Effects of Antinutritional Factors on Protein Digestibility and Amino Acid Availability in Foods

Abstract

The effects of the presence of some of the important antinutritional factors on protein and amino digestibilities of food and feed products was presented. Antinutritional factors may occur naturally, such as glucosinolates in mustard and rapeseed protein products, trypsin inhibitors and hemagglutinins in legumes, tannins in legumes and cereals, phytates in cereals and oilseeds, and gossypol in cottonseed protein products. It was observed that presence of high levels of dietry trypsin inhibitors from grain legumes caused reductions in protein and amino acid digestibilities of upto 50% in living systems. It was also observed that high levels of tannins in cereals resulted in reduced protein and amino acid digestibilities of upto 23% in living systems.

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SPECIAL GUEST EDITOR SECTION
Effects of Antinutritional Factors on Protein Digestibility and
Amino Acid Availability in Foods
G. S ARWAR GILANI,KEVIN A. COCKELL,andESTATIRA SEPEHR
Health Canada, Nutrition Research Division, Bureau of Nutritional Sciences, Banting Research Centre (AL: 2203 C),
Tunney’s Pasture, Ottawa, ON, K1A OL2, Canada
Digestibility of protein in traditional diets from
developing countries such as India, Guatemala,
and Brazil is considerably lower compared to that
of protein in typical North American diets (54–78
versus 88–94%). The presence of less digestible
protein fractions, high levels of insoluble fiber, and
high concentrations of antinutritional factors in the
diets of developing countries, which are based on
less refined cereals and grain legumes as major
sources of protein, are responsible for poor
digestibility of protein. The effects of the presence
of some of the important antinutritional factors on
protein and amino digestibilities of food and feed
products are reviewed in this chapter. Food and
feed products may contain a number of
antinutritional factors that may adversely affect
protein digestibility and amino acid availability.
Antinutritional factors may occur naturally, such as
glucosinolates in mustard and rapeseed protein
products, trypsin inhibitors and hemagglutinins in
legumes, tannins in legumes and cereals, phytates
in cereals and oilseeds, and gossypol in
cottonseed protein products. Antinutritional
factors may also be formed during heat/alkaline
processing of protein products, yielding Maillard
compounds, oxidized forms of sulfur amino acids,
D-amino acids, and lysinoalanine (LAL, an
unnatural amino acid derivative). The presence of
high levels of dietary trypsin inhibitors from
soybeans, kidney beans, or other grain legumes
can cause substantial reductions in protein and
amino acid digestibilities (up to 50%) in rats and
pigs. Similarly, the presence of high levels of
tannins in cereals, such as sorghum, and grain
legumes, such as fababean (Vicia faba L.), can
result in significantly reduced protein and amino
acid digestibilities (up to 23%) in rats, poultry, and
pigs. Studies involving phytase supplementation of
production rations for swine or poultry have
provided indirect evidence that normally
encountered levels of phytates in cereals and
legumes can reduce protein and amino acid
digestibilities by up to 10%. D-amino acids and LAL
formed during alkaline/heat treatment of proteins
such as casein, lactalbumin, soy protein isolate, or
wheat proteins are poorly digestible (less than
40%), and their presence can reduce protein
digestibility by up to 28% in rats and pigs. A
comparison of the protein digestibility
determination in young (5-week) versus old
(20-month) rats suggests greater susceptibility to
the adverse effects of antinutritional factors in old
rats than in young rats. Therefore, the inclusion of
protein digestibility data obtained with young rats,
as the recommended animal model, in the
calculation of PDCAAS (Protein Digestibility-
Corrected Amino Acid Score) may overestimate
protein digestibility and quality of products,
especially those containing antinutritional factors,
for the elderly. For products specifically intended
for the elderly, protein digestibility should be
determined using more mature rats.
Amounts of dietary essential amino acids (EAA),
digestibility of protein, and bioavailabilty of amino
acids are basic parameters in determining the quality
of a protein source. A joint Food and Agriculture
Organization/World Health Organization (FAO/WHO)
Expert Consultation on Protein Quality Assessment was held
in 1991 to review routine methods based on in vitro or animal
assays that correlate well with data from human studies (1).
The Consultation agreed that the Protein
Digestibility-Corrected Amino Acid Score (PDCAAS)
method was the most suitable approach for routine assessment
of protein quality for humans and recommended its adoption
as an official method at the international level. The validity of
the PDCAAS method was endorsed by FAO/WHO in 2001
(2) in the assessing protein quality of mixed diets and of
properly processed (containing minimal amounts of
antinutritional factors) and highly digestible (where
digestibility of protein is a good predictor of bioavailabilty of
amino acids) food products. However, it was recommended
that the impact of antinutritional factors associated with food
GILANI ET AL.: JOURNAL OF AOAC INTERNATIONAL VOL. 88, NO. 3, 2005 967
Guest edited as a special report on "Dietary Protein Quality For
Humans" by Paul J. Moughan.
Corresponding author's e-mail: sarwar_gilani@hc-sc.gc.ca.

proteins on protein digestibility and quality should be further
investigated.
Antinutritional factors may occur naturally or may be
formed during heat/alkaline processing. Some examples of
naturally occurring antinutritional factors include
glucosinolates in mustard and rapeseed protein products (3),
trypsin inhibitors and hemagglutinins in legumes (4), tannins
in legumes and cereals (5), phytates in cereals and
oilseeds (6), and gossypol in cottonseed protein
preparations (7), which would adversely affect nutrient
utilization and may contribute to growth depression in
animals.
During processing of foods, protein sources are treated
with heat, oxidizing agents (such as hydrogen peroxide),
organic solvents, alkalis, and acids for a variety of reasons
such as to sterilize/pasteurize; improve flavor, texture, and
other functional properties; deactivate antinutritional factors;
and prepare concentrated protein products (8–10). These
processing treatments may cause the formation of Maillard
compounds (reaction of sugars with the free e-NH2group of
lysine and other amino acids) or oxidized forms of sulfur
amino acids (such as methionine sulfoxide, methionine
sulfone, and cysteic acid); racemization of optically active
amino acids; and formation of cross links in the protein, such
as lysinoalanine (LAL) and lanthionine. All of these tend to
make the amino acids less available and, in general, the
protein less digestible (8, 11–14).
Based on extensive evaluation of existing in vitro and in
vivo methods in foods, the rat nitrogen balance method was
considered to be the most suitable and practical method for
predicting digestibility of protein by humans (1). Therefore,
when human balance studies cannot be used, the standardized
rat fecal balance method of Eggum (15) or McDonough et
al. (16) was recommended. However, the determination of
protein and amino acid digestibility by the balance method has
been criticized due to possible microbial modifications of
undigested and unabsorbed nitrogenous residues in the large
intestine (17). It is well known that the pattern of nitrogen
excretion is modified by the microflora in the large intestine.
This modification may result in overestimation of digestibility
of protein/amino acids, particularly in poorly digestible
products or those damaged by processing. Therefore, the
determination of protein/amino acid digestibility values based
on the analysis of digesta at the end of the small intestine
(terminal ileum) would increase the accuracy and sensitivity
of digestibility assays (17). However, further research,
including standardization of the ileal digestibility procedures
and generation of sufficient data on foods, is required to
permit replacement of the fecal method by the ileal
method (2). Studies should be undertaken to compare ileal
protein/amino acid digestibility values of humans and animal
models for identical foods.
The purpose of this study is to provide a review of the
published data on the effects of important dietary
antinutritional factors on protein digestibility and amino acid
availability in foods, as determined by human and animal
assays.
Protein Digestibility in Mixed Human Diets
Values for the digestibility of protein in diets for various
areas of the world have been reviewed (18–20). The overall
average true digestibility of protein in North American diets
(including lactovegetarian and vegetarian) was 92%, with
values for different types of diet ranging from 88 to 94%
(Table 1). However, considerably lower protein digestibility
values have been reported in the case of diets from some
968 GILANI ET AL.: JOURNAL OF AOAC INTERNATIONAL VOL. 88, NO. 3, 2005
Table 1. Human fecal digestibility values (%) for protein in diets from various areas/countries of the world
Area or country Diet Apparent digestibility, % True digestibility, %
North AmericaaMixed 84 94
North AmericaaLactovegetarian 83 88
North AmericaaVegetarian 79 93
IndiaaRice, red gram dahl, milk powder, vegetables 65 75
IndiaaRagi, red gram, potatoes, milk powder, vegetables 48 54
GuatemalaaBlack beans, corn tortilla, rice, wheat rolls, cheese, eggs, vegetables 69 77
NigeriaaCassava, rice, dried fish, vegetables 61 91
CeylonaRice, meat, fish, dairy products, breads, fruits, vegetables 82 87
BrazilbRice, beans, meat, eggs, vegetables 69 78
PhillippinesbMixed — 88
CamerooncSorghum flour, zebu meat, vegetables, baobab leaves 65 —
aData were abstracted from Hopkins (ref. 18).
bData were abstracted from FAO/WHO/UNU (ref. 19).
cData were abstracted from Carnu and Delpeuch (ref. 20). Values for apparent digestibility of protein in diets containing partially dehulled and
full-fiber sorghum meal were 60 and 57%, respectively.

