4)
(BV
(test)
/BV
(egg)
)£100
J. Boye et al.S186
British Journal of Nutrition

Table 1. Continued
Protein Quality Evaluation
Method
Abbrev Method Summary and/or Equation Unit Issues Reference
Protein Digestibility Corrected
Amino Acid Score
PDCAAS Relies on determination of protein contents, amino acid profile and
protein digestibility
% or dmnl †Proteins with higher than 100 % scores are rounded off to 100 %
(additional benefit in complementing less nutritious proteins is not
captured).
(5,6,8,9,86), and
references
therein
PDCAAS ¼((mg of limiting amino acid in 1 g of test protein)/(mg
of same amino acid in 1 g of reference protein or requirement
pattern)) £true faecal digestibility (%) £100
†Does not include impact of anti-nutritional factors associated with pro-
teins, including naturally occurring and those formed during
processing, on protein digestibility and quality.
†There may be a need to include corrections for the bioavailability of
individual amino acids and not just for digestibility of protein.
†Ileal vs faecal protein/amino acid digestibility (i.e., faecal digestibility
may overestimate due to microbial degradation).
†No standardized methods available for ileal digestibility.
†Effect of age on faecal and ileal protein/amino acid digestibility is not
known.
†Validity of the preschool-age child amino acid requirement values
(there is still a need for validation of the scoring pattern used for the
preschool-age child). Scoring pattern does not include conditionally
IAA.
†Humans consume proteins from varied protein sources. PDCAAS
values of single protein sources may not have practical significance.
a
Corrected for metabolic faecal protein loss
b
NPUstandardized and NPUoperative distinguish between studies at fixed or varying dietary protein concentrations, (WHO/FAO/UNU, 2007)
c
BV equation (definitions):
N
i
¼nitrogen intake in proteins on the test diet
N
e( f)
¼nitrogen excreted in faeces whilst on the test diet
N
e(u)
¼nitrogen excreted in urine whilst on the test diet
N
epf( f)
¼nitrogen excreted in faeces whilst on a protein free diet
N
epf(u)
¼nitrogen excreted in urine whilst on a protein free diet
d
dmnl – dimensionless
e
Example of an NB Equation using an arbitrary adjustment factor
NI ¼Protein intake (g/day) / 6·25
NL ¼Urinary urea nitrogen (g/day)
1
þ2to4
2
1
Obtained from 24 h urine sample;
2
Additional arbitary adjustment factor to account for other nitrogen losses (e.g., faecal, dermal, miscellaneous and non-urea nitrogen)
Protein Quality Evaluation S187
British Journal of Nutrition

and interaction of protein with other dietary factors before
absorption.
The PDCAAS method is based on a determination of protein
content, amino acid profile and protein digestibility. The limit-
ing amino acid score (i.e., ratio of first limiting amino acid in
1 g of protein from the target food to that found in a reference
protein or reference requirement) is multiplied by protein
digestibility (true faecal digestibility) which gives a value for
protein quality corrected for digestibility. The first limiting
amino acid is the IAA present at the lowest concentration in
a food. Depending on concentration, other IAA may be
described as the second, third, fourth, (etc.), limiting amino
acid. The recommended reference protein for infants under
1 year of age is that of human milk
(4)
. For all other age
groups, the FAO/WHO 1991 report recommended the use of
the amino acid scoring pattern for preschool children in the
2-5 year range. The 2007 WHO/FAO/UNU report, however,
revised this recommendation and has suggested the use of
the amino acid scoring pattern for preschool children in the
1-2 year range for preschool children, and the 3-10 yr pattern
when judging protein quality for schoolchildren and
adolescents.
Effects of bioactive compounds, preparation and
processing conditions on protein quality
Protein quality of foods is affected by methods used in food
preparation and processing and the presence of bioactive
compounds. Foods are rarely consumed raw in today’s fast
paced society, and are often processed to increase conven-
ience and safety, extend shelf life and improve taste. The qual-
ity of protein from both animal and plant sources is thus of
most interest after preparation and processing. Plant-based
foods are particularly of interest in this regard as they contain
bioactives which in the unprocessed form can reduce digest-
ibility and hence protein quality. Processing technologies
could, however, help to transform raw grains into useful pro-
ducts which maximise their inherent nutritional value to
ensure the nutrient security of populations, particularly, in
developing countries
(12)
. The impacts on protein quality of
bioactive compounds and some of the principal techniques
used in food preparation and processing are provided below.
Bioactive compounds
Antinutritional factors (ANF) can be naturally present in the
food matrix in which a protein is consumed, or be formed
during processing or storage. ANF can affect both the digest-
ibility and bioavailability of protein and AA. Examples of natu-
rally occurring ANF include phytates in cereals and oilseeds,
polyphenols in pulses, gossypol in cottonseed protein pro-
ducts and glucosinolates in mustard and rapeseed proteins
(8)
.
The limited digestibility of legume proteins has been ascribed
to the presence of ANF such as protease, trypsin and amylase
inhibitors (which can interact with digestive enzymes to form
inactive complexes), tannins (that have a high affinity for Pro
and His in proteins), lectins, phytic acid (which can form
complexes with proteins, proteases and amylases, inhibiting
proteolysis) and non-starch polysaccharides
(13,14)
.
Processing methods such as soaking, cooking and fermenta-
tion have been shown to lead to better AA digestibility, most
likely due to a reduction in ANF such as heat labile protease
inhibitors
(15)
.
Germination
Sprouting of seeds which involves soaking seeds until germi-
nation, is used in many countries as part of food preparation.
Germination significantly increases protein content and
decreases starch level in bambara groundnut (Voandzeia sub-
terranea)
(16)
. Compared to other cooking methods (wet cook-
ing and dry roasting), germination for 4-6 days significantly
decreased phytic acid content and increased in vitro protein
digestibility. The improvement of protein digestibility after
germination was attributed to a reduction of polyphenols and
phytic acid in the germinated seedling and an increase in
soluble proteins due to the action of proteolytic enzymes.
These enzymes were also effective in hydrolyzing protein-
polyphenol complexes in the seed.
Kannan et al.
(17)
also reported that germination increased
true protein digestibility (TPD) of cooked black bean pro-
ducts, however, the increase was not accompanied by an
increase in PDCAAS due to the limiting amino acid score.
Highest TPD and PDCAAS values were obtained for cooked
germinated beans combined with rice. Germination also had
little effect on the amino acid profile of cowpeas
(18)
. However,
in vitro protein quality and starch digestibility improved after
germination, resulting in a higher PDCAAS (still low) for a
weaning food prepared from 24 h ger minated cowpea flour
(56 %) compared to the control cowpea flour weaning
food (47 %).
In other studies, germination promoted an increase of 21 %
in the protein digestibility of sorghum proteins and an
increase in protein extractability
(19)
. Protein content of mung-
bean, chickpea and cowpea also increased by 9-11, 11-16 and
8-11 %, respectively after germination, and in vitro protein
digestibility similarly increased by 15-25, 6-17 and 6-17 %,
respectively with higher improvements observed with longer
times of germination
(12)
.
Higher increases in in vitro digestibility of Indian bean
(Dolichos lablab. var. lignosus) seeds in the early stage of ger-
mination were also reported
(20)
. PER values increased in rats
fed with the germinated bean, reaching comparable values
with a control group maintained on a casein diet after 32 h
of germination. Diets with germinated bean protein also
showed marked increases in both true and apparent nitrogen
digestibility, although the values were less than that observed
for casein fed rats.
In addition to protein, germination also caused significant
increases in thiamine, in vitro iron and calcium bioavailability
and in vitro protein digestibility of green gram (Phaseolus
aureus), cowpea (Vigna catjang), lentil (Lens culinaris) and
chickpea (Cicer arietinum)
(21)
. Dehulling the germinated
legumes yielded further increases in nutritive value. Phytic
acid and tannin were reduced by 18-21 % and 20-38 %,
J. Boye et al.S188
British Journal of Nutrition

respectively, on germination and further reduction was
observed on dehulling. The low levels of phytic acid and
tannin detectable in the cotyledons after dehulling, suggest
that most of the phytic acid and tannin were present in the
seed coat. Negative correlations were, however, reported
between antinutritional factors and nutrient bioavailability and
digestibility.
Osman
(22)
, however, noted that germination significantly
increased tannin content in another legume (Dolichos lablab
bean [Lablab purpuresus (L)sweet] ) compared with other tra-
ditional methods of food preparation, although germination
was more effective in improving protein digestibility than
soaking and cooking. Similarly, germination of kidney bean
(Phaseolus vulgaris L.) was less effective in reducing trypsin
inhibitors, saponins and phytohaemagglutinins than cooking/
autoclaving
(23)
. Germination, however, reduced stachyose,
raffinose, phytic acid and tannins. The combination of
germination followed by autoclaving resulted in a 100 %
reduction of phytic acid and tannin and a 9-18 % increase in
in vitro protein digestibility.
Wet or moist thermal treatment
In plant proteins heat processing, especially moist heat, may
sometimes improve protein digestibility by destroying pro-
tease inhibitors and denaturing proteins, which can open up
their structure allowing gastrointestinal enzymes greater
access for hydrolysis. Soaking, boiling, microwave cooking
and autoclaving increased total IAA content determined in
the seeds of cowpea, pea and kidney bean
(15)
. The magnitude
of the effect was in the following order: autoclaving .boiling
.microwave cooking .soaking. The determined level of
sulphur AA also slightly increased in all samples after micro-
wave cooking and autoclaving, and autoclaving was found
to be most effective for improving protein efficiency ratio
and amino acid score (based on assumption of CysþMet as
being first limiting), followed by micronization, microwave
cooking and fermentation. In vitro protein digestibility also
significantly improved after soaking, boiling, microwave
cooking, pressure cooking (autoclaving) and fermentation
(Table 2). The PDCAAS decreased in the order micro-
wave cooking .autoclaving .boiling .soaking, with some
Table 2. Effects of processing conditions on protein quality (adapted from Khattab et al.
(15)
)
Processing condition
Legume
a
CC EC CK EK CP EP
Raw
Minimum AAS
b
68·5 91·9 52·7 76·9 75·0 57·3
IVPD
c
82·3 81·6 70·5 78·0 78·4 80·1
PDCAAS
d
56 75 37 60 59 46
Soaking
Minimum AAS 53·1 73·8 51·9 81·2 52·7 75·4
IVPD 87·5 86·7 76·0 83·2 83·7 85·5
PDCAAS 46 64 39 68 44 64
Boiling
Minimum AAS 82·3 79·2 76·5 65·4 70·8 71·5
IVPD 98·1 97·2 87·4 94·2 94·3 95·8
PDCAAS 81 77 67 62 67 69
Roasting
Minimum AAS 83·8 75·4 66·5 65·0 68·1 58·8
IVPD 77·6 76·6 64·9 73·0 73·1 75·0
PDCAAS 65 58 43 47 50 44
Autoclaving
Minimum AAS 65·8108·190·489·285·476·2
IVPD 90·3 89·7 79·0 86·1 86·6 88·3
PDCAAS 59 97 71 77 74 67
Microwave
Minimum AAS 94·2 91·2 91·2 72·3 94·2 100·6
IVPD 92·8 92·2 81·7 88·6 89·1 90·9
PDCAAS 87 84 74 64 84 92
Fermentation
Minimum AAS 79·6 96·2 67·6 100·0 88·8 98·5
IVPD 85·1 84·3 73·4 80·9 81·4 82·9
PDCAAS 68 81 50 81 72 82
Micronization
Minimum AAS 98·5 100·0 99·2 88·5 101·9 110·8
IVPD 80·0 79·1 67·9 75·5 76·0 77·9
PDCAAS 79 79 67 67 77 86
Min PDCAAS
e
46 58 37 47 44 44
Max PDCAAS
f
87 97 74 81 84 92
a
CC: Canadian cowpea; EC: Egyptian cowpea; CK: Canadian kidney bean; EK: Egyptian kidney bean; CP: Canadian pea; EP: Egyptian pea.
b
AAS: amino acid score.
c
In vitro protein digestibility (IVPD) (%).
d
PDCAAS: protein digestibility corrected amino acid score.
e
Minimum PDCAAS value obtained for a particular legume.
f
Maximum PDCAAS value obtained for a particular legume.
Protein Quality Evaluation S189
British Journal of Nutrition

