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Food and Nutrition Bulletin, vol. 34, no. 2 (supplement) © 2013, The United Nations University.
Absorption studies show that phytase from
Aspergillus niger
significantly increases iron and zinc
bioavailability from phytate-rich foods
Abstract
Background. Iron and zinc deficiency are major public
health problems affecting many parts of the world,
including Southeast Asia. Infants, young children, and
women of reproductive age are particularly vulnerable
due to their high requirements. Even though iron and
zinc are present in significant amounts in the plant-based
diets typically consumed in developing countries, their
bioavailability is low due to high levels of absorption
inhibitors such as phytate. Phytase has been used in
animal nutrition for decades to improve the bioavail-
ability of certain minerals in feed.
Objective. To show the effect of phytase in human
nutrition based on evidence from human studies. Phytase
can be used either during processing or as an active food
ingredient degrading dietary phytate during stomach
transit time.
Methods. Evidence from human studies testing the
effect of phytase on iron and zinc bioavailability using
stable isotopes was reviewed.
Results. Twelve studies tested the effect of phytase on
iron and five tested its effect on zinc bioavailability. Most
of these studies used a phytase derived from Aspergillus
niger. They found a beneficial effect unless phytate con-
centrations were too low or levels of inhibitors or enhanc-
ers of iron absorption were too high. Twenty to 320
phytase units per 100 g of flour significantly improved
iron absorption, even though higher levels might fur-
ther increase iron bioavailability. For zinc, not enough
information is available to determine optimal activities.
Conclusions. Phytase clearly has a beneficial effect on
iron and zinc absorption from phytate-rich foods. It also
has the potential to increase the absorption of magne-
sium, calcium, and phosphorus in areas such as South-
east Asia where mineral deficiencies are widespread.
Key words: Iron bioavailability, iron deficiency,
phytase, stunting, zinc bioavailability, zinc deciency
Introduction
Iron and zinc deficiencies: Major public health
problems
Iron-deficiency anemia and zinc deficiency are major
public health problems worldwide [1, 2]. Globally,
anemia affects 1.62 billion people, of whom over 500
million are women of reproductive age and about 600
million are children [3]. Infants are among the most
vulnerable groups, and their development often begins
to falter with the introduction of complementary foods
or shortly afterwards [4, 5]. Iron requirements increase
during this period of development due to rapid growth,
and stores are quickly used up, eventually resulting
in iron deficiency unless sufficient iron is taken up
[1]. This then leads to iron-deficiency anemia and its
associated pathologies, including impaired cognitive
and psychomotor development [6–8] and, later in life,
reduced work capacity [9]. The prevalence of anemia,
of which around half is thought to be caused by iron
deficiency, is high in both pregnant and nonpregnant
women of reproductive age due to increased iron
requirements and menstrual blood losses, respectively
[3]. During pregnancy, iron deficiency significantly
increases the risk of maternal and perinatal mortality
[10]. Zinc deficiency also appears to be widespread in
Southeast Asia and increases the risk of adverse preg-
nancy outcomes [11] and stunting [12]. However, the
evidence linking zinc deficiency with cognitive and
physical development is somewhat inconsistent due to
a lack of reliable biomarkers for individual zinc status.
Barbara Troesch, Hua Jing, Arnaud Laillou, and Ann Fowler
Barbara Troesch and Ann Fowler are affiliated with DSM
Nutritional Products Ltd, Kaiseraugst, Switzerland; Hua Jing
is affiliated with DSM Nutritional Products Asia Pacific, Sin-
gapore; Arnaud Laillou is affiliated with the Global Alliance
for Improved Nutrition (GAIN), Geneva.
Please direct queries to the corresponding author: Barbara
Troesch, DSM Nutritional Products Ltd, Wurmisweg 576, 4303
Kaiseraugst, Switzerland; e-mail: barbara.troesch@dsm.com.
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Phytase human efficacy review
High level of phytate in diets in the developing world
Diets in developing countries are typically monotonous
and plant-based, containing little or no animal prod-
ucts. Use of the minerals from such diets is inefficient,
as their bioavailability is low due to the presence of
absorption inhibitors such as phytic acid. This also
applies to vegetarian and, to an even larger degree,
vegan diets in industrialized countries. The high-
est levels of phytic acid are found in grains, legume
seeds, and linseed. For example, a study among more
than 1,000 people in Andra Pradesh, India, where
a vegetarian diet is the norm, showed high levels
of phytate in the diet [13]. In 1988, Ferguson et al.
reported that the bioavailability of minerals in Eastern
Africa was low due to the high phytic acid content of
the staple foods [14], even among children [15]. This
hypothesis can be extended to many developing coun-
tries in Southeast Asia where diets are based on rice,
green vegetables, white meat (pork and chicken), and
green tea and consequently are rich in phytates and
polyphenols. In a comparison of women of reproduc-
tive age living in urban and rural Vietnam, those in
rural areas tended to consume more cereals (412.4 vs.