developing countries, where less-refined cereals and grain
legumes (such as pulses) are used as major sources of protein.
Values for the true digestibility of protein in diets from India,
Guatemala, and Brazil were 54–75, 77, and 78%, respectively.
The low true digestibility of protein in typical Indian diets has
also been demonstrated in experiments with children (18). For
example, values for true digestibility of protein in millet- and
ragi-based diets (consumed by low-income groups) were
63–65%. Differences in protein digestibility of diets
consumed in North America and developing countries
(Table 1) are due to inherent differences in the nature of
dietary components and not to differences in the digestive
physiology of the populations (18).
Protein Digestibility and Amino Acid Bioavailability
in Individual Foods
In the absence of information on the digestibility of protein
in a particular diet, the value can be estimated by using values
for individual foods and calculating a weighted mean
according to the proportion of protein consumed as these
foods (19).
Values for the true fecal digestibility of protein in some
common foods are summarized in Table 2. Animal protein
sources, low-fiber wheat flours or bread, wheat gluten, farina,
peanuts, and soy protein isolate are highly digestible
(94–99%) by man (18). Whole corn (except opaque-2 corn
containing high amylose), high-fiber wheat flour or bread,
polished rice, oat meal, triticale, cottonseed, soy flour, and
sunflower seeds have protein digestibilities of over
85% (18, 21). The ready-to-eat cereals based on corn, wheat,
rice or oats have low protein digestibilities (70–77%),
probably due to the processing involved in their
GILANI ET AL.: JOURNAL OF AOAC INTERNATIONAL VOL. 88, NO. 3, 2005 969
Table 2. True fecal protein digestibility values for
human adults in some common food productsa
Product
True protein digestibility, %
Mean Range
Animal
Meat, poultry, fish 94 90–99
Milk, casein, lactalbumen 95 90–100
Egg, egg albumen 97 92–106
Cereal
Wheatb, whole 79 —
Wheat, whole 87 80–93
Cornb, whole 76 —
Corn, whole 87 84–92
Corn, opaqueb93 92–95
Corn, opaqueb, high amylose 70 —
Rice, polished 89 82–91
Millet 79 —
Triticale 90 —
Bread, white wheat 97 95–101
Bread, whole wheat 92 91–92
Wheat, ready-to-eat 77 53–88
Rice, ready-to-eat cereals 75 75–85
Oats, ready-to-eat cereals 72 63–89
Oilseeds, flours, or isolates
Soybeansb78 —
Soybean flour 86 75–92
Soybean protein isolate 95 93–97
Cottonseed 90 70–98
Peanut 94 91–98
Sunflower flour 90 —
aAs summarized, except noted, by Hopkins (ref. 18).
bValues from UNU (ref. 21).
Table 3. Values (%) for the fecal protein digestibility of
the same samples of selected food products tested in 2
independent laboratories
Food product
True protein digestibility
(rat balance), %
Eggum et al.
(ref. 22)
Sarwar et al.
(ref. 23)
Casein + methionine NDa100
Beef salami 96 99
Casein 97 99
Skim milk 93 95
Tuna 93 97
Chicken franks 96 99
Sausage 94 94
Skim milk (overheated) 90 90
Peanut butter 92 98
Rolled oats 94 91
Soy protein isolate 92 98
Chickpeas 88 89
Pea concentrate 93 92
Kidney beans NDa81
Wheat cereal 91 91
Pinto beans 73 79
Lentils NDa84
Rice-wheat gluten 93 95
Animal-vegetable mixtures
Macaroni-cheese 95 94
Beef stew 86 89
aND = Not determined.

preparation (18). Millet also has a low protein digestibility
value of 79% (18).
True digestibilities of protein and amino acids were
determined (by the rat balance method) in the same samples of
selected foods in 2 independent laboratories (22, 23). As
noted in the case of humans (Table 2), the highest true protein
digestibility values (93–100%) were obtained for animal
foods by the rat balance method (Table 3). Sarwar et al. (23)
reported that true protein digestibility values for soybean
protein isolate, peanut butter, and rice-wheat gluten cereals
were 95–98%, while Eggum et al. (22) reported somewhat
lower values (92–93%) for these products (Table 3).
Intermediate true protein digestibility values (86–92%) were
obtained for chickpeas, beef stew, overheated skim milk,
rolled oats, whole wheat cereal, and pea protein concentrate.
The lowest true protein digestibility values (73–84%) were
obtained for pinto beans, kidney beans (Phaseolus vulgaris),
and lentils (Lens culinaris).
The low protein digestibility and amino acid bioavailabilty
of grain legumes or pulses (dried edible seeds belonging to the
family Leguminosae and subfamily Papilonoideae)iswell
documented (24, 25). The food legumes are major sources of
protein and other nutrients in many developing countries, and
they often have a significant role in desirable crop rotations.
The presence of less-digestible protein fraction(s), high
concentrations of insoluble fiber and tannins, and residual
amounts of antiphysiological factors (such as trypsin
inhibitors, amylase inhibitors, hemagglutinins, etc.) may be
responsible for the relatively low digestibility of the protein in
grain legumes. Although the antinutritional factors present in
grain legumes are generally inactivated by heat during
cooking, complexes formed between these substances and
bean proteins may not be completely dissociated and may
conceivably interfere with protein digestion (26). The
increased fecal excretion of deoxyribonucleic acid (DNA) and
nitrogen by rats fed cooked kidney beans compared with rats
fed a protein-free or casein diet was considered to be due to an
increased turnover of mucosal cells of the intestine rather than
low protein digestibility (27). The presence of residual
antiphysiological factors in cooked beans, peas, and lentils
may stimulate excretion of endogenous proteins, resulting in a
low digestibility of protein and amino acids (28).
A comparison of the digestibility data summarized in
Tables 2 and 3 would support the general belief that the
abilities of rats and humans to digest a variety of food proteins
are similar. Bodwell et al. (29) determined fecal protein
digestibility in the same preparations of 6 proteins
(spray-dried whole egg, cottage cheese, canned tuna, peanut
flour, soy protein isolate and wheat gluten) using human and
rat assays. True protein digestibility values (96–100%)
obtained with rats were similar to those obtained with
humans (94–104%).
Differences in the protein digestibility of various foods
may arise from inherent differences in the nature of food
protein (protein configuration, amino acid bonding), the
presence of nonprotein constituents that modify digestion
such as dietary fiber, and the presence of antinutritional
factors (occurring naturally or formed during processing) that
may alter the digestibility of the protein and the bioavailability
of amino acids.
Important Naturally Occurring Antinutritional
Factors
Trypsin Inhibitors
Inhibitors of enzymes, such as trypsin, chymotrypsin,
carboxpeptidases, elastase, and a-amylase, appear in many
food products, including legumes, cereals, potatoes, and
tomatoes (30). Adverse effects following short- and long-term
ingestion of raw soybean meal (the richest source of dietary
trypsin inhibitors) by mammals and birds on protein
utilization and growth have been attributed to the presence of
soybean trypsin inhibitors (30). It is now known that the
specificity of these inhibitors is not necessarily restricted to
trypsin, but some of these may, in fact, inhibit chymotrypsin,
elastase, and a number of other so-called serine proteases, that
is, proteases in which serine constitutes the active site (30).
970 GILANI ET AL.: JOURNAL OF AOAC INTERNATIONAL VOL. 88, NO. 3, 2005
Table 4. Trypsin inhibitor content of some common
food and feed products
Product
Trypsin inhibitor activity
mg/g sample mg/g protein
Whole soybeansa16.7–27.2 34.7–122.6
Whole soybeansb48.2 —
Raw soy floura28–32 57.8
Raw soy flourc52.1 104.26
Toasted soy floura7.9–9.4 15.9
Toasted soy flourc3.2–7.9 —
Soy protein concentratea5.4–7.3 8.4–11.2
Soy protein concentratec6.3–13.7 —
Soy protein concentrated4.4–7.3 6.8–11.2
Soy protein isolatea1.2–30.0 1.4–29.4
Soy protein isolatec4.4–11.0 —
Soy-based infant formulasd0.2–2.7 1.3–15.4
Soy tofua0.6 9.2
Soy tofub1.2–3.8 —
Soy milkb6.3 —
Soy saucea0.3 3.3
Soy misoa4.1 22.9
Pea (various cultivars)e2.0–12.5 —
Field bean (various cultivars)e1.4–5.7 —
aAs summarized by Anderson and Wolf (ref. 31).
bAs reported by Miyagi et al. (ref. 32).
cAs summarized by Liener (ref. 4).
dAs reported by Peace et al. (ref. 34).
eAs summarized by Gatel (ref. 33).

Protease inhibitors isolated from soybeans fall into 2 main
categories: those that have a molecular weight of about 21.5
[kilodalton (kDa)] with 2 disulfide bridges and possess a
specificity directed mainly against trypsin (the Kunitz
inhibitor), and those that have a molecular weight of about 8
kDa (6–10 kDa range for legumes other than soy) with a high
proportion of disulfide bonds and the capability of inhibiting
chymotrypsin and trypsin at independent binding sites (the
Bowman-Birk inhibitor; 4). Variants of these 2 types of
inhibitors have been isolated and characterized and have been
shown to possess minor differences in the length of the
protein, amino acid sequence, electrophoretic mobility, and
specificity and susceptibility to heat inactivation (4).
(a)Trypsin inhibitor contents of foods.—Among common
food and feed products, soybeans are the most concentrated
source of trypsin inhibitors (31, 32). The predominant trypsin
inhibitors in soybeans and derived materials are proteins, and
they are located, for the most part, with the main storage
proteins in the protein bodies of the cotyledon (31). Therefore,
trypsin inhibitors tend to fractionate with the milieu of storage
proteins as soybeans are processed into ingredients and foods.
In peas, trypsin inhibitors are also located mainly in the
cotyledons (33). Protease inhibitors make up 0.2–10.0% of
total seed protein of edible dry beans of various species.
Levels of trypsin inhibitors (mainly as the Kunitz trypsin
inhibitor) in soybeans have been reported to vary from
17–48 mg/g sample or from 37–123 mg/g protein (Table 4)
due to differences in varieties and strains and, perhaps, to use
of various methods of determination. The trypsin inhibitor
activity of peas and field beans and other grain legumes has
been reported to be 5–20 times lower than in raw
soybeans (33).
When soybeans are processed into raw defatted flour, none
of the trypsin inhibitors is removed, and they become
concentrated to levels of 28–52 mg/g flour, which
corresponds to levels of 58–104 mg/g of protein, assuming a
protein content of around 50% in soy flour (Table 4).
Owing to their proteic nature, protease inhibitors can be
inactivated by the heat-processing method, such as extrusion,
infrared radiation, micronizing, autoclaving, steam
processing, or flaking (33), or can be removed by
fractionation (30). The extent to which trypsin inhibitor
activity is reduced by heat depends on the initial level present
in the starting material, temperature, heating time, particle
size, moisture and, probably, crop species and
cultivar (30, 33).
Heated or toasted soy flours are produced, having a range
of trypsin inhibitor activities (Table 4), depending upon their
intended use. Most properly processed commercially
available soybean products intended for human consumption,
such as soy protein concentrate (about 70% protein), soy
protein isolate (about 90% protein), soy-based infant
formulas, soy milk, tofu, soy sauce, and miso, have received
sufficient heat treatment to cause inactivation of up to 80% of
the trypsin inhibitor activity present in raw soy flour (4).
Application of prolonged heating required to destroy all
inhibitor activity would adversely affect protein digestibility
and quality of the soybean products. Dietary surveys in the
United Kingdom (35) and the United States (36) have
reported that protease inhibitor activity is still present in many
GILANI ET AL.: JOURNAL OF AOAC INTERNATIONAL VOL. 88, NO. 3, 2005 971
Table 5. Effects of feeding raw soybean flour (Nutrisoy) and autoclaved Nutrisoy on apparent ileal and fecal
digestibilities (%) of protein and amino acids in growing pigsa
Product
Ileal digestibility Fecal digestibility
Nutrisoy Autoclaved Nutrisoy Nutrisoy Autoclaved Nutrisoy
Protein 37b77c77b90c
Arginine 45b90c85b96c
Histidine 44b83c85b95c
Isoleucine 40b86c74b91c
Leucine 37b86c75b92c
Lysine 41b80c80b90c
Methionined59b86c72b89c
Cysteine 35b68c77b86c
Phenylalanine 39b88c77b93c
Tyrosine 34b85c73b91c
Threonine 36b73c72b89c
Valine 38b84c74b91c
aAbstracted from Li et al. (ref. 44); 2 maize starch-based diets containing 200 g/kg diet of either Nutrisoy (a defatted soy flour containing active
trypsin inhibitors) or autoclaved Nutrisoy (containing reduced amounts of trypsin inhibitors) were tested.
b,c Means in the same row, within ileal or fecal digestibility, with different superscripts differ significantly (P< 0.01).
dDigestibility after correction for dietary supplementation of methionine.