exceptions. Very large differences in PDCAAS were observed
depending on processing: for example, the PDCAAS of raw
Canadian kidney beans was half the value of the PDCAAS
from microwave cooking.
Saleh and El-Adawy
(24)
similarly reported significant
decreases in antinutritional factors (trypsin inhibitor, haemag-
glutinin activity, tannins, saponins and phytic acid) of chick-
pea (Cicer arietinum L.) after cooking (i.e., using microwave
cooking and other traditional cooking methods). In vitro pro-
tein digestibility (IVPD) and protein efficiency ratio were
improved by all cooking treatments from 84 % (raw) to
approximately 90 % for IVPD and 2·3 (raw) to approximately
2·5 for PER. Chemical score and the amino acid determined
to be first limiting for chickpeas subjected to the various cook-
ing treatments, however, varied considerably, depending on
the type of treatment.
In other studies, saponins, trypsin inhibitors and phytohae-
magglutinins, diminished dramatically to undetectable amounts
when kidney beans (Phaseolus vulgaris L.) were cooked or
autoclaved
(23)
. Similarly, cooking of pre-soaked seed was
found to be the most effective method for reducing trypsin
inhibitor activity in Dolichos lablab bean
(22)
and a significant
increase in IVPD was also found when raw sprouts of mung-
bean, chickpea and cowpea were subjected to pressure cooking
and microwaving
(12)
.
Heat treatment can sometimes, however, be detrimental to
protein quality. As an example, digestibility and extractability
of sorghum proteins decreases, especially, on wet cook-
ing
(19,25)
. This occurs due to binding of the tannins to proteins
in tannin-containing sorghum cultivars. Tannins have antioxi-
dant properties and are bacteriostatic and/or bactericidal for
many bacteria species
(26)
. Although the positive benefits of
tannins are increasingly being recognised, their presence in
high amounts in plant based foods unfortunately has a detri-
mental effect on protein quality. The reduced digestibility of
cooked sorghum products has also been attributed to disul-
phide crosslinks occurring between g- and b-kafirin proteins
at the protein body periphery, which may impede digestion
of the centrally located major storage protein, a-kafirin
(25)
.
Milk sterilization (at 110-1208C, 20-30 min) also causes a
decline in protein quality, due to a decrease in Lys, Met and
Cys
(27)
. Maillard reactions involving Lys, and destruction of
the sulphonic group in the case of Met and Cys due to sterili-
zation, may be the cause. PDCAAS for sterilized semi-skimmed
milk was 34 % whereas it was much higher for pasteurized
semi-skimmed milk (76 %). In general, no appreciable Maillard
reaction is expected to occur during pasteurization, and only
very small losses of Lys have been reported ranging from
0-5 % on ultra high temperature (UHT) treatment
(28)
.
In another study where hot water (808C) reconstituted pow-
dered infant formulas were sterilized by autoclaving for 5 min
at 1058C, a 20 % reduction in total protein was found after auto-
claving compared with the conventional preparation, where
samples were reconstituted with warm water (378C) in glass bot-
tles but not autoclaved
(29)
. Concentrations of total free AA and in
particular some specific individual amino acids, Val (272 %),
Gln (260 %) and Lys (240 %), also decreased in the autoclaved
formulas. Higher concentrations of ammonia found after auto-
claving suggested degradation of protein and AA.
Dry heating
Dry heat treatment of protein flours includes processing treat-
ments such as dry roasting and micronization (a thermal treat-
ment based on infrared heating). Despite the heat processing
applied, dry roasting and micronization reduced the IVPD of
cowpea, pea and kidney bean by 5·72–7·96 and 2·75–
3·72 %, respectively, when compared with raw legumes
(15)
(Table 2). IAAs and PDCAAS, however, increased after roast-
ing and micronization for the majority of seeds studied. The
reduction in protein digestibility was attributed to non-
enzymatic browning (Maillard reaction) between the reducing
sugars from starch hydrolysis and the proteins, as well as
the thermal cross-linking that occurred during heating
(15)
.
For sorghum proteins a decrease of 4 % in protein digestibility
was observed after dry heating and protein extractability was
not affected
(19)
.
Smoking and broiling
Essential AA in rainbow trout were reported to be generally
much higher in raw samples than after smoking and broil-
ing
(30)
. Lys was particularly affected and in overheated fish,
was drastically reduced compared to untreated fish. Compared
with raw rainbow trout, broiling reduced the digestibility of
protein (i.e., % decrease) by 1·63-3·9 % and smoking by 4·2-
4·5 %. PDCAAS of raw trout was reduced by 6 % after smoking
and by 3 % after broiling. The decrease in protein quality was
attributed to an increase in SH (sulphydryl) groups and S-S
bonds, as well as complex chemical (cross-linking) reactions
such as protein-protein interactions or protein-fat interactions
when food was broiled at high temperatures. Smoking con-
ditions (time, temperature, compounds of wood smoke) are
all factors that can negatively influence protein digestibility.
Bender
(31)
showed that at the rather low temperature
needed to cook meat there is little loss of available Lys and
no loss of Met and Cys. No change in protein quality was
found after roasting meat in an open pan at 1638C when the
internal temperature did not rise above 808C; or when the
meat was browned in an oven for 30 min then sterilised in a
can
(31)
. When meat is roasted the outer part reaches a high
temperature and turns brown (Maillard reaction) which pro-
duces the desired roast flavour but since the roasted part is
only a small fraction of the total piece of meat and, especially,
when the internal temperature does not exceed about 808C,
there is no measurable change in the quality of the protein
as a whole
(31)
.
In another study
(32)
, intermediate moisture smoked beef
was prepared by cook-soak/equilibration, where samples
were either smoked for 18 h (heavy smoking) or for 4 h
(light smoking) at 508C. Smoking caused a marked decrease
in SDS-soluble protein and slightly decreased the available
Lys and percent conversion of the haemoproteins to the
cured nitrose forms. Smoking also caused increased darkening
J. Boye et al.S190
British Journal of Nutrition

and hardness of the samples and a slight loss of some of the
protein components.
Evans et al.
(33)
compared the protein quality of meats after
standard cooking procedures by assessing the effects on rela-
tive nutritive value (RNV) and amino acid composition. Boiled
tissue meats and processed meat showed higher RNV and
levels of essential AAs than fried or microwave cooked or
uncooked samples. However, in organ meats, cooking
did not change total protein content or total essential amino
acid contents relative to uncooked organ meats.
Spray-drying
Spray-drying is a downstream unit operation frequently used
in the food industry to extend the shelf life of foods. Liquid
foods are pumped through the nozzle of the spray dryer
and brought into intimate contact with a counter current
flow of hot air which causes flash vaporization of moisture
leaving a shelf stable powdered particulate material. Spray
drying is used for drying caseinates, whey protein, soya pro-
teins and a variety of other products and the temperatures
and times used can vary extensively for different food pro-
ducts. Concerns about the impact of spray-drying on protein
quality include the occurrence of Maillard reactions, degra-
dation of AA and possible conversion of L- to D- AA amongst
others.
In one study where the nutritional protein quality of lactose-
hydrolysed milk was studied with N balance experiments on
growing rats, the authors found that spray-drying under con-
ditions usually used for ordinary milk gave a considerable
reduction in protein quality, caused mainly or entirely by
loss of biologically available Lys
(34)
.
A higher spray drying temperature was also found to signifi-
cantly decrease protein quality and the contents of all AA in a
spray-dried protein hydrolysate from black tilapia fish
(35)
.
In vitro protein digestibility decreased from 92 % (1508C/768C
inlet/outlet temperature) to 88·4 % (1808C/908C inlet/outlet
temperature) and PDCAAS % decreased from 82 to 34, respect-
ively with increasing processing temperature. Additionally,
predicted protein efficiency ratios of the dried hydrolysates
decreased from 2·97 to 2·53.
Extrusion
Extrusion is a high temperature high shear process used in
food texturization which has grown in popularity in recent
times. It is used extensively in the processing of snacks, cer-
eals, meat and a variety of other products. The high tempera-
tures and shear pressures used during extrusion can denature
or degrade proteins and AA and impact protein quality either
positively or negatively.
Extrusion of pea seeds (Pisum sativum L. var. laguna)
increased protein recovery slightly and decreased trypsin
inhibitory activity (TIA) to negligible levels
(36)
. Val, Phe and
Lys contents decreased significantly (10– 22 %) when extru-
sion was carried out at 1298C, whereas Trp decreased only
when higher temperatures were used (1428C). Changes were
also observed in the concentrations of dietary dispensable
AA, with Pro undergoing the greatest reductions (28-38 %), fol-
lowed by Gly (10-15 %). Interestingly, biological indices for
protein quality (NPU, TD, BV, NPR), remained unchanged
after extrusion, but PDCAAS decreased from 66 (raw flour)
to 55, 61, 59 % for samples extruded at 129, 135 and 1428C,
respectively.
Digestibility of hard-to-cook flour of cowpea was also
improved by 56 % after extrusion
(37)
. Furthermore, extrusion
at 1508C significantly decreased antinutrients such as phytic
acid (33·2 %), lectin (100 %), a-amylase (100 %), and trypsin
inhibitors (38·2 %). In vitro protein digestibility of common
bean (Phaseolus vulgaris L.) was also improved by
72·3 –84·5 % after extrusion
(38)
.
Also, extrusion did not alter IVPD of soyabean and corn; how-
ever, it reduced the amount of trypsin inhibitors when a combi-
nation of the grains was extruded at 1208C
(39)
. Proximate
composition of extruded products from corn (Zea mays) and
lima bean (Phaseolus lunatus) flour blends showed increased
protein and ash contents whereas fat levels decreased. The
IVPD of the extrudates increased to 82 % compared to the raw
flours (77 %)
(40)
. Other reports in the literature suggest that
extrusion conditions targeting aflatoxin reduction in peanut
does not adversely affect protein nutritional quality.
In contrast, Silva et al.
(41)
found that extrusion of bovine
rumen protein containing about 96 % protein significantly
reduced true protein digestibility from 97·7 % to 93·1 %. The
limiting amino acid also changed after extrusion but scores
remained similar (i.e., 1·28 (Leu) for raw bovine rumen and
1·25 (Met þCys) for the extruded sample). Un-truncated pro-
tein digestibility-corrected amino acid scores decreased from
125 to 116 % after extrusion. In this particular example, for
both raw and extruded proteins, PDCAAS values were, how-
ever, excellent (100 %) and animal growth profiles using raw
and extruded rumen were also found to be comparable.
Irradiation
Food irradiation is a processing technique in which foods are
subjected to ionising radiation to destroy insects and other
pathogens of microbial origin and which affects food quality
as well as safety. Several countries permit the use of food
irradiation for safety reasons. Foods can be treated using
low dose (.2 kGy), medium dose (2-10 kGy) and high
dose (.10 kGy) radiation. Low dose irradiation is used to
delay sprouting of vegetables and aging of fruits. Medium
dose radiation helps to reduce levels of pathogenic organisms
whereas high doses are used for food sterilisation. Food
irradiation is not permitted for use to increase the nutritional
value of foods; nevertheless some authors have evaluated its
impact on protein quality. Bhat and Sridhar
(42)
studied the
impact of electron beam irradiation on the nutritional and
anti-nutritional properties of lotus seeds. Their results
showed a higher concentration of IAAs (Thr, Val, Leu, Tyr
þPhe, and Ly) after irradiation. PDCAAS, however, signifi-
cantly decreased after irradiation due to a decrease in IVPD
which went from 43 % for the untreated sample to 24 %,
Protein Quality Evaluation S191
British Journal of Nutrition

