Paper, Santos 2014

1 23
Biological Trace Element Research
ISSN 0163-4984
Biol Trace Elem Res
DOI 10.1007/s12011-014-0161-y
Trace Element Inhibition of Phytase
Activity
T. Santos, C. Connolly & R. Murphy

1 23
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Trace Element Inhibition of Phytase Activity
T. Santos & C. Connolly & R. Murphy
Received: 30 June 2014 /Accepted: 21 October 2014
# Springer Science+Business Media New York 2014
Abstract Nowadays, 70 % of global monogastric feeds con-
tains an exogenous phytase. Phytase supplementation has
enabled a more efficient utilisation of phytate phosphorous
(P) and reduction of P pollution. Trace minerals, such as iron
(Fe), zinc (Zn), copper (Cu) and manganese (Mn) are essential
for maintaining health and immunity as well as being involved
in animal growth, production and reproduction. Exogenous
sources of phytase and trace elements are regularly supple-
mented to monogastric diets and usually combined in a pre-
mix. However, the possibility for negative interaction between
individual components within the premix is high and is often
overlooked. Therefore, this initial study focused on assessing
the potential in vitro interaction between inorganic and organ-
ic chelated sources of Fe, Zn, Cu and Mn with three commer-
cially available phytase preparations. Additionally, this study
has investigated if the degree of enzyme inhibition was de-
pendent of the type of chelated sources. A highly significant
relationship between phytase inhibition, trace mineral type as
well as mineral source and concentration, p<0.001 was ver-
ified. The proteinate sources of OTMs were consistently and
significantly less inhibitory than the majority of the other
sources, p<0.05. This was verified for Escherichia coli and
Peniophora lycii phytases for Fe and Zn, as well as for Cu
with E. coli and Aspergillus niger phytases. Different chelate
trace mineral sources demonstrated diversifying abilities to
inhibit exogenous phytase activity.
Keywords Organic trace elements . Chelate sources .
Phytase . Inhibition . Interaction
Introduction
Mineral utilisation by animals primarily depends on their
absorption from the ingested feed. The term “bioavailability”
is generally used to describe both the absorption and the
ultimate metabolic utilisation of nutrients within the cell. In
the last decade, the feed industry has experienced a fine-tuning
in diet formulation, not only to match the animal’s nutritional
needs, but also to minimise pollution due to mineral excretion.
Feed additives can be used to increase the health status,
fertility and performance of farm animals. Improved nutrient
availability can be achieved through the use of feed additives
that improve nutrient digestibility such as organic trace ele-
ments and exogenous enzymes, respectively.
The bioavailability of many minerals is known to be affected
by phytic acid [1] (myo-inositol (1,2,3,4,5,6) hexaphosphoric
acid, IP6), a strong naturally occurring organic chelator and the
principal storage form of phosphorous (P) and other macro- and
microminerals in many plant tissues [2]. Phytic acid is the major
P storage compound in the plant seed where it can account for
up 80 % of the total P [1, 2]. It is also considered to be an
antinutritional factor for humans and animals as it may chelate
nutritionally important cations such as Cu2+
, Zn2+
, Co2+
,Cd2+
,
Mg2+
, Mn2+
, Fe2+
, Fe3+
, Ni2+
and Ca2+
[3–6].
Monogastric animals such as swine, poultry and fish re-
quire exogenous phytase to digest phytate to avoid P deficien-
cy [6]. Hence, undigested total P (organic and inorganic) is
excreted through the faeces, ultimately creating the potential
for eutrophication of fresh water streams [7, 8].
Phytase is an acid phosphohydrolase that catalyses the
hydrolysis of phosphate from phytic acid to inorganic phos-
phate and myo-inositol phosphate derivatives [9], and it has
emerged as one of the most effective and lucrative feed
additives [10].
Phytases can be classified depending on the position of the
first dephosphorylation of phytate. Within each class, not only
T. Santos (*) :C. Connolly :R. Murphy
Alltech Ireland, European Bioscience Centre, Dunboyne, Co. Meath,
Ireland
e-mail: tsantos@alltech.com
Biol Trace Elem Res
DOI 10.1007/s12011-014-0161-y
Author's personal copy

structural differences can be found, but also different mecha-
nisms for the hydrolysis of phytic acid. Microbial phytases,
when added to the animal diet, are able to hydrolyze the ester
bond between carbon 3 (in the case of 3-phytases) or carbon 6
(in the case of 6-phytases) and the associated phosphate
group, liberating the phosphate for the animal [11]. Three
different microbial sources of commercially available
phytases are expressly applied in animal nutrition including
Aspergillus niger, Escherichia coli and Peniophora lycii. The
A. niger enzyme is a 3-phytase (EC 3.1.3.8), and the E. coli
and P. lycii enzymes are 6-phytases (EC 3.3.26). In the same
way, the enzymes can be grouped in accordance with their
optimum pH of activity, as acidic, neutral or alkaline phos-
phatases [12]. The temperature and pH optima for A. niger,
P. lycii and E. coli phytases have been reported within the
range 50–65 °C and 4.5–5.5, respectively [10].
Many fungal phytases, such as A. niger and P. lycii, as well
as the E. coli phytase belong to the histidine acid phosphatases
(HAP). These enzymes share the same active-site sequence
(RHGXRXP), a catalytic dipeptide, and ten cysteine residues
[13].