of the products consumed in these countries. Doell et al. (35)
estimated an average daily intake of 330 mg trypsin
inhibitors/person in the United Kingdom, and certain groups
such as infants fed solely on soy-based products and
vegetarians or individuals consuming legume-based,
cholesterol-lowering diets, may be exposed to relatively
higher levels of trypsin inhibitors. Peace et al. (34) reported
that soy-based infant formulas may retain up to 28% of the
trypsin inhibitor activity. In the absence of regulatory upper
dietary safe limits of trypsin inhibitors, there is no guarantee
that each and every product would be properly processed and,
consequently, would contain minimum residual levels of
trypsin inhibitors, which may vary greatly with the extent of
heat and other processing conditions used in the preparation
of soybean products. An outbreak of gastrointestinal illness in
individuals who had consumed an unprocessed soy protein
extender in tuna fish salad (4) illustrated the fact that
inadequately processed soy products can find their way into
the human food chain.
(b)Mode of action of trypsin inhibitors.—The feeding of
raw soybean preparations or isolated inhibitors from soybeans
caused an enlargement of the pancreas in susceptible animals,
which could be described histologically as hypertrophy, that
is, an increase in the size of the acinar cells of the
pancreas (30). Concomitant with this increase in the size of
the pancreas was an increase in the secretion of digestive
enzymes, including trypsin, chymotrypsin, and elastase. This
provided support to the hypothesis that the growth depression
caused by the trypsin inhibitors was the consequence of an
endogenous loss of amino acids in the form of enzymes being
secreted by a hyperactive pancreas. The pancreatic enzymes,
such as trypsin and chymotrypsin, are particularly rich in
sulfur-containing amino acids. Therefore, the effect of a
hyperactive pancreas would be to divert these amino acids
from the synthesis of body tissue protein to the synthesis of
these enzymes, which are subsequently lost in the feces (30).
The trypsin inhibitor-induced pancreatic
hypertrophy/hyperplasia observed in susceptible animal
species has been explained by a negative feedback mechanism
in which enzyme secretion is inversely related to the level of
trypsin present in the small intestine (4). Therefore, when the
level of active trypsin in the gut is depressed due to the
presence of the inhibitor, the pancreas would respond in a
compensatory fashion by producing more enzyme. The
mediating agent between trypsin and the pancreas has been
reported to be the hormone cholecystokinin (CCK), which is
released from the jejunal endocrine cells when the level of
trypsin in the small intestine becomes depleted. Feeding rats
with raw soy flour or soybean trypsin inhibitor, in fact,
significantly increased the circulating level of CCK in the
blood (37), and repeated injections of CCK into rats caused
pancreatic enlargement. Moreover, the administration of a
CCK receptor antagonist inhibited the development of
pancreatic hypertrophy and hyperplasia of rats fed raw soy
flour (38). This does not seem to be true in pigs (39). The
trypsin inhibitor-induced hypertrophy/hyperplasia has also
not been observed in dogs, cows, adult rhesus monkeys, or
Cebus monkeys.
Trypsin and chymotrypsin are the major pancreatic serine
proteases present in the duodenum during the digestion
972 GILANI ET AL.: JOURNAL OF AOAC INTERNATIONAL VOL. 88, NO. 3, 2005
Table 6. True digestibility (%) of selected amino acids in thermally processed red kidney beans and a casein control,
as determined by the rat balance methoda
Processing treatmentsb
Amino acid
Red kidney beans
CaseinRaw Home-cooked Canned
Arginine 28c88d78e96
Histidine 32c86d80e98
Isoleucine 12b83d76d94
Leucine 4c86d74e97
Lysine 27c85d75e97
Methionine + cystine –19c68d40e93
Phenylalanine + tyrosine 8b85d79d98
Threonine 11b78d73d95
Tryptophan 13c84d63e97
Valine –8c82d68e96
aAbstracted from Wu et al. (ref. 45). Diets were formulated to contain 10% protein. A protein-free diet was fed to determine metabolic fecal
amino acid loss; used in the calculation of true digestibility.
bProcessing treatments: raw, uncooked dry beans; home, home-made beans (boiled in water, 100°C for 120 min); canned, commercially
canned beans, Progresso; Casein, Animal Nutrition Research Council (ANRC) casein.
c-e Means for bean diets in a row having different letters were significantly (P< 0.05) different.

process. An increase in their concentration at the ileum could
suggest enhanced pancreatic secretion and/or decreased
protein/amino acid digestion along the small intestine. A 4- to
6-fold increase in the ileal flow of active trypsin was reported
after 4 weeks of consumption of a milk-replacer containing
raw peas in preruminant calves (40). Salgado et al. (39)
determined the ileal flow and identified soluble proteins
present in large concentrations in ileal digesta of young pigs
fed soybean meal, peas, fababean (Vicia faba L.), blue lupin,
or chickpeas. Three protein bands at molecular masses of 25,
27, and 30 kDa had a significantly higher ileal flow in the pigs
fed the legume-based diets compared to those fed the casein
control diet. These proteins shared N-terminal amino acid
sequences with enzymes of the serine protease family,
including pig trypsin (25 kDa) and blood coagulation factor
IX or chymotrypsin (39).
The antinutritional effects of trypsin inhibitors have been
mostly studied in animals. Reports on the physiological
effects of feeding raw soy products or isolated trypsin
inhibitors in humans are limited. According to one study (41),
human subjects that were fed raw soybean flour containing
high levels of trypsin inhibitors had a nitrogen balance that
was less positive than those fed heated flour. The duodenal
instillation of a meal of raw soybeans into human subjects
stimulated the production of trypsin and chymotrypsin by the
pancreas. Similarly, the direct infusion of the purified
Bowman-Birk inhibitor into the duodenum of human subjects
induced a 2- to 3-fold stimulation in the production of the
enzymes trypsin, chymotrypsin, elastase, and amylase by the
pancreas (42). However, it is not known whether the chronic
ingestion of low levels of residual trypsin inhibitors in
soybean products and other legumes would pose a risk to
human health (4).
(c)Effects of trypsin inhibitors on protein and amino acid
digestibilities.—Protein and/or amino acid digestibility
have/has been reported to be negatively affected in animal
models by the presence of high levels of dietary trypsin
inhibitors and other antinutritional factors from
soybean (30, 43, 44), kidney beans (45), or other grain
legumes such as peas, lentil, black bean, pinto bean, seafarer
bean, and fababean (28).
True fecal protein digestibility of a diet based on raw soy
flour was considerably lower than that of the casein control
diet (93 versus 78%; 46). Similarly, the addition of raw ground
soybeans (causing an inhibition of 623 mg trypsin/100 g diet)
to a control diet reduced the true fecal protein digestibility
from 98 to 87% (43).
The effects of feeding a food-grade defatted soy flour
(Nutrisoy, Archer Daniels Midland, Decatur, IL) and
autoclaved Nutrisoy on protein and amino acid digestibilities
were compared in growing pigs (44). The trypsin inhibitor
activities in the Nutrisoy and autoclaved Nutrisoy diets were
13.4 and 3.0 g/kg, respectively. The ileal protein and amino
acid digestibilities in the diet containing the unautoclaved soy
flour were about 50% lower compared to those in the diet
containing the autoclaved soy flour (Table 5). The fecal
digestibilities of protein and amino acids in the diet containing
the unautoclaved soy flour were also lower compared to those
in the diet containing the autoclaved soy flour (Table 5).
However, the differences in the fecal digestibilities of amino
acids for the 2 diets were less pronounced than those observed
between the ileal digestibilities, due to the modifying action of
microflora in the large intestine (44). The lower ileal
digestibilities in pigs fed the unautoclaved soy flour diet may
be due to the formation of complexes between soybean trypsin
inhibitors and trypsin and chymotrypsin, which results in a
decrease in the available supply of these enzymes and a
decrease in the efficiency of protein digestion (44).
The effects of thermal processing (raw; home processing,
boiling in water at 100°C for 10 min; and commercial
canning) on amino acid digestibility in red kidney beans
(Phaseolus vulgaris L.) have been studied (45). The
GILANI ET AL.: JOURNAL OF AOAC INTERNATIONAL VOL. 88, NO. 3, 2005 973
Table 7. Values for the true fecal digestibility of protein and selected amino acids in peas, beans, and lentils as
determined by the rat balance methoda
Product Protein, % Lysine, % Methionine, % Cystine, % Threonine, % Tryptophan, %
Pea, century (autoclaved) 83 85 62 85 78 72
Pea flour 88 92 77 84 87 82
Pea protein concentrate 92 92 73 87 90 91
Chickpea (canned) 89 89 74 88 84 82
Lentils (canned) 84 84 41 40 77 73
Kidney bean (canned) 81 80 44 0 74 76
Pinto bean (canned) 79 78 45 56 72 70
Pinto bean (autoclaved) 80 78 61 59 68 58
Seafarer bean (autoclaved) 84 81 60 72 77 74
Black bean (autoclaved) 72 72 51 46 62 47
Fababean (autoclaved) 86 85 59 75 76 53
aAbstracted from Sarwar and Peace (ref. 28).