370.6 g/day, p < .01) [16] and thus more dietary phytate
(214 vs.166 mg/day, p < .05) (unpublished data). Simi-
lar trends were observed among Vietnamese children
aged 6 to 59 months, with phytate intakes of 136 mg/
day in rural areas versus 47 mg/day in urban areas
(p < .01) (unpublished data). These data indicate that
women of reproductive age and children, especially
children under 5 years of age, who live in rural areas
may be at higher risks for iron and zinc deficiencies
than those in urban areas, and thus would benefit more
from adequate release of these minerals from absorp-
tion inhibitors such as phytate in the food matrices.
Adequate degradation of phytate in staple foods may
play an important role in improving micronutrient
status worldwide and in Southeast Asia, where the
prevalence of anemia is 38% among women of repro-
ductive age and 65% among preschool children [3, 17].
In Vietnam, for example, zinc deficiencies are among
the most prevalent micronutrient deficiencies, with
more than 50% of women and children under 5 years
being deficient [18].
Phytase: A potential solution to increase
bioavailability
Given the varying affinity of phytic acid protons to
dissociate in the pH range of the stomach and intes-
tine, phytic acid is mostly found as negatively charged
phytate [19], often in the form of mono-, di-, or
trivalent metal salts [20]. These salts have a very low
solubility under the pH conditions of the upper gas-
trointestinal tract, where most minerals are absorbed,
and they tend to precipitate with increasing pH along
the intestine [21]. Iron–phytate complexes are insoluble
in the pH range from around 2.5 to 8 [22]. It was sug-
gested that the inhibition by phytate is not so much due
to the complexing itself, but because they tend to aggre-
gate in insoluble precipitates [23]. This is supported by
the finding that the solubility of inositol phosphates, as
well as the bioavailability of minerals, decreases with
increasing level of phosphorylation [21]. Even though
some level of exchange of ligands exists, it is unlikely
that iron dissociates from the aggregates in sufficient
quantities in the duodenum, where its absorption takes
place [24]. Phytic acid can be degraded by processes
such as fermentation, soaking, germination, and malt-
ing, whereby endogenous phytases in grains and seeds
are activated, or by adding an exogenous phytase [24,
25]. A sufficient degradation of phytate will prevent
the complexing and consequently also the precipitation
of iron into insoluble complexes, thereby increasing
its bioavailability. Enzymatic phytate (myo-inositol-
hexaphosphate) hydrolysis is shown in figure 1.
Phytase has been used in animal nutrition for two
FIG. 1. Enzymatic phytate (myo-inositol-hexaphosphate) hydrolysis
OH
O
OH
OH
OH
OH
P
O
O
O H
-
+ H
2
O
O
O
O
O
O
O
P P
P
P
P
P
O
O
O
O
O
HO
O
O
O
OH
O
O
O H
O
O
O
O
O
-
-
-
-
-
-
-
-
-
1 – 5 inorganic
orthophosphates
myo-inositol-hexaphosphate
myo-inositol-
penta, tetra, tri, di or mono
phosphate
+
Me
2+
(n) (e.g. Fe
2+
, Zn
2+
,
Ca
2+
, Mg
2+
)
n Me
2+
Phytase
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B. Troesch et al.
decades for sustainable mineral nutrition and is today
a standard ingredient in feed for monogastric animals.
Particularly in swine and poultry, dietary supplemen-
tation with phytases has had positive effects on the
availability of minerals such as phosphorus, calcium,
and zinc, and on reduction of fecal excretion of phos-
phorus [26, 27]. In human studies, the use of phytase
to date has mainly focused on the benefit for iron and
zinc absorption. The aim of this review is to summarize
the available evidence for the effect of phytase on the
bioavailability of iron and zinc. We primarily discuss
studies using stable isotopes to assess iron and zinc
absorption, as these provide the most direct measure
of changes in bioavailability and facilitate comparability
between the studies. In addition, we use evidence from
more long-term studies to support these findings and
to corroborate long-term benefits. The review focuses
on conditions of use, origins of the phytases, quantities
applied, and food matrices tested.
Conditions of use
Phytase can exert its effect during processing of
phytate-rich food, in which case it is not intended to
remain active at the time of consumption. Alternatively,
it can be used as a functional ingredient, for example,
in supplements intended for co-consumption with
phytate-rich foods. In both cases, the effect of phytase
is to improve the bioavailability of minerals from
phytate-rich foods.
Available evidence in humans
To our knowledge, 12 absorption studies investigat-
ing the effect of phytase on iron bioavailability in
humans had been published at the time of writing
[28–39] (table 1). Of these, nine used phytase to
degrade phytate during food processing [28–36], and
in three the phytase was used as a food ingredient, i.e.,
consumed as active enzyme [37–39]. The phytase was
derived from Aspergillus niger in eight of the food pro-
cessing studies [28–34, 36] and from Peniophora lycii
in one study [35]. Two of the studies feeding the active
enzyme used a phytase from A. niger [37, 39], while
the third did not state the organism that produced the
phytase [38].