after application of 30 kGy irradiation. The decrease in IVPD
was dose dependent: (irradiation dose kGy: IVPD % 0:43,
2·5:41, 5:40, 7·5:40, 10:40, 15:41 and 30:24) and was found
to be statistically significant only at 30 kGy. Radiation levels
ranging from 30 kGy to 75 kGy have been used for example
for microbial disinfection of spices and seasonings, sterilisa-
tion of frozen packaged meats for astronauts, and meals for
immuno compromised hospital patients.
Fermentation
Food fermentation has been used in many cultures since
ancient times to preserve food and improve taste. The fermen-
tation process involves the use of a variety of micro-organisms
such as bacteria, moulds and yeasts which may be naturally
present in or on the food or expressly added to induce fer-
mentation. Fermentation provides a technological alternative
for improving the nutritional value of a great variety of
legumes and cereals while maintaining acceptable sensory
properties
(43)
. The micro-organisms used in fermentation syn-
thesise enzymes which hydrolyse food constituents and con-
tribute to the development of products with desirable
organoleptic properties. Furthermore, the hydrolysis could
contribute to the decrease or elimination of anti-nutritional
factors which could help in improving nutritional quality of
the food.
Angulo-Bejarano et al.
(44)
reported an improved protein
digestibility of chickpea flour after solid-state fermentation
(SSF). In this study, chickpea seeds were soaked, seed coats
removed, and cotyledons were cooked at 908C for 30 min
and inoculated with a suspension of R. oligosporus and fer-
mented at 34·98C for 51·3 h followed by drying at 528C for
12 h and milling. Proteins from unfermented and fermented
flours had IVPD of 72 % and 83 %, respectively. True protein
digestibility (in vivo) increased form 84 % to 89 % and PER,
NPR and PDCAAS improved from 1·6 to 2·3, 2·7 to 3 and
from 73 to 92 %, respectively, as a consequence of the cook-
ing/fermentation process. The improvement of PER during
fermentation was attributed to better availability of AA and
greater digestibility of the proteins in the substrates. Total
sulphur AA (Met þCys) was the first limiting IAA in proteins
from untreated chickpea with an IAA score of 0·87. In the fer-
mented flour, Trp was the first limiting IAA with an IAA score
of 0·93. The essential AA content of untreated chickpea was
improved by the SSF process including levels of Ile, total
sulphur AA (Met þCys), total aromatic AA (Phe þTyr),
and Thr. The control sample for this study was, however,
raw uncooked flour. To quantify the effect of fermentation it
would have been useful if the authors had provided the pro-
tein quality results of the flour, cooked without the fermenta-
tion step.
In another similar study
(43)
, SSF increased the content of IAA
in maize from 41 to 49 g IAA/100 g protein. His, Ile, Leu, Lys
and Trp increased by 0·8, 0·5, 1·5, 1·5 and 0·12 g/100 g protein,
respectively. Total sulphur AA (Met & Cys) and total aromatic
AA (Tyr & Phe) increased by 0·6 and 3·5 g/100 g protein,
respectively. Some AA levels, notably Val, however decreased
from 6·1 to 4·3 g/100 g protein. First and second limiting IAAs
in the untreated flour were Lys (0·72) and Trp (0·73), but this
changed after fermentation to Trp (0·84) and Lys (0·98),
respectively. Overall, fermentation increased protein quality
indicators as follows: true protein digestibility from 76·6 to
86·8 %, protein efficiency ratio (PER) from 1·8 to 2·1 and
PDCAAS from 55 to 83 %.
Nicolau et al.
(45)
found the protein content in boiled rice
doubled after solid state fermentation using a strain of
Saccharomycopsis fibuligera, an amylase producing yeast.
The increase was attributed to the yeast biomass which con-
tributed protein rich in Lys (an amino acid limiting in rice),
Met and Trp. The fermented rice was enriched in B group vita-
mins (B1, B2, and B6) synthesized by the yeast and
phosphorus bioavailability also increased as result of fermen-
tation. Fermentation also promoted an increase of 39·6 % in
the protein digestibility of sorghum proteins and an increase
in protein extractability
(19)
.
A tempeh-type fermented product was susscesfully pre-
pared from fresh and hardened common beans
(46)
using
Rhizopus oligosporus. Soluble solids, total and soluble pro-
teins, soluble carbohydrates and pH of both bean samples
increased after fermentation whereas fat and fibre content
decreased. Trypsin inhibitor units (TIU/g d.b) decreased
from 120,000 – 130,000 in raw beans to 61,090 – 65,000
after soaking/dehulling/cooking (SDC) to 250 – 900 after fer-
mentation. Phytic acid levels did not change much by the SDC
treatment but significantly decreased from 2·1 to 1·4 g/100 g
d.b after fermentation. Cooking also reduced the lectin level
significantly to almost undetectable levels and as a result this
value did not change much after fermentation. The results
suggest that the Rhyzopus oligosporus used for fermentation
was capable of hydrolyzing the trypsin inhibitor and phytic
acid of the substrate which may explain the improvements
in protein quality in the previous studies reported above.
More recently, Fagbemi et al.
(47)
also found fermentation to
be the most effective processing method to reduce phytic acid
and trypsin inhibitor activity in mature seeds of breadnut,
cashew nut and fluted pumpkin, whereas boiling was most
effective in reducing tannin content. Boiling, fermentation,
germination and roasting reduced TIA of the seed flours by
20·4, 88·7, 0·9 and 26·8% (breadnut); 57·1, 67·1, 34·5, and
58·7 % (cashew nut); 100, 100, 94 and 100 % (pumpkin),
respectively. Boiled samples had the highest IVPD (most
digestible) followed by the fermented samples.
Interestingly, not all authors have observed increases in
nutritional quality after fermentation. Kannan et al.
(17)
found
no statistically significant increase in either TPD or PDCAAS
values upon fermentation of black bean products.
In the dairy sector, proteins in yogurt, acidophilus milk, and
bifidus milk have been found to be more digestible than those
in unfermented milk
(48)
. The enhanced digestibility is attribu-
ted to protein denaturation and hydrolysis during fermenta-
tion, which results in the formation of smaller, more
digestible curds. Pre-hydrolysis of milk proteins, as indicated
by increased levels of free AA, especially Pro and Gly,
occurs during the manufacture of yogurt
(49)
. Cultured yogurt
J. Boye et al.S192
British Journal of Nutrition

has a significantly higher protein quality than the mix from
which it is made as evidenced by in vivo digestibility, net pro-
tein ratio and computed protein efficiency ratio
(48)
. The
activity of proteolytic enzymes and peptidases is apparently
preserved throughout the shelf life of yogurt: the concen-
tration of free amino groups increases up to twofold during
the first 24 h and then doubles again during the next 21 d of
storage at 78C
(49)
. Some bacterial cultures have greater proteo-
lytic activity during milk fermentation and storage than others
(as indicated by elevated concentrations of peptides and free
AA after milk fermentation)
(49)
. Both heating and fermentation
reportedly contribute to the high protein quality of yogurt.
Overall, when a summary of the evidence is taken into con-
sideration, fermentation may be a useful processing technique
to reduce anti-nutritional factors in plant based proteins which
could contribute to improving protein digestibility as well as
protein quality. For animal protein as well, fermentation may
contribute to further improvements in quality.
Protein quality evaluation methods and recommended
intakes currently used in different countries
Various terminologies are used in the literature to describe
nutritional requirements. Table 3 provides a description of
some of the most frequently used ones. To promote adequate
nutrition, countries around the world provide recommen-
dations for intake of different nutrients including protein. As
proteins play a critical role in health, methods to assess their
quality, efficient methods of processing to enhance their nutri-
tive value and safe levels of intake need to be established.
Some country specific protein recommended intakes and
quality evaluation methods are provided in Table 4. Average
and safe levels of protein intake recommended by the
WHO/FAO/UNU
(6)
are shown in Table 5 and recommen-
dations for essential AA in Table 6.
It is important to note that the recommended dietary intake
(RDI) is set at 1·96 times the standard deviations (SD) above
the estimated average requirement (i.e., when requirement
for the nutrient is symmetrically distributed) in order to meet
the nutrient requirements of nearly all (97–98 %) healthy indi-
viduals in a particular life stage and gender group. The
National Health and Medical Research Council (NHMRC)
(50,51)
in Australia defines RDI as “the levels of intake of essential nutri-
ents considered,... on the basis of available scientific knowl-
edge, to be adequate to meet the known nutritional needs of
practically all healthy people ... they incorporate generous fac-
tors to accommodate variations in absorption and metabolism.
They therefore apply to group needs. RDIs exceed the actual
nutrient requirements of practically all healthy persons and
are not synonymous with requirements”.
Interpretation of the RDI especially in relation to evaluating
protein quality and determining appropriate amounts for label-
ing purposes can sometimes be challenging for consumers and
industry. The Codex Alimentarius and some countries (e.g.,
USA) therefore provide specific guidelines
(52,53)
. Furthermore,
as processing, matrix and the presence of bioactives can
Table 3. Different terminologies used in the literature to describe nutritional requirements
Acronym Definition and Description
a
AI Adequate Intake (used when EAR values are not available): Average daily nutrient intake estimated using the best approach
scientifically available (e.g., observed or experimentally determined approximations or estimates of observed median nutri-
ent intakes by a group (or groups) of healthy people) to describe an acceptable intake level or range.
AMDR Acceptable Macronutrient Distribution Ranges: Range of intakes for a particular energy source that is associated with reduced
risk of chronic disease while providing adequate intakes of essential nutrients. The AMDR is expressed as a percentage of
total energy intake. Recommended for calculating %DV of macronutrients (fat, protein, carbohydrate).
DRI Dietary Reference Intake: Includes the EAR, RDA, AI and UL. For the DRIs a requirement is defined as the lowest continuing
intake level of a nutrient that, for a specific indicator of adequacy, will maintain a defined level of nutrition in an individual.
DRV Daily Reference Value: DRVs are label reference values originally established for eight nutrients for which there were no
National Academy of Sciences (NAS) Recommended Dietary Allowances (RDAs) at the time. Based on a body of scientific
literature linking diet and the risk of chronic disease, the Food and Drug Administration (FDA) of the United States of
America established DRVs as label reference values for total fat, saturated fat, cholesterol, total carbohydrate, dietary fiber,
sodium, potassium, and protein based on a 2,000 calorie diet.
%DV Percent Daily Value: Percentage of the Recommended Daily Allowance provided by a specified amount of food (i.e., serving
and/or portion size) on a nutrition label. Intended not only to help individuals compare different products within a food type,
but also to help them understand nutrition information about foods. The %DV is based on population-weighted EARs or AIs.
EAR Estimated Average Requirement: A daily nutrient intake level expected to satisfy the needs of half of the apparently healthy
individuals in that age and gender group.
RDA/RDI Recommended Dietary Allowance/ Recommended Dietary Intake: Estimate of the minimum daily average dietary intake of a
nutrient which is considered sufficient to meet the requirements of nearly all (97– 98 %) healthy individuals in a particular
life-stage and gender group. This value is usually 2SD above the EAR when the requirement is symetrically distributed in a
population.
RDI Reference Daily Intake: Denote(s) those nutrients whose label reference values have been derived from the NAS RDA and
Estimated Safe and Adequate Daily Dietary Intakes.
RNI Reference Nutrient Intake: Provides an estimate of the amount of nutrient that should meet the needs of most of the group
(97·5 %) to which they apply (Similar to RDA/RDI).
SL Safe Level: Defined as the 97·5th percentile of the population distribution of requirement (average protein requirement of the
individuals in the population, plus twice the SD).
UL Upper Level: The highest level of consumption shown to be safe based on current data. At intakes above the UL, the potential
risk of adverse effects may increase.
a
Source: Ref 88; 89; 6
Protein Quality Evaluation S193
British Journal of Nutrition

