The Importance of Lactic Acid Bacteria for Phytate Degradation
during Cereal Dough Fermentation
ANNA REALE,†URSULA KONIETZNY,‡RAFFAELE COPPOLA,†
ELENA SORRENTINO,†AND RALF GREINER*,§
Distaam, Universita ` degli Studi del Molise, Via De Sanctis, 86100 Campobasso, Italy, Waldstrasse 5c,
76706 Dettenheim, Germany, and Centre for Molecular Biology, Federal Research Centre for
Nutrition and Food, Haid-und-Neu-Strasse 9, 76131 Karlsruhe, Germany
Lactic acid fermentation of cereal flours resulted in a 100 (rye), 95-100 (wheat), and 39-47% (oat)
reduction in phytate content within 24 h. The extent of phytate degradation was shown to be
independent from the lactic acid bacteria strain used for fermentation. However, phytate degradation
during cereal dough fermentation was positively correlated with endogenous plant phytase activity
(rye, 6750 mU g-1; wheat, 2930 mU g-1; and oat, 23 mU g-1), and heat inactivation of the endogenous
cereal phytases prior to lactic acid fermentation resulted in a complete loss of phytate degradation.
Phytate degradation was restored after addition of a purified phytase to the liquid dough. Incubation
of the cereal flours in buffered solutions resulted in a pH-dependent phytate degradation. The optimum
of phytate degradation was shown to be around pH 5.5. Studies on phytase production of 50 lactic
acid bacteria strains, previously isolated from sourdoughs, did not result in a significant production of
intra- as well as extracellular phytase activity. Therefore, lactic acid bacteria do not participate directly
in phytate degradation but provide favorable conditions for the endogenous cereal phytase activity
by lowering the pH value.
KEYWORDS: Inositol phosphates; lactic acid bacteria; phytase; phytate
Wholemeal bread is a staple food in many countries, because
whole cereal flours provide fiber, complex carbohydrates,
proteins, vitamins, and minerals. However, the presence of
significant amounts of phytate in the flours and the wholemeal
breads produced thereof interferes with mineral absorption.
Phytate [myo-inositol(1,2,3,4,5,6)hexakisphosphate], the major
storage form of phosphorus in the plant seed (1), forms insoluble
complexes with numerous divalent and trivalent metal cations,
particularly at slightly alkaline pH values (2), as prevailing in
the small intestine, the major site of mineral absorption in the
human gastrointestinal tract. The formation of insoluble metal
cation-phytate complexes at physiological pH values is re-
garded as the major reason for poor mineral availability, because
these complexes are essentially nonabsorbable from the gas-
trointestinal tract. Minerals of concern in this regard would
include Zn2+, Fe2+/3+, Ca2+, Mg2+, Mn2+, and Cu2+(3, 4).
Especially, zinc and iron deficiencies were reported as a
consequence of high phytate intakes (5, 6).
Because excessive amounts of phytate in the diet can lead to
mineral deficiencies, phytate should be avoided among vulner-
able groups and eliminated by extraneous processing efforts.
Lactic acid fermentation was reported to significantly reduce
the phytate content in plant-based foods (7-13) with a
concomitant improvement of mineral solubility (7, 8, 14). The
dephosphorylation of phytate is initiated by a class of enzymes
called phytases (15), and extracellular phytase activity was
suggested to be responsible for the observed reduction in phytate
content during lactic acid fermentation (8-11). However, the
majority of studies revealed that lactic acid bacteria did not show
significant extracellular phytase activities (11, 12, 16-18) and
the very low phytase activities observed were suggested to be
due to nonspecific acid phosphatases (17). So far, only Lacto-
bacillus amyloVorus and Lactobacillus plantarum were reported
to produce significant extracellular phytase activities (19). The
observed reduction in phytate content during lactic acid
fermentation might therefore be due to an activation of endog-
enous plant phytases or a coprecipitation of phytate and proteins
as a consequence of a fall in pH during fermentation. The
majority of plant grains and seeds exhibits phytase activity from
pH 3 to 10 with maximal activity at pH values between 5 and
5.5 (20). The pH of the unfermented plant material is in general
close to 7 and reaches a value of about 4 after complete lactic
acid fermentation, thus passing through the interval that was
shown to be optimal for plant phytases. Coprecipitation of
phytate with proteins has been demonstrated to occur in a whey
protein system when the pH was lowered simply by addition
* To whom the correspondence should be addressed. Tel: +49(0)721/
6625 479. Fax: +49(0)721/6625 457. E-mail: email@example.com.