Four studies of zinc absorption used phytase as a
food processing aid [34, 40–42], and in one the phytase
was used as a food ingredient directly given with the
test meal* (table 2). The phytases used were derived
from A. niger [34, 41]* or from cereals [40, 42]. A. niger
* The study has only been presented at a conference so far:
Brnic M. Effect of the enzyme phytase and EDTA on human
zinc absorption from maize porridges fortified with ZnSO
4
or
ZnO. Bioavailability Conference 2010. Asilomar Conference
Grounds, Pacific Grove, California, USA, 2010.
TABLE 1. Summary of absorption studies using phytase to improve iron bioavailability
Study
Added iron
compound Phytase (dose) Test meal
Study
population
Phytate-to-iron molar ratio
Absorption
ratio Outcome
Original
After dephyti-
nization
Used in food processing
Hurrell et al.
1992 [28]
Native iron with
extrinsic label
Aspergillus niger
(NA)
Soy protein isolate Adults (n = 8) 4.2 < 0.006 4.8 p < .001
Adults (n = 7) 3.0 < 0.005 4.0 p < .05
Davidsson et al.
1994 [29]
FeSO
4
A. niger (NA) Soy formula Infants (n = 10) 2.1 0.4 1.2 p < .05
Infants (n = 10) 2.1 < 0.007 2.2 p < .001
Davidsson et al.
1997 [30]
FeSO
4
A. niger (NA) White wheat- and milk powder–
based formula
Infants (n = 12) 0.6 0.08 1.0 NS
Hurrell et al.
1998 [31]
Native iron with
extrinsic label
A. niger (NA) Soy formula Adults (n = 9) 2.1 < 0.007 2.5 p < .05
Davidsson et al.
2001 [32]
FeSO
4
A. niger (NA) Pea formula Adults (n = 10) 8.0 < 0.02 1.6 p < .0001
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Phytase human efficacy review
Hurrell et al.
2003 [33]
Native iron with
extrinsic label
A. niger (NA) Wheat porridge Adults (n = 11) 0.12%
a
≤ 0.002%
a
11.6 p < .0001
Wheat, milk NA NA 1.3 NS
Rice porridge Adults (n = 9) 0.16%
a
≤ 0.002%
a
3.1 p < .001
Oat porridge Adults (n = 10) 0.67%
a
≤ 0.002%
a
8.4 p < .0001
Maize porridge 0.26%
a
≤ 0.002%
a
5.0 p < .0001
Sorghum porridge
b
Adults (n = 8) 0.87%
a
≤ 0.002%
a
1.3 NS
Sorghum porridge
c
0.89%
a
≤ 0.002%
a
1.8 p < .01
Sorghum porridge
c
Adults (n = 9) 0.43%
a
≤ 0.002%
a
2.0 p < .001
Sorghum porridge
c
0.71%
a
≤ 0.002%
a
1.9 p < .01
Wheat porridge with milk Adults (n = 6) NA NA 2.5 p < .05
Wheat porridge with milk and
ascorbic acid
NA NA 2.5 p < .05
Wheat and soy porridge Adults (n = 9) 0.30%
a
0.02% 3.3 p < .005
Wheat and soy porridge with
ascorbic acid
NA NA 3.5 p < .005
Davidsson et al.
2004 [34]
FeSO
4
A. niger (NA) Soy formula Infants (n = 9) 3.1 < 0.1 1.3 NS
Zhang et al.
2007 [35]
Ferric ammonium
citrate
Peniophora lycii
(NA)
Oat drink Adults (n = 20
e
) 3.7
d
0.6
d
1.8 p < .05
Petry et al. 2010
[36]
FeSO
4
A. niger (100
FTU/batch)
Bean porridge Adults (n = 49
e
) 5.8 < 0.1 1.2 NS
Dehulled bean porridge 5.7 < 0.1 4.3 p < .001
Used as active enzyme
Sandberg et al.
1996 [37]
FeSO
4
A. niger (~ 20
FTU/100 g
flour)
Bread rolls with white wheat flour Adults (n = 10) 0.6
d
NA 2.0 p < .0001
Layrisse et al.
2000 [38]
FeSO
4
NA (~ 300
FTU/100 g
flour)
Bread with white wheat flour Adults (n =14) 3.2 NA 2.0 p < .05
Iron bis-glycine
chelate
3.2 NA 1.7 p < .05
Troesch et al.
