International Journal of Food Microbiology 87 (2003) 259 270

www.elsevier.com/locate/ijfoodmicro

Phytase activity in sourdough lactic acid bacteria:

purification and characterization of a phytase
from Lactobacillus sanfranciscensis CB1
Maria De Angelis a, Giovanna Gallo b, Maria Rosaria Corbo c, Paul L.H. McSweeney d,
Michele Faccia e, Marinella Giovine b, Marco Gobbetti b,*
b

a
Istituto di Scienze delle Produzioni Alimentari, CNR, Bari, Italy
Dipartimento di Protezione delle Piante e Microbiologia Applicata, Facolta` di Agraria, Universita` degli Studi di Bari,
Via G. Amendola 165/a, Bari 70125, Italy
c
Istituto di Produzioni e Preparazioni Alimentari, Facolta` di Agraria, Universita` degli Studi di Foggia, Italy
d
Department of Food Science, Food Technology and Nutrition, University College Cork, Ireland
e
Dipartimento di Produzioni Animali, Facolta` di Agraria, Universita` degli Studi di Bari, Bari, Italy

Received 5 August 2002; received in revised form 15 November 2002; accepted 8 January 2003

Abstract
The phytase activity of 12 species of sourdough lactic acid bacteria was screened. It was intracellular only, largely
distributed among the species and strains of Lactobacillus sanfranciscensis possessed the highest levels of activity. A
monomeric ca. 50-kDa phytase was purified to homogeneity from L. sanfranciscensis CB1 by three chromatographic steps. L.
sanfranciscensis CB1 exhibited the highest hydrolysing activity on Na-phytate after reaching the stationary phase of growth (ca.
12 h). Cells cultivated in the presence of maltose and fructose showed an increase of the phytase activity of ca. 35% with respect
to the other carbon sources used. The phytase was optimally active at pH 4.0 and 45 jC. The enzyme was strongly inhibited by
2 mM of phenylmethylsulfonyl fluoride (PMSF), and 2 mM Hg2 + and Fe2 +. It had a pI of ca. 5.0. The substrate specificity was
dependent on the type of phosphate ester; a very low activity was detected on a-D-glucose-1-phosphate and D-fructose-6- and
1,6-phosphate, while the highest hydrolysis was found towards adenosine-5V-tri-, di- and mono-phosphate. Compared to these
substrates, the activity on Na-phytate was also relevant. The enzyme was thermo-stable after exposure to 70 jC for 30 min; the
D value calculated at 80 jC was ca. 10 min. As shown by the Central Composite Design (CCD) applied to study the individual
and interactive effects of pH, temperature and NaCl, acidic conditions and elevated temperatures were indispensable for the
enzyme adaptation to high NaCl concentrations. L. sanfranciscensis CB1 cells or the correspondent cytoplasmic extract were
used to ferment a sourdough for 8 h at 37 jC; a marked decreased (64 74%) of the Na-phytate concentration was found
compared with the unstarted dough. The sourdough started with L. sanfranciscensis CB1 cells was re-used for several times and
the phytase activity was maintained to a considerable level.
D 2003 Elsevier Science B.V. All rights reserved.
Keywords: Sourdough; Lactobacillus sanfranciscensis; Phytase

* Corresponding author. Tel.: +39-80-5442949; fax: +39-80-5442911.

E-mail address: gobbetti@agr.uniba.it (M. Gobbetti).
0168-1605/03/$ - see front matter D 2003 Elsevier Science B.V. All rights reserved.
doi:10.1016/S0168-1605(03)00072-2

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M. De Angelis et al. / International Journal of Food Microbiology 87 (2003) 259270

1. Introduction
Phytic acid [myo-inositol hexakis(dihydrogenphosphate)] constitutes 1 4% by weight of cereal grains
and oilseed mails, being a source of myo-inositol and
the major storage form of phosphorus. This molecule
is highly charged with six phosphate groups extending
from the central myo-inositol ring. For this property,
phytic acid is considered to be an antinutritional factor
for humans and animals as it acts as an excellent
chelator of cations such as Ca2 +, Mg2 +, Fe2 + and
Zn2 + and as it complexes the basic amino acid group
of proteins, thus decreasing the dietary bioavailability
of these nutrients (Wodzinski and Ullah, 1996; Dvorakova, 1998).
Phytase [myo-inositol hexakis(dihydrogenphosphate) phosphohydrolase, EC 3.1.3.8] catalyses the hydrolysis of phytic acid to myo-inositol and phosphoric
acid via penta- to mono-phosphates. This enzymatic
activity produces available phosphate and a non-metal
chelator compound. Phytases are considered to be
enzymes of great value in upgrading the nutritional
quality of phytate-rich foods and feeds (Martinez et al.,
1996).
Phytases can be derived from a number of sources including plants, animals and microorganisms.
Recent research has shown that microbial sources
are more promising for the production of phytases
on a commercial level and on cereal based foods.
Microbial phytases are easily produced and extracted when synthesized extracellularly in a culture
medium, and, in general, they may be synthesized
by the same microbial starter used for food processing. Natural or genetically modified strains of
bacteria such as Escherichia coli, Bacillus subtilis,
Bacillus amyloliquefaciens and Klebsiella sp.;
yeasts such as Schwanniomyces castellii, Schwanniomyces occidentalis, Hansenula polymorph and
Rhodotorula gracilis; and fungi such as Aspergillus
niger and Aspergillus ficuum are some of the most
important species used for the production of microbial phytases (for review, see Pandey et al., 2001).
Wholemeal bread is a staple fermented food in
many countries due to its nutritional value. Nevertheless, the presence of a high concentration of
phytic acid compromises the bioavailability of the
mineral fraction especially. Endogenous phytase
activity may be contained in the wheat and rye