digestibility was determined as fecal true digestibility in rats
fed red kidney beans as the sole source of dietary protein. As
expected, the raw bean diet had the lowest amino acid digestibility
values (Table 6). The digestibility values for methionine + cystine
(first-limiting amino acid) and valine in the raw bean diet were
negative, while the values for other EAA ranged from 4.5 to
27.0% (Table 6). Home processing and commercial canning of the
raw beans caused a substantial improvement in the digestibility of
amino acids. However, the amino acid digestibility values for the
canned beans were significantly lower than those for the home
processed beans (40–76 versus 68–85%), suggesting an adverse
effect of the severe heat treatment used during canning on amino
acid digestibility.
The true digestibility of protein and individual amino acids
in diets containing autoclaved samples of peas, lentil, pinto
bean, seafarer bean, black bean, or fababean have been
determined by the rat balance method (28). True digestibility
values of the legume diets were considerably lower than those
of the casein control (100 vs 72–90%). In the legume diets, the
true digestibility values of methionine (51–82%), cystine
(46–85%), tryptophan (47–90%), and threonine (62–84%)
were considerably lower than the true digestibility values of
protein (72–90%; Table 7). These data suggest that protein
digestibility may not a be a good predictor for the
bioavailability of dietary limiting amino acids in grain legumes.
Tannins
Tannins are naturally occurring water-soluble
polyphenolic compounds with molecular weights between 0.5
and 3 kDa, and they possess the ability to precipitate proteins
in aqueous solutions (46). They are present in various plant
species, including cereal grains and legume seeds (47). In
diets for humans and monogastric animal species, tannins can
reduce the digestibility of protein, carbohydrates, and
minerals; may lower the activity of digestive enzymes; and
may cause damage to the mucosa of the digestive tract or exert
systemic toxic effects (47).
The chemistry, occurrence, and nutritional effects of
tannins on monogastric animals have been reviewed (47, 48).
In general, tannins are classified into hydrolyzable and
condensed tannins. The hydrolyzable tannins are readily
hydrolyzed by acids, alkalis, or some enzymes, yielding
glucose or some other polyhydroxy alcohol and gallic acid or
some related phenolic acids. Condensed tannins, mainly
polymerized products of flavan-3-ol (catechin) and
flavan-3,4-diol or a mixture of these, are also referred to as
flavolans or procyanidins, and they are resistant to hydrolysis.
Condensed tannins are most commonly found in
dicotyledonous plants. Moreover, in commonly consumed
food products, the condensed tannins are the major
polyphenols, and hydrolyzable tannins are present only in
trace amounts (48). Both types of dietary tannins exhibit the
ability to complex and precipitate proteins (47, 48), therefore,
both have antinutritional properties.
(a)Tannin contents of foods and feeds.—The limited data
on tannin contents of various foods and feedstuffs have been
summarized (Table 8). Certain varieties of some important
crops used as human or animal food, such as sorghum, millet,
barley, and a number of beans and peas, may contain
considerable amounts of tannins (5, 48, 49). Condensed
tannins are mainly present in the testa of colored seeds (47).
When tannins are measured as total phenols, considerable
amounts are also found in the cotyledon fraction. This
observation could, however, be attributed to the presence of
some nontannin phenolics, such as phenolic amino acids, in
this part of the seed. In general, most beans, various types of
peas, barley, and finger millet may contain up to 20 g/kg
tannins (Table 8). However, the levels in sorghum grains (up
to 72 g/kg) and forage products, such as browse legumes,
could be much higher (up to 111 g/kg). In the economically
disadvantaged countries of the world, cereals and legumes
form the main source of nutrients in daily life. The total
dietary intake of tannins can, therefore, be considerably
higher among these populations. The prevalence of protein
malnutrition in these areas can further aggravate the
antinutritional effects of dietary tannins.
(b)Mode of action of tannins.—It is well known that
tannins are potential protein precipitants (5) and that they
reduce protein digestibility in animals (48–50). Increased
fecal nitrogen associated with ingestion of tannin-containing
feeds is ascribed largely to interactions between either tannins
and dietary proteins or tannins and digestive enzymes, or
both (48–50).
Diets containing vegetable tannins, predominantly
hydrolyzable gallotannins, at levels of 13.5, 25, and 50 g/kg
were fed to growing broiler cockerels to investigate their
974 GILANI ET AL.: JOURNAL OF AOAC INTERNATIONAL VOL. 88, NO. 3, 2005
Table 8. Tannin content of some food and feed
products
Product Tannin content, g/kg
Chickpea (Cicer arietinum) 0.8–2.7a
Cowpea (Vigna sinensis) 1.4–10.2a
Pea (Pisum sativum L.) 0.6–3.5a
Pea (Pisum sativum L.) 5.0–10.5a
Pigeonpea (Cajanus cajan) 3.8–17.1a
Winged bean (Psophocarpus
tetragonolobus)
0.3–7.5a
Dry beans (Phaseolus vulgaris L.) 0.3–12.6a
Fababean (Vicia faba L.) 0.5–24.1a
Sorghum (Sorghum vulgare) 0.5–72.0a
Barley (Hordeum sativum) 5.5–12.3a
Finger millet (white seed) 0.3–0.6b
Finger millet (brown and dark brown seed) 5.7–20.0b
Browse legumes 0.0–110.7a
Sal (Shorea robusta) seed meal 26.0c
aAs summarized by Jansman and Longstaff (ref. 49).
bAs summarized by Salunkhe et al. (ref. 48).
cAs reported by Ahmed et al. (ref. 5).

effects on enzymes in the pancreas, intestinal lumen, and
intestinal mucosa (5). Pancreatic weight showed a significant
increase with increasing level of dietary tannins, and trypsin
and a-amylase activities in the pancreas of birds fed at the
highest level of tannins were more than double compared to
those fed the tannin-free control diet. In the intestinal lumen,
inhibition of trypsin activity increased with increasing level of
dietary tannin. Similarly, dipeptidase and sucrose
a-glucosidase in the intestinal mucosa were both inhibited by
tannins. The digestibility of protein and growth of birds were
adversely affected by the tannin-containing diets. Similarly,
the feeding of high tannin fababean hulls significantly
reduced aminopeptidase activity in the jejunal mucosal
homogenates in young piglets (51). The reduced
aminopeptidase activity was associated with lower protein
digestibility.
The pancreatic enlargement induced by the
tannin-containing diets may be mediated by hormones
transported in the blood (5). Several investigators have
demonstrated the roles of gastrointestinal hormones,
particularly CCK and secretin, on pancreatic growth (52, 53).
The type of pancreatic enlargement induced by dietary
tannins (5) has also been reported in response to dietary trypsin
inhibitors (4). This may indicate a common mode of action of
these antinutritional factors, at least at a cellular level.
The consumption of diets containing high tannin sorghum,
isolated and purified condensed tannins from sorghum, or
tannic acid was shown to increase specifically the size of the
parotid glands and the synthesis and secretion of proline-rich
proteins in rats (54). It was hypothesized that proline-rich
proteins are secreted with the saliva and are bound to dietary
tannins in the oral cavity to protect dietary protein. Binding of
tannins to both dietary and endogenous proteins, such as
digestive enzymes and proteins located at the luminal side of
the intestinal tract, has been used to explain the reduced
apparent digestibility of protein in tannin-containing
diets (55). Clear evidence for systemic effects in animals after
feeding condensed tannins does not exist. Condensed tannins
are hypothesized to be resistant to intestinal degradation and
too large for intact absorption.
In a dose-response experiment, increasing the level of
tannin-rich fababean hull extract in the diet (providing 0.0,
0.08, 0.16, 0.33, 0.66, 1.32, and 1.99% tannins as catechin
equivalents) resulted in a linear increase in both the relative
size of parotid glands in the rat (multiple regression
correlation coefficient, r2= 0.90) and the quantity of
proline-rich proteins in the glands (r2= 0.89; 55).
(c)Effects of tannins on protein and amino acid
digestibilities.—Presence of dietary tannins in cereals, such as
sorghum, and grain legumes, such as field beans and
fababeans (Vicia faba L.), has been reported to reduce protein
GILANI ET AL.: JOURNAL OF AOAC INTERNATIONAL VOL. 88, NO. 3, 2005 975
Table 9. Apparent fecal digestibility values for amino acids in diets containing varying amounts of tannin from
tannin-rich fababean (Vicia faba L.) extract, as determined by the rat balance methoda
Amino acid
Diet
1 (0.0)b2 (0.08)b3 (0.16)b4 (0.33)b5 (0.66)b6 (1.32)b7 (1.99)b
Arginine 90c86d85d,e 83e73f62g43h
Histidine 94c92d91d90e86f82g60h
Isoleucine 89c88c,d 87c,d,e 87c,d,e 85d,e 85d,e 64f
Leucine 94c93c,d 92d92d90e88f71g
Lysine 93c92c92c92c90d87e71f
Phenylalanine 89c87c,d 86c,d 87c,d 85c,d 80e66f
Threonine 89c85d85d85d81e77e54f
Valine 92c90c,d 90c,d 89d87e85e63f
Alanine 83c81d81c,d 81d81c,d 81c,d 38e
Aspartic acid 87c85d84d82d77e69f46
Glutamic acid 93c91d91d,e 90e83f74 71h
Glycine 79c70d64d55e20f–29h–20g
Proline 96c92c93c86d72e50f52f
Serine 87c84d83d82d79e73f57
Tyrosine 93c92c90c,d 90c88d,e 86e67f
Total amino acids 91c89d89d,e 87e82f74g60h
aAbstracted from Jansman et al. (ref. 55); all diets contained casein (200 g/kg diet) and supplemental DL-methionine (3 g/kg diet).
bPercentage catechin equivalents in parentheses, calculated contents based on the analyzed content of the tannin extract.
c-h Values in the same row with different superscripts differ significantly (P< 0.05).