2009 [39]
FeSO
4
A. niger (~ 320
FTU/100 g
flour)
Maize porridge Adults (n = 17) 8 NA 1.7 p < .05
NaFeEDTA Adults (n = 18) 8 NA 1.2 p < .05
NaFeEDTA Maize porridge with ascorbic acid Adults (n = 16) 8 NA 1.8 p < .01
FTU, unit of phytase activity (amount that liberates 1 µmol of inorganic phosphorus per minute from an excess of sodium phytate at 37° C and a pH of 5.5); NA, not available, NaFeEDTA, sodium iron
ethylenediaminetetraacetate; NS, not significant
a. Phytate content only, as no information was available on native iron content.
b. High-tannin sorghum.
c. Low-tannin sorghum.
d. Calculated from phytate phosphorus content assuming that all phytate is present as 6-inositol phosphate.
e. Interstudy comparison.
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B. Troesch et al.
phytase was shown to be active over a broad pH range,
including the physiological pH of the stomach [43]
and to be stable during the expected residence time at
gastric pH [39]. It is therefore a suitable candidate for
phytate degradation in the stomach as well as during
processing. Phytase from wheat, on the other hand, is
thought to be less effective for use as an active ingredi-
ent because of its low activity and stability in the lower
pH range found in the stomach in pockets where the
buffering from food is low [44].
Improvement of bioavailability and nutritional status
Table 1 shows that the majority of studies found statis-
tically significant improvements in iron absorption [28,
29, 31–33, 35–39]; the improvement in iron absorption
achieved following phytase treatment ranged from
1.0- to 11.6-fold. In the studies in which no significant
differences were observed, the low intrinsic phytate
content of the test meal, in combination with the iron
absorption-enhancing effect of added ascorbic acid
[30, 34], the inhibiting effect of milk [30, 33], or the
high levels of polyphenols [33, 36], may account for
the lack of effect. All five zinc studies showed statisti-
cally significant improvements in zinc absorption [34,
40–42]* (table 2), with improvements ranging from
1.4- to 2.0-fold.
These results are supported by a study comparing
the effects of feeding normal and dephytinized maize
flour for 12 days, in which the use of dephytinized
maize flour resulted in a decrease in soluble transferrin
receptor and zinc protoporphyrin levels [45]. In the
study with the longest feeding period to our knowledge,
phytase was used over approximately 6 months [46].
The enzyme was used as part of a home fortification
powder with 2.5 mg of iron from sodium iron ethyl-
enediaminetetraacetate (NaFeEDTA), 2.5 mg of zinc
from zinc oxide, ascorbic acid, and other nutrients in
a maize porridge compared with a control porridge
without added nutrients. Even though the beneficial
effect cannot be attributed to phytase alone, a signifi-
cant decrease in iron and zinc deficiency was observed
despite the low levels of the two minerals in the powder,
TABLE 2. Summary of absorption studies using phytase to improve zinc bioavailability
Study
Added zinc
compound
Phytase
(dose) Test meal
Study
population
Phytate-to-zinc
molar ratio
Absorp-
tion
ratio OutcomeOriginal
After
dephytinization
Used in food processing
Egli et al.
2004 [40]
Native zinc
with extrinsic
label
Endogenous
wheat
phytase
Wheat
and soy
porridge
Adults
(n = 9)
6.4 0.15 1.5 p = .005
Davidsson
et al. 2004
[34]
ZnSO
4
Aspergillus
niger (NA)
Soy-based
formula
Infants
(n = 9)
3.6 < 0.007 1.4 p = .03
Kim et al.
2007 [41]
Native zinc
with extrinsic
label
A. niger (10
FTU/100 g
brown rice;
20 FTU/
100 g soy-
bean curd
residue)
High- vs.
low-phytate
diet
a
Adults
b
(n = 7)
21.4 9.1 2.0 p < .001
Adults
c
(n = 10)
23.5 10.5 1.7 p < .001
Thacher et
al. 2009
[42]
Zn(O
2
CCH
3
)
2
with labeled
ZnCl
2
Wheat
phytase
(0.2 g/120
g
d
)
Maize
porridge
Children
(n = 34)
3.9 mg/g
e
2.8 mg/g
e
1.7 p < .001
Used as active enzyme
Brnic 2010
*
ZnSO
4
A. niger
(NA)
Maize
porridge
Adults
(n = 15)
9 NA 1.9 p < .001
FTU, unit of phytase activity (amount that liberates 1 µmol of inorganic phosphorus per minute from an excess of sodium phytate at 37°C
and a pH of 5.5); NA, not available
a. In addition to the use of phytase, cooked brown rice in the diet was also replaced by white rice to reduce phytate content.
b. Aged 22 to 24 years.
c. Aged 66 to 75 years.
d. No information was available on activity of this phytase.
e. Phytate content only, as no information on native zinc content was available.
* Brnic M. Effect of the enzyme phytase and EDTA on human zinc absorption from maize porridges fortified with ZnSO4 or ZnO. Bioavail-
ability Conference 2010. Asilomar Conference Grounds, Pacific Grove, California, USA, 2010.