flours but the level of it greatly varies with the

variety and crop year, and, generally, is considered
to be insufficient to significantly decrease the
amount of phytic acid (Cossa et al., 2000). Bread
making by sourdough fermentation may result in a
more suitable pH condition for the degradation of
phytic acid by endogenous phytases and sourdough
may be a source of microbial phytases. Svanberg et
al. (1993) found that lactic acid fermentation of
maize or sorghum can shift a low iron availability
diet into an intermediate to high iron bioavailability.
Two commercial strains of bakers yeast (Saccharomyces cerevisiae) were shown to express phytase
activity (Turk et al., 2000) and fungal phytases
were used to improve the nutritional value and
the bread making performance of whole wheat
bread (Haros et al., 2001). Although lactic acid
bacteria represent the great part of the sourdough
microorganisms and positively influence several
dough and bread properties, very few studies have
been carried on their phytase activity. These studies
dealt with lactic acid bacteria which were rarely
found in sourdough such as Lactobacillus amylovorus (Sreeramulu et al., 1996); with strains of lactic
acid bacteria which decreased the phytate concentration in a medium where it was the only source of
phosphates (Shirai et al., 1994); or with strains of
Lactobacillus plantarum not isolated from sourdough (Zamudio et al., 2001). To our knowledge,
only Lopez et al. (2000) have considered the
phytase activity of L. plantarum, Lactobacillus
acidophilus and Leuconostoc mesenteroides subsp.
mesenteroides sourdough strains in a whole-wheat
flour medium. Lactobacillus sanfranciscensis is
considered as a key sourdough lactic acid bacterium
due to its very frequent isolation from sourdoughs
and due to its fermentative and enzymatic properties
(Gobbetti, 1998; Corsetti et al., 2001). A wide
characterization of the phytase activity from sourdough lactic acid bacteria, including L. sanfranciscensis strains, is needed to elucidate their role in
the degradation of phytic acid during the fermentative process.
After a preliminary screening of the phytase
activity in sourdough lactic acid bacteria, this
study reports the purification, characterization and
use of a phytase enzyme from L. sanfranciscensis
CB1.

M. De Angelis et al. / International Journal of Food Microbiology 87 (2003) 259270

2. Materials and methods

2.1. Sourdough lactic acid bacteria and culture
conditions
Lactobacillus alimentarius 5Q, L. sanfranciscensis CB1, Lactobacillus brevis 14G, Lactobacillus
fermentum 6E, Leuconostoc citreum 23B, Lactococcus lactis subsp. lactis 11M, Weissela confusa 14A,
L. acidophilus 16A, L. plantarum DC400, Lactobacillus farciminis I2, Lactobacillus hilgardii 52B and
Lactobacillus fructivorans DA106 corresponds to the
species most frequently isolated from Italian sourdoughs (Gobbetti, 1998; Corsetti et al., 2001). These
species together with other 11 strains of L. sanfranciscensis were routinely propagated in modified SDB
(Kline and Sugihara, 1971) broth containing maltose
(27 mM) (Sigma, M-5885, Milan, Italy), glucose (55
mM) (Sigma, G-8270), and Na-phytate (3 mM)
(Sigma, P-8810) at 30 jC or 37 jC for 24 h.
Twenty-four-hour-old cells were used to inoculate
(4% v/v) fresh modified SDB broth and the incubation at 30 or 37 jC was allowed for different times.
In the standard conditions, the cells were harvested
by centrifugation (7000
g for 10 min at 4 jC) after
24 h of incubation, washed two times in sterile bidistilled water (Carlo Erba Reagenti, 307585, Milan,
Italy), re-suspended in sterile bi-distilled water (ca.
109 cfu/ml) and used for the enzyme assay. The
enzyme assay was also carried out by using the
cell-free supernatant and the cytoplasmic extract
(see below).
To assay the effect of the various carbon sources on
the phytase activity, the cells were also cultivated in
modified SDB broth containing maltose (28 or 14
mM), maltose (14 mM) plus glucose (14 mM),
maltose (14 mM) plus fructose (14 mM) (Sigma, F2543), or maltose (14 mM) plus sucrose (14 mM).
These soluble carbohydrates are those usually found
in the wheat flour (Gobbetti, 1998).
2.2. Preparation of the cytoplasmic extracts
After cultivation at 30 or 37 jC for 24 h in
modified SDB, the cells were harvested by centrifugation (7000
g for 10 min at 4 jC), re-suspended in
0.05 M Tris HCl pH 7.5 (Trizma Base, Sigma, T6066), containing 0.1 M CaCl2 (Carlo Erba Reagenti,