†Universita ` degli Studi del Molise.
§Federal Research Centre for Nutrition and Food.
of lactic acid (21), and precipitation of complexes formed by
phytate and proteins has been reported for several other food
The objective of this work was to elucidate the role of lactic
acid bacteria in the degradation of phytate during lactic acid
fermentation in general and during sourdough fermentation in
MATERIALS AND METHODS
Materials. L. plantarum (DSM20174T, DSM2601), L. amyloVorus
DSM20531, Lactobacillus acidophilus DSM20079, and Lactobacillus
sanfranciscensis (DSM 1721) were obtained from the German Col-
lection of Microorganisms and Cell Cultures (Braunschweig, Germany).
L. plantarum (S18, S29), L. acidophilus S37, and Leuconostoc
mesenteroides (S18, S38, and S50) were kindly provided by Lopez
(INRA, Clermont-Ferrand, France). All other strains were obtained from
the Strain Collection of the Department of Science and Technology of
Agro-food, Environment and Microbiology, Universita ` degli Studi del
Molise. These strains were previously isolated from southern Italian
sourdoughs. Phytic acid dodecasodium salt was purchased from Aldrich
(Steinheim, Germany). Ultrasep ES 100 RP18 was obtained from
Bischoff (Leonberg, Germany), and AG1 X-8, 100-200 mesh, resin
was purchased from Bio-Rad (Mu ¨nchen, Germany). All reagents were
of analytical grade.
Screening for Phytate-Degrading Activity. Phytase production was
studied in PSM broth, MRS broth containing 3 mM Na-phytate, and
SDB broth containing 3 mM Na-phytate and glucose (20 g/L) or maltose
(20 g/L) or both glucose (10 g/L) and maltose (5 g/L) as a carbohydrate
source. PSM broth consisted of glucose (2 g/L), sodium citrate (0.1
g/L), Na-phytate (5 g/L), MgSO4× 7H2O (0.075 g/L), MnSO4× 1H2O
(0.01 g/L), Tween 80 (1 g/L), FeSO4 (0.005 g/L), L-glutamate (0.15
g/L), alanine (0.2 g/L), L-arginine-HCl (0.05 g/L), L-asparagine (0.2
g/L), L-cysteine (0.05 g/L), L-phenylalanine (0.04 g/L), L-histidine (0.04
g/L), L-isoleucine (0.06 g/L), L-leucine (0.06 g/L), l-lysine (0.05 g/L),
L-methionine (0.05 g/L), L-proline (0.04 g/L), L-tyrosine (0.002 g/L),
L-threonine (0.025 g/L), L-tryptophane (0.025 g/L), thiamine (0.0005
g/L), L-valine (0.015 g/L), vitamin B12 (0.001 g/L), biotin (0.001 g/L),
pantothenic acid (0.01 g/L), folic acid (0.001 g/L), niacin (0.01 g/L),
riboflavin (0.005 g/L), adenine (0.025 g/L), guanine (0.025 g/L), uracil
(0.025 g/L), and thymidine (0.025 g/L). The pH was adjusted to 6.
After cultivation at 30 °C, the cells were collected by centrifugation
at 7000g and 4 °C for 10 min and resuspended in 0.05 M Tris-HCl,
pH 7.5, containing 0.1 M CaCl2. The cells were collected again by
centrifugation at 8000g and 4 °C for 10 min and resuspended in 0.05
M Tris-HCl, pH 7.5, preheated to 30 °C. The suspension was incubated
at 30 °C for 30 min. Thereafter, the cells were collected by centrifuga-
tion at 9000g and 20 °C for 20 min, resuspended in 0.05 M Tris-HCl,
pH 7.5, containing 24% sucrose and 10 mM MgCl2, and incubated for
30 min at 37 °C. A lysozyme solution (5 mg/mL) was added to the
cell suspension to give a final of 0.1% (v/v), and the suspension was
incubated at 37 °C for 45 min. After centrifugation at 9000g and 20
°C for 20 min, the resulting pellets were resuspended in 0.05 M Tris-
HCl, pH 7.5, at 4 °C. Sphaeroplasts were disrupted by two cycles of
sonication (20 s for each treatment) and incubation for 30 min at 37
°C. The suspension was centrifuged at 14000g and 4 °C for 30 min.
