JFS:

Sensory and Nutritive Qualities of Food

Phytase and Citric Acid Supplementation in

Whole-Wheat Bread Improves Phytatephosphorus Release and Iron Dialyzability
J.M. PORRES, P. ETCHEVERRY, D.D. MILLER, X.G. LEI

ABSTRACT: Conditions were established for maximizing phytate breakdown in whole-wheat flour (wwf) during
bread baking and for assessing the effects of dephytinization on dialyzability of intrinsic and added iron in the
bread. Three different sources of phytase (Aspergillus niger, A. fumigatus, and Escherichia coli) with various levels of
citric acid (0 to 6.25 g/kg wwf) were used. Supplementing citric acid at 6.25 g/kg wwf enhanced phytate degradation
catalyzed by intrinsic phytase from 42% in the untreated bread to 69% (P , 0.05). Supplementation of microbial
phytase (285 units/kg) plus 3.125 or 6.25 g citric acid/kg wwf further enhanced phytate reduction up to 85%. Compared with the untreated bread, citric acid alone and the combination of citric acid and phytase enhanced total iron
dialyzability by 12- and 15-fold, respectively, while the combination of phytase, citric acid, and ascorbic acid
improved total iron dialyzability in the mixture by 24-fold.
Keywords: whole-wheat bread, phytase, citric acid, iron, phytate.

Introduction

to be a reliable indicator of the availability of iron from different foods (Schricker and others 1981; Miller and Berner
areas around the world and its negative health conse- 1989; Kapsokefaulou and Miller 1991). Therefore, our objecquences in the general population demand constant re- tives for this study were: (1) to determine the minimal
search for ways to improve iron availability from foods. amounts of extrinsic phytase from three different sources,
Legumes and cereals are significant sources of iron, but and the amount of citric acid needed to maximize phytate
their nutritional importance is generally compromised by degradation and free phosphorus release during wholethe presence of phytate. This compound forms complexes wheat bread preparation; and (2) to determine if there was a
with di- and trivalent cations at the physiological pH con- synergistic effect of phytase and citric and ascorbic acids,
ditions of the small intestine, rendering them unavailable two well-documented enhancers of iron absorption, on total
for absorption (Cheryan 1980; Lnnerdal and others 1988). dialyzability of added and intrinsic iron in bread.
High levels of phytate in whole-wheat bread are associatMaterials and Methods
ed with nutritional deficiencies of iron and other essential
minerals (Reinhold 1971; Haghshenass and others 1972).
Whole-wheat flour and bakers yeast contain intrinsic Ingredients
phytase that can significantly reduce phytate concentraWhole-wheat flour with an extraction rate of 100% [extion during bread making if pH conditions are favorable traction rate is defined as the proportion (%) of the whole(Larsson and Sandberg 1991; Trk and others 1996).
wheat grain obtained as the finished flour], dry yeast (New
Phytases are a group of enzymes that sequentially Hope Mills, Moravia, N.Y., U.S.A.), and other ingredients
cleave orthophosphate groups from the inositol ring of used for the bread dough were purchased in local supermarphytic acid, increasing the amount of available free phos- kets. Ultrapure water (Barnsted Thermolyne Corporation,
phorus and decreasing phytates affinity for different cat- Dubuque, Iowa., U.S.A.) was used for all the breads preions (Lei and others 1993). In recent years, we have char- pared. Citric acid (granular, monohydrate, FW 210.14) was
acterized several phytases with different biochemical from J.T. Baker (Phillipsburg, N.J., U.S.A.). Aspergillus niger
properties and demonstrated their effectiveness in ani- phytase was a gift from BASF (Mt. Olive, N.J., U.S.A.) (5000
mal feeding (Han and others 1998; Stahl and others U/g). Aspergillus fumigatus and Escherichia coli phytases
1999). But the potential of these phytases for improving (350 and 1,500 U/mL, respectively) were expressed and purimineral availability of human food such as whole-wheat fied in our laboratory (Rodriguez and others 1999, 2000).
bread remains to be determined. Other researchers have One unit of phytase activity (PU) is defined as the amount of
used relatively high levels of microbial phytase and or- enzyme that liberates 1 mmol of inorganic phosphorus from
ganic acids during bread preparation to reduce its sodium phytate per minute at pH 5.5 and 37 8C.
phytate content ( Trk and Sandberg 1992). Although they
have obtained a good reduction of phytate in the bread, Dough formulation and analysis
there are concerns about the cost associated with high
A standard bread formula was used: whole-wheat flour
levels of supplemental phytase and the impact of high (425 g), double-distilled water (315 mL), dry yeast (9 g), salt (6
levels of acids on the bread palatability for certain g), and sugar (41 g). Citric acid and phytase were added to
groups of consumers.
the water prior to mixing all components. Dough was mixed,
The percentage of total dialyzable iron has been described proofed and baked in a commercial bread maker (Regal