261

328257), and centrifuged again at 8000
g for 10

min at 4 jC. After washing and re-suspension in 0.05
M Tris HCl, pH 7.5, at 30 jC, the cells wereincubated at 30 jC for 30 min, centrifuged at
9000
g for 20 min at 20 jC and re-suspended in
0.05 M Tris HCl pH 7.5, containing 24% sucrose
and 10 mM MgCl2 (Carlo Erba Reagenti, 349357).
After incubation for 30 min at 37 jC, lysozyme
(Sigma, L-6876) was added to the cell suspension
and incubation was carried out for 45 min at 37 jC.
After centrifugation at 9000
g for 20 min at 20 jC,
the pellets were re-suspended in 0.02 M Tris HCl,
pH 7.5, at 4 jC. Sphaeroplasts were disrupted by two
cycles of sonication (ca. 20 s for each treatment)
(Sony Prep, Model 150, Sanyo, UK) and after incubation for 30 min at 37 jC the supernatant (cytoplasmic extract) was recovered by centrifugation at
14,000
g for 30 min at 4 jC. The supernatant was
dialyzed (Dialysis Tubing, cut off 12000 Da; Sigma,
D-9527) against distilled water for 24 h at 4 jC and
freeze-dried.
2.3. Enzyme purification
Five liters of a 24-h-old culture of L. sanfranciscensis CB1 cultivated in modified SDB broth were
harvested and the cytoplasmic extract was prepared as
described above. After freeze-drying, the cytoplasmic
extract was re-suspended in 50 mM Na-acetate
(Sigma, S-5889) buffer, pH 5.6, and applied to a
DEAE-cellulose anion exchange column (55
1.6
cm inside diameter) (Amersham Pharmacia Biotech,
Uppsala, Sweden). Proteins were eluted with a linear
NaCl (Carlo Erba Reagenti, 479687) gradient (0 0.3
M) in 50 mM Na-acetate buffer, pH 5.6, at a flow rate
of 42 ml/h. Fractions with the highest phytase activity
were pooled, dialyzed against 5 mM Na-acetate
buffer, pH 5.6, concentrated ca. 10-times by freeze
drying and subjected to gel filtration on a FPLC
Superose 12 HR 10/30 column (Pharmacia Biotech).
Elution with 50 mM Na-acetate buffer, pH 5.6,
containing 0.15 M NaCl, was at a flow rate of 18
ml/h. Active fractions were pooled, dialyzed against
50 mM Na-acetate buffer, pH 5.6, and, finally,
applied to a FPLC Mono-Q HR 5/5 column (Pharmacia Biotech) by eluting with a linear NaCl gradient
from 0 to 0.5 M in the same buffer at a flow rate of
24 ml/h.

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M. De Angelis et al. / International Journal of Food Microbiology 87 (2003) 259270

2.4. Phytase assay

Phytase activity was measured in terms of inorganic orthophosphate released from the phytic acid by
phytase, using a modification of the method of Fiske
and Subbarow (1925). A reaction mixture containing
150 Al of enzyme preparation or cell suspension and
600 Al of substrate (3 mM Na-phytate in 0.2 M Naacetate, pH 4.0) was incubated at 45 jC (Shimizu,
1992). The reaction was stopped by adding 750 Al of
5% trichloroacetic acid (Sigma, T-6399). The released
inorganic phosphate was measured by adding with
750 Al of colour reagent prepared daily by mixing four
volumes of 1.5% (w/v) ammonium molybdate
(Sigma, A7302) in a 5.5% (v/v) sulphuric acid solution (Carlo Erba Reagenti, 410301) and one volume
of a 2.7% (w/v) ferrous sulphate (Sigma, F-7002)
solution. The absorbance was measured at 700 nm.
One unit of phytase activity was defined as the
amount of enzyme required to liberate 1 nmol of
phosphate per min under the assay conditions. The
specific phytase activity was defined as U per mg of
protein. Protein concentration in the enzyme preparations and during the purification steps was determined
by the method of Bradford (1976), using bovine
serum albumin as standard.
For the substrate specificity, phytase activity was
also determined by monitoring the rate of hydrolysis
of p-nitrophenyl phosphate ( p-NPP) (Sigma, 104-0).
The assay mixture contained 200 Al of 1.5 mM p-NPP
(final concentration) in 0.2 M Na-acetate, pH 5.2, and
400 Al of enzyme preparation or cell suspension. The
mixture was incubated at 45 jC and the reaction was
stopped by adding 600 Al of 0.1 M NaOH. The pnitrophenol released was determined by measuring the
absorbance at 405 nm.
The phytase activity during sourdough fermentation
was assayed through the determination of the phytic
acid concentration by HPLC method using a refractive
index detector. Samples were re-dissolved in 10%
methanol (mobile phase) and first purified by High
Performance Size Exclusion Chromatography by using
a Ultrahydrogel DP GPC column, 7.8
300 mm
(Waters Ass., USA). Elution was at a flow rate of 0.5
ml/min. The peak containing phytic acid (ca. 1 ml) was
recovered, diluted to 5 ml with methanol and immediately injected in a second HPLC apparatus equipped
with a Spherisorb S5C1 column, 4.6
250 mm