Supplementation of animal diets with microbial
phytase is an environmentally friendly solution [14] that
reduces the need for phosphorous supplements and po-
tentially increases the bioavailability of proteins as well
as of essential minerals whilst improving animal perfor-
mance [15]. Another strategy for reducing mineral con-
centrations in diets is the inclusion of mineral sources
that may display greater bioavailability than the conven-
tional inorganic form. Over the last 20 years, research
has shown that using highly bioavailable sources of
trace minerals has positive effects on performance and
health of farm animals [16–19]. The principle is to bind
minerals to organic molecules (ligands), allowing the
formation of structures with unique characteristics and
high bioavailability (metal complexes or chelates). The
different classes of organic trace minerals which include
metal amino acid complexes; metal (specific amino ac-
id) complexes; metal amino acid chelates; metal
proteinates and metal polysaccharide complexes applied
in animal nutrition have been defined by the Associa-
tion of American Feed Control Officials [20] and are
shown in Table 1. Chelation has a clear influence on
bioavailability of trace elements and organic trace min-
erals. While phytate decreases bioavailability, the use of
organic trace minerals increases absorption of trace ele-
ments. Chelation is the ability of a ligand or chelating
agent to form a complex containing a heterocyclic ring
structure with a metal ion [21]. An important feature of
metal chelates is their high stability due to the confor-
mation in which the metal is held by coordinating
groups. The strength of the interaction between organic
ligands and metals is usually expressed in terms of a
stability constant, also called an equilibrium, formation
or binding constant. Knowledge of stability constants
enables the behaviour of a metal ion with one or more
ligands to be modelled as a function of pH and reactant
concentration [22]. Reported differences in the bioavail-
ability of organic and inorganic minerals have been
attributed to differences in dissociation rates of the
mineral from the organic or inorganic substrate to which
they are bound, or to differences in mineral-chelate
solubility [23]. One of the characteristics considered
important to the physiological function of chelated and
complexed metals is the degree to which the organic
ligands remain bound to the metal under physiological
conditions [24].
The role of phytase in increasing the bioavailability
of zinc (Zn), copper (Cu) and other minerals has been
widely observed [25–28]. Consequently, microbial
phytase and trace elements are usually supplied in com-
bination within the premix. On the other hand, it is also
well-known that metal ions can be enzyme inhibitors,
and research has shown that phytases have been
inhibited by Cu, Zn, Fe and Mn [4] [5]. However, very
little information is available comparing the effective-
ness of the different organic chelate sources and phytase
in general.
The objective of this initial study was to verify the
effects of mineral sources on phytase activity by
assessing the potential in vitro effect between inorganic
(sulphates) and chelated organic forms of Cu, Zn, Fe
and Mn (proteinates, glycinates, polysaccharide com-
plexes and amino acid chelates) on the activity of three
different sources of commercially available phytases
(A. niger, E. coli and P. lycii). The degree of phytase
inhibition relative to the type of mineral chelate and the
way in which enzymes from different source organisms
behave in their presence was also investigated. The
biochemichal/chemical mechanisms behind the enzymes
or the chelated minerals tested were not investigated in
the current work.
Methods
Sources of Phytase
Commercially available phytase from three different microbi-
al sources was used in this study including A. niger
(Natuphos® 10,000 G, BASF Aktiengesellschaft, 67056
Ludwigshafen/Germany), E. coli (Phyzyme® XP 5000 G,
Danisco (UK) Limited, Marlborough, UK) and P. lycii
(Ronozyme® NP (M), DSM Nutritional Products,
Wurmisweg 576, CH-4303 Kaiseraugst, Switzerland). The
phytases were supplied from industrial sources.
Santos et al.

Sources of Minerals
Four commercial organic Fe, Zn and Cu products and three
organic Mn sources from various manufacturers, as well as the
respective ACS reagent-grade inorganic sulphate salts FeSO4·
7H2O (Fe SO4), CuSO4·5H20 (Cu SO4), ZnSO4·7H2O (Zn
SO4) and MnSO4·H2O (Mn SO4) (Sigma Aldrich, St. Louis,
USA) were assessed in laboratory assays.
Organic mineral sources included Fe, Cu, Zn and Mn
proteinates (Fe, Zn, Cu and Mn PRO); Fe, Cu, Zn and Mn
glycinates (Fe, Zn, Cu and Mn GLY); Fe, Cu, Zn and Mn
polysaccharide complexes (Fe, Zn, Cu and Mn PSC); and Fe,
Cu and Zn amino acid chelates (Fe, Zn and Cu ACH). The
proteinates were kindly supplied by Alltech Ireland Limited,
and all the other sources were obtained from independent
distributors, rather than the manufacturers of the products.
Mineral Analysis
Mineral concentrations of Fe, Zn, Cu and Mn sources were
analysed using inductively coupled plasma-mass spectrome-
try (ICP-MS) (Agilent Technologies, Waldbronn, Germany).
Approximately 0.1 g of each source was weighed in triplicate
and digested with 10 mL of HNO3 for 35 min at 180 °C in a
CEM Discover microwave (CEM Corporation, Matthews,
NC). After digestion, the samples were diluted with >18
MΩcm water to the expected mineral concentration. Samples
and standards were matrix-matched to 2 % HNO3 prior to
analysis.
Assay for Phytase Activity
Samples were examined for total phytase activity using a
modification of the assay described by Engelen et al. [29].