The clear solutions (cytoplasmatic extracts) were dialyzed against 0.02
M sodium acetate buffer, pH 5, and used for enzyme assays.
Fermentation. For fermentation, 10 g of cereal flour was suspended
in 100 mL of water. Inactivation of the endogenous cereal phytases
was achieved by autoclaving these mixtures for 30 min at 121 °C.
Fermentation was started by inoculation with 109cfu of the individual
lactic acid bacteria strain. The inocula were collected by centrifugation
at 5000g for 10 min and washed twice with 0.9% NaCl. Fermentation
was performed at 30 °C on a rotary shaker at 200 rpm. Controls were
run at different pH values without inoculation.
Assay of Phytase and Acid Phosphatase Activities. Enzyme
activities were measured at 37 °C. The phytase activity in the cereal
flours was determined by suspending 1 g of dry-milled cereal grains
in 20 mL of 100 mM sodium acetate buffer, pH 4.5, containing 100
µmol of sodium phytate preincubated at 37 °C (25). After an incubation
period of 40 min, 400 µL of the incubation mixtures was removed and
1.5 mL of a freshly prepared solution of acetone/5 N H2SO4/10 mM
ammonium molybdate (2:1:1 v/v) and thereafter 100 µL of citric acid
were added (26). Any cloudiness was removed by centrifugation prior
to the measurement of absorbance at 355 nm. To calculate the enzyme
activity, a calibration curve was produced over the range of 5-600
nmol of phosphate. Activity (U) was expressed as µmol phosphate
liberated per minute. Correction for the initial phosphate content of
the samples was made. The incubation mixture for the lactic acid
bacteria phytase activity determination consisted of 350 µL of 0.1 M
sodium acetate buffer, pH 4.5, containing 2 µmol of sodium phytate.
The enzymatic reactions were started by adding 50 µL of the enzyme
preparations (cytoplasmatic extract, incubation broth) to the assay
mixtures. After an incubation period of 30 min, the liberated phosphate
was measured by addition of 1.5 mL of a freshly prepared solution of
acetone/5 N H2SO4/10 mM ammonium molybdate as described above.
Acid phosphatase was determined in 200 µL of 50 mM citrate-
NaOH, pH 4.5, containing 1 µmol of p-nitrophenyl phosphate. After
15 min, the reaction was stopped by adding 1.0 mL of 1 N NaOH.
The acid phosphatase activity was determined by measuring the
absorbance of the formed p-nitrophenolate at 405 nm. One unit of
enzyme was defined as the amount of acid phosphatase releasing 1
µmol p-nitrophenolate per minute.
myo-Inositol Phosphate Analysis. myo-Inositol phosphates were
extracted from the fermentation mixtures or the cultivation broths by
addition of HCl to a final concentration of 2.4% and shaking for 3 h
at room temperature. Quantification of myo-inositol phosphates was
performed as described by Sandberg and Ahderinne (27). The slurries
were centrifuged at 30000g for 30 min, and 1 mL of the supernatants
was diluted 1:25 with water and applied to a column (0.7 cm × 15
cm) containing AG1-X8, 100-200 mesh resin. The column was washed
with 25 mL of water and then with 25 mL of 25 mM HCl. The myo-
inositol phosphates were eluted with 25 mL of 2 M HCl. The eluates
obtained were concentrated in a vacuum evaporator to complete dryness.