(Waters Ass.). The operating conditions were the same

as those used for purification. Purification and quantitative analysis of the phytic acid were carried by using a
standard of phytic acid (Sigma).
2.5. Enzyme characterization
The apparent molecular mass of the purified
enzyme was estimated both by FPLC gel filtration
on a FPLC Superose 12HR 10/30 column (50 mM
Na-acetate buffer, pH 5.6, containing 0.15 M NaCl)
(Pharmacia Biotech) and by sodium dodecyl sulphate
(SDS)-PAGE according to Laemmli (1970). The gel
contained 12% acrylamide (separation distance, 10
cm; gel thickness, 1 mm) and electrophoresis was
performed with the Mini Protein II (Bio-Rad, Milan,
Italy). The proteins were stained with Coomassie
brilliant blue. Molecular mass marker proteins (low
range 14.4 97.4 kDa, Bio-Rad) were used as references. The zymogram was prepared by soaking the
gel first in 1% Triton X-100 for 1 h at room temperature and then 0.1 M sodium acetate buffer (pH 4.5)
for 1 h at 4 jC. Phytase activity was detected by
incubating the gel for 16 h in a 0.1 M sodium acetate
buffer (pH 4.5) containing 0.4% (w/v) Na-phytate.
Activity bands were visualized by immerging the gel
in a 2% (w/v) aqueous cobalt chloride (Sigma, C2644) solution. After a 5-min incubation at room
temperature, the cobalt chloride solution was replaced
with a freshly prepared solution containing equal
volumes of a 6.25% (w/v) aqueous ammonium
molybdate solution and 0.42% (w/v) ammonium
vanadate (Sigma, A-1183) solution. Phytase activity
was evident as zones of clearing in an opaque background (Bae et al., 1999).
The isoelectric point (pI) of the enzyme was determined by two-dimensional (2D) electrophoresis and
using amyloglucosidase (pI 3.8), ovalbumin (pI 5.1),
carbonic anhydrase (pI 7.0), and myoglobin (pI 7.6) as
markers. The purified enzyme was re-suspended
directly in the denaturating buffer containing 8 M
urea (Pharmacia, 17-1319-01), 4% CHAPS (3-3-cholamidopropyl dimethylammonium-1-propane sulfonate) (Sigma, C-9426), 40 mM Tris base and 65
mM dithiothreitol (DTT) (Sigma, D-9163). Two-D
electrophoresis was performed using the immobiline/
polyacrylamide system as described by De Angelis et
al. (2001). The protein mapped was scanned with a

M. De Angelis et al. / International Journal of Food Microbiology 87 (2003) 259270

laser densitometer (Molecular Dynamics 300s) and

analysed with the Image Master 2D elite computer
software (Pharmacia Biotech).
The optimum pH for the purified enzyme was
determined at 45 jC in the range of 3.0 6.0 by using
20 mM Na-acetate buffer. The temperature dependence was determined, at pH 4.0, in the range of 10 70
jC.
To check the thermal stability of the enzyme, equal
volumes of the pure enzyme preparation in 20 mM
Na-acetate buffer, pH 4.0, were heated in capillary
glass tubes at 60 to 80 jC for 1 to 30 min, and the
residual activity was assayed at 45 jC and expressed
as a percentage of the activity of an unheated sample.
Decimal reduction time (D) was calculated by plotting
the log of the residual activity versus time.
Inorganic phosphate released from a number of 3
mM phosphate esters (final concentration), incubated
with the pure enzyme preparation at pH 4.0 and 45
jC, was determined, as described above, to measure
the substrate specificity.
To assay the effect of inhibitors and divalent cations, a mixture containing solutions of the purified
enzyme and 2 mM (final concentration) chemical
reagents or divalent cations in 20 mM Na-acetate
buffer, pH 4.0, was incubated for 30 min at 45 jC.
Reaction was initiated by adding the appropriate substrate, and enzyme activity was assayed under standard
conditions. Controls to eliminate the interference of
inhibitors or divalent cations were included.
2.6. Experimental design and statistical analysis
The effects of temperature, pH and NaCl on the
enzyme activity on Na-phytate were determined by
modulating the variables according to a three factor,
five level Central Composite Design (CCD). The 17
combinations obtained for each enzyme activity are
shown in Table 1. Two replicates of each combination
were used.
Modelling was aimed at describing the enzyme
activity as a function of the independent variables of
the CCD. A software package (Statistica for Windows, Statsoft, Tulsa, OK) was used to fit the secondorder model to the independent variables using the
following equation:
X
X
X
c
B i vi
Bii v2i
Bij vi vj

263

Table 1
Composition of the various runs of the Central Composite Design
and specific activity (U*/mg of protein) of the phytase from L.
sanfranciscensis CB1
Run

Temperature
(jC)

pH

NaCl
(%)

Specific activity
(U/mg)

1
2
3
4
5
6
7
8
9
10
11
12
13
14
15
16
17

26
26
26
26
38
38
38
38
32
32
32
32
32
32
20
44
32

4.1
5.5
4.1
5.5
4.1
5.5
4.1
5.5
4.8
4.8
3.4
6.2
4.8
4.8
4.8
4.8
4.8

1.2
1.2
3.6
3.6
1.2
1.2
3.6
3.6
2.4
2.4
2.4
2.4
0
4.8
2.4
2.4
2.4

30.4
5.1
14.3
2.8
72.8
8.5
18.2
6.2
27.2
27.0
1.1
1.9
48.1
6.3
3.1
100.2
26.4

The specific activity was the average of three replicates of each

combination.
* U, is the amount of enzyme required to liberate 1 nmol of
phosphate per min under the assay conditions.

where c is the dependent variable to be modelled,

Bi, Bii and Bij are regression coefficients of the
model, and vi and vj are the independent variables
in coded values. The variables with a significance
lower than 95% ( P>0.05) were not included in the
final model. The experimental data ere modelled
using Stepwise Regression with Backwards selection
procedure; the system begins with all variables in
the model and the nonsignificant terms are omitted
one-by-one, consequently only the terms significant
at 95% were considered. The three dimensional
surface plot was drawn to illustrate the main and
interactive effects of the independent variables (temperature, pH and NaCl) on the dependent one
(enzyme activity).
2.7. Sourdough fermentation
The characteristic of the wheat flour used were:
moisture 12.8%, protein (N
5.70) 10.7% of dry
matter (d.m.), fat 1.8% of d.m. and ash 0.6% of
d.m. Wheat flour (100 g) treated with microwave
(2
1 min at 900 W and then 30 s at 900 W), 45