HE WIDESPREAD PREVALENCE OF IRON DEFICIENCY IN MANY

Sensory and Nutritive Qualities of Food

614 JOURNAL OF FOOD SCIENCEVol. 66, No. 4, 2001

2001 Institute of Food Technologists

Phytase and Citrate in Whole-Wheat Bread. . .

Table 1Description of the different treatments applied
Treatments
Phytase source
Phytase amount
(PU/kg wwf)a
Citric acid
(g/kg wwf)

(1)
(2)
(3)
C CA/3.125 CA/6.25

(4)
CA/8

(5)
AN1

A. niger
1,125

3.125

6.25

(6)
AN2

(7)
AN3

A. niger A. niger
285
285

3.125

6.25

(8)
EC1

(9)
EC2

E. coli
285

E. coli
285

3.125

6.25

(10)
F1

(11)
F2

(12)
F3

A. fumigatus A. fumigatus A. fumigatus

285
285
570

3.125

6.25

3.125

a Phytase units added per kg of whole-wheat flour.

Kitchen Pro, Regal Ware, Inc. Kewaskum, Wis., U.S.A.). We

chose the bread machines whole-wheat bread program,
which consisted of a primary knead and rise for 10 and 25
min, respectively, and a secondary knead and rise for 20 and
30 min, respectively, followed by a 30-s punchdown and a final rise for 70 min. Baking time was 65 min. Temperature inside the bread dough during the first rise was 30 8C. This rose
to 37 8C after the secondary knead and remained constant

through the secondary rise, punch down, and final rise. Temperatures in the interior of the loaf reached 100 to 110 8C
during baking.
After baking, breads were cooled to room temperature,
cut into small pieces, frozen, and freeze-dried (Labconco
Corp, Kansas City, Mo., U.S.A.). Freeze-dried breads were
ground in a domestic grinder (Oster Corporation, Milwaukee, Wis., U.S.A.), while samples taken for the time-course
experiment were ground with a mortar and a pestle. All the
samples were stored at 4 8C until analysis. Dry matter content of the bread was determined by drying for 48 h at
105 8C. Dough pH in samples collected at different times of
the proofing process was measured using an IQ 240 pH / mV
/ Thermometer fitted with a model pH16-SS 3.5 mm Micro
Probe (IQ Scientific Instruments, Inc., San Diego, Calif.,
U.S.A.). Loaf volumes of the different breads were determined using millet displacement.

Phytase and citric acid treatments

A total of 11 treatments were tested in normally processed whole-wheat bread (treatments 1 -11, Table 1). A 12th
treatment was included for a time-course study. Treatments
1 to 4 were included to determine the amount of citric acid
needed to provide an optimal pH condition for maximal
phytate hydrolysis catalyzed by the intrinsic phytase in the
flour. Four levels of citric acid (CA): 0, 3.125, 6.25 and 8 g/kg
whole-wheat flour (wwf ) (C, CA/3.125, CA/6.25, CA/8), were
added to the formulation. Treatment 5 was to test the effect
of a high level of supplemental A. niger phytase (1,125 PU/kg)
alone on phytate hydrolysis in dough (AN1). Treatments 6 to
11 were to determine the effects of three different phytases
(A. niger, E. coli, and A. fumigatus) at 285 PU/kg combined
with two levels of citric acid (3.125 or 6.25 g/kg) (AN2 and
AN3, EC1 and EC2, F1 and F2, respectively).