Aliquots (0.5 mL) of the samples were appropriately diluted in
5 mM sodium acetate buffer, pH 5.5, and added to 0.5 mL of
substrate solution (2.5 mM phytic acid sodium hydrate from
rice in 0.2 M sodium acetate buffer pH 5.5) for 10 min at 50 °C
in a water bath. The reaction was stopped with the addition of
2 mL of ice-cold colour stop solution (10 mM ammonium
molybdate/5 N sulphuric acid/acetone, in the ratio 1:1:2),
followed by the addition of 100 μL of 1.0 M citric acid.
Triplicate assay samples were carried out for each treatment.
After the incubation and subsequent substrate hydrolysis, the
enzyme activity was quantified from the amount of orthophos-
phate released by the hydrolysed substrate upon determining
the increase in absorbance at λ380 nm (Shimadzu 160-A). The
results of the unknowns were compared with a standard curve
prepared with inorganic phosphate (K2HPO4), and the phytase
activities of the test samples were determined. One phytase
unit (PU per gram) is defined as the amount of enzyme that
will liberate 1 μmol of inorganic phosphate per minute and is
calculated as outlined below.
PU=g ¼ ΔA380 Â F Â 2 Â Dð Þ=10
Where
ΔA380 is the difference in absorbance between the sample
and the blank
F is the phosphate concentration (micromoles per
milliliter) corresponding to the absorbance (λ380nm)
1.0 obtained from the standard curve
2 is a multiplication to a standard of 1.0 mL
10 is the time of the reaction
D is the required dilution to be within the limits range
of the assay.
Enzyme stock solutions of 40 PU/ml were prepared in
5.0 mM sodium acetate buffer, pH 5.5, for all tested enzymes.
A temperature of 50 °C was selected after consideration of the
best compromise for optimal phytase activity for each of the
enzymes and the standardisation of the study.
Mineral Sources Effect on Phytase Activity
Triplicates of organic and inorganic samples were accu-
rately weighed and made up to a suitable volume with
5.0 mM sodium acetate buffer (pH 5.5) to give a final
Table 1 AAFCO definitions for organic mineral complexes
Metal amino acid complex The product resulting from complexing a soluble metal salt with an amino acid (<300 Da)
Metal (specific amino acid) complex The product resulting from complexing a soluble metal salt with a specific amino acid
Metal amino acid chelate The product resulting from the reaction of a metal ion from a soluble salt with amino acids
with a mole ratio of one mole of metal to one to three (preferably two) moles of amino
acids to form coordinate covalent bonds. The average weight of the hydrolysed amino
acids must be approximately 150 Da and the resulting molecular weight of the chelate
must not exceed 800 Da
Metal proteinate The product resulting from the chelation of a soluble salt with amino acids and/or
partially hydrolysed protein
Metal polysaccharide complex The product resulting from complexing of a soluble salt with a polysaccharide
solution declared as an ingredient as the specific metal complex

concentration of 1,000 ppm of each metal sample stock
solution. The necessary calculations made were based
on the metal concentrations of the metal chelates con-
firmed by the mineral analysis using ICP-MS, or on the
molecular formula in the case of the inorganic sul-
phates. The mineral sources were then extracted in for
30 min at 250 rpm. Phytase activities were studied in
the presence of different concentrations of Fe, Zn, Cu
and Mn ions ranging from 0.1 to 25 ppm in the reac-
tion mixture. The range of concentrations used represent
10 % of the higher and lower limits of those concen-
trations typically applied in the poultry and swine nu-
trition industry. The reaction mixture (4 mL) contained
80 μL of enzyme with variable concentrations (0.1–
25 ppm) of the metal tested, as well as a corresponding
volume of sodium acetate buffer, pH 5.5. The effect of
the metal ions was determined by incubating triplicates
of each initial mineral sample for 15 min at 50 °C in a
water bath. After incubation, the samples were immedi-
ately placed on an ice-cold bath (5 min). Finally, the
samples were assayed promptly for phytase activity as
described in the previous section. The relative activity
was calculated by comparing the remaining activity after
each treatment to that of the untreated enzyme.
The parameters of 50 °C temperature and pH 5.5 were
selected taking into consideration the best compromise for
optimal phytase activity for each of the three enzymes [3]
and standardisation of the study.
The pH of the all the samples was assessed, and no signif-
icant shift was verified after the mineral addition; hence, there
was no need to include a pH control.
Statistical Analysis
Data are expressed as the mean±SD (n=3) and with a 95 %
confidence interval. GraphPad PRISM, version 6.03 for Win-
dows (GraphPad Software, San Diego California USA), was
used for all the statistical analysis, including the determination
of IC50 values for inhibition of enzyme activity. The data were
fitted by nonlinear regression to the variable slope sigmoidal
dose–response curves with a confidence interval of 95 %. Y is
the percent activity, and X is the corresponding Log (concen-
tration of the metal ion). The interpolated IC50 parameter is the
absolute IC50, and it is defined as the concentration giving an
inhibition of 50 % of phytase activity. A one-way analysis of
variance (ANOVA) followed by Tukey’s honestly significant
differences (HSD) post hoc tests were carried out to compare
IC50 concentrations. Significance of results was considered at
p<0.05.
Data excluding the control were further analysed as a
factorial arrangement of treatments (n source×n concentra-
tion) by two-way ANOVA with a model that included the
main effects of the source of metal, its concentration, as well
as their interaction. The difference between sources at exact
concentrations was assessed using Tukey’s HSD post hoc
tests. Significance of results was considered at p<0.05.