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M. De Angelis et al. / International Journal of Food Microbiology 87 (2003) 259270

ml of sterile water, containing 12 mM sodium

phytate, and 15 ml of cellular suspension (108
cfu/ml) or the equivalent cytoplasmic extract were
used to produce a 160 g of dough (dough yield
160) with a continuous high speed mixer (dough
mixing time, 5 min). Doughs were incubated at 37
jC for 8 h. After fermentation, the sourdough
started with L. sanfranciscensis CB1 cells was
directly re-used to inoculate (20%) a new dough
of 160 g which was fermented for further 8 h. This
sourdough was then re-used for several times under
the conditions described above. The treatment of the
wheat flour by microwave to deactivate the endogenous phytase activity and the addition of Naphytate were in agreement with the protocol
described by Lopez et al. (2000).
After fermentation, aliquots (2 g) of sourdoughs
were homogenized with 100 ml 0.5 M HCl, heated
at ca. 90 jC for 5 min to deactivate the enzyme and
centrifuged at 4000
g for 10 min at 20 jC. The
supernatant was diluted with 15 ml of 1 M HCl,
added of methanol in a ratio 3:7 and centrifuged at
4000
g per 10 min at 4 jC. After recovery, the
pellet was used for HPLC analysis as described
above.

3. Results
3.1. Phytase activity of sourdough lactic acid bacteria
Preliminarily, the cell suspensions (ca. 109 cfu/ml)
of 12 species of lactic acid bacteria isolated from
sourdoughs were screened for the phytase activity
(Table 2). Although with some differences, the
activity was largely distributed in all the species.
After 2 h of incubation at 45 jC, the highest values
were found for L. sanfranciscensis CB1 (420.8 U/
ml), L. fructivorans DA106 (295.3 U/ml), W. confusa
14A (259.6 U/ml), Lc. lactis subsp. lactis 11 M
(249.2 U/ml) and L. alimentarius 5 Q (230.7 U/
ml). The other species had values of activity less
than 173 U/ml. The activity increased slightly by
using the cytoplasmic extracts. None of the species
used showed phytase activity in the cell-free supernatant (data not shown).
Eleven other strains of L. sanfranciscensis were
assayed for the phytase activity. Nine of them had

Table 2
Phytase activity (U*) of sourdough lactic acid bacteria after 2 h of
incubation at 40 jC in modified SDB broth containing 3 mM of Naphytate
Lactic acid bacteria

Phytase activity (U/ml)

L. farciminis 2I
L. alimentarius 5Q
Leuc. citreum 23B
L. acidophilus 16A
L. plantarum DC400
L. fermentum 6E
L. hilgardii 52B
L. fructivorans DA106
Lc. lactis subsp. lactis 11M
W. confusa 14A
L. sanfranciscensis CB1
L. sanfranciscensis 22E
L. brevis 14G

116.3g
230.7d
172.6e
89.4i
110.6g,h
130.6f
159.1e
295.3b
249.2c
259.6c
420.8a
7.6l
105.4h

The phytase activity was the average of three replicates twice analysed.
al
Values in the same column with different superscript letters
significantly differed (P < 0.05).
* U, is the amount of enzyme required to liberate 1 nmol of
phosphate per min under the assay conditions.

values of activity ranging from 217 to 420 U/ml and

only one (strain 22E) had an activity less than 10 U/
ml.
Based on these preliminary results and considering that L. sanfranciscensis CB1 is a key sourdough
lactic acid bacterium, well characterized for other
metabolic properties (Gobbetti, 1998; De Angelis et
al., 2001, 2002), this strain was used for the further
characterization of the phytase activity.
L. sanfranciscensis CB1 exhibited the highest
hydrolysing activity on Na-phytate after reaching
the stationary phase of growth (ca. 12 h), then it
maintained constant until 24 36 h of incubation.
After the sub-cellular fractionation, it was shown
that the total activity was contained in the cytoplasmic fraction. To elucidate the influence of various
carbon sources on the expression of the phytase
activity, L. sanfranciscensis CB1 cells were cultivated in the presence of maltose alone (28 and 14
mM) and in combination (14 + 14 mM) with fructose, glucose or sucrose, and then assayed for the
enzyme activity. Cells cultivated in the presence of
maltose and fructose showed an increase of the
phytase activity of ca. 35% with respect to the other
conditions (data not shown).

M. De Angelis et al. / International Journal of Food Microbiology 87 (2003) 259270

265

3.2. Purification and characterization of the phytase

enzyme of L. sanfranciscensis CB1
The purification to homogeneity of the phytase of
L. sanfranciscensis CB1 required three chromatographic steps: anion exchange on DEAE-cellulose,
gel filtration on Superose 12 HR 10/30 and again
anion exchange on Mono-Q HR 5/5 columns. The
enzyme was purified 160-fold with a recovery of
18%. A single band running at an apparent molecular
mass of ca. 50 kDa was found by SDS-PAGE (Fig. 1).
The same apparent molecular mass was determined by
gel filtration on Superose 12 HR 10/30 column. As
shown by the zymogram (Fig. 1), the phytase activity
corresponded to the protein band located at ca. 50
kDa. The pure preparation of the enzyme was used for
further characterization.
The activity of the phytase was optimal at pH 4.0,
with a rapid decrease at both pH 3.5 and 4.5. It
maintained only ca. 30% of the maximum activity in
the range of pH 5.5 6.0. The optimum temperature
was found to be 45 jC, with ca. 40% of residual
activity at 30 and 60 jC.
As determined by two-dimensional (2D) electrophoresis, the isoelectric point (pI) of the enzyme was
found to be ca. 5.0.