and amino acid digestibilities in various animal
models (55–57). Similarly, a negative correlation (r = – 0.85)
was reported between in vitro protein digestibility and dietary
tannin content (58).
Increasing the level of tannin-rich fababean hull extract
resulted in a linear decrease in the apparent digestibility of
total (r2= 0.97) and individual (r2=0.27to0.99)aminoacids
(Table 9). The apparent digestibility of most indispensable
amino acids was affected to a lesser degree than that of some
of the nonessential amino acids, particularly proline, glycine,
and glutamic acid. It was suggested that the more pronounced
reduction in digestibility for these amino acids was primarily
due to the interactions of tannins with the proline-rich proteins
that were secreted by the parotid glands, as these 3 amino
acids make up 73% of the weight of isolated proline-rich
proteins from parotid glands of tannin-fed rats (57).
Jansman et al. (57) also studied the effects of feeding
fababean hulls with a low or high content of condensed
tannins on fecal and/or ileal and fecal digestibilities of protein
and amino acids in pigs. Inclusion of high tannin (3.3%)
instead of low tannin (<0.1%) beans decreased the apparent
and true ileal digestibility of protein from 83 to 74 and 94 to
90%, respectively, and increased the excretion of endogenous
protein from 22 to 32 and 13 to 23 g/kg of dry matter intake at
the ileal and fecal level, respectively. Feeding of the high
tannin hulls also reduced the apparent ileal digestibility of
most individual amino acids (Table 10). The reductions in
amino acid digestibilities ranged from 4–23%. Based on these
observations, it was concluded that condensed tannins in
fababean hulls adversely affect the digestibility of protein and
amino acids in pigs. This could have resulted from the
increased amounts of both dietary and endogenous protein in
ileal digesta and feces of pigs (57). In this respect, the effect of
condensed tannins from fababeans in pigs seems to differ
from that in rats because, in rats fababean tannins mainly
increase the secretion of salivary (endogenous) protein.
The comparative effects of high tannin and low tannin
fababean varieties on protein and amino acid digestibilities in
various animal species have been reviewed (33). Two varieties
of fababean (high tannin and low tannin) were compared. Total
nitrogen digestibility, as determined by apparent fecal
digestibility and apparent ileal digestibility in pigs and by
apparent fecal digestibility in chickens, was significantly lower
in the high tannin variety than in the low tannin variety
(Table 11). Similarly, lysine, methionine + cystine and
threonine digestibilities, as determined by apparent and true
976 GILANI ET AL.: JOURNAL OF AOAC INTERNATIONAL VOL. 88, NO. 3, 2005
Table 10. Apparent ileal digestibility of amino acids in experimental diets containing high- and low-tannin fababean
hulls as determined in young pigsa
Amino acid HSCbLSCbHSHTbLSHTbLSLTb
Arginine 90c85d,e 83e79f88c,d
Histidine 84c74d74d65e71d
Isoleucine 86c80d77d72e78d
Leucine 89c80d82d73e79d
Lysine 85c69e,f 77d63f73d,e
Methionine 84c76d,e 72e,f 68f78d
Cystine 32c,d 43c2e20d44c
Phenylalanine 88c81d81d73e80d
Threonine 73c67d63d,e 60e68c,d
Valine 85c77d78d69e76d
Alanine 78c74c63d63d72c
Aspartic acid 84c72d,e 75d68e70e
Glutamic 90c80d86c75e81d
Glycine 66c58c,d 44e50d,e 62c
Proline 84c56d,e 71c,d 41e66d
Serine 81c72d71d64e73d
Tyrosine 89c80d79d70e76d
Total amino acids 85c72d77d66e75d
aAdapted from Jansman et al. (ref. 57).
bHSC = Control diet containing protein sources with a high solubility (casein and fababean cotyledons); LSC = control diet containing protein
sources with a low solubility (fish meal, soy protein concentrate, meat meal, potato protein, and sunflower meal); HSHT = diet containing high
solubility proteins plus high tannins (3.3% catechin equivalent); LSHT = diet containing low solubility proteins plus high tannins; LSLT = diet
containing low solubility proteins plus low tannins (<0.1% catechin equivalent).
c-f Values with a different superscript within a row differ significantly (P< 0.05).

ileal digestibility methods, were significantly lower in the high
tannin compared to the low tannin variety.
Phytates
Phytic acid, or myo-inositol 1,2,3,5/4,6-hexakis
(dihydrogenphosphate), is a naturally occurring compound
found primarily in seeds, nuts and grains, where it functionsas
a store of mineral nutrients and inositol to be used during
germination (59, 60). Phytic acid is typically found in plant
tissues as salts of mono- and divalent cations, collectively
known as phytate, with much of it associated with proteinsin a
globoid particle or aleurone grain (61). In human and animal
nutrition, phytate, with its abundance of negatively charged
phosphate groups, is best known for its ability to chelate
several nutritionally essential minerals in the gastrointestinal
tract, making them less bioavailable (62). Phytate interferes
with zinc homeostasis and also affects the bioavailability of
other essential mineral nutrients (63). Although generally
recognized as nutritionally adverse interactions, chelation of
minerals by phytate has also been suggested to provide some
protection from colon carcinogenesis (64, 65) and to account
for the apparent antioxidant activity of phytate (66). Phytate
can also bind with proteins in the gastrointestinal tract, as
discussed below.
(a)Phytate contents of foods and feeds.—Phytate is found
in many commonly consumed nuts, seeds, and grains at
concentrations of one to several percent on a dry weight
basis (60). It is not evenly distributed within grains, as most of
the phytate can be found in the germ of corn, the bran of
wheat, and the pericarp of rice (61). Processing methods, such
as milling or refining, that remove these portions can,
therefore, result in dramatic declines in phytate content of the
finished product (67). In beans and peas, the greatest
proportion of the phytate is found in the edible cotyledon, so
mechanical processing does not lead to much reduction in
phytate level. In addition, phytate is relatively heat-stable,
with only a small fraction being destroyed during cooking or
the heat processing of foods, while the phytase enzymes that
might break down the phytate are heat-labile (68). Prolonged
soaking, fermentation, or germination, however, expose the
phytic acid to endogenous, bacterial, or yeast phytases, with
consequent reduction in phytate levels in the food as
consumed (61). Data summarizing phytate content and
processing effects in large numbers of foods are available, for
example, in Harland and Oberleas (59) and Reddy (61).
Phytate is synthesized in plants by successive
phosphorylation of inositol (69). Breakdown of phytate
involves successive dephosphorylation by phytases present in
plants, microorganisms, and certain animal tissues (70). A
range of partially phosphorylated compounds can, thus, exist,
from penta- to mono-phosphoinositol, and these vary in their
capacity for cation or protein binding. Older methods of
phytate analysis, such as those based on phytate precipitation
with iron, do not differentiate well between inositol
compounds of varying degrees of phosphorylation; newer
liquid chromatography (LC)-based methods allow for
determination of specific compounds (59, 71).
(b)Mode of action of phytates.—Phytate can negatively
influence the activity of digestive enzymes through chelation
of mineral cofactors or interaction with the protein (either
enzyme or substrate) at either acidic or alkaline pH (68). Some
digestive enzymes require metal cofactors, such as zinc or
calcium, for full activity; examples include a-amylase,
alkaline phosphatase, carboxypeptidases, and
aminopeptidases. Binding of phytate to proteins may be direct
(phytate:protein) or indirect (via a cation bridge).
GILANI ET AL.: JOURNAL OF AOAC INTERNATIONAL VOL. 88, NO. 3, 2005 977
Table 11. Literature reports comparing nitrogen and amino acid digestibilities for high- and low-tannin fababean
(Vicia faba L.) varietiesa
Animal MethodbComponent High tannin, % Low tannin, %
Pig AFD Nitrogen 72c80d
Pig AFD Nitrogen 77c85d
Piglet AID Nitrogen 74c79d
Piglet AID Methionine + cystine 59c62d
Piglet AID Threonine 72c77d
Piglet AID Nitrogen 75c85d
Pig TID Nitrogen 82c85d
Pig TID Lysine 87c88c
Pig TID Threonine 81c82c
Chicken AFD Nitrogen 67c83d
Chicken AFD Nitrogen 69c84d
aAbstracted from Gatel (ref. 33).
bAFD = Apparent fecal digestibility; AID = apparent ileal digestibility measured with postvalvular T caecum cannula; TID = true ileal digestibility
measured with ileo-rectal anastomosis.
c,d Means in the same row with different superscripts differ significantly (P< 0.05).

Phytate:protein and phytate:cation:protein interactions are
complex, and they vary with pH, time, and relative
concentrations (68). At low pH (for example in the stomach),
positively charged side groups of basic amino acids in
proteins can bind to the negatively charged phytate due to
strong electrostatic interactions (67, 72). Above its isoelectric
point (pI), a protein carries a net negative charge, and
multivalent cation bridging (typically involving calcium)
appears to be involved in complex formation between phytate
and proteins. Phytate:cation:protein interaction would be
expected to predominate at the higher pH found in the small
intestine (73). Another indirect mechanism of phytate
inhibition of digestive enzyme activity measured in vitro has
been suggested to involve complex interactions among
phytate, digestive enzymes, and other proteins present in the
solution (74).
(c)Effects of phytate on protein and amino acid
digestibilities.—Phytic acid interferes with the proteolytic
action of pepsin on a variety of plant and animal proteins as
determined in vitro, presumably through the formation of
phytate:protein complexes at low pH (75–77). Decreasing the
phosphate content of phytate by partial or complete hydrolysis
reduces the inhibitory effect (76). The effect of phytate on
trypsin activity is less clear; in some studies, phytate has been
shown to significantly inhibit trypsin proteolysis in vitro
(78, 79), while in others it has not (77). Differences in assay
conditions may account for the lack of consistency between
these studies. The mechanism proposed for phytate inhibition
of tryptic activity involves a complexing of calcium, leading
to lowered trypsinogen activation and increased autocatalytic
degradation of trypsin (79).
Studies of the effect of phytate addition to multienzyme
proteolytic assay systems have shown significant (up to
20–25%) inhibition of casein digestion in vitro (80).
Decreasing the phytate content by 23–26% through
fermentation treatment of finger millet was associated with a
14 to 26% increase in in vitro digestibility of the finger millet
protein using pepsin and pancreatin (81).
Investigations of the effects of phytate on protein and
amino acid digestibility in vivo have primarily involved
studies of phytase supplementation to production rations for
poultry and swine (73). Microbial phytase is added to animal
978 GILANI ET AL.: JOURNAL OF AOAC INTERNATIONAL VOL. 88, NO. 3, 2005
Table 12. Effect of microbial phytase addition on the apparent ileal digestibility of nitrogen and amino acids for a
marginally lysine-deficient diet for broiler chicksa
Microbial phytase activity added, FTU/kg dietb
Pooled SEMc
Nitrogen/amino acid 0 125 250 375 500 750 1000
Nitrogen 78 79 79 80 81 81 82 0.51d
Essential amino acid
Arginine 82 82 83 85 86 85 86 0.45d
Histidine 808080838283830.76
d
Isoleucine 767576777979800.56
d
Leucine 76 77 77 79 79 80 81 0.82d
Lysine 79 81 82 82 83 83 84 0.46d
Phenylalanine 77 77 78 78 81 80 82 0.58d
Threonine 75 76 77 76 78 78 80 0.66d
Tryptophan 76 77 76 78 79 79 80 0.47d
Valine 777576748180820.59
d
Nonessential amino acid
Alanine 77 77 77 80 80 82 83 0.70d
Asparticacid 777778798182810.43
d
Glutamic acid 81 81 82 85 85 85 86 0.58d
Glycine 777777808079820.47
d
Serine 76 77 76 78 79 80 81 0.63d
Tyrosine 76 75 77 76 79 78 79 0.52d
Overall mean 77 78 78 79 81 81 82 0.36d
aAdapted from Ravindran et al. (ref. 83).
bOne phytase unit (FTU) = quantity of phytase that releases 1 mmol of inorganic phosphorus/min from 150 mmol/L sodium phytate at pH 5.5
and 37°C.
cSEM = Standard error of the mean; each mean represents 6 pens of 10 birds each.
dLinear dose effect significant, P< 0.001.