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Phytase human efficacy review
indicating potentially increased bioavailability even
with the high levels of phytate in the food matrix. The
estimated increase in bioavailability corresponds well
with the findings of a series of absorption studies con-
ducted in preparation for this long-term study, which
showed a clear additional benefit of the phytase on the
bioavailability of iron, even in the presence of ascorbic
acid and EDTA [39]. Overall, the reviewed studies pro-
vide strong evidence for a beneficial effect of phytase
on the bioavailability of iron and zinc.
Effect of subject’s age
Of the 12 studies on iron, 9 were performed in adults
[28, 31–33, 35–39] and 3 in infants [29, 30, 34]
(table 1). The infants used for comparison by Hurrell
et al. [31] were the ones reported on in the study by
Davidsson et al. [29] and are therefore not counted
again. The authors showed that the results from stud-
ies in adults can be extrapolated to assess the impact of
phytate on iron absorption in infants, since the relative
inhibitory effect was similar for both, at least for such
formula-type meals [31]. Data from infants suggest
that, whereas homeostatic regulation of iron absorption
is not fully developed in young infants at around 4 to
6 months, it is present in older infants [47]. Similarly,
in rat pups, the intestinal iron transporters divalent
metal transporter 1 and ferroportin were appropriately
regulated in response to iron supplementation by day
20, equivalent to older human infants [47].
Of the five zinc studies, three were performed in
adults [40, 41, 43]*, one was performed in children
[42], and one was performed in infants [34] (table 2).
No information on the zinc status of the infants par-
ticipating in this study is available. Therefore, it cannot
be established whether a correlation exists between
zinc status and level of absorption. However, evidence
from rat pups indicates that homeostatic regulation of
zinc absorption develops during mid to late infancy
but might not be adequate if zinc deficiency of the diet
persists [47]. One study comparing zinc absorption in
elderly and young adults showed no difference in the
inhibitory effect of phytate between the two groups
[48]. This corresponds with results from studies in
animal nutrition, where phytase has been used for
several decades and was shown to increase mineral bio-
availability in pigs independently of their age [27]. In
summary, the available evidence suggests that phytase
has a beneficial effect on iron and zinc absorption inde-
pendently of the age of the target population.
Effect of food matrix
Evidence from an in vitro study suggests that, depend-
ing on the food matrix, 50% to 80% phytate degrada-
tion can be achieved with the use of a phytase from A.
niger [44]. It was proposed that this variation stems
from differences in accessibility of the substrate based
on its location in the plant seed or on the mineral con-
tent of the matrix [44]. The test meals used to assess
the impact of phytase on iron bioavailability consisted
of soy [28, 29, 31, 34], peas [32], beans [36], or a range
of cereals [30, 33, 35, 37–39], including rice [33],
and were given in the form of drinks, infant formula,
porridges, or bread rolls (table 1). The range of food
matrices used in the studies indicates that the effect of
enzymatic dephytinization is not limited to a particu-
lar food type but can be generalized to phytate-rich
foods, at least as long as phytate is the only or the main
inhibitor of iron absorption present in the food [50]. If
foodstuffs also contain other iron absorption inhibitors,
e.g., polyphenols or milk, ascorbic acid should be used
in addition [33]. Ascorbic acid is a known enhancer
of iron absorption and has been recommended for
milk-containing diets fortified with iron [50]. Only
one study investigated the influence of milk on the
effect of phytase, with or without ascorbic acid, and
found somewhat inconsistent results [33]. One, but
not the other, substudy showed a slight but statistically
significant effect of dephytinization on iron absorption.
The effect of the phytase remained in the presence of
ascorbic acid, despite the enhanced absorption already
seen in the meal with the native phytate content due
to the presence of ascorbic acid. This added benefit of
phytase in the presence of ascorbic acid was also shown
for iron absorption from a maize-based porridge [39]
as well as from a wheat- and soy-based porridge [33].
The inhibitory effect of phytate and polyphenols is
not additive, and it is thought that phytate and poly-
phenols affect iron absorption by similar mechanisms
[36]. As a consequence, the impact of phytate removal
will be limited if the inhibiting effect of polyphenols
persists. However, polyphenols are a heterogeneous
group of compounds that vary greatly in their ability
to chelate iron [51, 52]. The inhibitory effect of poly-
phenols from tea, red wine, and cocoa is thought to be
stronger than that of polyphenols from beans, but even
among beans, differences in polyphenol composition
and content lead to varying effects on iron bioavail-
ability [36]. The importance of such intervariety dif-
ferences in polyphenol content was also highlighted
by Hurrell et al. [33], who found a positive effect of
dephytinization of low-tannin but not of high-tannin
sorghum on iron absorption.