Table 4. Country specific recommended protein intakes and quality evaluation methods
Country
Protein Recommended Intake
Age
EAR (g/kg
body
weight)
EAR
(g/day)
RDA/RDI or AI*
(g/kg body
weight)
RDA/RDI
or AI*
(g/day) AMDR
Reference
body weight
(kg)
Protein Quality
Assessment Method Reference
Australia and 0 – 6 months 1·43* 10* Upper bound of AMDR is recommended 7
a
Not found (51)
New Zealand 7-12 months 1·6* 14* for those aged 14 years and above 9
b
1-3 yr 0·92 12 1·08 14 13
4-8 yr 0·73 16 0·91 20 22
9-13 yr (M) 0·78 31 0·94 40 40
9-13 yr (F) 0·61 24 0·87 35 40
14-18 yr (M) 0·76 49 0·99 65 64
14-18 yr (F) 0·62 35 0·77 45 57
19-70 yr (M) 0·68 52 0·84 64 76
19-70 yr (F) 0·60 37 0·75 46 61
.70 yr (M) 0·86 65 1·07 81 76
.70 yr (F) 0·75 46 0·94 57 61
Canada 0-6 months 1·52* 9·1* AMDR for protein is set at 5 – 20 % of 6
a
PDCAAS / PER (90)
7-12 months 1·0 9 1·2 11 total calories for 1 – 3 yr; 9
1 – 3 yr 0·87 10·4 1·05 13 12
4 – 8 yr 0·76 15 0·95 19 10 – 30 % for 4 – 18 yr; 20
9-13 yr (M) 0·76 27 0·95 34 10 – 35 % for .18 yr. 36
9-13 yr (F) 0·76 28 0·95 34 37
14 – 18 yr (M) 0·73 44·5 0·85 52 61
14 – 18 yr (F) 0·71 38 0·85 46 57
.19 yr (M) 0·66 46 0·8 56 70
.19 yr (F) 0·66 38 0·8 46 57
UK
c
1-3 yrs 15 Not found Not found (92)
4-6 yrs 20
7-10 yr 28
11-14 yr 42
15-18 yr 55
19-50 yr 55
.50 yr 53
USA 0-6 months 9·1* AMDR for protein is set at 5 – 20 % of PDCAAS (PER, (52,91)
7-12 months 13·5 total calories for 1 – 3 yr; reference casein)
1 – 3 yr 1·05 13 10 – 30 % for 4 – 18 yr;
4 – 8 yr 0·95 19 10 – 35 % for .18 yr.
9-13 yr (M) 0·95 34
9-13 yr (F) 0·95 34
14 – 18 yr (M) 0·85
d
52
.14 yr (F) 0·8 46
.19 yr (M) 0·8 56
Pregnancy 1·1 71
Lactation 1·3 71
a
2-6 months;
b
7-11 months;
c
Reference Nutrient Intake (RNI) is set at 0·75g of protein/kg per day for adults;
d
Similar for female in Ref 91
J. Boye et al.S194
British Journal of Nutrition

influence protein extraction, AA recovery, AAS, the first limit-
ing amino acid and AA digestibility, it is important that these
be taken into consideration in the evaluation of protein quality
and in establishing labelling guidelines.
Protein quality of some key food groups
Tables 7–9 provide a list of protein quality indices for various
food groups and food blends. Foods are frequently consumed
in complement with other foods which raises questions as to
the significance of the PDCAAS values of single protein
sources. Practically all animal sources of protein have
PDCAAS values equal to or above 1 (or 100 %). The excess
IAA provided by these foods could be useful in complement-
ing negative IAA balances in other foods with lower protein
quality.
Although some plant sources of protein, such as soya pro-
tein isolate and soyabean have PDCAAS values close to
100 %, many other sources of plant protein have PDCAAS
values that are much lower. Large variations in PDCAAS are
evident even within food groups: for example, where pearl
millet and sorghum have PDCAAS values as low as 20 %,
one variety of quinoa was reported to have a PDCAAS as
high as 100 % (Table 7). Similarly, among tree nuts, almonds
(Prunus dulcis L.) have a PDCAAS of ,25 % while Baru
almonds (Dipteryx alata Vog.) and tropical almonds
(Terminalia catappa) have a value that is almost three times
higher. The advantage of combining food proteins is evident
from Table 9: by mixing sorghum and finger millet with
30 % mung bean flour and 10 % nonfat milk, the PDCAAS
values are increased 2-3 fold with respect to those for the
grains on their own.
Concerns have been raised by some about the adequacy of
plant sources of protein to provide dietary protein require-
ments. The American Dietetic Association
(54)
has indicated
that an assortment of plant foods eaten over the course of a
day can provide all dietary essential AA and ensure adequate
nitrogen retention and use in healthy adults, thus complemen-
tary proteins do not need to be consumed at the same meal.
The ADA further clarifies that although some vegan women
have protein intakes that are marginal, typical protein intakes
of lacto-ovo-vegetarians and of vegans when well planned
appear to meet and exceed requirements and can also provide
adequately for the protein needs of athletes.
Key issues still requiring consideration in protein quality
evaluation
There still remains a myriad of issues requiring consideration
in relation to the evaluation of protein quality. A few of
these issues are provided below.
Amino acid scoring patterns
The amino acid scoring patterns in use at the present time
determine the effectiveness with which absorbed dietary nitro-
gen can meet the indispensable amino acid requirement at the
safe level of protein intake
(5,6)
. The safe level of intake as indi-
cated above is set at 1·96 times the SD above the estimated
average requirement. Further discussions and considerations
are required as to whether the scoring patterns should be
based on amino acid requirement values divided by the
mean protein requirement or the safe level of protein intake.
Further reflection is also required on the AA requirements of
infants vs. adults (i.e., growth vs. maintenance). Infants and
children have need for positive nitrogen balance to sustain
growth whereas adults need nitrogen equilibrium to maintain
health. Questions have also been raised about specific
requirements for each of the sulphur containing IAA. Further-
more, from a practical standpoint, food regulators need to
consider that it may be beneficial for industry if PDCAAS
could be calculated using only one reference pattern (i.e.,
1-2 yr or 3-10 yr pattern).
PDCAAS truncation
For higher quality proteins having PDCAAS values greater
than 1 or 100 %, the current recommendation is to truncate
Table 5. WHO/FAO/UNU (2007)
6
recommended average and safe
levels of protein intake
Age Average requirement Safe level
(yr) (g protein/kg body weight/day) ( þ1·96SD)
0·5 1·12 1·31
1 0·95 1·14
1·5 0·85 1·03
2 0·79 0·97
3 0·73 0·90
4 0·69 0·86
5 0·69 0·85
6 0·72 0·89
7 0·74 0·91
8 0·75 0·92
9 0·75 0·92
10 0·75 0·91
Adolescent Girls
11 0·73 0·90
12 0·72 1·89
13 0·71 1·88
14 0·70 0·87
15 0·69 0·85
16 0·68 0·84
17 0·67 0·83
18 0·66 0·82
Adolescent Boys
11 0·75 0·91
12 0·74 0·90
13 0·73 0·90
14 0·72 0·89
15 0·72 0·88
16 0·71 0·87
17 0·70 0·86
18 0·69 0·85
Adults 0·66 0·83
Pregnant
*
1
st
trimester 1 g protein/day
2
nd
trimester 9 g protein/day
3
rd
trimester 31 g protein/day
Lactating
*
,6 months 19 g protein/day
.6 months 12·5 g protein/day
* Additional protein intake to that recommended for adults.
Protein Quality Evaluation S195
British Journal of Nutrition

the value to the maximum value of 1. As a mixture of proteins
may be consumed in the diet over any given period of time in
the course of a day, the extra IAA provided by higher quality
proteins could complement proteins lacking IAA. The descrip-
tion of this benefit is lost with truncation. There are also
questions in regards to which value to truncate, i.e., the
PDCAAS or the amino acid score prior to multiplying by the
digestibility factor. The FAO/WHO 1991 Expert Consultation
recommended the former whereas the WHO/FAO/UNU 2007
Expert Consultation argued, that on the basis that digestibility
is first limiting, the PDCAAS value should be calculated from a
truncated amino acid score value. From a biological and prac-
tical perspective, AA released after digestion of a given
amount of protein will be based on the total amount of AA
initially present in the protein and not on a truncated
amount, thus the FAO/WHO 1991 recommendation to trun-
cate the PDCAAS score rather than the AA score seems
valid. Further discussions are evidently required on this issue.
Calculation of the amino acid score for a dietary protein
mixture
In calculating the amino acid score for a food protein mixture,
where the digestibility of individual constituents varies, the
FAO/WHO 1991 Expert Committee recommended calculating
the PDCAAS using a weighted average protein digestibility
and AA score calculated from the weighted amino acid con-
tent per gram of dietary protein. The WHO/FAO/UNU 2007
report recommended that the composition and amino acid
score of the absorbed available AA in a mixture will reflect
the relative digestibility of the individual food protein constitu-
ents, thus the amino acid score for food mixtures should be
calculated from the weighted average digestible amino acid
content. This is valid. However, a concern with the example
provided in the WHO/FAO/UNU 2007 report is that digestibil-
ity could be construed to have been used “twice” not “once” in
calculating the PDCAAS (first in calculating available protein,
and then secondly to calculate the PDCAAS). A simpler rec-
ommendation would be to base the digestible amino acid
value on the original amount of protein present in the diet
rather than on the digestible protein which would make the
second use of the digestibility function unnecessary.
Use of faecal vs. ileal digestibility
The FAO/WHO 1991 Expert Committee recommended the
standardized rat faecal-balance method as the most suitable
practical method for predicting protein digestibility. As
pointed out by the WHO/FAO/UNU 2007 report, although
faecal digestibility is probably the most appropriate measure
of overall nitrogen digestibility, it is unlikely to be a true
measure of amino acid digestibility. Digestibility measure-
ments at the ileal level may provide a better measure of
amino acid digestibility, however this may pose significant
challenges for many researchers.
Use of
in vivo
vs.
in vitro
techniques in calculating protein
digestibility
Animal studies to determine true protein digestibility can be
expensive, thus, cheaper in vitro methods that accurately esti-
mate true protein digestibility are needed. Hsu et al.
(55)
and
Satterlee et al.
(56)
developed a multienzyme in vitro system
consisting of trypsin, chymotrypsin and peptidase. The pH
of a protein suspension immediately after 10 min digestion
with the multienzyme solution at 378C
(55)
, or after an
additional l0 min incubation with microbial protease at
558C
(56)
was highly correlated with the in vivo apparent
faecal digestibility of rats (0·90 with a standard error of esti-
mate of 2·23 for the first study). Pedersen and Eggum
(57)
developed a pH-stat assay in which initial rate of alkali con-
sumption is used to calculate a rate of hydrolysis of peptide
bonds. McDonough et al.
(58)
also standardized a pH-stat
method for in vitro digestibility.
Various workers have reported good correlations between
some of the in vitro methods proposed and in vivo digestibil-
ity
(30,59 – 62)
. Some legumes, however, appear to have higher
in vitro values compared to the in vivo values. Carias et al.
(63)
Table 6. Suggested patterns of human dietary indispensible amino acid requirements
Amino Acid
(mg/g crude protein)
FAO/WHO/UNU 1985 WHO/FAO/ UNU 2007
Infant
Pre-School Child
(2-5 yrs)
School Child
(10-12 yrs) Adult
b
Adult
c
recalculated from 1985
Adult
c
(average)
Adult
d
(Safe level)
His (IAA
a
)2619 1916 15 1519
Ile (IAA) 46 28 28 13 15 30 38
Leu (IAA) 93 66 44 19 21 59 73
Lys (IAA) 66 58 44 16 18 45 56
Met þCys (IAA) 42 25 22 17 20 22 27
Met (16)20
Cys (6)7
Phe þTyr (IAA) 72 63 22 19 21 38 47
Thr (IAA) 43 34 28 9 11 23 29
Trp (IAA) 17 11 9 5 5 6 7
Val (IAA) 55 35 25 13 15 39 48
IAA-Indispensible amino acid;
a
Conditionally indispensable (children);
b
Based on a safe level of intake of 0·75 g protein/kg per day);
c
Based on a mean nitrogen requirement of
105 mg nitrogen/kg per day (0·66 g protein/kg per day);
d
Based on the assumption that the inter-individual coefficient of variation of the requirements for amino acids is the
same as that for total protein, i.e. 12 %; thus safe levels of intake recommended as being 24 % higher than average values
(6)
.
J. Boye et al.S196
British Journal of Nutrition