A study was conducted to determine the time-course of

phytate degradation and free phosphorus release by intrinsic
and (or) extrinsic phytases at different levels of citric acid.
Samples were collected initially and at six different timepoints of proofing (15, 30, 60, 90, 120, and 150 min), and assayed for phytate and free phosphorus. Five treatments were
included: C, CA/6.25, AN3, F1, and F3 (Table 1). Treatment
F3 was to assess the effect of a relatively high dose of phytase
(570PU/kg wwf) combined with 3.125 g CA/kg wwf on the efficiency of phytate degradation during proofing time.
Figures 1a and 1bTime-course of phytate degradation
and free phosphorus release from bread dough (mg/g DM)
Each point in a curve is the mean of three different replicates. The different treatments are presented in Table 1.
Time-repeated measurement analysis showed a time effect (P , 0.0001) and a timetreatment interaction
(P , 0.0001)

Iron dialyzability
Iron dialyzability was assayed according to Kapsokefaulou
and Miller (1991). In all treatments, 279 g Fe/g bread was
added as the extrinsic iron. The source was from an
Fe(NO3)3 solution with a concentration of 1,000 mg Fe/L
Vol. 66, No. 4, 2001JOURNAL OF FOOD SCIENCE

615

Sensory and Nutritive Qualities of Food

Time-course study

Phytase and Citrate in Whole-Wheat Bread. . .

Table 2Phytate content and free phosphorus released
(mg/g DM) from different breads
Phytate
(mg/g DM)
WWF
(1)
C
(2) CA/3.125
(3) CA/6.25
(4) CA/8
(5) AN1
(6) AN2
(7) AN3
(8) EC1
(9) EC2
(10) F1
(11) F2

15.26 6 0.75
8.84 6 0.37a
7.21 6 0.56b
4.64 6 0.25e
6.88 6 0.42bc
6.51 6 0.56c
4.04 6 0.11f
2.23 6 0.09g
5.70 6 0.25d
3.65 6 0.21f
2.54 6 0.37g
2.28 6 0.20g

Results

% Phytate
reduction

Phosphorus *
(mg/g DM) *

Effects of citric acid and extrinsic phytase

supplementation

0%
42%
52.8%
69.6%
54.9%
57.3%
73.5%
85.4%
62.6%
76.1%
83.4%
85%

0.18 6 0.04
1.47 6 0.10a
1.94 6 0.04bc
2.05 6 0.06cd
1.87 6 0.02b
1.85 6 0.11b
2.24 6 0.02e
2.69 6 0.06f
2.08 6 0.08d
2.33 6 0.06e
2.53 6 0.07g
2.75 6 0.04f

Bread making without supplementation with either

citric acid or phytase (C) resulted in a 42% reduction in
phytate content ( Table 2). Adding different concentrations of citric acid to the dough enhanced phytate degradation (P , 0.05). Specifically, the level of 3.125, 6.25, and
8 g CA/kg wwf (CA/3.125, CA/6.25, CA/8) gave a total of
52, 69, and 55% phytate reduction in bread, respectively.
Probably due to the inhibition on yeast fermentation by
the lowered pH and (or) high levels of citric acid, 6.25
and 8 g CA/kg wwf caused 10.2 and 18.1% decrease in the
final volume of the bread compared to the control, respectively. Supplementation of 1,125 PU/kg wwf in the
absence of citric acid (AN1) reduced the levels of phytate
similarly to CA/3.125 and CA/8 compared to the untreated bread (C). In contrast, supplementation of 285 PU/kg
wwf with either 3.125 or 6.25 gCA/kg wwf (AN2 and AN3)
significantly improved phytate degradation. Similar
phytate reductions (83.4 against 85%) were produced by
A. fumigatus phytase at these two levels of citric acid addition (F1 and F2), whereas A. niger phytase was more effective at 6.25 (AN3) than 3.125 g CA/kg wwf (AN2). At either level of citric acid, E. coli phytase was less effective
than the other two phytases in hydrolyzing phytate. The
effects of all these treatments on free phosphorus release
in the breads were consistent with their effects on
phytate degradation.

* Values are expressed as means 6 standard deviation (n = 3).

a, b, c, d, e, f, g Means within the same column without same superscript are

significantly different (P , 0.05).

(Certified Atomic Absorption Standard , Fisher Sl124-100).