Results
The effect of trace minerals on phytase activity was studied by
adding various sources of Fe, Zn, Cu and Mn with concentra-
tions ranging from 0.1 to 25 ppm. The results indicate that
source of mineral and concentration affected each of the three
phytase sources (two-way ANOVA; mineral source and con-
centration as variables, p<0.001).
The Effect of Mineral Sources on E. coli Phytase Activity
The effect of Fe (a), Zn (b), Cu (c) and Mn (d) on E. coli
phytase activity is illustrated in Fig. 1 which shows the vari-
able slope sigmoidal dose–response curves of the relative
phytase activity (percent) versus Log (concentration of the
metal ion) (parts per million (ppm)). The absolutes IC50 were
calculated when applicable and statistically compared by
Tukey’s multiple comparisons tests (Table 2). Significance
of results was considered at p<0.05. Differences in mineral
sources at specific concentrations for E. coli phytase activity
were evaluated by Tukey’s HSD post hoc tests and are shown
in Table 3. This enzyme was dramatically inhibited by Cu
(Fig. 1c). It can be observed that Cu PRO was the mineral
source that produced the lowest phytase inhibition. The Cu
PRO had a significantly higher IC50 of 0.7±0.6 ppm, p<0.05
(Table 2), as well as higher residual phytase activities com-
pared with the other sources tested (Table 3). Copper IC50
differed significantly from Fe and Zn IC50 (p<0.05) requiring
approximately ten times lower concentration to inhibit
phytase. No significant reduction in E. coli phytase activity
was found when Mn was present (Fig. 1d). Hence, IC50 values
for Mn were not applicable, because phytase inhibition did not
reach 50 % for any of the sources tested. Further analysis
showed that Mn SO4 and Mn Gly significantly increased
(p<0.05) the activity of the E. coli enzyme by 7 and 13 %,
whereas Mn GLY and Mn PRO significantly decreased
(p<0.05) phytase activity by approximately 9 and 12 % (Ta-
ble 3). Both Fe and Zn substantially reduced E. coli phytase
activity (Fig. 1a and b) and generated identical IC50, with the
exception of IC50 from GLY and ACH that were significantly
lower for Fe, p<0.05 (Table 2). Notably, Fe ACH was highly
inhibitory decreasing phytase activity by approximately 42 %
at a concentration of 0.5 ppm. The enzyme activity was
moderately decreased with 2.5 ppm of Fe ACH, levelling
out between the concentrations 5 and 25 ppm to a residual
activity of 22.0±1.0 % (Fig. 1a and Table 3). Overall,
proteinates caused the lowest inhibition and had the highest
Santos et al.

IC50 for Fe, Zn and Cu. IC50 concentrations may be ranked as
follows: Fe PRO>>Fe SO4>Fe PSC>Fe GLY>>Fe ACH;
Zn PRO>Zn ACH>Zn GLY>Zn PSC>Zn SO4; and Cu
PRO>>Cu ACH>Cu PSC>Cu SO4>Cu GLY.
The Effect of Mineral Sources on P. lycii Phytase Activity
The sigmoidal dose–response curves representing the effect of
Fe (a), Zn (b), Cu (c) and Mn (d) mineral sources on P. lycii
phytase activity are shown in Fig. 2. The enzyme activity was
gradually decreased by Fe (a) and Zn (b), whereas Cu (c)
supplementation moderately reduced activity and the enzyme
remained relatively stable in the presence of Mn (d) (Fig. 2).
Fe PRO displayed the highest IC50 (16.7±2.5 ppm), p<0.05,
followed by Fe PSC (10.4±1.3 ppm); these IC50 were sub-
stantially and significantly higher (p<0.05) than any other Fe
sources (Table 4). Although Zn PRO IC50 was slightly higher
than the other Zn sources (6.2±1.0 ppm), none of the IC50
were statistically different (p>0.05), which supports the iden-
tical trends of the curves visualised in Fig. 2b. P. lycii’s activity
Table 2 IC50 concentration values (ppm) of mineral sources for inhibition of E. coli phytase
IC50 concentration (ppm)
PRO GLY ACH PSC SO4
Fe 6.7±1.1 A 1 3.4±0.3 B C 1 0.9±0.1 C 1 3.7±0.1 B 1 4.1±0.4 B 1
Zn 8.6±0.5 A 1 6.1±0.5 B C 2 6.7±0.4 A B 2 5.4±0.4 B C 1 4.9±0.5 C 1
Cu 0.7±0.06 A 2 0.3±0.03 B 3 0.5±0.06 B 1 0.4±0.05 B 2 0.3±0.03 B 2
Mn N/A N/A N/A N/A N/A