feeds to break down phytate, improve phosphorus
bioavailability, and reduce the environmental impacts of
intensive animal production arising from high phosphate
manures. Significant improvements in apparent digestibilities
of proteins and amino acids have also been noted in several
studies. Mechanisms suggested include alleviation of the
inhibitory effects of phytate on digestive enzymes, release of
protein from endogenous phytate:protein complexes in the
feedstuffs, and prevention of complex formation between
phytate and proteins or amino acids in the gut (73). Only a few
studies have been published on the effects of phytase
supplementation on ileal digestibility of amino acids in pigs.
Methodological differences render comparisons among these
difficult, but improvements of 3–10% in ileal digestibilities of
at least some amino acids have been noted, which appear to
correspond with the magnitude of improved growth rates and
protein retention observed by others (73).
Phytase supplementation improves the digestibilities of
proteins and amino acids for poultry, from a variety of
feedstuffs including cereals, cereal byproducts, and oilseed
meals (82). In the latter study, crude protein digestibility,
measured as apparent ileal digestibility in broiler chicks, was
improved by 2–6% through the addition of phytase to the
different diets at a level of 1200 FTU/kg diet. One FTU is the
quantity of phytase that releases 1 mol inorganic
phosphorus/min from a solution of 0.00015 mol/L sodium
phytate at pH 5.5 and 37°C. Mean apparent ileal digestibilities
of 15 amino acids improved by 2–9% with phytase addition to
the diets. For individual indispensable amino acids, average
improvements across the range of feedstuffs tested were
4–8%, with the greatest effects being noted for threonine and
valine. Dietary phytate concentration was negatively
correlated with the inherent protein digestibility (r = –0.81;
P< 0.001 from Student’s t-test) and inherent mean amino acid
digestibility (r = –0.85; P< 0.001) of the feedstuffs (82).
Addition of increasing levels of microbial phytase to a
marginally (91% of the recommended level) lysine-deficient
wheat-soybean meal-sorghum diet significantly improved the
apparent ileal digestibilities of nitrogen and amino acids for
broiler chicks (83). Improvements of 3–5% were noted in
digestibilities of nitrogen and for 15 amino acids (Table 12).
Significant linear dose responses were found for digestibilities
of nitrogen and all 15 amino acids using phytase levels up to
1000 FTU/kg diet. Phytase also increased apparent
metabolizable energy in that study, reaching a plateau effect at
750 FTU/kg diet (83).
Protein-Bound D-Amino Acids and LAL
Exposure of food proteins to certain processing conditions,
such as heat and/or alkaline treatments, induces 2 major
chemical changes: racemization of amino acids to
D-enantiomers and concurrent formation of LAL (84, 85).
Racemization of L-amino acids to their D-isomers in proteins is
pH-, time-, and temperature-dependent. Although
racemization rates of various amino acids in a protein vary, the
relative rates in different proteins are similar (Table 13).
The chemistry, nutritional quality, and safety of D-amino
acids have been recently reviewed (85). Racemization impairs
protein and amino acid digestibility and nutritional quality.
The nutritional utilization of different D-amino acids varies
widely in animals and humans (85).
LAL (an unnatural amino acid derivative) is formed during
the alkaline treatment of proteins, mainly by the addition of an
e-amino group of a lysine residue to the double bond of a
GILANI ET AL.: JOURNAL OF AOAC INTERNATIONAL VOL. 88, NO. 3, 2005 979
Table 13. D-Amino acid composition (%) of 8 alkali/heat-treated protein sourcesa
Amino acid Casein Lactalbumin Wheat gluten Zein Fish Soybean Bovine albumin Hemoglobin
Serine 41 47 42 44 42 44 43 44
Cystine — 32 32 44 23 21 23 30
Methionine 25 32 33 30 29 24 30 26
Threonine 29 29 30 36 33 28 28 31
Phenylalanine 24 24 24 32 28 25 28 30
Asparticacid29 23 264225312719
Glutamic acid 20 19 32 35 19 21 18 20
Tyrosine 15 19 19 35 16 14 15 23
Alanine 15 14 19 22 19 16 22 17
Lysine 8 7 9 8 11 11 13 10
Leucine 7 5 7 8 7687
Isoleucine 33 454465
LALb4.4 5.4 0.9 0.3 2.8 3.2 8.5 4.4
aAbstracted from Friedman (ref. 85); conditions: 0.1M NaOH, 75°C, 3 h.
bMixture of (LD +LL) lysinoalanine isomers in g/16 g N.

dehydroalanine residue that has been generated by the
b-elimination reaction of cystine, phosphoserine, or
glycoserine residues (86). The quantity of LAL formed is
dependent upon many factors, such as temperature,
concentration of alkali, time of exposure to alkali, type of
protein, and type of cations in the solution (87, 88). The
formation of LAL was also reported in a variety of proteins
when heated under nonalkaline conditions (87).
(a)LAL contents of foods and feeds.—Protein-containing
foods and feeds are commonly processed with alkaline/heat
treatments. However, information on the levels of LAL found
in foods that are part of the everyday diet is limited. The data
published vary widely according to the treatments applied.
The most representative results, as summarized by Finot et
al. (89) and Friedman (84), are presented in Table 14.
Domestic cooking may produce up to 1820 ppm LAL in
foodstuffs that are initially free of LAL, such as sausage,
chicken meat, and eggs. Commercial preparations that have
undergone technological treatments may contain LAL in
variable amounts according to the type of product and the
conditions of preparation, including alkaline/heat treatment.
Sterilized milk and milk powders have been reported to
contain up to 1620 ppm LAL. Special attention has been paid
to the high LAL contents of sterilized liquid infant formulas,
which may contain as much as 2120 ppm LAL. Because such
formulas are often the sole source of protein for infants over a
significant time period, it has been recommended that the
LAL content of infant formulas be kept under 200 ppm (84).
Protein-rich ingredients, such as dried egg white, soy protein
isolate, and sodium caseinates, have been reported to contain
significant amounts of LAL (up to 6900 ppm; Table 14).
Among the common food products, whipping agents are
known to contain the highest levels of LAL (up to
53 150 ppm).
(b)Antinutritional effects of LAL.—The nutritional
quality and safety effects of dietary LAL have been recently
reviewed (85, 90). The health concerns associated with LAL
are 2-fold: LAL formation in processed foods results in a loss of
essential amino acids (such as lysine, cysteine, and threonine)
and reduced protein digestibility and quality, and LAL
consumption may lead to kidney damage (86, 88, 91, 92).
Nephrocytomegaly (enlarged kidney cells) and
nephrokaryomegaly (enlarged nuclei in kidney cells) are
unique lesions induced in rats by LAL. The level of dietary
LAL needed to induce nephrocytomegaly depends on whether
LAL is fed as the free amino acid or bound in a protein.
Enlarged nuclei have been reported in rats fed as little as
1200–1400 ppm of protein-bound LAL (93). In comparison,
feeding of free LAL was found to produce nephrocytomegaly
at much lower levels (100–250 ppm; 94, 95).
L-lysyl-D-alanyl-LAL, the most potent isomer of the 4 optical
isomers of LAL, induced nephrocytomegaly when fed at
levels as low as 30 ppm, whereas D-lysyl-D-alanyl-LAL did
not produce lesions at less than 1000 ppm (96).
LAL, which is a strong chelator of mineral nutrients such
as calcium, iron, copper, and zinc, may exert its toxic effect by
metal binding in renal tubule cells. The human kidney is more
susceptible to damage by LAL than is the kidney of several
other animal species (97). In an in vivo study conducted at
Health Canada), we saw altered mineral (iron and copper)
status in rats due to consumption of LAL from processed
foods (90). Liver and kidney iron levels were greatly reduced;
this is of particular concern because iron is often already
reduced in the elderly and infants, who are likely to consume
diets composed of exclusively formula-type foods.
The susceptibility of humans to the nephrotoxic effect of
LAL is unknown. Nephrotoxic effects were not observed after
long-term feeding of alkali-treated soy protein to baboons,
therefore, consumption of low amounts of dietary LAL is
probably safe for humans (30). However, further analytical
data are needed for assessment of the actual quantity of LAL
ingested (91). In addition, the influence of chronic
consumption of alkaline-treated foods high in LAL on the
balance of copper and other minerals in humans should be
examined.
(c)Mode of action of D-amino acids and
LAL.—Protein-bound D-amino acids formed during
processing, especially at alkaline pH, may have adverse
effects on protein digestibility and the quality and safety of
processed foods (84, 85). When absorbed, D-amino acids may
be made utilizable by the action of racemases or epimerases or
D-amino acid oxidases (85). The amino acid oxidase system
(which varies in the amounts and specificity of oxidases in
different animal species) may become saturated when high
concentrations of D-amino acids are consumed. Although
980 GILANI ET AL.: JOURNAL OF AOAC INTERNATIONAL VOL. 88, NO. 3, 2005
Table 14. LALacontents of some common foods and
food products
Food/product LAL, ppm
Frankfurterb0–170
Chicken thighb0–200
Chicken meatc370
Eggsc160–1820
Corn chipsc390
Pretzelsb220–500
Processed riceb1000
Tortillasb200
Infant formula, dryb150–920
Infant formula, liquidc160–2120
Evaporated milkb150–860
Milk powdersc150–1620
Sterilized milkb200–1160
Soy protein isolatec370–1300
Sodium caseinatec430–6900
Whipping agentsb,c 6500–53150
aLAL = Lysinoalanine.
bAbstracted from Finot (ref. 89).
cAbstracted from Friedman (ref. 84).