Fig. 2. Thermal stability of the phytase of L. sanfranciscensis CB1

determined at 60 (.), 70 (o) and 80 jC (z) in 20 mM Na-acetate
buffer, pH 4.0.

The thermal stability of the phytase was determined in 20 mM Na-acetate buffer, pH 4.0. It retained
the 100% of the maximum activity after heating at 60
jC for 30 min, ca. 70% of the maximum activity at 70
jC for 30 min and decreased to 10% of the maximum
activity after a treatment of 80 jC for 10 min (Fig. 2).
Indeed, the D value calculated at 80 jC was ca. 10
min.
The activity of the purified enzyme on various
phosphate esters and on p-NPP substrate is reported in
Table 3. The phytase exhibited the highest activity on

Table 3
Substrate specificity (U*/mg of protein) of the phytase from L.
sanfranciscensis CB1 on different substrates

Fig. 1. SDS-PAGE and zymogram for the intracellular phytase of L.

sanfranciscensis CB1. Lanes: S, reference proteins; 1, cytoplasmic
extract; 2, enzyme preparation after anion exchange on DEAEcellulose; 3, enzyme preparation after gel filtration on Superose
12HR 10/30; 4, enzyme preparation after anion exchange on MonoQ HR 5/5; 5, zymogram developed for phytase activity.

Substrates

Phytase activity (U/mg)

p-Nitrophenyl phosphate
a-D-Glucose-1-phosphate
D-Fructose-6-phosphate
D-Fructose-1,6-phosphate
Adenosine-5V-monophosphate
Adenosine-5V-diphosphate
Adenosine-5V-triphosphate
Na-phytate
Acetyl phosphate

120.2a
3.7f
8.7e
11.7d
93.4c
95.6b,c
96.8b
96.8b
6.2e,f

The phytase activity was the average of three replicates twice

analysed.
af
Values in the same column with different superscript letters
significantly differed (P < 0.05).
* U, is the amount of enzyme required to liberate 1 nmol of
phosphate per min under the assay conditions.

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p-NPP, adenosine-5V-tri-, di- and mono-phosphate and

on Na-phytate.
The effect of inhibitors and divalent cations on
phytase activity is shown in Table 4. The enzyme
activity was almost completely inhibited in the presence of the serine proteinase inhibitor such as phenylmethylsulfonyl fluoride (PMSF), it was slightly
affected by DL-penicillamine and not influenced by
the other inhibitors. Among divalent cations, only
Hg2 + and Fe2 + markedly decreased the enzyme activity.
3.3. Experimental design and statistical analysis
The CCD was used because it reduces the number
of possible combinations to a manageable size, since
the use of only a fraction of the total number of factor
combinations for experimentation. In statistical literature, this technique is known as confounding
(Gacula, 1988). Ranges of temperature (20 44 jC),
pH (3.4 6.2) and NaCl (0 4.8%) which are compatible with the sourdough processing (Gobbetti, 1998)
were considered. The polynomial equation describing

Table 4
Effect of inhibitors and divalent cations on the phytase from L.
sanfranciscensis CB1
Inhibitors and cationsa
NEM
Iodoacetamide
PMSF
EDTA
DL-Penicillamine
DTT
Mg2 +
Cu2 +
Co2 +
Hg2 +
Ni2 +
Mn2 +
Ca2 +
Fe2 +
Zn2 +

the effects of the different variables on the enzyme

activity of L. sanfranciscensis CB1 was the following:  10.61[T] + 81.41[pH]  8.35[NaCl]  9.57
[pH2] + 0.20[T2], with a regression coefficient, R of
0.915; an F-value of 12.344 and a standard error of
residuals, S.E. of 17.067.
The effects indicated by the equation are better
shown by considering the three-dimensional plots in
Fig. 3. They were obtained by imposing a constant
value (e.g., the central point of the CCD) to one
variable at time. As shown in Fig. 3a and b, the
increase of the temperature had always a positive
effect on the enzyme activity; this effect was only in
part reduced when pH and NaCl were at the highest
values. The optimum pH around 4.0 was confirmed
(Fig. 3a and c), and, apart from the variation of
temperature and NaCl, the activity was higher at the
lowest value of pH 3.4 with respect to the highest
value of pH 6.2. The increase of the NaCl concentration had always a negative effect on the enzyme
activity. Nevertheless, at 44 jC and pH 4.8 (Fig. 3b)
the activity still maintained at ca. 60 (U/mg) also in
the presence of 4.8% NaCl. By considering the
interactive effects of the three variables, the maximum
activity (ca. 90 U/mg) occurred at 0% NaCl, pH 4.8
and 44 jC.
3.4. Hydrolysis of sodium phytate during sourdough
fermentation

Relative activityb (%)

92 F 3.0
99 F 1.0
4 F 0.2
92.5 F 5.4
88 F 2.0
94.5 F 1.0
98.6 F 2.0
77 F 1.0
98 F 3.0
8 F 1.5
87 F 2.0
94 F 1.5
98 F 3.5
1 F 0.2
92 F 2.0

The phytase activity was the average of three replicates twice

analysed.
a
NEM, N-ethylmaleimide; PMSF, phenylmethylsulfonyl fluoride; EDTA, ethylenediaminetetra acetic acid; DTT, dithiotreitol.
b
The activity of the control in the absence of chemical reagents
and divalent cations was taken as 100%.