In vitro studies of enzymatic degradation of poly-
phenols confirmed that the presence of high amounts
of these compounds can mask the beneficial effect of
dephytinization [53]. However, at lower polyphenol
levels, the addition of an exogenous phytase even
reduced the amount of iron bound to polyphenols,
*Brnic M. Effect of the enzyme phytase and EDTA on
human zinc absorption from maize porridges fortified with
ZnSO4 or ZnO. Bioavailability Conference 2010. Asilomar
Conference Grounds, Pacific Grove, California, USA, 2010.
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S96
B. Troesch et al.
thereby potentially increasing iron bioavailability
beyond the benefit of liberating phytate-bound iron
[49]. The authors suggest that phytate hydrolysis prod-
ucts might complex polyphenols and consequently
reduce their iron-binding capacity. Addition of ascorbic
acid can also counter inhibition of iron absorption by
polyphenols [54]. Other options would be to remove
polyphenols from foods by soaking, cooking, or dehull-
ing them, or by breeding low-polyphenol varieties [36].
The zinc studies used soy-based [34, 40, 41] or
cereal-based [40–43]* meals, and all of them showed
a beneficial effect of dephytinization on zinc absorp-
tion or status (table 2). Even though less evidence is
available, the results from a rat study indicate that zinc
absorption might be inhibited by cow’s milk, most
likely by casein with a potential contribution of cal-
cium [55]. The effect of ascorbic acid on zinc absorp-
tion has not been studied as extensively as its effect
on iron absorption. The results from an absorption
study suggest that ascorbic acid does not enhance zinc
bioavailability [56]. Although the evidence is limited,
the results from Caco2-cell models indicate that some
degree of inhibition of zinc absorption by polyphenols
exists but that it is less extensive than for iron [57]. The
inhibition of absorption by polyphenols also seems to
be brought about by different mechanisms for zinc
than for iron.
In summary, phytase improves mineral bioavail-
ability from various phytate-rich foods, both when it is
used as a food ingredient, acting in the stomach of con-
sumers, and when it is used as a food processing aid,
acting during food manufacturing. The food matrix
appears to be less relevant, unless high concentrations
of other potent inhibitors or enhancers of absorption
are present.
Effect of conditions of use
In the studies that showed significant improvements
in iron absorption, phytate-to-iron molar ratios of
approximately 2:1 to 8:1 were reduced to 0.6:1 to
< 0.005:1 (table 1). In a slightly longer study, a signifi-
cant improvement in iron status was already achieved
when the ratio was reduced from 11:1 to 4:1 [45]. Based
on the evidence available from studies investigating
the effect of dephytinization with various methods,
it has been recommended that, in order to achieve a
relevant increase in iron absorption, the phytate-to-
iron molar ratio should be decreased at least to < 1:1,
and preferably to < 0.4:1 [50]. The studies that did not
show a significant improvement in iron absorption
found reductions from 0.6:1 to < 0.08:1 [30], from 3:1
to 0.1:1 [34], and from 5.8:1 to 0.1:1 [36]. In one of the
studies, the lack of effect was likely due as much to the
comparatively low initial phytate levels [30] as to the
relatively high ascorbic acid [30, 34] or polyphenol
[36] contents of the test meals. A recent review judged
a reduction to a phytate-to-iron ratio of < 6:1 in meals
containing some enhancers of iron absorption to be
sufficient [58].
The zinc studies cited show that statistically sig-
nificant effects were seen with residual phytate-to-
zinc ratios ranging from < 0.07:1 to 10.5:1 (table 2).
Since the reduction from 23.5:1 to 10.5:1 resulted in
a significant improvement of zinc absorption [41], a
relative reduction might already be beneficial, even
if the residual phytate content remains at an elevated
level. This is supported by a study investigating the
effect of dephytinization on serum zinc levels, which
found that a reduction from a phytate-to-zinc ratio
as low as 0.8:1 to 0.05:1 still had a positive impact on
apparent zinc absorption [59]. Thus, phytate inhibition
of zinc absorption may follow a dose–response rela-
tionship without a specific threshold for inhibition, as
was proposed for iron. However, zinc absorption only
increased up to twofold in the study with relatively
low phytate reduction and might further increase with
more complete phytate degradation. More evidence is
needed to draw firm conclusions.
In the three iron studies published at the time of
writing, amounts of approximately 20, 300, and 320
FTU (unit of phytase activity: the amount that liber-
ates 1 µmol of inorganic phosphorus per minute from
an excess of sodium phytate at 37°C and a pH of 5.5)
were added to 100-g portions of flour [37–39]. Despite
the differences in the amount of added enzyme, the
fold increase in iron absorption from iron sulfate was
comparable. However, this is likely due to the much
lower phytate-to-iron ratio in the study using the low
level of phytase. The rationale given in one study is that
the quantity of phytase needed was calculated assuming
that the meal contained approximately 1 g of phytate
[39]. The authors assumed that this had to be degraded
sufficiently, i.e., up to four phosphate units had to be
released per phytate molecule during the stomach resi-
dence time of approximately 60 minutes. It is reported
that phytates with five or six phosphate groups bind
iron sufficiently strongly to inhibit its absorption [60],
but there is also some evidence for an inhibitory effect
of phytates with only three or four groups, probably by
complexing iron between more than one molecule of
phytate [60]. As the phytase activity at the pH in the
stomach was estimated to be around 50% compared
with its activity under optimal conditions, 8 FTU
would be necessary to adequately degrade 1 µmol or
approximately 0.7 mg of phytate during a stomach resi-
dence time of approximately 1 hour [39]. Assuming a
phytate content of about 1 mg per 100 g of whole grain
flour, this would result in the addition of 320 FTU.