proteins containing D-amino acids can be hydrolyzed at
peptide bonds containing L-amino acids, the hydrolysis rates
may be slower than those for corresponding native proteins.
Such changes adversely affect the nutritional quality and
safety of foods by generating biologically nonutilizable forms
of amino acids through the creation of D-D,D-L,andL-D peptide
bonds that are partly or fully inaccessible to proteolytic
enzymes (85). Moreover, these racemized proteins may
compete with proteins that do not have racemized amino acids
for the active site of digestive proteinases in the gut and, thus,
impair the biological utilization of the unracemized proteins.
The slower absorption of free and peptide-bound D- compared
with L-amino acids may contribute to the decrease in protein
digestibility (98). It is not known whether D-amino
acid-containing oligopeptides can change the microflora of
the digestive tract.
Alkali-treated proteins containing LAL have lower
digestibility compared to untreated proteins. For instance, an
inverse relationship between the LAL content of casein and
the extent of in vitro proteolysis by trypsin has been
demonstrated (99). The biological utilization of LAL as a
source of lysine in a mouse growth assay was 3.8% of that of
crystalline lysine (100). Similarly, LAL was completely
unavailable as a source of lysine to the rat, although it was
37% available to the chick (101). Possible causes for the
reduction in digestibility and nutritional quality following
heat/alkaline treatment include destruction of arginine,
cystine, and lysine; isomerization of L-amino acids to less
digestible D-isomers; formation of inter- and intramolecular
cross links; and inhibition of proteolytic enzymes (30).
(d)Effects of D-amino acids and LAL on protein and
amino acid digestibilities.—Alkali treatment of proteins
GILANI ET AL.: JOURNAL OF AOAC INTERNATIONAL VOL. 88, NO. 3, 2005 981
Table 15. Amino acid composition, true fecal protein digestibility, and protein quality of untreated and
alkaline/heat-treated lactalbumin and SPIa
Amino acid
g/100 g protein
Lactalbumin, untreated Lactalbumin, treatedbSPI, untreated SPI, treatedb
Lysinoalanine 0.1 4.42 0.03 1.94
Arginine 3.43 3.12 7.84 7.56
Histidine 2.2 1.9 2.51 2.77
Isoleucine 6.02 6.25 5.08 5.38
Leucine 13.68 14.29 8.15 8.97
Lysine 10.5 8.35 6.08 4.94
Methionine 2.71 2.6 1.47 1.5
Cyst(e)ine 2.81 0.76 1.16 0.27
Phenylalanine 4.26 4.64 5.42 5.98
Tyrosine 4.45 4.69 3.85 4.25
Threonine 5.73 3.13 3.98 2.57
Tryptophan 2.12 2.12 1.35 1.3
Valine 6.29 6.62 5.12 5.78
Alanine 5.81 6.17 4.17 4.63
Aspartic acid 11.2 12.41 11.05 11.96
Glutamic acid 17.6 12.42 18.84 20.6
Glycine 2.24 2.52 3.93 4.45
Proline 5.69 5.77 5.21 5.71
Serine 4.93 3.46 5.12 4.16
True protein digestibilityc99 73 96 68
RPERc89 0 56 0
RNPRc91 0 64 0
aAbstracted from Sarwar et al. (ref. 90).
bProtein sources were subjected to alkaline treatment with 0.1M NaOH at room temperature for 1 h, followed by heat treatment at 75ECfor
3 h, neutralization with 10M HCl to pH 7.5, ultrafiltration to remove salts, and spray drying of the ultrafiltrate retentate.
cDetermined in rats; RPER (relative protein efficiency ratio; casein + Met = 100) and RNPR (relative net protein ratio; RNR + Met = 100)
values were calculated using the following equations: RPER = [PER of test diet/PER of control diet] ´100; RPNR = [NPR of test diet/NPR of
control diet] ´100, where PER = weight gain of test rat/protein consumed by test rat and NPR = (weight gain of test rat + weight loss of
nonprotein rat)/(protein consumed by test rat); Met = methionine; RNR = net protein ratio.

reduces the nutritional quality of the treated proteins in
monogastric animals (84, 85, 90, 102). The alkaline treatment
of soy protein reduced fecal protein digestibility from 97 to
83% in rats and lowered body weight gain in baboons (84).
Similarly, alkaline/heat treatment had significant negative
effects on the true fecal protein digestibility of lactalbumin (99
versus 73%) and soy protein isolate (96 versus 68%) in
rats (90). The protein quality of these sources as predicted by
rat growth was also significantly reduced (Table 15). The
treated proteins contained considerably higher amounts of
LAL compared to the untreated proteins. The amount of LAL
in the treated lactalbumin was more than 2-fold higher than in
the treated soy protein isolate. As expected, the formation of
LAL in the treated proteins was associated with a loss of
lysine (19–20%), cystine (73–77%), and serine (18–30%).
There was also a loss of threonine (35–45%) in the treated
proteins (Table 15). Most other amino acids were not greatly
affected by the alkaline treatment of the 2 proteins. Because
the methodology used did not distinguish between the D-and
L-forms of amino acids, the influence of the processing
conditions on the formation of D-amino acids could not be
determined in this study (90).
Protein digestibility and the quality of some enteral
products based on caseinates and soy protein isolate (SPI)
were inferior to casein (103). For example, the true fecal
protein digestibility of 5 commercial enteral products, as
determined in rats, was significantly lower than that of casein
(89–92 versus 95%). The enteral products also contained
higher levels of LAL than casein (998–2333 versus 0 mg/g
protein). Because the formation of LAL was also reported to
occur in a variety of proteins under nonalkaline conditions,
the lower protein digestibility of the enteral products could be
explained by the presence of LAL in these products. Similarly,
heat treatment at pH 12.2 was reported to cause a significant
reduction in protein digestibility and the net protein utilization
of casein and soybean (84).
In comparing the protein nutritional value of milk-based
infant formulas sold in Europe, Pompei et al. (104) found that
the formation of LAL caused by heat processing was one of
the most sensitive predictors of protein damage in infant
formulas. They found that liquid forms of milk-based
formulas contained up to 10 times more LAL than powder
forms, suggesting a more severe heat treatment in the
preparation of liquid forms compared to powders. Similarly,
Sarwar et al. (105) reported that the true digestibility of
protein and indispensable amino acids in liquid concentrates
was up to 13% lower than in powder forms of milk-based
infant formulas (Table 16). Protein quality (as predicted by rat
growth methods) of liquid concentrates were also up to 25%
lower than that of powders. Reduction in protein and amino
acid digestibilities of liquid concentrates compared to
powders may be due to the formation of Maillard compounds,
oxidized forms of sulfur amino acids, and crosslinked
peptides such as LAL.
de Vrese et al. (102) studied the formation of D-amino acids
and LAL and their effects on protein digestibility in 3 protein
sources (casein, b-lactoglobulin, and wheat protein) subjected
to heat and alkaline treatments(heatingfor6or24hat65°C,
pH 10.5–11.5). Treatment of these proteins for 24 h increased
levels of D-amino acid residues. For example, about 11–15%
of L-asparagine and aspartic acid, the most susceptible amino
acids, underwent racemization in the 3 protein sources (102).
Similarly, the alkaline/heat treatment increased levels of LAL,
and about 12–15% of total lysine was converted to LAL in
these protein sources (102).
982 GILANI ET AL.: JOURNAL OF AOAC INTERNATIONAL VOL. 88, NO. 3, 2005
Table 16. True rat fecal digestibility (%) of protein and selected amino acids in milk-based infant formulas sold in
Canadaa
Formula Protein Lysine Methionine Cystine Threonine Tryptophan
Casein + Met (control) 98b98b100b97b,c 97b100b
Formulas of manufacturer 1
Powder 94d96c93d92d93c,d 95c,d
Liquid concentrate 88e,f 87 86 85 90e92e
Formulas of manufacturer 2
Powder 93d93d98c96c90e96c
Liquid concentrate 88 85g89 89e87 96c
Formulas of manufacturer 3
Powder 97b,c 98b99b,c 96c94c96c
Liquid concentrate 90b85f,g 92d91d87 94d
Formulas of manufacturer 4
Liquid concentrate 90e89e89e89e93c92e
aAbstracted from Sarwar et al. (ref. 105).
b-g Means in each column without a common superscript differ significantly (P< 0.05).