Ascorbic acid (Sigma, St. Louis, Mo., U.S.A.) was added at 4.4
mg/g bread as an iron dialyzability enhancer. Bread samples
were digested with pepsin for 2 h in a 50 mL digestion vial at
37 8C in a shaking water bath. Afterwards, a dialysis bag containing 10 mL of 0.2 N Pipes (pH 7.0) was placed in each digestion vial for 30 min. Then, 5 mL of pancreatin-bile mixture (2 mg/mL pancreatin, 12 mg/mL bile mixture) (Sigma,
St. Louis, Mo., U.S.A.) was added to each digestion vial and
incubated for another 2 h. At the end of this incubation, dialysis bags were removed from the digestion vials and rinsed
by dipping in water. Bag contents were transferred to beakers and their contents weighed. Total and ferrous Fe were
measured in the dialysates using ferrozine according to Kapsokefalou and Miller (1991). Total dialyzable Fe was expressed as percentage of the total Fe present in each vial.

Phytate and free phosphorus determination

Phytate and free phosphorus content were measured in
bread and dough as described by Latta and Eskin (1980) and
Chen and others (1956). Samples were extracted with 2.4%
HCl for 90 min and centrifuged for 15 min at 2,000g (Model
GS-6KR, Beckman, Palo Alto, Calif., U.S.A.). The supernatant was diluted with ultrapure water in a ratio 1:2.5 or 1:3
depending on phytate concentration, and 15 mL of this dilution were run through a column packed with DOWEX 14
to 400 (200 to 400 mesh) strong basic anion exchange resin.
After this, the column was subsequently washed with 15 mL
of water and 15 mL of 0.1 M NaCl. All these three washes
were pooled together and free phosphorus was assayed in
that mix. Phytic acid was finally eluted from the column using a 0.7 M NaCl solution and measured using the Wade Re- Figure 2Effect of phytase, citric acid, and ascorbic acid
agent (0.3% sulfosalicylic acid and 0.03% FeCl3 6H2O).
on total dialyzable Fe from whole-wheat bread. Each bar

Sensory and Nutritive Qualities of Food

Statistics
Data was analyzed using one-way ANOVA (SAS Institute, Inc.
1988). Duncans Multiple Range test was applied to determine
significance of differences in iron dialyzability, phytate, and free
phosphorus content of whole-wheat bread between various
treatments. Time-repeated measurement analysis was applied
to the time-course data. Significant differences between individual points of the various bread treatments were analyzed using Duncans Multiple Range test. The level of significance was
set at 0.05.

616 JOURNAL OF FOOD SCIENCEVol. 66, No. 4, 2001

represents the mean 6 SD of three different replicates.

Values without sharing a common letter differ (P , 0.05).
Abbreviations: HClControl without sample, phytase or
Fe added. FeControl with the same volume of Fe standard added to all the samples. wwf + Fe, C + Fe, AN1 +
Fe, CA/6.25 + Fe, CA/8 + Fe, AN3 + FeAs expressed in
Table 1 plus iron standard (279 mg Fe/g bread) added
prior to pepsin digestion. wwf + Fe + AA, C+ Fe + AA,
AN3 + Fe + AAAs expressed in Table 1 plus iron standard (279 mg Fe/g bread) and ascorbic acid (4.4 mg AA/
g bread) added prior to pepsin digestion. Fe + AAControl with the same volume of Fe standard and ascorbic
acid as in all other samples.

Phytase and Citrate in Whole-Wheat Bread. . .

Table 3Phytase activity present in whole-wheat flour and
different bread doughs after 2 h leavening (Units/kg DM)
Treatment

Phytase activity*

WWF
Bread doughs
C (1)**
CA/6.25 (3)
AN3 (7)
F1 (10)
F3 (12)

505.95 6 18.73
706.53 6 10.04
678.94 6 4.92
972.41 6 45.77
1047.06 6 70.25
1435.66 6 102.00

* Values are expressed as mean 6 standard deviation (n = 3).