Since the increase in the absorption was less than in
*Brnic M. Effect of the enzyme phytase and EDTA on
human zinc absorption from maize porridges fortified with
ZnSO4 or ZnO. Bioavailability Conference 2010. Asilomar
Conference Grounds, Pacific Grove, California, USA, 2010.
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S97
Phytase human efficacy review
some of the studies in which phytate was degraded by
other means [28, 31, 33], a higher dose for meals with
comparable phytate levels might be indicated.
A study in pigs given feed containing 0, 15, and 90
FTU/100 g found a clear dose–response relationship,
with 7%, 22%, and 52% of phytate being degraded,
respectively, at the duodenal site [61]. With the higher
dose, around half of the inositol-penta-phosphate
was further hydrolyzed, and inositol-tri- and di-
phosphates dominated, whereas with the lower dose,
mainly penta-, tetra-, and tri-phosphates were found
[61]. Therefore, an increase in the dose of added
phytase might result in a more complete hydrolysis
with a further shift toward less phosphorylation and
consequently, an additional increase in bioavailability.
Degradation of higher inositol-phosphates further
along the small intestine is probably low, given the
pH conditions, the low solubility of phytate, and the
enzymatic digestion of the added phytase occurring in
the small intestine [62]. However, since iron is mostly
absorbed in the proximal part of the duodenum, later
degradation of phytate will not have a major impact
on its absorption. Evidence from a further study in
pigs indicates that complete phytate degradation, even
through the addition of exogenous phytases, is not
possible, as around one-third of it is protected from
hydrolysis due to its binding to the food matrix [21].
In summary, approximately 20 to 320 FTU per 100 g
of flour was demonstrated to improve iron bioavail-
ability significantly. Higher doses of supplementary
phytase may improve mineral bioavailability even
further, as suggested by the results of studies achiev-
ing complete dephytinization of the foods. Further
studies are warranted to explore the potential for more
complete phytate degradation during stomach transit
time. For zinc, no information on the dose in the one
study that used the active phase was published, and the
effect of dosage should be studied in future studies to
optimize the use of phytase. Some indication can be
derived from a study in rats, in which the addition of
1,000 FTU of an A. niger phytase resulted in a doubling
of apparent absorption [63].
Outlook and conclusions
Iron and zinc deficiencies remain major public health
problems, especially in developing countries, and for-
tification programs often do not achieve the expected
results due to the low bioavailability of the minerals
resulting from high levels of inhibitors of absorption,
such as phytate. For example, the median intakes of
dietary iron and zinc met less than 50% of the require-
ments for Vietnamese children [18]. Fortified rice and
wheat flour intakes provided less than 60% and 10% of
the iron and zinc requirements, respectively, to children
in Vietnam [18]. The beneficial effect of phytase will
be most pronounced on diets with very high levels of
phytate containing little or no enhancers of mineral
absorption, such as diets based on plant staples such as
maize and wheat. Although the typical complementary
foods consumed in developing countries were shown
to contain significant amounts of minerals, including
iron and zinc, these are often poorly available because
of the presence of high levels of phytate. They may
provide 2.5 to 12.0mg of iron as well as 1.0 to 10.4 of
mg zinc daily [64] that could potentially be made at
least partially available by adding phytase either during
preparation or just before consumption. This could
reduce the amount of minerals added to the diet via
fortification and supplementation. Besides cost savings,
such an approach might also have advantages from a
safety point of view, since concerns have been raised
about negative health effects of supplemental iron in
the presence of infections, particularly malaria and
diarrheal diseases [65]. Providing adequate amounts of
bioavailable iron while minimizing the intake of added
iron might therefore be advisable.
Moreover, evidence is emerging that unabsorbed
iron has a negative effect on the gut flora, shifting its
composition toward a more pathogenic profile [66].
Even though the iron complexed by phytate is not
available for absorption, it appears to be available to
the bacteria in the large intestine. Extensive phytate
degradation by microbial phytases in the colon of pigs
was shown, despite the fact that most of the phytate was
in the solid phase by that time and therefore was less
accessible to enzymes [62]. When normal and germ-
free rats were compared, a 56% difference in phytate
degradation was observed [67]. Colonic phytate degra-
dation in pigs was actually increased when less phytate
was degraded in the stomach and small intestine [62].