Digestibility of protein and amino acids in the alkaline/heat
treated proteins was also determined by a true ileal method
with 15N-labeling in minipigs (102). True protein digestibility
in the treated casein, b-lactoglobulin, and wheat protein
decreased by 13% (80 versus 93%), 14% (83 versus 97%),
and 17% (76 versus 93%), respectively. Similarly, heat and
alkali treatment of casein caused significant reductions in
apparent protein digestibility (up to 17%) for aspartate
(aspartic acid + asparagine), serine, and glycine (Table 17).
However, digestibility of other amino acids, such as
L-phenylalanine and L-tyrosine, was not affected. The
apparent digestibilities of D-aspratate (asparagine + aspartic
acid, D-glutamate (glutamic acid + glutamine),
D-phenylalanine, and LAL were between 29 and 39% (102).
This reduction in digestibility was, however, not correlated to
the degree of racemization because the reduction in
digestibility was already maximal after 6 h of heat/alkaline
treatment, although racemization increased further with the
longer time of treatment. This study provided evidence that
even small amounts of D-amino acids and LAL within a
protein can adversely affect protein/amino acid digestibility.
The question of whether either D-amino acids and D-amino
acid-containing peptides, or crosslinks such as LAL, are
individually responsible for lower protein digestibility, or
whether these effects are additive, remains unresolved. Both
racemization and crosslinks were shown to inhibit proteolysis
and decrease protein and peptide digestibility in in situ
experiments using isolated loops of rat intestine (106).
Experiments with guanidinated caseins in which theloss of
protein digestibility was independent of LAL strongly suggest
that D-amino acids are mainly responsible for the impaired
digestibility while LAL may play a minor role (102). This
observation is, however, in contrast to other reports on
inhibitory effects of LAL on proteolysis (107, 108).
Because pepsin and chymotrypsin attack the polypeptide
backbone mainly at bonds involving phenylalanine or
tyrosine, proteolysis of dietary proteins in the stomach and
upper section of the small intestine creates mainly peptides
with phenylalanine or tyrosine as the terminal amino
acids (102). Further degradation of the peptides by carboxy-
and aminopeptidases may proceed up to the point at which
there is a D-amino acid at the end of the peptide chain,
inhibiting further activity of the exopeptidases and the release
of absorbable peptides and free amino acids (109). Therefore,
digestibility of indispensable amino acids, which show only a
low susceptibility toward racemization, may, nevertheless, be
markedly impaired by the presence of adjacent D-amino
acids (85, 102).
To investigate the influence of adjacent amino acids on the
bioavailability of methionine, Sarwar et al. (110) determined
the relative bioavailability of methionine (L-methionine =
100) in tripeptides found in b-casein (Ala-Met-Ala,
GILANI ET AL.: JOURNAL OF AOAC INTERNATIONAL VOL. 88, NO. 3, 2005 983
Table 17. Effects of alkaline/heat treatment on apparent ileal digestibility of selected L-amino acids in casein fed to
adult miniature pigsa
Treatment L-Asxb,% L-Ser, % L-Glxc,% L-Phe, % L-Tyr, %
None 77e67e86e84e89e
6h,65EC, pH 11.0 69f52f77f85e88e
24 h, 65EC, pH 11.0 65f50f76f85e88e
aAbstracted from de Vrese et al. (ref. 102).
bAsx = Aspartic acid + asparagine.
cGlx = Glutamic acid + glutamine.
dValues are means (n=9).
e,f Values within columns not sharing a superscript are significantly different (P< 0.05).
Table 18. NPR and methionine bioavailability values
for some tripeptides containing L-andD-methioninea
DietbNPRcMethionine
bioavailability, %
Basal 1.17 —
Basal plus L-Met (reference) 3.82d100d
Basal plus D-Met 3.81d100d
Basal plus tripeptides
Ala-Met-Ala 3.05e71e
Ala-D-Met-Ala 2.37f45f
Val-Met-Phe 2.71g58g
Val-D-Met-Phe 1.09f0h
Thr-Met-Arg 2.64g55g
Thr-D-Met-Arg 1.15f0h
Thr-Met-Lys 1.13f0h
aAbstracted from Sarwar et al. (ref. 110).
bAmino acid abbreviations: Met = methionine; Ala = alanine; Val =
valine; Phe = phenylalanine; Thr = threonine; Arg = arginine; Lys =
lysine.
cMeans (n= 8); the NPR (net protein ratio) of the casein + Met diet
was 5.60, suggesting that the experimental conditions of the
feeding study were properly controlled.
d-g Means within the same column bearing different superscripts differ
significantly (P< 0.05).

Ala-D-Met-Ala, Val-Met-Phe, and Val-D-Met-Phe) and in soy
glycinine (Thr-Met-Arg, Thr-D-Met-Arg, and Thr-Met-Lys;
see Table 18 for definitions of amino acid abbreviations) by a
rat growth method using net protein ratio (NPR) as the
performance index. Crystalline D-Met was completely
available to the growing rat but, in the tripeptide form D-Met
(0–45%) was considerably less available than L-Met
(38–71%; Table 18). The bioavailability of Met was also
influenced by the side chain of the adjacent amino acids. Met
in tripeptides with bulky amino acids (Val-Met-Phe,
Thr-Met-Arg, and Thr-Met-Lys) was less available than in
those with the lighter amino acids (Ala-Met-Ala). D-Met in the
tripeptides with bulky amino acids (Val-D-Met-Phe and
Thr-D-Met-Arg) was completely unavailable for rat growth.
The lower bioavailability of D-Met when compared to L-Met
in Ala-Met-Ala or Val-Met-Phe (Table 18) may be due to
lower hydrolysis (by peptidases of the brush border
membranes of the intestinal mucosa) of the respective peptide
containing D-Met than that containing L-Met (109). These
observations suggest that protein-bound D-Met (formed
during processing) may be considerably less bioavailable than
protein-bound L-Met. Moreover, bioavailability of
protein-bound Met may be influenced by the amino acids
adjacent to Met in the polypeptide chain.
These findings concerning the adverse effects of D-amino
acids and LAL formed during heat processing of proteins
may have considerable implications in animal
nutrition (84, 85, 102). For example, feeding roller-dried milk
powder to calves may impair their growth (102). Similarly,
alkaline/heat detoxification of aflatoxins in some feedstuffs
may result in the formation of considerable amounts of
D-amino acids and LAL (102). The diminished nutritive value
for the racemized, heat and alkali-treated dietary proteins may
also have relevance for human nutrition (84, 85, 102). This
reduction in protein nutritional value may not only be due to a
reduction in L-amino acid content but also to diminished
digestibility.
(e)Effect of age on protein digestibility of protein in
products containing antinutritional factors using rat
assay.—The PDCAAS method (1, 2) involves the use of
young rats for predicting protein digestibility of foods for all
ages, including the elderly. To assess the usefulness of protein
digestibility measured in mature rats in the calculation of
PDCAAS for the elderly, the influence of age on the
digestibility of protein in 5-week and 20-month old rats by the
balance method has been studied (111). Fifteen protein
products were tested, each fed as the sole source of dietary
protein (10%). A protein-free diet was also included to obtain
an estimate of metabolic fecal protein. Protein digestibility
values (corrected for metabolic fecal protein loss) in the
mature rats were significantly (P< 0.05) lower than in young
rats for most products (Table 19). However, these differences
were small (up to 5%) for optimally processed animal and
vegetable protein products. But, digestibility values in the
mature rats were considerably lower (7–17%) than for the
young rats when fed products containing antinutritional
factors, i.e., mustard flour containing glucosinolates,
alkaline/heat-treated soy protein isolate and lactalbumin
containing LAL, raw soybean meal and black beans
containing trypsin inhibitors, or heated skim milk powder
containing Maillard compounds. Therefore, the inclusion of
protein digestibility data obtained using young rats in the
calculations of PDCAAS may overestimate protein
digestibility and quality of these products for the elderly. For
products specifically intended for the elderly, protein
digestibility should be determined using older rats.
The digestibility of protein is considered a good
approximation of the bioavailability of amino acids in mixed
diets and properly processed food products that contain
minimal amounts of residual antinutritional factors (1, 2).
However, there often are quite large differences between the
digestibility for protein and the individual amino acids,
especially in coarse cereals and grain legumes and in those
products that contain antinutritional factors present naturally
or formed during processing (112) or storage (113).
Therefore, there may be a need to include corrections for the
bioavailability of individual amino acids in calculating
PDCAAS values of such products. Although the effect of
animal age on the determination of the bioavailability of
individual amino acids has not been studied, it is quite
possible that the differences due to age in bioavailability of
individual amino acids may even be greater than the
984 GILANI ET AL.: JOURNAL OF AOAC INTERNATIONAL VOL. 88, NO. 3, 2005
Table 19. Protein digestibility values (%) for some
animal and vegetable protein products as affected by the
age of rata
Diet
Protein digestibilityb-h
5-week-old rats
20-month-old
rats
Casein 99b96b
Whey protein concentrate 100b97b
Whey protein hydrolysate 99b98b
Lactalbumin (ALBM) 98b96b
ALBM, alkaline/heat-treated 71h64f
Skim milk powder (SMP) 93c,d 92c
SMP, heated 79f,g 70d
Soy protein isolate (SPI) 95c93c
SPI, alkaline/heat-treated 66i49h
Soybean meal, autoclaved 81e–g 78d
Soybean meal, raw 80f,g 72e
Black beans, autoclaved 83e78d
Black beans, raw 71h60g
Fababeans, autoclaved 82e,f 77d
Mustard protein flour 92d79d
aAbstracted from Sarwar and Sepehr (ref. 111).
b-h Within each column, digestibility values (attributed to source of
protein) with different superscripts differ significantly (P<0.05).

differences in digestibility of protein in products containing
antinutritional factors.
The effect of aging on pancreatic digestive enzymes in
3-month- and 20-month-old Fischer-344 rats has been
studied (114). Although trypsinogen concentrations did not
change, pancreatic amylase concentration was reduced by
41% and lipase concentration was increased by 29% in the
aging animals. Only in young rats, lipase concentration
increased 25% during the consumption of a high-fat (72%)
diet compared to the intake of a high sucrose (75%) diet.
Feeding a high-starch diet induced a 26% increase in amylase
in young rats, but not in the older animals. Trypsinogen
concentration was unchanged by dietary manipulation, while
jejunal enteropeptidase concentration was modestly reduced
in the aging rat. Postprandial luminal concentrations of trypsin
and amylase did not differ in the 2 age groups. It was,
therefore, concluded that aging may induce modest changes in
pancreatic digestive enzymes and in jejunal enteropeptidase
that may not be physiologically important to protein
digestibility. It was further concluded that the pancreas of
older rats did not adapt to changes in dietary conditions as well
as young rats. The protein source tested in the study (114) was
lactalbumin, a highly digestible and high-quality protein
product. Therefore, further information is needed to
determine the influence of aging on pancreatic digestive
enzymes in rats fed proteins containing antinutritional factors.
Although it is generally recognized that the abilities of rats
and humans to digest a variety of foods are similar (1), human
studies are needed to confirm the adverse effect of age on
protein digestibility of products containing antinutritional
factors, as reported by Gilani et al. (111).
Conclusions
The influence of important antinutritional factors present
in common food and feed products on the digestibility of
protein and availability of amino acids is reviewed in this
section. The antinutritional factors included those present
naturally, such as trypsin inhibitors in legumes, tannins in
legumes and cereals, phytates in cereals, and glucosinolates in
mustard protein products, or those formed during
heat/alkaline processing, such as LAL, D-amino acids, and
Maillard compounds. The presence of high levels of these
dietary antinutritional factors has been reported to cause
significant reductions (up to 50%) in protein digestibility
and/or amino acid bioavailability values in animal models
and/or humans. The adverse effects of the antinutritional
factors on protein digestibility were more pronounced in
mature rats compared to young rats, suggesting the need for
the use of mature rats in determining protein digestibility and
the PDCAAS values of products intended for the elderly.
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