** See Table 1 for an explanation of the numbered treatments.

Time-course study

dough caused by different organic acids has been reported

previously (Trk and others 1996). The pH optimum described for wheat phytase is 5.2 (Peers 1953), although an effective phytate breakdown has been observed between pH
4.6 and 5.1 (Larsson and Sandberg 1991; Trk and others
1996). In the present study, we conducted a titration study
with various amounts of citric acid added to the dough to establish the pH and citric acid concentrations that optimize
the activity of intrinsic phytase from the whole-wheat flour.
The largest reduction in phytate levels was obtained when
6.25 g CA/kg wwf were added (CA/6.25), yielding a dough pH
of 4.6 to 4.8. Adding more (8 g/kg wwf and a dough pH of 4.3
to 4.5) or less (3.125 g/kg wwf and a dough pH of 5.2 to 5.5)
citric acid also enhanced phytate reduction, but to a lesser
degree. While pH is clearly an important factor in phytase
activity, the improvement in phytate breakdown by citric
acid supplementation could be partially due to the capacity
of the organic acid to complex some of the minerals bound
to the phytate molecule, rendering it more susceptible to
phytase attack (Maenz and others 1999). Several groups (Reinhold 1975; Harland and Harland 1980; Harland and Frlich
1989; Trk and others 1996; Trk and others 2000) have reported that yeast phytase contributes to phytate breakdown
during bread making. In our study, phytase activity was
greater in the doughs without any extrinsic phytase supplementation than phytase activity in the whole-wheat flour
used (Table 3). But it is unclear whether this increment of
phytase activity was from the yeast or from the activation of
intrinsic phytase.

There was a time effect (P , 0.0001) and a timetreatment

interaction (P , 0.0001) on phytate degradation and free
phosphorus release from whole-wheat bread catalyzed by
phytase in the presence of citric acid (Figures 1a and 1b). After 90 min, the improvements by phytase and citric acid over
the untreated control were significant. The combination of
570 PU of A. fumigatus phytase with 3.125 g CA/kg wwf (F3)
(Table 1) was the most effective treatment, and the sharpest
reduction of phytate occurred between 60 and 90 min proofing. No significant changes in these two parameters were observed beyond 120 min. There was an increase of phytase activity in the dough of untreated (C) and supplemental 6.25 g
CA/wwf (CA/6.25) after 2 h leavening. When extrinsic
phytases from different sources were supplemented, a conExtrinsic phytase is needed to maximize phytate
comitant increase in activity was detected (Table 3).

degradation in bread dough

Iron dialyzability

Trk and Sandberg (1992) have demonstrated the potential of microbial phytase for phytate breakdown in wholewheat bread. However, the high concentrations of phytase
and lactic acid used in their study may limit the possible applications of this approach. Thus, we were interested in supplementing with lower concentrations of both organic acid
and extrinsic phytase in the bread that still produce significant reductions in its phytate levels.
Our results clearly indicate that this goal is achievable by
using the appropriate combination of microbial phytase and
citric acid. Supplementation with high levels of phytase in the
absence of citric acid (AN1) yielded a phytate breakdown
that was only slightly better than the untreated bread (C).
Therefore, supplementation of bread dough with organic acids is required to achieve maximal activity of extrinsic
Discussion
phytase, as is the case with intrinsic phytase. This is not surIGH LEVELS OF PHYTATE IN WHOLE - WHEAT BREAD (T ER - prising in view of the high pH of bread doughs (5.7 to 6.8)
Sarkissian and others 1974) can dramatically inhibit the which is not optimal for the phytase present in the flour or
absorption of some minerals. This may seriously compro- the added extrinsic phytases. Phytase from A. fumigatus was
mise the health status of individuals who rely on it as staple the most efficient enzyme at both concentrations of citric
food (Hagshenass and others 1972; Reinhold and others acid. This enzyme has a pH optimum closer to the condi1973; Brune and others 1992). In recent years, phytases with tions for bread making used in this study than the other two
different biochemical properties have been developed and phytases, and is more effective in the hydrolysis of inositol
extensively applied in the field of animal nutrition (Han and phosphates with lower degrees of phosphorylation (Wyss
others 1998; Stahl and others 1999). But there are few reports and others 1999). In contrast, the pH optimum of E. coli
actually dealing with their applications to human nutrition phytase is more acidic than the lowest dough pH in our
(Trk and Sandberg 1992; Sandberg and others 1996; Greiner study, precluding its full action.
and Konietzny 1998, 1999).