The residues were dissolved in 500 µL of water, and 20 µL of these
solutions was chromatographed on Ultrasep ES 100 RP18 (2 mm ×
250 mm, 6.0 µm). The column was run at 45 °C and 0.2 mL min-1of
an eluant consisting of formic acid/methanol/water/TBAH (tetrabuty-
lammonium hydroxide) (44:56:5:1.5 v/v), pH 4.25. A mixture of the
individual myo-inositol phosphate esters (IP3-IP6) was used as a
Purification of the Rye Phytase. Purification of the phytase rye
was performed as described previously (28). The phytase was purified
to apparent homogeneity according to denaturing and nondenaturing
polyacrylamide gel electrophoresis.
Screening for Phytase Activities. Fifty strains of lactic acid
bacteria were screened for intra- as well as extracellular phytate-
degrading activity using different growth media. Neither
phosphate reduction (PSM) nor addition of sodium phytate
(MRS and SDB) or modification of the carbon source (SDB)
resulted in a significant production of intra- as well as
extracellular phytase activities in the studied strains. The
intracellular phytase activities determined ranged between 0.3
and 5.7 mU/mL, whereas the extracellular phytase activity could
not be observed. However, using p-nitrophenyl phosphate as a
substrate, significant intra- as well as extracellular acid phos-
phatase activities were detectable (data not shown). Evaluation
of phytate degradation during cultivation of the lactic acid
bacteria strains by high-performance liquid chromatography
confirmed the absence of significant extracellular phytase
activity. No phytate degradation could be established even after
10 days of cultivation at 30 °C in all of the growth broths used
(data not shown).
Lactic Acid Fermentation of Cereal Flours. Four lactic acid
bacteria strains [L. plantarum (DSM20174T, DSM2601), L.
amyloVorus (DSM20531), and L. acidophilus (DSM20079)],
which have been previously reported as phytase producers (19),
were used for fermentation experiments. Lactic acid fermenta-
tion of cereal flours resulted in a significant reduction in phytate
content with a concomitant increase in the concentrations of
the lower myo-inositol phosphates within 24 h (Table 1). All
strains showed similar pH and growth patterns. After 24 h, the
phytate reduction was determined to be 100% in rye, 95-100%
in wheat, and 39-47% in oat flour.
No significant phytate degradation was observed during
fermentation of heat-treated cereal flours. The heat treatment
resulted in a complete inactivation of the endogenous cereal
acid phosphatases including phytases. During fermentation, a
significant decrease in the content of the partially phosphorylated
lower myo-inositol phosphate esters (IP5and IP4) occurred in
the nonheat-treated inoculated samples but not in the nonin-
oculated controls. After 24 h of fermentation, IP5reduction was
determined to be 40-63% in rye, 48-61% in wheat, and 52-
79% in oat flour, whereas the reduction in IP4was found to be
78-91% in rye, 87-93% in wheat, and 82-91% in oat flour
within 24 h.
Phytate degradation during lactic acid fermentation after
addition of 6.75 U of purified rye phytase per gram of heat-
treated rye flour (Table 2) was not significantly different to
that observed with the native rye flour (contains 6.75 U/g of
endogenous phytase activity) (Table 1).
Incubation of cereal flours in buffered solutions resulted in a
pH-dependent phytate degradation (Table 3). The optimum of
phytate degradation by the endogenous cereal phytases was
shown to be within the pH range of the broth during lactic acid
Phytase production by lactic acid bacteria is still controver-
sially discussed within the scientific community. Particularly
regarding the importance of lactic acid bacteria phytase for
phytate degradation during sourdough fermentation, the scien-
tific data are interpreted as supporting the hypothesis that either
lactic acid bacteria phytase is significantly involved in phytate
degradation during sourdough fermentation (8, 10, 11) or the
intrinsic cereal phytases are responsible for phytate degradation
after being activated by a fall in pH due to lactic acid production
by the lactic acid bacteria (7, 12, 16, 29). To act on phytate,
phytates must have access to the phytate in the dough. Our
studies revealed that none of the 50 lactic acid bacteria strains
included produced measurable extracellular phytase activity in
MRS and SDB medium as well as in the phosphate-reduced
media PSM. A phosphate-reduced medium was included,
because a tight regulatory inhibition of the formation of phytases
by phosphate levels was generally observed in all microorgan-
isms (30). Phosphate was shown to exert its effect on the
synthesis of phytases at the level of transcription. The efficient
derepression of phytase formation by phosphate starvation in
most microorganisms suggests a possible role for these enzymes
in providing the cell with phosphate. The assumption could also
explain why, with the exception of sourdough bacteria, there is
no clear evidence for lactic acid bacteria with the ability to
degrade phytate. Lactic acid bacteria are adapted to environ-
ments rich in nutrients and energy where evolutionary selection
pressure would not favor the capability to produce a phytase.