IC50 values are the means SD (n=2) that represent the upper and lower limits of the interpolated dose–inhibition curves with 95 % confidence. For each
IC50, source (columns) means marked by different italicized letters significantly differ (p<0.05; Tukey’s HSD post hoc tests). For each IC50, mineral
means (lines) marked by different italicized numbers significantly differ (p<0.05; Tukey’s HSD post hoc tests)
N/A not applicable, PRO proteinate, GLY glycinate, ACH amino acid chelate, PSC polysaccharide complex, SO4 sulphate
a b
- 0 .5 0 - 0 .2 5 0 .0 0 0 .2 5 0 .5 0 0 .7 5 1 .0 0 1 .2 5 1 .5 0
0
1 0
2 0
3 0
4 0
5 0
6 0
7 0
8 0
9 0
1 0 0
L o g F e ( p p m ) C o n c e n tr a t io n
%PhytaseRelativeActivity
Fe PRO
F e G LY
F e A C H
Fe PSC
F e S O 4
- 0 .5 0 - 0 .2 5 0 .0 0 0 .2 5 0 .5 0 0 .7 5 1 .0 0 1 .2 5 1 .5 0
0
1 0
2 0
3 0
4 0
5 0
6 0
7 0
8 0
9 0
1 0 0
L o g Z n (p p m ) C o n c e n tr a tio n
Zn P RO
Z n G L Y
Z n A C H
Zn PSC
Z n S O 4
c d
- 1 .2 5 - 1 .0 0 - 0 .7 5 - 0 .5 0 - 0 .2 5 0 .0 0 0 .2 5 0 .5 0 0 .7 5 1 .0 0
0
1 0
2 0
3 0
4 0
5 0
6 0
7 0
8 0
9 0
1 0 0
L o g C u (p p m ) C o n c e n tr a tio n
Cu PRO
C u G LY
C u AC H
Cu PSC
C u S O 4
0 .0 0 .5 1 .0 1 .5
8 0
9 0
1 0 0
1 1 0
1 2 0
L o g M n (p p m ) C o n c e n tr a t io n
Mn PRO
M n G LY
Mn PSC
M n S O 4
Fig. 1 Sigmoidal dose–response
curves representing the effect of
Fe (a), Zn (b), Cu (c) and Mn (d)
mineral sources on E. coli phytase
activity (%) Data are presented as
means SD (n=3). Fe PRO, Fe
proteinate; Zn PRO, Zn
proteinate; Cu PRO, Cu
proteinate; Mn PRO, Mn
proteinate; Fe GLY, Fe glycinate;
Zn GLY, Zn glycinate; Cu GLY,
Cu glycinate; Mn GLY, Mn
glycinate; Fe PSC, Fe
polysaccharide complex; Zn PSC,
Zn polysaccharide complex; Cu
PSC, Cu polysaccharide
complex; Mn PSC, Mn
polysaccharide complex; Fe
ACH, Fe amino acid chelate; Zn
ACH, Zn amino acid chelate; Cu
ACH, Cu amino acid chelate; Fe
SO4, Fe sulphate; Cu SO4, Cu
sulphate; Zn SO4, Zn sulphate;
Mn SO4, Mn sulphate

was not considerably affected by Cu PSC (±10 %) (Table 5).
No difference greater than ±5 % was observed in P. lycii’s
activity with Mn.
The Effect of Mineral Sources on A. niger Phytase Activity
Data are presented as means±SD (n=3), Fe PRO representing
Fe proteinate; Zn PRO, Zn proteinate; Cu PRO, Cu proteinate;
Mn PRO, Mn proteinate; Fe GLY, Fe glycinate; Zn GLY, Zn
glycinate; Cu GLY, Cu glycinate; Mn GLY, Mn glycinate; Fe
PSC, Fe polysaccharide complex; Zn PSC, Zn polysaccharide
complex; Cu PSC, Cu polysaccharide complex; Mn PSC, Mn
polysaccharide complex; Fe ACH, Fe amino acid chelate; Zn
ACH, Zn amino acid chelate; Cu ACH, Cu amino acid che-
late; Fe SO4, Fe sulphate; Cu SO4, Cu sulphate; Zn SO4, Zn
sulphate; and Mn SO4, Mn sulphate.
Copper (Fig. 3c) and Fe (Fig. 3a) effected a significant
decrease in phytase activity (p<0.05). The concentrations of
Cu PRO and Fe PRO required to inhibit phytase by 50 % were
2.2±0.4 and 10.9±1.0 ppm, respectively (Table 6). Cu PRO
had the highest IC50 and was statistically different from inor-
ganic CuSO4 (p<0.05). Fe PSC had an IC50 of 12.0±1.3 ppm,
which was significantly higher (p<0.05) than the other Fe
sources, with the exception of Fe PRO. Both Zn PSC and Zn
SO4 did not affect A. niger phytase activities enough to cause
50 % inhibition within the concentrations tested. The inhibi-
tion curves for these minerals also displayed identical trends
(Fig. 3b), which correlated with no statistical difference
(p>0.05) in their activities at the concentrations of 0.5 and
25 ppm (Table 7). Mn GLY significantly reduced phytase
activity (p<0.05) for the concentrations analysed in Table 6.
For the A. niger phytase, Mn acted as an inhibitor for all tested
sources (Fig. 3d and Table 7).
Discussion
Results from this study revealed a significant relation-
ship between phytase inhibition, trace mineral type as
well as source and concentration, p<0.001. This con-
firmed the premise on which this study was based that
different mineral sources can affect enzyme activity in
different ways, depending on mineral type, mineral
source and phytase enzyme.