After sourdough incubation for 8 h at 37 jC, the

two sourdoughs started with L. sanfranciscensis
strains had a pH value of 3.8 4.0. The cell concentration of both bacteria strains reached 109 cfu/g. The
same values of pH and cell concentration were always
found when the sourdough fermented with L. sanfranciscensis CB1 was re-used as starter. The unstarted
sourdough had a value of pH of 5.6.
During sourdough fermentation with L. sanfranciscensis CB1 cells, a marked decrease of the concentration of Na-phytate was found (4.29 mM)
compared with the unstarted dough (11.85 mM)
(Table 5). On the contrary, the use of L. sanfranciscensis 22E cells did not cause a significant hydrolysis
of the Na-phytate (11.26 mM). This strain had a very
low activity, as determined during the initial screening
within L. sanfranciscensis strains. The use of the
cytoplasmic extract, which corresponded to 109 cfu/g

M. De Angelis et al. / International Journal of Food Microbiology 87 (2003) 259270

267

Table 5
Variation in the concentration (mM) of Na-phytate after fermentation of various dough samples for 8 h at 37 jC
Dough

Na-phytate (mM)

Unstarted dough
Sourdough started with L.
sanfranciscensis 22E
Sourdough started with L.
sanfranciscensis CB1
Sourdough added of the
cytoplasmic extract of L.
sanfranciscensis CB1
Re-use* I of the sourdough started
with L. sanfranciscensis CB1
Re-use II of the sourdough started
with L. sanfranciscensis CB1
Re-use III of the sourdough started
with L. sanfranciscensis CB1

11.85a
11.26a
4.29d
3.10e

6.15c
8.80b
8.70b

The concentration of the phytic acid was the average of three

replicates twice analysed.
ae
Values in the same column with different superscript letters
significantly differed (P < 0.05).
* For the conditions of re-use of the sourdough started with L.
sanfranciscensis CB1, see Materials and methods.

L. sanfranciscensis CB1 cells, caused a further

decrease of the Na-phytate concentration (3.1 mM).
When the sourdough fermented with L. sanfranciscensis CB1 cells was re-used as starter (20%) for a
new dough, a consistent hydrolysis of Na-phytate was
shown for several times.

4. Discussion

Fig. 3. Phytase activity of L. sanfranciscensis CB1. Threedimensional plots of the interactions of: (a) temperature
pH; (b)
temperature
NaCl; and (c) pH
NaCl.

Although microbial phytases are considered of a

great value in upgrading the nutritional quality of
plant foods, very few studies have dealt with lactic
acid bacteria. Nineteen strains of lactic acid bacteria of
the genera Lactobacillus and Streptococcus collected
from different culture collections were screened by
Sreeramulu et al. (1996) for the production of extracellular phytase. Nine of them exhibited this enzyme
activity in the fermentation medium but L. amylovorus B4552 produced the maximum amounts of phytase extracellularly. It was evident that lactic acid
bacteria used in milk and milk-based products such
as strains of L. acidophilus, Lactobacillus casei,
Lactococcus lactis, Lactobacillus delbrueckii and
Streptococcus sp. were very poor producers of phy-

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M. De Angelis et al. / International Journal of Food Microbiology 87 (2003) 259270

tase, whereas L. amylovorus and L. plantarum, which

are present in a variety of microbial systems of plant
origin, resulted in the maximum yield of enzyme. An
extracellular nonspecific acid phosphatase activity
which had the higher hydrolysis rate with monophosphorylated compounds such as acetyl phosphate
but low level of activity towards Na-phytate was
characterized in another L. plantarum strain (Zamudio
et al., 2001).
None of the above strains were isolated from
sourdoughs. This report shows that the capacity of
hydrolysing Na-phytate was largely distributed among
sourdough lactic acid bacteria, especially in L. sanfranciscensis. The phytase activity of the sourdough
lactic acid bacteria was intracellular only. The only
other study which dealt with lactic acid bacteria isolated from sourdough showed the hydrolysis of phytate by L. plantarum, L. acidophilus and Leuconostoc
mesenteroides subsp. mesenteroides strains in white
flour medium by using whole cells (Lopez et al.,
2000). No great differences were found in the levels
of phytic acid hydrolysis, suggesting that phytase
enzymes were similar among these bacteria.
After the initial screening, we purified to homogeneity and characterized the phytase of L. sanfranciscensis CB1, a well-known and characterized sourdough strain (Gobbetti, 1998). As determined by
SDS-PAGE and gel filtration, the apparent molecular
mass of the enzyme was ca. 50 kDa. Overall, microbial phytase are considered as monomeric proteins
ranging from 40 to 100 kDa (Pandey et al., 2001). The
bacterial phytases characterized from Bacillus subtilis,
Escherichia coli and Klebsiella terrigena had apparent molecular masses of 36 to 45 kDa (Pandey et al.,
2001). The only other nonspecific acid phosphatase
enzyme purified from a lactic acid bacterium had an
apparent molecular mass of 52 kDa (Zamudio et al.,
2001). The enzyme of L. sanfranciscensis CB1
showed pH and temperature optima of 4.0 and 45
jC, respectively. By using an experimental CCD, it
was shown that in the presence of acidic conditions
and at the more elevated temperatures which characterized the sourdough process, the enzyme also adapted to high NaCl concentration. The other phytase
characterized from bacteria other them lactic acid
bacteria had some different properties, mainly regarding the optimum pH. The extracellular phytases of B.
subtilis N-77 and VTT E-68013 strains had pH and