The increase in bioavailability as a result of the addition
of phytase makes a reduction in iron intake possible
while keeping the amount of absorbed iron constant.
Whether this reduction in unabsorbed iron translates
into a relevant impact on the gut microbiota needs to
be shown by in vitro and long-term studies.
A potential risk associated with phytase is increased
bioavailability of heavy metals such as arsenic, cad-
mium, lead, and mercury. However, data from rats
indicate no increase in cadmium uptake and only a
tendency toward increased bone lead levels following
a diet with high levels of these heavy metals as well as
a microbial phytase [68]. Blood lead levels measured
during an intervention study with phytase as part of
a micronutrient powder were unaffected by the use
of phytase (unpublished data from a study described
by Troesch et al. [46]). Moreover, improving iron
status by increasing iron bioavailability is thought to
have a positive effect on lead levels, possibly by down-
regulating transport proteins used for lead and iron
uptake [69]. The bioavailability of arsenic is already
quite high [70], and it is highly unlikely that phytase
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S98
B. Troesch et al.
will contribute significantly to the intake of inorganic
arsenic. Mercury exposure is predominantly associated
with the consumption of fish and other seafood. Expo-
sure to mercury in cereals and a potential increase in
bioavailability with the use of phytase is therefore not
relevant. Consequently, the risk of increased exposure
to heavy metals due to the inclusion of phytase into the
largely plant-based diets in developing countries can be
regarded as minimal.
Iron and zinc deficiencies are thought to affect cog-
nitive development, and evidence, albeit somewhat
inconsistent and limited, from humans and animals
exists to support a causal relationship [71, 72]. A recent
systematic review based on available studies found a
mildly positive effect on cognitive outcomes from iron
supplementation of infants, children, and adolescents
[73]. Improving iron and zinc status, by the addition
of phytase or by other means, may therefore have a
beneficial impact on brain and mental development.
Even though the prevalence of childhood stunt-
ing decreased from approximately 40% in 1990 to
approximately 27% in 2010, it is estimated that around
171 million children aged 0 to 5 years still suffer from
impaired growth worldwide, most of them in develop-
ing countries [17]. Around 70 million preschool chil-
dren in South Central Asia and 15 million preschool
children in Southeast Asia are affected. Moreover,
stunting is estimated to be the cause of approximately
15% of deaths in children under 5 years of age, cor-
responding to 1.5 million lives lost in 2004 [74]. For
normal growth and formation of the skeleton, adequate
supplies of macronutrients, various vitamins, as well
as minerals such as calcium, phosphorus, magnesium,
and zinc, are essential [75]. Because these so-called
type II nutrients are not stored in the body outside
functional tissue and are lost if tissue is lost (such as
during weight loss in times of malnutrition), they have
to be replaced in balance to allow for tissue synthesis
needed for growth [76]. Consequently, the effect of one
of these nutrients on growth can only be studied if all
the others are available in adequate amounts [76]. Zinc
intervention studies aiming at improving growth often
show only a small benefit [77]; a potential reason for
this is that other nutrients essential for growth, such as
phosphorus, are often missing or are poorly available
from the typically monotonous, plant-based diets with
little or no animal products. Yet, specific data on the
effect of phosphorus on growth in humans are very
limited, and studies evaluating the intake of this min-
eral hardly seem to take bioavailability into account.
Phytate serves as the most important phosphorus
storage compound of the plant [19], with 60% to 80%
of phosphorus bound to this molecule [78]. Phytase is
added to animal feed to reduce the amount of added
phosphorus by improving the absorbability of phytate–
phosphorus, while still allowing for rapid growth of
the animals [78]. This makes a strong case for the
essentiality of phosphorus for physical development.
Its role in catch-up growth as well as the prevention of
stunting should be studied in humans in more detail,
as this might be another application where the addition
of phytase is potentially beneficial. To our knowledge,
no study has examined the effect of the phosphorus
released from phytate by the addition of phytase on
growth in infants, but, given its importance in bone
formation [75], a beneficial effect is conceivable.
The evidence summarized in this review clearly
shows the beneficial effect on iron and zinc absorp-
tion of phytase added either during processing or as
an active food ingredient. It makes a strong case for
using the enzyme to improve mineral bioavailability
from plant-based foods in developing countries, but
also from vegetarian diets in the Western world. Fur-
ther studies can be expected to strengthen the case for
phytase use to improve growth and cognitive develop-
ment thanks to its impact on the absorption of iron,
zinc, calcium, magnesium, and phosphorus, especially
in Southeast Asia, one of the regions most affected by
iron and zinc deficiencies.
Acknowledgments
Barbara Troesch, Hua Jing, and Ann Fowler are
employed by DSM Nutritional Products Ltd., a pro-
ducer of enzymes for human consumption, including
a phytase from Aspergillus niger.
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