Table 7. Protein digestibility-corrected amino acid score (PDCAAS) for cereals, meat proteins, vegetables and tree nuts
a
Food Food Processing
Total protein
g/100 g edible
portion,
unless
indicated.
Protein
digestibility (%)
LAA (as per
reference)
PDCAAS (%)
reported
PDCAAS (%)
re-calculated using
reference pattern
for 1-2 yr child,
and LAA
b
PDCAAS (%)
re-calculated using
reference pattern
for 3-10 yr child,
and LAA
c
Methods used
(reference pattern
and digestion) Reference
Cereals and cereal proteins
Barley Air-dried at 408C 11·9 (DM) 75·3 (data not
given)
(data not given) 49, Lys 53, Lys In vivo (pig). Trp nd. (93)
d
Buckwheat
(var. Siva
dolenjska)
(data not given) 12·3 (DM) 79·9 (data not
given)
(data not given) 77, Leu, Lys 80, Leu In vivo (rat). (94)
Buckwheat
(var. Bednja 4n)
(data not given) 12·2 (DM) 78·8 (data not
given)
(data not given) 75, Leu, Lys 77, Leu In vivo (rat). (94)
Maize (Corn) Meal 6·9 82·4 Lys 37 41, Lys 45, Lys 2-5 yr child. In vivo
(rat). Trp nd.
(95)
e
Maize, Quality
Protein Maize,
V-537
Nixtamalized extruded flour. 10·8 (DM) 80·9 Lys 57 64, Lys 69, Ile 2-5 yr child. In vitro. (96)
Oats Ground 13·6 (DM) 85·7 (data not
given)
(data not given) 71, Lys 77, Lys In vivo (rat) and
in vitro. In vivo used
when PDCAAS
re-calculated.
(97)
f
(10·0-16·1) (83-90)
93·3 (91-95) in vitro
simulated total
tract; 90·7 (88-92)
in vitro simulated
ileal
Oats Rolled oats. Finely ground,
autoclaved 15 min at
1218C, freeze dried.
16·8 (DM) 90 Lys 66 (AA data not
given)
(AA data not
given)
NRC 1980. In vivo
(rat).
(98)
g
Pearl Millet
(cv. Dempy)
Raw flour 13·0 73·6 Lys 24 21, Met þCys 23, Met þCys,
Lys
FAO/WHO 1973.
In vitro. His and Trp
nd.
(99)
h
Pearl Millet
(cv. Dempy)
Cooked flour (flour mixed
with water and boiled
20 min. Cooked gruel
dried at 658C and
reground).
12·9 57·6 Lys 14 15, Lys, Met þ
Cys
16, Lys FAO/WHO 1973.
In vitro. His and Trp
nd.
(99)
Quinoa
(var. 40057)
Raw flour 14·1 (DM) 91·7 Phe þTyr 79 85, Val, Lys 89, Val FAO/WHO/UNU 1985,
but values slightly
different. In vivo
(rat).
(100)
i
Quinoa
(var. 40057)
Washed flour (washed
20 min with tap water,
rinsed and dried, then
milled)
14·2 (DM) 91·6 Phe þTyr 78 100, Lys 109, Lys FAO/WHO/UNU 1985,
but values slightly
different. In vivo
(rat).
(100)
Rice (parental
rice)
Hulled, raw, whole grain
ground
8·2 92·0 Lys 53 58, Lys 63, Lys 2-5 yr child. In vivo.
Ileal (pig)
(101)
j
Rice-genetically
modified
(expressing
human lactofer-
rin gene)
Hulled, raw, whole grain
ground
8·1 93·4 Trp 54 71, Trp, Lys 76, Lys 2-5 yr child. In vivo.
Ileal (pig)
(101)
Protein Quality Evaluation S197
British Journal of Nutrition

Table 7. Continued
Food Food Processing Total protein
g/100 g edible
portion,
unless
indicated.
Protein
digestibility (%)
LAA (as per
reference)
PDCAAS (%)
reported
PDCAAS (%)
re-calculated using
reference pattern
for 1-2 yr child,
and LAA
b
PDCAAS (%)
re-calculated using
reference pattern
for 3-10 yr child,
and LAA
c
Methods used
(reference pattern
and digestion)
Reference
Sorghum, cv.
Orbit (non-
tannin), white
Whole grain flour, raw 7·6 80·6 Lys 29 29 (Lys) 32 (Lys) 1-2 yr child. In vitro.
Only Lys analyzed.
(102)
k
Sorghum, cv.
Orbit (non-
tannin),
“Ugali” unfermented thick
porridge, made from
sorghum and water
2·2 64·6 (assumed
Lys)
26 26 (Lys) 28 (Lys) 1-2 yr child. In vitro.
Only Lys analyzed.
(102)
Sorghum, cv. NS
5511, red
Whole grain flour, raw 9·9 61·8 Lys 19 19 (Lys) 21 (Lys) 1-2 yr child. In vitro.
Only Lys analyzed.
(102)
Sorghum, cv. NS
5511
“Ugali” unfermented thick
porridge, made from
sorghum (cv. orbit) and
water
2·8 32·6 (assumed
Lys)
10 10 (Lys) 11 (Lys) 1-2 yr child. In vitro.
Only Lys analyzed.
(102)
Wheat Flour 11·6 89·4 Lys 40 45, Lys 48, Lys 2-5 yr child. In vivo
(rat). Trp nd.
(95)
Wheat Bread, dried and ground 12·8 89·0 (86·1) Lys 37 41, Lys 45, Lys 2-5 yr child. In vivo
and in vitro (brack-
ets). His not given.
(103)
l
Wheat
(var. Cranich)
12·3 (DM) 96·0 42, Lys 46, Lys In vivo (rat). (94)
Zein (protein) Isolated protein from maize 77·3 63·0 Lys 1 1, Lys 2, Lys 2-5 yr child. In vivo
(rat).
(8)
m
Milk and milk proteins
Milk, Full cream,
pasteurized
Pasteurized 3·03 86·4 Trp 76 113 (using values
for Trp)
127 (using values
for Trp)
2-5 yr child. In vitro. (27)
n
Milk, Full cream
UHT
UHT 3·0 87·1 Trp 82 123 (using value
for Trp)
138 (using values
for Trp)
2-5 yr child. In vitro. (27)
Milk, Full cream,
sterilized
Sterilized 3·2 87·1 Met þCys 73 70 (using value for
Met þCys)
76 (using values
for Met þCys)
2-5 yr child. In vitro. (27)
Milk, Semi-
skimmed
pasteurized
Pasteurized 3·1 86·5 Trp 76 113 (using value
for Trp)
127 (using value
for Trp)
2-5 yr child. In vitro. (27)
Milk, Semi-
skimmed UHT
UHT 3·1 87·3 Trp 91 136 (using value
for Trp)
153 (using value
for Trp)
2-5 yr child. In vitro. (27)
Milk, Semi-
skimmed steri-
lized
Sterilized 3·1 86·5 Met þCys 34 33 (using value for
Met þCys)
36 (using values
for Trp)
2-5 yr child. In vitro. (27)
Milk, Skimmed
UHT
UHT 3·2 88·2 Trp 100 148 (using value
for Trp)
166 (using value
for Trp)
2-5 yr child. In vitro. (27)
Milk, Skim Powder 29·5 (33·9) 94·0 Trp 100 115, Met þCys 124, Met þCys 2-5 yr child. Faecal
(rat).
(8)
Milk, Skim heated Powder, autoclaved at
1218C for 1 h
29·9 (33·8) 77·0 Lys 31 34, Lys 37, Lys 2-5 yr child. In vivo
(rat).
(8)
Alpha-lactalbu-
min
74·1 (85·2) 99·0 Thr 100 (166
untruncated)
Lys, 200 Lys, 217 2-5 yr child. In vivo
(rat).
(8)
Casein 81·6 93·3 His 93 99, His 111, His 2-5 yr child. In vivo
(rat). Trp nd.
(96)
Casein 80·2 (89·9) 99·0 Trp 100 (120
untruncated)
131, Met þCys 142, Met þCys 2-5 yr child. In vivo
(rat).
(8)
J. Boye et al.S198
British Journal of Nutrition

Table 7. Continued
Food Food Processing Total protein
g/100 g edible
portion,
unless
indicated.
Protein
digestibility (%)
LAA (as per
reference)
PDCAAS (%)
reported
PDCAAS (%)
re-calculated using
reference pattern
for 1-2 yr child,
and LAA
b
PDCAAS (%)
re-calculated using
reference pattern
for 3-10 yr child,
and LAA
c
Methods used
(reference pattern
and digestion)
Reference
Whey protein
concentrate
Air-dried 83·3 100·0 (97·0) (data not
given)
100 (100) (AA data not
given. Authors
have truncated
PDCAAS)
2-5 yr child. In vivo
(rat)
(9)
o
Whey protein
hydrosylate
Air dried 84·4 99·0 (98·0) (data not
given)
100 (100) (AA data not
given. Authors
have truncated
PDCAAS)
2-5 yr child. In vivo
(rat)
(9)
Egg Lyophilized powder 48·6 90·1 (His) (104) 101, Ile 101, Ile 2-5 yr child. In vivo
(rat).
(95)
Meat and Poultry
Beef Dried beef, ground. 81·8 92·4 (Val) (114) 95, Val 99, Val 2-5 yr child. In vivo
(rat). Trp nd.
(95)
Chicken Flour prepared from fresh
chicken breast meat,
dried.
77·3 95·2 (Val) (92) 109 Val 115, Val PDCAAS calculated
from reference pat-
tern given in paper.
In vivo (rat). Trp nd.
(104)
p
Fish and fish
products
Herring Smoked 19·5 98·1 Phe þTyr 109 118, Leu 121, Leu 2-5 yr child. In vitro. (105)
q
Herring fillets Salted 12·8 97·9 Trp 97 121, Val 127, Leu 2-5 yr child. In vitro. (105)
Herring fillets Marinated 15·4 97·8 Trp 93 122, Val 128, Leu 2-5 yr child. In vitro. (105)
Herring Smoked 19·5 98·1 Phe þTyr 109 118, Leu 121, Leu 2-5 yr child. In vitro. (105)
Mackerel Smoked 19·7 98·5 Trp 94 100, Leu 103, Leu 2-5 yr child. In vitro. (105)
Mackerel Canned in oil 13·7 93·5 Phe þTyr 139 148, Leu 153, Leu 2-5 yr child. In vitro. (105)
Mackerel Fried, in vinegar 15·8 97·0 Trp 96 123, Leu 127, Leu 2-5 yr child. In vitro. (105)
Sprats Canned in oil 13·2 93·0 Met þCys(?) 127 (?) 122, Met þCys 133, Met þCys 2-5 yr child. In vitro. (105)
Sprats Smoked 22·0 97·7 Trp 76 105, Leu 108, Leu 2-5 yr child. In vitro. (105)
Sardine Canned in oil 16·7 95·0 Phe þTyr 124 131, Leu 136, Leu 2-5 yr child. In vitro. (105)
Tuna Canned in oil 15·7 93·7 Phe þTyr 124 135, Leu 139, Leu 2-5 yr child. In vitro. (105)
Vegetables
Kale Cooked, freeze-dried 17·9 (DM) 77·3 Lys 81 90, Lys 98, Lys 2-5 yr child. In vivo
(rat).
(106)
r
Potato (organic
cultivation,
2002)
Cooked, freeze-dried 10·7 (DM) 102·0 Leu 67 70, Leu 73, Leu 2-5 yr child. In vivo
(rat).
(106)
Potato (conven-
tional cultiva-
tion, 2002)
Cooked, freeze-dried 7·8 (DM) 101·8 Leu 81 85, Leu 88 2-5 yr child. In vivo
(rat).
(106)
Tree nuts
Almond (Prunus
dulcis L.), var.
Carmel
Raw, whole almond flour. 20·6 88·6 Met þCys 23 22, Met þCys 24, Met þCys FAO/WHO 2-5 yr.
In vivo (rat)
(107)
s
Almond (Prunus
dulcis L.), var.
Mission
Raw, whole almond flour. 23·3 92·3 Met þCys 24 23, Met þCys 25, Met þCys FAO/WHO 2-5 yr.
In vivo (rat)
(107)
Almond (Prunus
dulcis L.), var.
Nonpareil
Raw, whole Almond flour. 21·0 82·6 Met þCys 22 21, Met þCys 23, Met þCys FAO/WHO 2-5 yr.
In vivo (rat)
(107)
Protein Quality Evaluation S199
British Journal of Nutrition