Total iron dialyzability from wwf and untreated bread

(C 1 Fe) was 0 and 1.8%, respectively. Supplementation of
1,125 PU in the bread in the absence of citric acid (AN1 + Fe)
did not increase dialyzability (Figure 2). When 6.25 or 8 g CA/
kg wwf were added to the dough (CA/6.25 1 Fe, CA/8 1 Fe),
iron dilalyzability of the bread was 12-fold higher than C 1
Fe. The combination of phytase and 6.25 g CA/kg wwf (AN3
1 Fe) further increased iron dialyzability to a value that was
15-fold higher than the untreated bread. Ascorbic acid improved (P , 0.05) iron dialyzability in the wwf 1 Fe and C 1
Fe groups. The combination of phytase, citric, and ascorbic
acid (AN3 1 Fe 1 AA) resulted in a 24-fold increase in iron
dialyzability compared to C 1 Fe.

The intrinsic phytase-catalyzed phytate breakdown in

bread is increased by citric acid
The improved effectiveness of intrinsic phytase from
whole-wheat flour in response to acidification of the bread

Maximal degradation of phytate by phytase requires

2 h of leavening
The effects of phytase and citric acid on phytate breakdown and phosphorus release were time-dependent. The
combination of 570 PU (A. fumigatus) and 3.125 g CA/kg wwf
Vol. 66, No. 4, 2001JOURNAL OF FOOD SCIENCE

617

Sensory and Nutritive Qualities of Food

Phytase and Citrate in Whole-Wheat Bread. . .

(F3) was the most effective treatment, and no appreciable
difference was observed for phytate hydrolysis or free phosphorus release between time-points 120 and 150 min. This
suggested that leavening time of 120 min was adequate for
maximal hydrolysis of phytate by phytase.
Differences in free phosphorus release between F1 and
AN3 observed during the last points of the time-course study
are most probably due to the higher affinity of A. fumigatus
phytase for inositol phosphates with lower degree of phosphorylation (Wyss and others 1999). This characteristic of A.
fumigatus phytases may be used to produce cooperative or
synergistic phytate hydrolysis with other types of phytases.

Phytase, citric acid, and ascorbic acid collectively

improve iron dialyzability

Sensory and Nutritive Qualities of Food

Iron deficiency is a widespread nutritional problem in

many areas of the world (Fairbanks 1999) and can have very
serious consequences for the welfare and health status of the
general population. To study how our treatments could affect the availability of iron from whole-wheat bread, we have
used an in vitro technique based on the expected improvement of iron absorption when the solubility of this mineral is
increased. Citric acid improves iron dialyzability under our
experimental conditions. This positive effect of citric acid has
been previously described not only for iron but also for other trace minerals (Clydesdale 1983; Hazell and Johnson 1987;
Anand and Seshadri 1995; Walter and others 1998). With its
capacity to form complexes with minerals at pHs common
in the lumen of the small intestine, citric acid may help
maintain iron in a soluble form. Phytate present in foods of
vegetable origin is a well-recognized inhibitor of nonheme
iron absorption (Gillooly and others 1983; Stahl and others
1999), and its reduction has consistently improved the availability of this mineral from bread (Brune and others 1992).
Phytate hydrolysis is also desirable to increase the availability
of other nutritionally important minerals like zinc and phosphorus (Lei and others 1993; Han and others 1998). The ability of phytase supplementation to further reduce phytate
present in the bread supplemented with 6.25 g CA/kg wwf
(AN3 + Fe) gave rise to a significant increase in iron dialyzability compared to (CA/6.25 1 Fe), and reached a value 15fold higher than (C 1 Fe).
The beneficial effects of ascorbic acid as an enhancer of
iron absorption (Cook 1983; Monsen 1988; Kapsokefalou and
Miller 1991) are clearly observed in our experiments. Adding
ascorbic acid caused a significant increase in the dialyzability
of iron from all the breads studied. Most importantly, the
combination of citric acid, ascorbic acid, and phytase offers
a great potential in preventing nutritional iron deficiency, as
it dramatically improves iron dialyzability without the need
of costly or technologically advanced food processing operations. These results should, however, be corroborated with
an in vivo study.
In conclusion, the combination of phytase and citric acid
yields a significant reduction of phytate in whole-wheat
bread. Supplementation with phytase, citric acid, and ascorbic acid greatly improved iron dialyzability from this meal,
and could be a promising approach to prevent nutritional
iron deficiency and anemia.