Phytases exhibit differences in the way they react to
metal ions. Analysis of the effect of the tested trace
minerals on phytase activities revealed that Fe was a
strong inhibitor for E. coli, P. lycii and A. niger phytase
activities. Zinc severely repressed P. lycii and E. coli
phytases. It also inhibited A. niger phytase, though the
affect on activity was markedly less. Copper demon-
strated a potent inhibitory effect on E. coli and A. niger
Table3ComparisonofmineralsourcesdifferencesforspecificconcentrationsforE.coliphytaseactivity
Mineralconcentration(ppm)
FeZnCuMn
0.57.5250.57.5250.51551025
SourcePRO93.4±0.2A48.5±1.4A26.7±1.1A97.8±0.7A53.5±1.0A23.7±0.2A53.1±0.4A45.1±0.9A17.2±0.4A101.8±0.6A98.6±0.5A87.9±0.3A
GLY86.5±0.8B29.1±1.8B2.6±0.1B92.4±2.0B47.6±1.4B19.5±0.1B42.0±0.6B29.1±0.7B3.5±0.3B102.7±0.7AB108.1±0.5B112.9±0.6B
ACH57.9±0.2C25.4±0.6C22.0±1.0C90.0±1.6C47.3±1.0B21.2±0.7B48.0±0.4B37.0±0.8C7.1±0.7CN/AN/AN/A
PSC83.5±0.7D40.8±0.5D20.1±1.7D92.5±0.6BC43.2±0.7C4.7±0.4C43.2±1.2C31.0±1.1D0.0±1.5D110.3±0.3C104.3±1.5C90.5±0.7C
SO485.8±0.5B33.5±1.5E2.4±0.4B90.8±0.3BC39.9±1.2D14.7±0.5D39.6±1.0D30.1±0.5BD10.7±0.4E103.4±0.3B105.2±0.8C106.7±0.3D
DataarethemeansSD(n=3)thatcorrespondtoE.coliphytaserelativeactivity(%).Foreachconcentrationofmineral(columns),sourcemeansmarkedbydifferentitalicizedletterssignificantlydiffer
(p<0.05;Tukey’sHSDposthoctests)
PROproteinate,GLYglycinate,ACHaminoacidchelate,PSCpolysaccharidecomplex,SO4sulphate
Santos et al.

phytases; however, it only moderately inhibited P. lycii
phytase. Manganese was the metal ion that had the least
effect on phytase activity. The tested phytases were
quite stable in the presence of Mn with the exception
of the A. niger phytase that showed a slight inhibition.
These findings suggest a difference between the mech-
anisms of the tested HAP phytases because they
displayed different responses to the same cation. De-
pending on the source and/or expression host, phytases
are known to show distinctive biophysical and biochem-
ical properties [30]. A number of works have described
differences between phytases [30–33]. This variation is
attributed to physicochemical differences, including mo-
nomeric (A. niger and E. coli) versus dimeric (P. lycii)
proteins, different amino acid sequences and isoelectric
points, in addition to possible glycosylation differences.
Moreover, other studies focusing on the characterisa-
tion of phytases illustrated similar modulation of
phytase activity. For example, Greiner et al. [34]
characterised two phytases from E. coli in which the
effect of metal ions on the enzyme activity revealed that
while Mn2+
was slightly deactivating, Cu2+
and Zn2+
a b
- 0 .5 0 - 0 .2 5 0 .0 0 0 .2 5 0 .5 0 0 .7 5 1 .0 0 1 .2 5 1 .5 0
0
1 0
2 0
3 0
4 0
5 0
6 0
7 0
8 0
9 0
1 0 0
L o g F e ( p p m ) C o n c e n tr a t io n
Fe PRO
F e G LY
F e A C H
Fe PSC
F e S O 4
- 0 .5 0 - 0 .2 5 0 .0 0 0 .2 5 0 .5 0 0 .7 5 1 .0 0 1 .2 5 1 .5 0
0
1 0
2 0
3 0
4 0
5 0
6 0
7 0
8 0
9 0
1 0 0
Zn P RO
Z n G L Y
Z n A C H
Zn PSC
Z n S O 4
c d
- 1 .2 5 - 1 .0 0 - 0 .7 5 - 0 .5 0 - 0 .2 5 0 .0 0 0 .2 5 0 .5 0 0 .7 5 1 .0 0
0
1 0
2 0
3 0
4 0
5 0
6 0
7 0
8 0
9 0
1 0 0
Cu PRO
C u G LY
C u AC H
Cu PSC
C u S O 4
0 .0 0 .5 1 .0 1 .5
8 0
8 5
9 0
9 5
1 0 0
1 0 5
1 1 0
Mn PRO
M n G LY
Mn PSC
M n S O 4
mineral sources on P. lycii relative
phytase activity (%) Data are
presented as means±SD (n=3).
Fe PRO, Fe proteinate; Zn PRO,
Zn proteinate; Cu PRO, Cu
complex; Mn PSC, Mn
Mn SO4, Mn sulphate
Table 4 IC50 concentration values (ppm) of mineral sources for inhibition of P. lycii phytase
PRO GLY ACH PSC SO4
Fe 16.7±2.5 A 1 3.8±0.8 B 1 1.4±0.2 B 1 10.4±1.3 C 1 2.9±0.4 B 1
Zn 6.2±1.0 A 2 4.9±0.6 A 1 4.9±0.3 A 1 4.6±0.3 A 2 4.6±0.3 A 1
Cu N/A N/A N/A N/A N/A
IC50 values are the means SD (n=2) that represent the upper and lower limits of the interpolated dose-inhibition curves with 95 % confidence
For each IC50, source means (columns) marked by different italicized letters significantly differ (p<0.05; Tukey’s HSD post hoc tests). For each IC50,
mineral means (lines) marked by different italicized numbers significantly differ (p<0.05; Tukey’s HSD post hoc tests)
N/A not applicable, PRO proteinate, GLY glycinate, ACH amino acid chelate, PSC polysaccharide complex, SO4, sulphate

showed strong inhibitory effects. Reduced phytase ac-
tivity was also found in the presence of Fe2+
being
attributed to a lower phytate concentration because of
the appearance of a Fe-phytate precipitate. The same
researcher studied the effect of metal ions on the
phytase activity of Klebsiella terrigena and verified yet
again that Mn2+
was only slightly inhibitory, whereas
Cu2+
, Zn2+
and Fe2+
showed strong inhibitory effects
[35]. Our results for the A. niger phytase differ some-
what from those previously reported for other
Aspergillus-derived phytases. As reported by Dvorakova
et al. [36], Mn ions are known to stimulate the phytase
of A. niger 92, while Cu2+
and Zn2+
ions are highly
inhibitory. Furthermore, A. niger 11T53A9 phytase ac-
tivity was vigorously inhibited by Zn2+
and Fe2+
[37].