In addition, our findings are in agreement with the majority of
studies on phytase production by lactic acid bacteria (11, 12,
16-18). So far, only L. amyloVorus and L. plantarum were
reported to produce significant extracellular phytase activity (19),
but we have been unable to reproduce this finding even when
using the same L. amyloVorus strain (DSM20531) under
identical growth conditions.
A low intracellular phytase activity was determined in all
lactic acid bacteria included in the study. This is in accordance
with the results reported by De Angelis et al. (11). The
intracellular phytase activity might be found in almost every
cell, since phytate is a common cellular constituent with a
significant turnover (31). However, it is very unlikely that
intracellular phytases are involved in extracellular phytate
dephosphorylation even if it could not be ruled out that phytate
is taken up by bacterial cells. It is not known, for example,
Table 1. myo-Inositol Phosphate Content of Cereal Flours during
Lactic Acid Fermentationa
12.0 ± 0.43 7.0
11.6 ± 0.4
9.2 ± 0.7
5.8 ± 0.3
0.2 ± 0.05 0.3 ± 0.02 3.9 ± 0.4
14.7 ± 0.70.5 ± 0.1
12.4 ± 0.41.7 ± 0.15 0.8 ± 0.05 0.4 ± 0.1
8.3 ± 0.3 3.4 ± 0.2
0.7 ± 0.1 2.1 ± 0.2
0.1 ± 0.02 0.2 ± 0.05 5.3 ± 0.3
12.9 ± 0.50.4 ± 0.1
12.5 ± 0.40.6 ± 0.1
11.4 ± 0.70.9 ± 0.1
9.6 ± 0.3 1.2 ± 0.2
7.2 ± 0.2 1.4 ± 0.3
0.4 ± 0.03
1.5 ± 0.2
1.9 ± 0.4
0.8 ± 0.1
2.5 ± 0.2
0.3 ± 0.04 11.8 ± 1.05 6.4
1.7 ± 0.211.9 ± 1.1
5.1 ± 0.4 9.5 ± 0.87 4.4
3.2 ± 0.34.9 ± 0.4
ND15.2 ± 0.8
15.3 ± 0.7
0.9 ± 0.115.7 ± 0.9
5.0 ± 0.4 14.9 ± 1.1
6.1 ± 0.5 11.7 ± 0.87 4.2
ND13.3 ± 0.6
ND13.2 ± 0.53 6.7
0.1 ± 0.01 13.0 ± 0.91 5.1
0.3 ± 0.1 13.0 ± 0.8
0.9 ± 0.112.9 ± 0.9
241.7 ± 0.1
3.1 ± 0.3
7.1 ± 0.4
0.1 ± 0.03
0.6 ± 0.1
1.9 ± 0.2
3.4 ± 0.3
aND, not detectable. The data are mean values ± standard deviation of three
independent experiments with each lactic acid bacteria strain used.