temperature optima of 6.0 7.0 and 60 70 jC, respectively (Shimizu, 1992; Kerovuo et al., 1998); the
phytases of E. coli and Klebsiella sp. were characterized by optimal activity at pH 5.0 6.0 and 58 60
jC (Greiner et al., 1997; Golovan et al., 2000). The
nonspecific acid phosphatase of L. plantarum had an
optimum activity at pH 5.5 and 65 jC (Zamudio et al.,
2001). Based on these considerations, it seemed that
the phytase of L. sanfranciscensis CB1 was specifically adapted for the acidic conditions of the sourdough fermentation. The phytase of L. sanfranciscensis CB1 had a pI of ca. 5.0 and a sensitivity to divalent
cations which did not differ substantially from those
reported for other bacterial phytases (Pandey et al.,
2001). The insensitivity to EDTA as well as the
complete inhibition by PMSF distinguished this
enzyme. Indeed, only the phytase purified from E.
coli was not affected by EDTA also (Golovan et al.,
2000). The hydrolysing activity of the enzyme of L.
sanfranciscensis CB1 seemed to be dependent on the
type of phosphate ester since a very low activity was
detected on a-D-glucose-1-phosphate and D-fructose-6
and 1,6-phosphate, while the highest hydrolysis was
found towards adenosine-5V-tri-, di- and mono-phosphate. Compared to these substrates, the activity on
Na-phytate was also relevant but not that on acetylphosphate. Since this rather large substrate specificity,
which includes the Na-phytate also, and since the high
activity on Na-phytate which was detected during
sourdough fermentation also, it may be concluded that
this enzyme is a true phytase and not a general
phosphatase with some residual activity on phytic
acid. This is in contrast with the other enzyme partially
purified from L. plantarum which was defined as a
nonspecific acid phosphatase due to the very high
hydrolysing activity on acetyl phosphate but very
low on Na-phytate (Zamudio et al., 2001).
Thermostability is considered an important and
useful criterion for industrial application of phytase.
The Aspergillus fumigatus phytase which was
expressed in Pichia pastoris retained 20 40% of
residual activity in sodium acetate buffer after 20
min of exposure to 65 90 jC (Rodriguez et al.,
2000). The phytase from B. amyloliquefaciens DS11
had an apparent half-life of 42 min at 80 jC (Kim et
al., 1999). Certain characteristics of thermostability
were also found for the phytase of L. sanfranciscensis
CB1. It maintained 70% of residual activity after

M. De Angelis et al. / International Journal of Food Microbiology 87 (2003) 259270

exposure to 70 jC for 30 min in 50 mM Na-acetate

buffer, pH 4.0, meaning that it may keep active also
during the first period of dough baking.
The highest yield of phytase activity in L. sanfranciscensis CB1 was found after reaching the stationary phase of growth and when the cells were
cultivated in modified SDB containing maltose and
fructose which promoted an increase of the cell yield
(8.0
109 cfu/ml) compared to the growth in the
presence of other carbon sources (ca. 2
109 cfu/
ml). This strain was shown to be able to co-metabolise
maltose and fructose by using the latter as an external
acceptor of electrons, thus producing an extra mole of
ATP via acetate kinase (Gobbetti et al., 1995). The
highest phytase activity of L. amylovorus was found
after 32 h of growth and declined steadily after 44 h of
cultivation. Most of the enzyme was synthesized
during the exponential phase of growth. The effect
of different carbon sources on the growth and phytase
production by L. amylovorus was also studied, showing that the growth was excellent on glucose medium
along with the maximum yield of phytase activity
(Sreeramulu et al., 1996).
After inoculating with Leuc. mesenteroides subsp.
mesenteroides strain 38 a whole wheat flour medium
naturally rich in phytic acid for 9 h, Lopez et al. (2000)
established that the degradation of phytic acid and the
production of lactic acid lead to a greater Ca2 + and
Mg2 + solubility than in the control medium. In this
study, there was an evidence of the phytase activity of
L. sanfranciscensis CB1 also during sourdough fermentation. After 8 h of incubation with cells or with
the correspondent cytoplasmic extract, there was a
decrease of 64 74% of the Na-phytate concentration
compared to the unstarted dough. By re-using this
sourdough for several times, the phytase activity was
maintained to a considerable and constant level. The
kinetics of hydrolysis and the intensity of Na-phytate
degradation were in agreement with the results by
Lopez et al. (2000) with the selected Leuc. mesenteroides subsp. mesenteroides strain 38.
This report is the first to show that L. sanfranciscensis has a potential in improving the nutritional
quality of cereal based products due to its phytase
activity. Overall, since lactic acid bacteria from bakery
leavened products promote an acidification, which
may activate wheat flour endogenous phytases, and
degrade phytic acid, which decreases the chelating

269

activity towards cations and proteins, the mineral

availability in fermented cereal foods (bread) should
be higher than that of unleavened products (extruded
cereals). A comparison between the phytase activity
of sourdough lactic acid bacteria and bakers yeasts is
under study.

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