References
Anand A, Seshadri S. 1995. A quantitative model for prediction of iron availability from Indian meals: An experimental study. Int J Food Sci Nutr 46(4):335342.
Brune M, Rossander-Hultn L, Hallberg L, Gleerup A, Sandberg AS. 1992. Iron
absorption from bread in humans: Inhibiting effects of cereal fiber, phytate,

618 JOURNAL OF FOOD SCIENCEVol. 66, No. 4, 2001

and inositol phosphates with different numbers of phosphate groups. J Nutr

122(3):442-449.
Chen PS, Toribara TY, Warner H. 1956. Microdetermination of phosphorus. Anal
Chem 28(11):1756-1758.
Cheryan M. 1980. Phytic acid interactions in food systems. CRC Crit Rev Food Sci
Nutr 13(4):297-336.
Clydesdale FM. 1983. Physicochemical determinants of iron availability. Food
Technol 37(10):133-138, 144.
Cook JD. 1983. Determinants of nonheme iron absorption in man. Food Technol
37(10):124-126.
Fairbanks VF. 1999. Iron in medicine and nutrition. In: Shils ME, Olson JA, Shike
M, Ross AC, editors. Modern nutrition in health and disease. 9 th ed.
Baltimore(Md): Williams and Wilkins. P 193-221.
Gillooly M, Bothwell TH, Torrance JD, MacPhail AP, Derman DP, Bezwoda WR,
Mills W, Charlton RW. 1983. The effects of organic acids, phytates, and polyphenols on the absorption of iron from vegetables. Brit J Nutr 49(3):331-342.
Greiner R, Konietzny U. 1998. Endogenous phytate-degrading enzymes are
responsible for phytate reduction while preparing beans (Phaseolus vulgaris). J Food Process Preserv 22(4):321-331.
Greiner R, Konietzny U. 1999. Improving enzymatic reduction of myo-inositol
phosphates with inhibitory effects on mineral absorption in black beans
(Phaseolus vulgaris var. preto). J Food Process Preserv 23(3):249-261.
Haghshenass M, Mahloudji M, Reinhold JG, Mohammadi N. 1972. Iron-deficiency anemia in an Iranian population associated with high intakes of iron. Am
J Clin Nutr 25(11):1143-1146.
Han YM, Roneker KR, Pond WG, Lei XG. 1998. Adding wheat middlings, microbial
phytase, and citric acid to corn-soybean meal diets for growing pigs may replace inorganic phosphorus supplementation. J Anim Sci 76(10):2649-2656.
Harland BF, Frlich W. 1989. Effects of phytase from three yeast on phytate reduction in Norwegian whole-wheat flour. Cereal Chem 66(4):357-358.
Harland BF, Harland J. 1980. Fermentative reduction of phytate in rye, white,
and whole-wheat breads. Cereal Chem 57(3):226-229.
Hazell T, Johnson IT. 1987. In vitro estimation of iron availability from a range
of plant foods: influence of phytate, ascorbate, and citrate. Brit J Nutr 57(2):223233.
Kapsokefalou M, Miller DD. 1991. Effects of meat and selected food components
on the valence of nonheme iron during in vitro digestion. J Food Sci 56(2):352355, 358.
Larsson M, Sandberg AS. 1991. Phytate reduction in bread containing oat flour,
oat brand, or rye bran. J Cereal Sci 14(2):141-149.
Latta M, Eskin M. 1980. A simple and rapid colorimetric method for phytate determination. J Agric Food Chem 28(6):1315-1317.
Lei XG, Ku PK, Miller ER, Ullrey DE, Yokoyama MT. 1993. Supplemental microbial
phytase improves bioavailability of dietary zinc to weanling pigs. J Nutr
123(6):1117-1123.
Lnnerdal B, Bell JG, Hendrickx AG, Burns RA, Keen CL. 1988. Effect of phytate
removal on zinc absorption from soy formula. Am J Clin Nutr 48(5):1301-1306.
Maenz DD, Engele-Schaan CM, Newkirk RW, Classen HL. 1999. The effect of
minerals and mineral chelators on the formation of phytase-resistant and
phytase-susceptible forms of phytic acid in solution and in a slurry of canola
meal. Anim Feed Sci Technol 81(3-4):177-192.