Table 7. Continued
Food Food Processing Total protein
g/100 g edible
portion,
unless
indicated.
Protein
digestibility (%)
LAA (as per
reference)
PDCAAS (%)
reported
PDCAAS (%)
re-calculated using
reference pattern
for 1-2 yr child,
and LAA
b
PDCAAS (%)
re-calculated using
reference pattern
for 3-10 yr child,
and LAA
c
Methods used
(reference pattern
and digestion)
Reference
Baru almond
(Dipteryx alata
Vog.)
Roasted in electric oven for
30 min at 1408C and then
ground.
25·8 79·4 Met þCys;
Lys (1 tree)
73 (66-82) 67 (61-75), Met þ
Cys; Lys
(1 tree)
73 (66-82), Met þ
Cys; Lys
(1 tree)
WHO/FAO/UNU 2007
pattern, 4-18 yrs.
In vivo (rat).
(108)
t
(23·8-28·1) (66-82)
Tropical almond
(Terminalia
catappa)
Defatted seed flour 55·9 92·0 Tyr 31 (AAS mis-
calculated.
PDCAAS
should have
been 76)
60 Lys, Val 63, Val 2-5 yr child. in vivo
(rat)
(109)
u
a
Where authors did not give PDCAAS, it was calculated using amino acid score (AAS) of limiting amino acid and digestibility values given. Abbreviations: cv. cultivar; DM: dry matter; LAA: Limiting amino acid; nd: not determined;
NRC: National research council. PDCAAS: Protein digestibility corrected amino acid score. Further details on the specific method used for the in vivo digestibility measurement (i.e., true vs apparent, faecal vs ileal) can be found
in the references provided.
b
PDCAAS re-calculated using LAA and reference pattern for 1-2yr child
(6)
. Neither the AAS nor PDCAAS were truncated. A few studies had digestibility values for individual amino acids as well as for protein. In such cases, the pro-
tein digestibility value was used. Trp and His were not determined (nd) in some studies. In all the in vivo digestibility studies with the exception of Ahrens et al. 2005 (where rats were fed raw whole almond flour), the diet fed to
animals was not the individual food item but included corn starch, sucrose, oil, vitamins, minerals, cellulose etc.
c
PDCAAS re-calculated using LAA and reference pattern for 1-2yr child
(6)
as per reference, pg 181; “Thus for children aged over 2 years and adolescents, given the minor contribution that growth makes to the requirement for
these age groups, the scoring pattern differs from that of adults to only a minor extent. For this reason, when judging protein quality for schoolchildren and adolescents, it is probably more practical to use just one pattern, i.e. that
derived for the age group 3– 10 years.”
d
Diets were fed as grain mono-diets with mineral, vitamin supplementation and some amino acids, to meet requirements.
e
The authors include digestibility and PDCAAS data for unusual products such as frog meat. Trp has apparently not been determined in the AA analyses. Diets included soyabean oil, corn starch, dextrinised corn starch cellulose,
sucrose, vitamins and minerals. The article is in Portuguese.
f
Oat cultivars used were Adamo, Sang, Svea, Vital, Freja and Sanna. Diets contained the oat samples as the main ingredient and were kept constant in N-concentration by adjusting with the N-free mixture to obtain 1.5 % N in DM.
g
Reference pattern used to calculate PDCAAS was National Research Council (1980) Recommended Dietary Allowances. National Academy of Sciences, Washington, DC.
h
The authors note the reduction in protein digestibility of pearl millet on wet cooking.
i
Diets contained sucrose, maize starch, cellulose, maize oil, minerals and choline chloride.
j
Amino acids not given per total protein, so calculated using other data in paper, from AAS and scoring pattern. Diet included soyabean oil, vitamins, minerals and dichromium trioxide.
k
The tannin-free sorghum variety (both raw and cooked) had better digestibility and higher PDCAAS when compared with the tannin containing variety. For both sorghum cultivars the digestibility of the raw flour was higher than
that of the cooked products: the digestibility of sorghum proteins are reported to decrease on wet cooking due to exogenous factors (grain organisational structure, polyphenols, phytic acid, starch and non-starch polysaccharides)
and endogenous factors (disulphide and non-disulphide crosslinking, kafirin hydrophobicity and changes in protein secondary structure)
(25,105)
. Only Lys analyzed.
l
Diets included corn oil, starch, vitamins and minerals.
m
Diets contained sucrose and corn starch. The protein content in all diets was adjusted to ,8 g/100 g. Total protein per item was calculated from diet data. As data (g/100 g protein) were not given, values were calculated using
data in Table 5 of reference, assuming reference pattern (WHO/FAO 1992) used was Lys 58, Met þCys 25, Thr 34 and Trp 11 mg/g protein. Values for protein content in parenthesis were obtained from Gilani and Sepehr,
2003. Although zein has a digestibility of 63, the PDCAAS is just 1 because of its extremely low AAS (limiting AA Lys, AAS: 2).
n
Skimmed UHT has highest PDCAAS. Sterilization leads to reduced protein quality, with sterilized semi-skimmed milk having a PDCAAS of 34 c.f. 76 for semi-skimmed pasteurized milk, due to significant reductions in Met (44%)
and Cys (49 %). Lys also decreased by 18-23 %. An increase in AA content and digestibility in skimmed milk c.f. full cream and semi-skimmed milk is reported, which the authors say may be due to more effective proteolytic
action in the in vitro method (due to elimination of heterogeneity in the various (fat-water) states. As amino acid data were not given per total protein, AAS and scoring pattern for limiting AA were used in order to obtain them.
o
Digestibility and PDCAAS for young 5-wk-old rats given with data for 20-mo-old rats in parenthesis. Authors have truncated PDCAAS. It was not possible to recalculate the PDCAAS as individual AA data were not given. Diets
contained sucrose, corn starch, lard, soyabean oil, vitamins and minerals.
p
Diets included sucrose, corn oil, cellulose, corn starch, minerals and vitamins. Trp not determined. Reference pattern given was cited to be WHO/FAO/UNU 1985, but values given are incorrect.
q
PDCAAS for more fish items are available in reference, only a selection is shown in table above. AA data (g/100 g protein) not given for His.
r
Data are for diets containing sucrose, maize starch, cellulose, soyabean oil, minerals and vitamins. The protein content in all diets was adjusted to 15 g N per kilogram of dry matter. The potato diets also included 5.1% casein in
order to obtain the required N content and increase palatability
(109)
: vegetables and fruit grown and harvested in two different years, using 3 different cultivation methods. A clear significant effect of the cultivation method was not
generally seen (when both cultivation years were considered) with the exception of potato, where the conventional cultivation method gave a higher PDCAAS. Growing year influenced the protein quality more than the cultivation
system.
s
Test diet was raw, whole almond flour. Tannins (expressed as catechin equivalents) were present at 0.12-0.18 g/100g, while trypsin inhibitory activity and hemagglutinating activity was not detected. The AAS score was only 26 %
for all three varieties, leading to a PDCAAS of 22-24: although almond proteins are highly digestible, the protein is of poor quality due to low AAS.
t
Diets included cellulose, vitamins and minerals, choline bitartrate, corn starch and, for peanut diet, soyabean oil. Nuts were from 6 different sub-populations of almond trees in the south-eastern region of Goias State (Brazil).
Digestibility for individual samples is not given. Protein content varied by 6g/100 g and PDCAAS varied from 66-82% for trees from different sub-populations in the same region. The AAS (%) varied from 83-103, with the limiting
AA being Met þCys for five trees but Lys for the sixth tree. The PDCAAS for peanut and almond from this study are comparable, because the AAS of peanut was only 76 % (c.f. average score of 92%) for Baru almond, even
though the digestibility of peanut was much higher.
u
Diet included corn starch, dextrinised corn starch, sucrose, soyabean oil, vitamins and minerals, L-Cys, choline bitartrate and butylated hydroxytoluene.
J. Boye et al.S200
British Journal of Nutrition

Table 8. Protein digestibility-corrected amino acid score (PDCAAS) for some legumes
v
Food Food Processing
Total protein
g/100 g
edible por-
tion, unless
indicated.
Protein
digestibility
(%)
LAA (as per
reference)
PDCAAS
(%) reported
PDCAAS (%) re-
calculated using
reference pattern
for 1-2 yr child,
and LAA
w
PDCAAS (%) re-
calculated using
reference pattern
for 3-10 yr child,
and LAA
x
Methods used
(reference
pattern and
digestion) Reference
Beans var. Pe
´rola Flour of boiled, oven-dried,
milled beans.
20·3 78·7 Met þCys 63 60, Met þCys 65, Met þCys 2-5 yr child.
In vivo (rat).
Trp nd.
(95)
Black beans,
common, var.
Talamanca
Soaked 12 h, then cooked in
water for 45 min, drained
and lyophilized.
28·1 80·9 Met þCys,
Trp
38 61, Met þCys 67, Met þCys FAO/WHO/UNU
1985, Infant
requirements.
In vivo (rat)
(17, 110)
y
Black beans,
common, var.
Talamanca
Cooked fermented beans:
ground bean grits soaked
16 h, cooked 1008C, 22 min;
inoculated with Rhzopus
oligosporus and fermented
25 h, 378C, homogenized
and lyophilized.
28·7 80·3 Met þCys,
Trp
38 61, Met þCys 66, Met þCys FAO/WHO/UNU
1985, Infant
requirements.
In vivo (rat)
(17, 110)
Black beans,
common, var.
Talamanca
Cooked germinated beans:
germinated for 72 h at 308C
at 100 % relative humidity.
Germinated bean paste
mixed with cooked beans
and water (1:1:1·5).
Lyophilized.
29·2 86·8 Met þCys,
Trp
39 62, Met þCys 68, Met þCys FAO/WHO/UNU
1985, Infant
requirements.
In vivo (rat)
(17, 110)
Black beans Raw, ground 18·9 (21·5) 71·0 Met þCys 72 69, Met þCys 75, Met þCys 2-5 yr child.
In vivo (rat).
(8)
Black beans Soaked when raw, then auto-
claved, dried and ground.
19·6 (22·7) 83·0 Trp 84 81, Met þCys 88, Met þCys 2-5 yr child.
In vivo (rat).
(8)
Chickpea Extruded flour. (23, DM) 82·6 Met þCys 69 66, Met þCys,
Val
72 (Met þCys) 2-5 yr child.
In vitro.
(97)
Chickpea Defatted flour from seeds
soaked, decorticated, and
dried.
25·0 78·4 Trp 44 59, Met þCys 64, Met þCys 2-5 yr child.
In vivo (rat).
(14)
z
Cowpea, var.
Bechuana white
Whole grain flour, raw 21·2 91·3 80 (not
given)
80 (not given) 87 (Lys) 1-2 yr child.
In vitro. Only
Lys analyzed.
(102)
Cowpea, Canadian Soaked, 22 h. Dried overnight
at 558C and ground.
26·3 87·5 Met þCys 35 46 Met þCys 50 Met þCys FAO/WHO
1973. In vitro.
(15)
Cowpea, Canadian Microwaved 15 mins, with
water. Dried overnight at
558C and ground.
26·3 92·8 Met þCys 65 87 Met þCys 95 Met þCys FAO/WHO
1973. In vitro.
(15)
Cowpea, Egyptian,
var. Asmerly
Roasted for 15 min at 1808C,
in sand bath. Dried overnight
at 558C and ground.
26·3 76·6 Met þCys 43 58 Met þCys 63 Met þCys FAO/WHO
1973. In vitro.
(15)
Cowpea, Egyptian,
var. Asmerly
Autoclaved, with water. Dried
overnight at 558Cand
ground.
26·3 89·7 Met þCys 72 97 Met þCys 105 Met þCys FAO/WHO
1973. In vitro.
(15)
Fava bean Autoclaved 27·2 82·0 (77·0) (not given) 73 (69) (AA data not
given.)
(AA data not
given.)
2-5 yr child.
In vivo (5-wk-
old rats)
(9)
Protein Quality Evaluation S201
British Journal of Nutrition