A rather contradictory result was the fact that Cu2+
only
had a slight effect on P. lycii phytase, particularly be-
cause it was such a strong inhibitor for the other tested
phytases. However, other investigations had comparable
results with phytases from other sources [37, 38]. In
addition, P. lycii is a basidiomycete fungus, and a num-
ber of these fungi are known to remove and immobilise
Cu2+
[39, 40].
Conjointly, it was observed that different sources of the
same mineral influenced phytase differently. We should con-
sider two main assessments including, organic versus inor-
ganic; and proteinates versus glycinates versus amino acid
chelates versus polysaccharide complexes. Furthermore, we
have to regard effects within the chelates, the metal effect and
a potential ligand effect.
Overall, the source that appeared to show the least
inhibitory effect across all the metals and phytases test-
ed was the proteinate. The proteinates were consistently
and significantly less inhibitory than the majority of the
other sources. This was verified for E. coli and P. lycii
phytases for Fe and Zn, as well as for Cu with E. coli
and A. niger phytases. Some exceptions occurred: the
Cu interaction with P. lycii in which the polysaccharide
complex and the enzyme demonstrated to be quite sta-
ble; the A. niger phytase displayed less inhibition for Zn
and Fe polysaccharide complex, although, for Fe, the Fe
PSC was not significantly different from Fe PRO,
p>0.05. The reason for these results is not clear, but
it may be connected with the different stabilities of the
chelates and/or a ligand effect. The chelation strength of
an organic mineral source and its behaviour under phys-
iological conditions is critical in determining the value
of products used as supplements in animal nutrition
[41]. Ligand sources, such as proteins, amino acids,
peptides or polysaccharides have an effect on chelating
properties and strength of the chelation bond. According
to the extensive research done by Cao et al. [42] on the
characterisation of organic supplemental Zn sources, the
Table5ComparisonofmineralsourcesdifferencesforspecificconcentrationsforP.lyciiphytaseactivity
FeZnCuMn
0.57.5250.57.5250.51551025
GLY89.5±0.8B25.9±0.6B4.8±0.7B88.6±1.6B40.9±0.5A23.3±0.4B88.7±0.9B84.5±1.2B67.1±0.4B94.1±0.6B91.0±0.4B89.6±1.0B
ACH70.4±0.2C23.8±0.8C9.9±0.6C90.7±0.3C35.0±0.5B14.5±1.5C88.3±0.8B86.5±0.4A71.9±1.0CN/AN/AN/A
PSC94.1±1.4D54.43±0.7D38.2±0.9D89.0±0.2B39.8±0.6C15.9±0.2C94.4±0.3C91.6±0.9C90.1±0.7D96.3±1.5C98.1.3±0.7A100.0±0.5C
SO481.2±0.4E20.3±0.5E0.9±0.2E89.7±0.2BC36.8±0.6C15.9±0.2C90.5±0.9D87.3±0.3A68.4±0.5B96.9±1.8C98.8±1.2A99.4±0.3C
Dataarethemeans±SD(n=3)thatcorrespondtoE.coliphytaserelativeactivity(%)
Foreachconcentrationofmineral(columns),sourcemeansmarkedbydifferentitalicizedletterssignificantlydiffer(p<0.05;Tukey’sHSDposthoctests)
PROproteinate,GLYglycinate,ACHaminoacidchelate,PSCpolysaccharidecomplex,SO4sulphate
Santos et al.

amount of zinc remaining bound in the complexed products
was not as great as that in the chelated products. This conclu-
sion was supported by their results in which they showed that
the amount of Zn chelated in the tested products, including
three Zn proteinates, three Zn specific amino acid complexes,
a Zn polysaccharide complex and a Zn amino acid chelate,
was in agreement with their chelation effectiveness. The
higher the chelation quotient (Qf), higher the amount of Zn
still chelated in water. The Zn complexed products displayed
weak chelation (Qf<10); two of the Zn proteinates showed a
moderately strong chelation (10≤Qf≤100), and one of the Zn
proteinates was strongly chelated (Qf>100). These facts can
be used as a suggestion that the tested organic sources in our
study reacted differently because of their differing stabilities.