Miller DD, Berner LA. 1989. Is solubility in vitro a reliable predictor of iron
bioavailability? Biol Trace Elem Res 19(1-2):11-24.
Monsen ER. 1988. Iron nutrition and absorption: Dietary factors which impact
iron availability. J Am Diet Assoc 88(7):786-790.
Peers FG. 1953. The phytase of wheat. Biochem J 53(1):102-110.
Reinhold JG. 1971. High phytate content of rural Iranian bread: A possible cause
of human zinc deficiency. Am J Clin Nutr 24(10):1204-1206.
Reinhold JG. 1975. Phytate destruction by yeast fermentation in whole-wheat
meals. J Am Diet Assoc 66(1):38-41.
Reinhold JG, Lahimgarzadeh A, Nasr K, Hedayati H. 1973. Effects of purified
phytate and phytate-rich bread upon metabolism of zinc, calcium, phosphorus,
and nitrogen in man. Lancet 1:283-288.
Rodriguez E, Mullaney EJ, Lei XG. 2000. Expression of the Aspergillus fumigatus
phytase gene in Pichia pastoris and characterization of the recombinant enzyme. Biochem Biophys Res Com 268(2):373-378.
Rodriguez E, Porres JM, Han, YM., Lei XG. 1999. Different sensitivity of recombinant Aspergillus niger phytase (r-PhyA) and Escherichia coli pH 2.5 acid phosphatase (r-AppA) to trypsin and pepsin in vitro. Arch Biochem Biophys
365(2):262-267.
Sandberg AS, Rossander L, Trk M. 1996. Dietary Aspergillus niger phytase increases iron absorption in humans. J Nutr 126(2):476-480.
SAS. 1988. SAS/STAT users guide (Release 6.03). SAS Inst Inc, Cary, NC.
Schricker BR, Miller DD, Rasmussen RR, Van Campen D. 1981. A comparison of
in vivo and in vitro methods for determining availability of iron from meals.
Am J Clin Nutr 34(10):2257-2263.
Stahl CH, Han YM, Roneker KR, House WA, Lei XG. 1999. Phytase improves iron
bioavailability for hemoglobin synthesis in young pigs. J Anim Sci 77(8):21352142.
Ter-Sarkissian N, Azar M, Ghavifekr H, Ferguson T, Hedayat H. 1974. High phytic
acid in Iranian breads. J Am Diet Assoc 65(6):651-653.
Trk M, Carlsson NG, Sandberg AS. 1996. Reduction in the levels of phytate
during wholemeal bread making; effect of yeast, and wheat phytases. J Cereal
Sci 23(3):257-264.
Trk M, Sandberg AS. 1992. Phytate degradation during breadmaking: Effect of
phytase addition. J Cereal Sci 15(3):281-294.
Trk M, Sandberg AS, Carlsson NG, Andlid T. 2000. Inositol hexaphosphate hydrolysis by bakers yeast: Capacity, kinetics, and degradation products. J Agric
Food Chem 48(1):100-104.
Walter A, Rimbach E, Most E, Pallauf J. 1998. Effect of citric acid supplements to a
maize-soya diet on the in vitro availability of minerals, trace elements, and

Phytase and Citrate in Whole-Wheat Bread. . .

The authors thank Deborah A. Ross, Orlena Cheng, Daniel Omar Maizon, Carol Roneker,
William A. House, and Wilson G. Pond for their valuable assistance. Dr. Porres was funded

in part by a grant from the University of Granada, Spain. The project was supported by the
Cornell Biotechnology Program.

Authors Porres and Lei are with the Department of Animal Science, and
authors Etcheverry and Miller are with the Department of Food Science,
Cornell University, Ithaca, N.Y. Please direct inquiries to X.G. Lei, Dept. of
Animal Science, 252 Morrison Hall, Cornell Univ., Ithaca, NY 14853 (e-mail:
x120@cornell.edu).

Vol. 66, No. 4, 2001JOURNAL OF FOOD SCIENCE

619

Sensory and Nutritive Qualities of Food

heavy metals. J Vet Med A. 45(9):517-524.

Wyss M, Brugger R, Kronenberger A, Rmy R, Fimbel R, Oesterhelt G, Lehmann
M, Van Loon APGM. 1999. Biochemical characterization of fungal phytases
(myo-inositol hexakisphosphate phosphohydrolases): Catalytic properties. Appl
Environ Microbiol 65(2):367-373.
Ms. 20000720