Table 8. Continued
Food Food Processing Total protein
g/100 g
edible por-
tion, unless
indicated.
Protein
digestibility
(%)
LAA (as per
reference)
PDCAAS
(%) reported
PDCAAS (%) re-
calculated using
reference pattern
for 1-2 yr child,
and LAA
w
PDCAAS (%) re-
calculated using
reference pattern
for 3-10 yr child,
and LAA
x
Methods used
(reference
pattern and
digestion)
Reference
Fava bean (cv.
Diana)
Soaked 18 h; drained; auto-
claved 10 min at 1218C;
cooled and freeze dried.
Finely ground.
31·5 (“moist-
ure free”
basis)
86·0 Trp 55 (AA data not
given)
(AA data not
given)
NRC 1980.
In vivo (rat).
(98)
aa
Kidney bean, red,
Canadian
Raw 24·9 70·5 Met þCys 28 37 Met þCys 40 Met þCys FAO/WHO
1973. In vitro.
(15)
Kidney bean, red,
Canadian
Cooked in microwave oven for
15 min, with water.
24·9 81·7 Met þCys 55 74 Met þCys 81 Met þCys FAO/WHO
1973. In vitro.
(15)
Kidney bean,
white, var. Giza
3 (Egyptian)
Roasted for 20 min at 1808C,
in sand bath. Dried overnight
at 558C and ground.
28·1 73·0 Met þCys 32 47 Met þCys 51 Met þCys FAO/WHO
1973. In vitro.
(15)
Kidney bean,
white, var. Giza
3 (Egyptian)
Fermented for 24 h with dry
active yeast. Dried overnight
at 558C and ground.
28·1 80·9 Met þCys 60 81 Met þCys 88 Met þCys FAO/WHO
1973. In vitro.
(15)
Lentil (Lens culi-
naris, cv. Medik)
Soaked 18 h; drained; auto-
claved 10 min at 1218C;
cooled and freeze dried.
Finely ground.
24·8 (“moist-
ure free”
basis)
85·0 Trp 52 (AA data not
given)
NRC 1980.
In vivo (rat).
(98)
Pea (Organic culti-
vation, 2002)
Cooked (and freeze-dried) 25·9 (DM) 90·3 Trp 75 82 Met þCys 2-5 yr child.
in vivo (rat).
(106)
Pea, Canadian Soaked 18 h. Dried overnight
at 558C and ground.
21·6 83·7 Met þCys 33 44 Met þCys 48 Met þCys FAO/WHO
1973. In vitro.
(15)
Pea, Canadian Cooked in microwave oven for
15 min, with water. Dried
overnight at 558Cand
ground.
21·6 89·1 Met þCys 62 84 Met þCys 91 Met þCys FAO/WHO
1973. In vitro.
(15)
Pea, var.
Nebraska,
(Egyptian)
Roasted at 15 min at 1808C, in
sand bath.
23·1 75·0 Met þCys 33 44 Met þCys 48 Met þCys FAO/WHO
1973. In vitro.
(15)
Pea, var.
Nebraska,
(Egyptian)
Cooked in microwave oven for
15 min, with water. Dried
overnight at 558Cand
ground.
23·1 90·9 Met þCys 72 92 Ile 92 Ile FAO/WHO
1973. In vitro.
(15)
Peanut Roasted in electric oven for
30 min at 1408C and then
ground.
32·6 91·8 Lys 70 65, Lys 70, Lys WHO/FAO/UNU
2007 pattern,
4-18 yrs.
In vivo (rat).
(108)
Peanut (Arachis
hypogaea L.) cv.
Ranferi Diaz
Defatted flour 26·6 87·1 Thr 30 41, Thr 45, Thr 2-5 yr child.
In vitro.
(111)
bb
Peanut (Arachis
hypogaea L.) cv.
VA-81-B
Defatted flour 24·8 84·0 Thr 50 66, Thr 71, Thr 2-5 yr child.
In vitro.
(111)
Peanut (Arachis
hypogaea L.) cv.
Florunner
Defatted flour 24·9 87·2 Thr 30 32, Thr 34, Thr 2-5 yr child.
In vitro.
(111)
J. Boye et al.S202
British Journal of Nutrition

Table 8. Continued
Food Food Processing Total protein
g/100 g
edible por-
tion, unless
indicated.
Protein
digestibility
(%)
LAA (as per
reference)
PDCAAS
(%) reported
PDCAAS (%) re-
calculated using
reference pattern
for 1-2 yr child,
and LAA
w
PDCAAS (%) re-
calculated using
reference pattern
for 3-10 yr child,
and LAA
x
Methods used
(reference
pattern and
digestion)
Reference
Soyabean
(conventional)
Dry heat 898C, 5 min. Beans
processed to flour.
41·9 71·8 Met þCys 54 52, Met þCys 56, Met þCys 2-5 yr child.
In vivo (rat).
Trp nd.
(95)
Soyabean (kunitz
trypsin inhibitor
and lipoxygen-
ase free)
Dry heat 898C, 5 min. Beans
processed to flour.
40·0 74·3 Met þCys 64 61, Met þCys 66, Met þCys 2-5 yr child.
In vivo (rat).
Trp nd.
(95)
Soyabean Meal, raw 36·8 (41·9) 80·0 Trp 80 88, Met þCys 96, Met þCys 2-5 yr child.
In vivo (rat).
(8)
Soyabean Meal, autoclaved 37·5 (41·9) 83·0 Lys 83 89, Met þCys 97, Met þCys 2-5 yr child.
In vivo (rat).
(8)
Soyabean Protein isolate 75·6 (81·9) 96·0 Lys, Met þ
Cys
100 (101
untrunca-
ted)
97, Met þCys 105, Met þCys 2-5 yr child.
In vivo (rat).
(8)
Soyabean Textured soyabean protein.
Ground to obtain meal.
53·3 86·4 Met þCys 65 62, Met þCys 67, Met þCys 2-5 yr child.
In vivo (rat).
Trp nd.
(95)
v
Where authors did not give PDCAAS, it was calculated using AAS of limiting amino acid and digestibility values given. Abbreviations: cv. cultivar; DM: dry matter; LAA: Limiting amino acid; nd: not determined; NRC: National
research council. PDCAAS: Protein digestibility corrected amino acid score. Further details on the specific method used for the in vivo digestibility measurement (i.e., true vs apparent, faecal vs ileal) can be found in the
references provided.
w
PDCAAS re-calculated using LAA and reference pattern for 1-2yr child. Neither the AAS nor PDCAAS were truncated. A few studies had digestibility values for individual amino acids as well as for protein. In such cases, the
protein digestibility value was used. Trp and His were not determined in some studies. In all the in vivo digestibility studies with the exception of Ahrens et al.
(110)
(where rats were fed raw, whole, almond flour), the diet fed to
animals were not the individual food item but included corn starch, sucrose, oil, vitamins, minerals, cellulose etc.
x
PDCAAS re-calculated using LAA and reference pattern for 1-2yr child
(6)
.
y
Diets included cellulose, corn oil, corn starch, choline bitartrate, vitamins and minerals.
z
Diets included sucrose, fat, vitamins and minerals, fibre, choline bitartrate and corn starch.
aa
Diets contained corn starch, corn oil, cellulose, minerals and vitamins.
bb
Data available for six peanut cultivars. The AAS varied from 30-60 % among the cultivars, digestibility 84-87 %, leading to large variations in PDCAAS, 30-50. Only data for Lys and Thr were given. Therefore, in recalculating the
PDCAAS, it was not possible to check if the limiting AA (and PDCAAS) had changed.
Protein Quality Evaluation S203
British Journal of Nutrition

Table 9. Protein digestibility-corrected amino acid score (PDCAAS) of some composite products
a
Food Food Processing
Total
protein
g/100 g
edible
portion,
unless
indicated.
Protein
digestibility
(%)
LAA (as per
reference)
PDCAAS
(%)
reported
b
PDCAAS (%)
re-calculated
using reference
pattern for 1-2
yr child, and
LAA
c
PDCAAS (%)
re-calculated
using reference
pattern for 3-10
yr child, and
LAA
d
Methods used
(reference pattern
and digestion) Reference
Herring in tomato sauce canned 12·0 90·6 Phe þTyr 105 110, Leu 114, Leu 2-5 yr child. In vitro.
His AA data not
given per 100 g
protein.
(105)
Mackerel in tomato sauce canned 12·7 92·2 Phe þTyr 95 100, Leu 103, Leu 2-5 yr child. In vitro.
His AA data not
given per 100 g
protein.
(105)
Sprats in tomato sauce canned 11·5 91·2 Met þCys 97 94, Met þCys 101, Met þ
Cys
2-5 yr child. In vitro.
His AA data not
given per 100 g
protein.
(105)
Sorghum (cv. orbit) 1
cowpea (70:30 w/w), raw
Flour, raw 11·9 (13·2
DM)
85·8 (assumed
Lys)
57 57 62 (Lys) 1-2 yr child. In vitro.
Only Lys ana-
lyzed.
(102)
e
Sorghum (cv. NS 5511) 1
cowpea (70:30 w/w), raw
Flour, raw 13·4 (14·9
DM)
76·0 (assumed
Lys)
50 50 54 (Lys) 1-2 yr child. In vitro.
Only Lys ana-
lyzed.
(102)
“Ugali” unfermented thick
porridge (African), made
from sorghum (cv. orbit)
1chickpea flour and
water
cooked 3·3 (13·2
DM)
72·2 (data not
given)
48 48 52 (Lys) 1-2 yr child. In vitro.
Only Lys ana-
lyzed.
(102)
“Ugali” unfermented thick
porridge (African), made
from sorghum (cv. NS
5511) 1chickpea flour
and water
cooked 3·7 (14·8
DM)
56·7 (data not
given)
37 37 40 (Lys) 1-2 yr child. In vitro.
Only Lys ana-
lyzed.
(102)
“Uji” fermented thin por-
ridge (African), made from
sorghum (cv. orbit) 1
chickpea flour and water
Cooked 1·6 (13·3
DM)
77·0 (data not
given)
44 44 48 (Lys) 1-2 yr child. In vitro.
Only Lys ana-
lyzed.
(102)
“Uji” fermented thin por-
ridge (African), made from
sorghum (cv. NS 5511) 1
chickpea flour and water
Cooked 1·8 (15·1
DM)
61·0 (Assumed
Lys)
35 35 38 (Lys) 1-2 yr child. In vitro.
Only Lys ana-
lyzed.
(102)
“Injera” fermented flat bread
(African), made from sor-
ghum (cv. orbit) 1chick-
pea flour, dried baker’s
yeast and sugar.
Cooked, then freeze dried.
Protein content in yeast
was 38 g/100 g and Lys
8 g/100 g protein.
5·8 (14·2
DM)
79·8 (Assumed
Lys
60 60, assumed
Lys
65 (Lys) 1-2 yr child. In vitro.
Only Lys ana-
lyzed.
(102)
“Injera” fermented flat bread
(African), made from sor-
ghum (cv. NS 5511) 1
chickpea flour, dried
baker’s yeast and sugar.
Cooked, then freeze dried.
Protein content in yeast
was 38 g/100 g and Lys
8 g/100 g protein.
6·4 (15·6
DM)
62·5 (Assumed
Lys
41 41, assumed
Lys
44 (Lys) 1-2 yr child. In vitro.
Only Lys ana-
lyzed.
(102)
J. Boye et al.S204
British Journal of Nutrition