Table 2. myo-Inositol Phosphate Content of Heat-Treated Rye Flour
during Lactic Acid Fermentation after Addition of Purified Rye Phytasea
11.7 ± 0.6
9.5 ± 0.4
6.1 ± 0.2
0.4 ± 0.15
0.4 ± 0.05
1.4 ± 0.2
1.7 ± 0.3
0.2 ± 0.04
0.9 ± 0.11
2.2 ± 0.2
3.7 ± 0.3
1.6 ± 0.15
0.3 ± 0.07
1.6 ± 0.2
5.0 ± 0.4
3.0 ± 0.3
12.1 ± 0.65
12.1 ± 0.78
11.6 ± 0.9
9.3 ± 0.89
4.6 ± 0.45
aND, not detectable. The data are mean values ± standard deviation of three
Table 3. myo-Inositol Phosphate Content of Cereal Flours during
Incubation at Different pH Values without Inoculationa
10.9 ± 0.6
2.1 ± 0.2
7.1 ± 0.5
12.7 ± 0.7
5.1 ± 0.4
6.9 ± 0.5
2.8 ± 0.3
1.9 ± 0.2
2.9 ± 0.4
0.5 ± 0.08
1.5 ± 0.11
1.2 ± 0.1
1.2 ± 0.15
4.2 ± 0.3
1.4 ± 0.12
3.3 ± 0.2
2.1 ± 0.4
1.8 ± 0.2
0.3 ± 0.06
0.7 ± 0.1
5.7 ± 0.2
0.5 ± 0.04
4.6 ± 0.5
3.7 ± 0.2
3.1 ± 0.3
15.2 ± 1.11
0.7 ± 0.1
13.9 ± 0.9
11.9 ± 1.06
7.9 ± 0.7
13.2 ± 0.78
12.4 ± 1.11
13.0 ± 1.1
aIncubation was performed for 24 h at 30 °C on a rotary shaker at 200 rpm.
ND, not detectable. The data are mean values ± standard deviation of three
how bacteria with an apparent lack of extracellular phytase
activity, such as some Pseudomonas strains, either grow in the
absence of a readily utilizable phosphate source or acquire
phosphate. Because no phosphate was detected in the growth
medium either initially or throughout the growth period (32),
phytate might be transported into the bacterial cells. Further-
more, Wang et al. (33) suggested that in Klebsiella pneumoniae,
the gene encoding the phytase is cotranscribed from a polycis-
tronic mRNA, which also acts as a template for an inositol
Because of the apparent lack of lactic acid bacterial extra-
cellular phytase activity, the observed reduction in phytate
content during lactic acid fermentation might be due to an
activation of intrinsic cereal phytases as a consequence of a
fall in pH during fermentation. This suggestion was supported
by the findings that with all lactic acid bacteria used in this
study a similar decrease in phytate content was observed during
sourdough fermentation and the extent of phytate degradation
correlates very well with the intrinsic phytase activity of the
cereal flours. In addition, no significant phytate degradation was
observed during fermentation after heat treatment of the cereal
flours prior to fermentation. The heat treatment (autoclaving at
121 °C for 30 min) resulted in a complete inactivation of the
endogenous cereal acid phosphatases including phytases. Au-
toclaving was chosen for enzyme inactivation, because treatment
of the flours by microwave as described by Lopez et al. (8)
and de Angelis et al. (11), respectively, did not result in a
complete loss of the intrinsic phytase activity. Because a
significant decrease in the content of the partially phosphorylated
myo-inositol phosphate esters still occurred in the inoculated
samples, but not in the noninoculated controls, lactic acid
bacteria produced extracellular phosphatases capable of acting
on partially phosphorlyated myo-inositol phosphates. The pres-
ence of extracellular phosphatases, which do not act on phytate,
is in agreement with results obtained from screening studies.
The lack of phytate degradation could be overcome by addition
of a purified phytase to the fermentation broth. A further
confirmation for the importance of the intrinsic cereal phytases
came from the incubation of the cereal flours in buffered
solutions, which resulted in a pH-dependent phytate degradation.
The optimum of phytate degradation was shown to be close to
a pH value of 5.5. It was already reported that cereal phytases
exhibit maximal activity at pH values between 5 and 5.5 (20).
Lactic acid fermentation significantly reduces phytate content
in plant-based foods. This report clearly demonstrates that
phytate reduction is mainly due to the activity of the intrinsic
plant phytases. The importance of lactic acid bacteria for phytate
dephosphorylation is limited to providing favorable conditions
for the endogenous cereal phytases by lowering pH value. All
lactic acid bacteria strains used in this study lacked extracellular
phytase activity. However, even if a wild-type lactic acid
bacterium produces extracellular phytase activity, it is very
unlikely that its production will be sufficient to allow significant
phytate dephosphorylation during fermentation.
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Received for review December 5, 2006. Revised manuscript received
February 19, 2007. Accepted February 26, 2007.