Table 9. Continued
Food Food Processing Total
protein
g/100 g
edible
portion,
unless
indicated.
Protein
digestibility
(%)
LAA (as per
reference)
PDCAAS
(%)
reported
b
PDCAAS (%)
re-calculated
using reference
pattern for 1-2
yr child, and
LAA
c
PDCAAS (%)
re-calculated
using reference
pattern for 3-10
yr child, and
LAA
d
Methods used
(reference pattern
and digestion)
Reference
Mixed diet of pea (43 %),
potato (30 %), rapeseed oil
(13 %), carrot (5 %) apple
(1 %), kale (1 %) and
DL-Met, vitamins and
minerals.
Potatoes, peas and kale
cooked.
16·1 (DM) 73·0 Trp (lowest
PDCAAS)
64 (Amino acid
composition
of mixture
not given)
2-5 yr child. In vivo
(rat).
(106)
f
(72-74) (63-64)
Chickpea (79 %) and maize
(21 %)
Extruded chickpea flour,
var. Blanco Sinaloa 92;
nixtamlized extruded
maize flour, quality pro-
tein maize V-537. Mix-
ture containing sucrose
and water was heated
908C for 8 min.
20·1 (DM) 84·5 Trp (lowest
PDCAAS)
78 76, Val 80, Val 2-5 yr child. In vitro. (96)
Cooking banana fruit and
bambara (groundnut)
seeds (70:30)
Banana, oven dried (608C,
24 h) flour. Groundnut
seeds roasted in hot
sand at 1808C, flour.
(not
given)
45·0 (Val) (9·3) 9, Val 10, Val PDCAAS calculated
using 1-2 yr pat-
tern and AA data
given by authors.
In vivo (rat). Trp
nd.
(112)
Cooking banana fruit and
bambara (groundnut)
seeds (60:40)
Banana, oven dried (608C,
24 h) flour. Groundnut
seeds roasted in hot
sand at 1808C, flour.
(not
given)
58·1 (Val) (16) 16, Val 17, Val In vivo (rat). Trp nd.
PDCAAS calcu-
lated by using AA
data given by
authors and 1-2 yr
pattern.
(112)
Table bread fortified with
8 % defatted soyabean
meal
Bread dried at 508Cfor
24 h, then ground
15·8 87·4 (84·9) Lys 47 53, Lys 57, Lys 2-5 yr child. In vivo
and in vitro
(brackets). His not
given.
(103)
g
Table bread fortified with
12 % defatted soyabean
meal
Bread dried at 508Cfor
24 h, then ground
17·3 86·4 (84·5) Lys 53 59, Lys 64, Lys 2-5 yr child. In vivo
and in vitro
(brackets). His not
given.
(103)
Table bread fortified with
8 % defatted soyabean
meal and 4 % defatted
sesame meal
Bread dried at 508Cfor
24 h, then ground
17·6 85·8 (83·2) Lys 46 51, Lys 56, Lys 2-5 yr child. In vivo
and in vitro
(brackets). His not
given.
(103)
Bean and rice, 53 % bean
protein, 47 % rice protein
Cooked bean and cooked
rice. Lyophilized.
20·8 84·3 Ile and Trp 47 73, Ile 73, Ile FAO/WHO/UNU
1985, Infant
requirements.
In vivo (rat).
(17, 110)
h
Protein Quality Evaluation S205
British Journal of Nutrition

Table 9. Continued
Food Food Processing Total
protein
g/100 g
edible
portion,
unless
indicated.
Protein
digestibility
(%)
LAA (as per
reference)
PDCAAS
(%)
reported
b
PDCAAS (%)
re-calculated
using reference
pattern for 1-2
yr child, and
LAA
c
PDCAAS (%)
re-calculated
using reference
pattern for 3-10
yr child, and
LAA
d
Methods used
(reference pattern
and digestion)
Reference
Bean and rice, 53 % bean
protein, 47 % rice protein
Cooked, fermented bean
and cooked rice.
Lyophilized.
19·8 84·6 Ile and Trp 47 73, Ile 73, Ile FAO/WHO/UNU
1985, Infant
requirements.
In vivo (rat)
(17, 110)
Bean and rice, 53 % bean
protein, 47 % rice protein
Cooked, germinated bean
and cooked rice. Lyophi-
lized.
19·6 91·0 Ile and Trp 51 81, Ile 81, Ile FAO/WHO/UNU
1985, Infant
requirements.
In vivo (rat)
(17, 110)
Pearl millet (NW 305, blue
seeds), mung bean (Vigna
radiata L.) and nonfat dry
milk (60:30:10)
Flour of decorticated mung
bean splits toasted at
908C, 1 h. Mung bean
flour, milled millet and
milk extruded.
19·0 82·8 (85·6) Lys 68 74, Lys 80, Lys 2-5 yr child. In vivo
and in vitro
(brackets).
PDCAAS recalcu-
lated using in vivo
digestibility. Trp
not given.
(113)
Finger millet (Indaf, brick
red seeds), mung bean
(Vigna radiata L.) flour
and nonfat dry milk
(60:30:10)
Flour of decorticated mung
bean splits toasted at
908C, 1 h. Mung bean
flour, milled millet and
milk extruded
13·1 79·6 (72·1) Lys 69 69, Met þCys 75, Met þCys 2-5 yr child. In vivo
and in vitro
(brackets).
PDCAAS recalcu-
lated using in vivo
digestibility. Trp
not given.
(113)
Sorghum (SPV 475, white
seeds), mung bean (Vigna
radiata L.) flour and nonfat
dry milk (60:30:10)
Flour of decorticated mung
bean splits toasted at
908C, 1 h. Mung bean
flour, milled millet and
milk extruded
17·0 72·9 Lys 57 61, Met þCys 66, Met þCys 2-5 yr child. In vitro.
Trp not given.
(113)
a
In all reports, digestibility studies were carried out on actual mixture. With the possible exception of Jorgensen et al. 2008, all studies obtained the AAS from amino acid analysis done on actual mixture rather than calculating from
mixture ratio of individual ingredients. Abbreviations: cv. cultivar; DM: dry matter; LAA: Limiting amino acid; nd: not determined; NRC: National research council. PDCAAS: Protein digestibility corrected amino acid score. Further
details on the specific method used for the in vivo digestibility measurement (i.e., true vs apparent, faecal vs ileal) can be found in the references provided.
b
Where authors did not give PDCAAS, it was calculated using AAS of limiting amino acid and digestibility values given.
c
PDCAAS re-calculated using LAA and reference pattern for 1-2yr child
6
. Neither the AAS nor PDCAAS were truncated. A few studies had digestibility values for individual amino acids as well as for protein. In such cases, the
protein digestibility value was used. Trp and His were not determined in some studies. In all the in vivo digestibility studies the diet fed to animals were not the individual food item but included corn starch, sucrose, oil, vitamins,
minerals, cellulose etc.
d
PDCAAS re-calculated using LAA and reference pattern for 1-2yr child
6
.
e
Values for total protein content were provided by authors, personal communication. Addition of protein-rich cowpea was shown to improve both the digestibility and the PDCAAS of sorghum foods (between 2- and 3-fold higher),
ascribed to the increase in Lys content and improved protein digestibility.
f
The authors measured the digestibility in adult rats of a mixed diet and compared the PDCAAS thus obtained with the predicted PDCAAS from the parameters determined using young rats on single ingredients, according to FAO/-
WHO/UNU 1992. It is not stated if the amino acid analysis was carried out again for the mixture (mixed diet), or was calculated from the composition previously experimentally determined for the individual ingredients, as per
FAO/WHO/UNU 1992. The predicted digestibility of the mixed diets was at least 5% higher than the determined digestibility. The authors concluded that the use of young rats may result in an overestimation of the protein digest-
ibility and quality for adult animals.
g
Breads also contained corn oil, starch, vitamins and minerals. Unlike the PDCAAS values shown above, authors found that weight gain, in vivo PER and in vitro PER in “table bread fortified with 8 % defatted soyabean meal and
4 % defatted sesame meal” were only slightly lower than in “table bread fortified with 12% defatted soyabean meal”, which showed the highest values for all these factors.
h
Diets included cellulose, corn oil, corn starch, vitamins, minerals and choline bitartrate
(110)
.
J. Boye et al.S206
British Journal of Nutrition

used three in vitro methods (pH drop, pH stat and pepsin
digestibility) and two in vivo methods (true and apparent
faecal digestibility in rats) to compare the protein digestibility
of casein, soya protein isolate, fish meal, black beans, corn
meal and wheat flour. All methods were in agreement for
highly digestible proteins but less so for proteins with digestibil-
ities below 85 %. They recommended that for non-conventional
proteins or for known proteins subjected to processing, protein
digestibility should be measured in vivo. Further studies are
needed to ascertain the conditions under which in vitro digest-
ibility methods can estimate apparent and/or true protein
digestibility. Furthermore, the FAO/WHO 1981 recommended
that amino acid scores be adjusted for “true” protein digestibil-
ity, thus the relevance of using “apparent” protein digestibility
(or equivalents) should be considered. Similarly, as some
studies have shown that apparent digestibility varies with the
level of protein intake, some consideration should be given as
to how in vitro digestibility methods estimating apparent digest-
ibility are affected by protein concentration. In addition, some
emerging research suggests that the rate of protein digestion
may also be of importance in protein quality
(7)
.
Protein vs. amino acid digestibility
Digestibility and bioavailability of AA influence protein quality.
There are concerns that protein digestibility measurements
may not provide accurate estimation of the digestibility of
specific IAA. Thus, research is needed to determine to what
extent protein digestibility as a whole (in vivo or in vitro)
reflects the digestibility of specific IAA and how this may be
affected by processing, matrix effects and other biotic and
abiotic factors. Further research is also needed on the impact
of processing on the bioavailability of specific essential AA.
Impact of processing and anti-nutritional components on
protein quality
Several workers have reported that the presence of anti-
nutritional components in some protein sources can influence
their digestibility. Gilani and Sepehr
(9)
concluded from protein
digestibility studies using young and old rats that the use of
young rats may overestimate protein quality for the elderly
for proteins containing antinutritional factors. The magnitude
of the effect varied by protein type as well by processing treat-
ment when the same protein source was subjected to different
treatments, which suggests both protein and processing
effects. Interestingly, research is also emerging on potential
health benefits of some of these “bioactive” compounds
which raises questions about whether their consumption to
some extent may be beneficial.
Formation of tannin-protein complexes, as an example,
have shown antioxidant properties acting as potent radical
cation scavengers which could make them radical sinks in
the gastrointestinal tract
(64)
. Tannins also have antimicrobial
properties. Various mechanisms for the tannin antimicrobial
activity have been suggested, including inhibition of extra-
cellular microbial enzymes, deprivation of substrates required
for microbial growth, or direct action on microbial metabolism
through the inhibition of oxidative phosphorylation
(26)
.
In vivo and in vitro experiments have demonstrated marked
anticancer (preventive as well as therapeutic) effects of inosi-
tol hexaphosphate (IP
6
, phytic acid)
(65)
.IP
6
reduced cell pro-
liferation and increased differentiation of malignant cells
resulting sometimes in reversion to the normal phenotype.
Phytic acid may also be beneficial by reducing the bioavail-
ability of toxic heavy metals such as cadmium and lead, and
reducing excessive oxidation activity of iron and copper
through chelation
(66)
. Kunitz trypsin inhibitors and Bowman-
Birk inhibitors isolated from legumes have been shown to
function as therapeutic agents against digestive system
cancer and in ulceratitis prevention, and may contain anti-
inflammatory activity and anti-viral activity
(67)
.
As it is still unknown at what concentrations these intakes
may be beneficial and the specific mechanisms at play, further
research specifically regarding how the presence of these so-
called antinutritional compounds are affected by processing
and the impact of both factors on protein quality will be
useful.
Conclusion
Significant progress has been made in the last half century in
defining protein quality and establishing appropriate levels of
intake to support growth and maintain health. As results from
new research emerge, recommendations may need to be
updated or revised to maintain relevance. Changes in lifestyle,
energy expenditure and new challenges in populations with
high disease burdens require constant surveillance. To keep
guidelines and legislations relevant, new scientific data will
be required to support policy and inform expert recommen-
dations at the global level. Some studies, for example, suggest
that calorie intake and frequency of protein consumption
influences nitrogen retention and should be considered in
protein quality evaluation. Several studies have also shown
that over and above that of basic nutrition, additional health
benefits may be provided by specific amino acids and bio-
active peptides
(7,68 – 81)
. On the other hand, disease risks
associated with over consumption of protein and potential
differences in effects of different proteins in stimulating dis-
ease is poorly understood and requires further research
(82)
.
Additionally, an important issue that will require consideration
is whether the recommended protein intakes for public health
purposes are based on assumptions of a particular protein
quality of the diet. This is of interest as dietary protein quality
can vary depending on social, economic and other geographic
factors. Further discussions are also needed on the practical
interpretation and use of the term ‘safe’ or ‘upper level’ to
describe recommended protein intakes. For example, the
2007 WHO/FAO/UNU Report
(6)
notes that “the term ‘safe
intake’ also includes the concept that there is no risk to indi-
viduals from excess protein intakes up to levels considerably
above the safe intake”. As we move towards the year 2050
and beyond, particular challenges around the paradox of
under-nutrition/malnutrition and over-nutrition, especially as
Protein Quality Evaluation S207
British Journal of Nutrition

related to protein intake and consequent disease burdens, will
also need to be given greater attention.
Acknowledgements
a) Contributions made by each author to the manuscript: BB,
JIB – Conceived and designed outline of manuscript. RW, JIB–
Wrote the manuscript. BB, RW, JIB – Revised manuscript.
b) Conflict of interest: The authors state that there are no
conflicts of interest.
c) Funding: This work received no specific grant from any
funding agency in the public, commercial or not-for-profit
sectors.
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