Our results therefore imply that the proteinates were more
stable than the other sources tested. Similarly to our work,
Pang and Applegate [43] conducted an in vitro study
where they assessed the effect of copper source and
concentration on phytate phosphorous hydrolysis by
phytase. Like us, they concluded that the effect of Cu
on phytase was dependent on the Cu source. They at-
tributed the difference between sources to the different
solubility of Cu sources and less insoluble copper-
phytate being formed.
a b
- 0 .5 0 - 0 .2 5 0 .0 0 0 .2 5 0 .5 0 0 .7 5 1 .0 0 1 .2 5 1 .5 0
0
1 0
2 0
3 0
4 0
5 0
6 0
7 0
8 0
9 0
1 0 0
L o g F e (p p m ) C o n c e n tr a tio n%PhytaseRelativeActivity
Fe PRO
F e G LY
Fe AC H
Fe PSC
Fe SO 4
- 0 .5 0 - 0 .2 5 0 .0 0 0 .2 5 0 .5 0 0 .7 5 1 .0 0 1 .2 5 1 .5 0
0
1 0
2 0
3 0
4 0
5 0
6 0
7 0
8 0
9 0
1 0 0
Zn P RO
Z n G L Y
Z n A C H
Zn PSC
Z n S O 4
c d
- 1 .2 5 - 1 .0 0 - 0 .7 5 - 0 .5 0 - 0 .2 5 0 .0 0 0 .2 5 0 .5 0 0 .7 5 1 .0 0
0
1 0
2 0
3 0
4 0
5 0
6 0
7 0
8 0
9 0
1 0 0
Cu PRO
C u G LY
C u AC H
Cu PSC
C u S O 4
0 .0 0 .5 1 .0 1 .5
5 0
6 0
7 0
8 0
9 0
1 0 0
1 1 0
Mn PRO
M n G LY
Mn PSC
M n S O 4
mineral sources on A. niger
relative phytase activity (%). Data
are presented as means±SD (n=
3). Fe PRO, Fe proteinate; Zn
PRO, Zn proteinate; Cu PRO, Cu
complex; Mn PSC, Mn
Mn SO4, Mn sulphate
Table 6 IC50 concentration values (ppm) of mineral sources for inhibition of A. niger phytase
PRO GLY ACH PSC SO4
Fe 10.9±1.0 A 1 6.0±0.9 B 1 1.1±0.1 C 1 12.0±1.3 A 1 8.5±1.0 A B 1
Zn 26.5±0.1 A 2 14.0±2.1 B 2 12.9±1.5 B 2 N/A N/A
Cu 2.2±0.4 A 3 1.4±0.2 AB 3 1.5±0.3 AB 1 1.4±0.3 AB 2 0.9±0.1 B 2
IC50 values are the means±SD (n=2) that represent the upper and lower limits of the interpolated dose–inhibition curves with 95 % confidence
For each IC50, source means (columns) marked by different italicized letters significantly differ (p<0.05; Tukey’s HSD post hoc tests). For each IC50,
mineral means (lines) marked by different italicized numbers significantly differ (p<0.05; Tukey’s HSD post hoc tests)
N/A not applicable, PRO proteinate, GLY glycinate, ACH amino acid chelate, PSC polysaccharide complex, SO4 sulphate

The findings of this study are a good indication of what
potential interactions can occur within a premix. The levels of
trace minerals used in poultry and pig nutrition are still those
recommended by the NRC [44, 45], though manufacturers of
organic supplements recommend lower amounts. The range
of concentrations used in this work represent 10 % of the
higher and lower limits of those concentrations typically ap-
plied in the industry for these animals.
Our results demonstrated that different chelate sources
present different abilities to inhibit phytase. Additionally, this
study also suggests that enzyme inhibition can be a possible
indication of chelation stability. Further studies in vitro can
potentially focus on mineral combinations and/or stabilisation
studies within the premix.
Acknowledgments The support, both financially and professionally,
offered by Alltech is greatly appreciated.
Conflict of Interest The authors declare that they have no conflict of
interest.
The manuscript does not contain clinical studies or patient data.
References
1. Lopez HW, Leenhardt F, Coudray C, Remesy C (2002) Minerals and
phytic acid interactions: is it a real problem for human nutrition? Int J
Food Sci Technol 37(7):727–739
2. Kumar V, Sinha AK, Makkar HP, De Boeck G, Becker K (2012)
Phytate and phytase in fish nutrition. J Anim Physiol Anim Nutr
96(3):335–364
3. Kim Y-O, Kim H-K, Bae K-S, Yu J-H, Oh T-K (1998) Purification
and properties of a thermostable phytase from Bacillus sp. DS11.
Enzym Microb Technol 22(1):2–7
4. Tran TT, Hashim SO, Gaber Y, Mamo G, Mattiasson B, Hatti-Kaul R
(2011) Thermostable alkaline phytase from Bacillus sp. MD2: effect
of divalent metals on activity and stability. J Inorg Biochecm 105(7):
1000–1007
5. Persson H, Turk M, Nyman M, Sandberg AS (1998) Binding of
Cu2+, Zn2+ and Cd2+ to inositol tri-, tetra-, penta-, and
hexaphosphates. J Agric Food Chem 46(8):3194–3200
6. 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
7. Rimbach G, Brandt K, Most E, Pallauf J (1995) Supplemental phytic
acid and microbial phytase change zinc bioavailability and cadmium
accumulation in growing rats. J Trace Elem Med Biol 9(2):117–122
8. Mallin MA, Cahoon LB (2003) Industrialized animal production—a
major source of nutrient and microbial pollution to aquatic ecosys-
tems. Popul Environ 24(5):369–385
9. Bohn L, Meyer AS, Rasmussen SK (2008) Phytate: impact on
environment and human nutrition. A challenge for molecular breed-
ing. J Zhejiang University-Science B 9(3):165–191
10. Lei XG, Weaver JD, Mullaney E, Ullah AH, Azain MJ (2013)
Phytase, a new life for an “old” enzyme. Annu Rev Anim Biosci
1(1):283–309
11. Roopesh K, Ramachandran S, Nampoothiri KM, Szakacs G, Pandey
A (2006) Comparison of phytase production on wheat bran and
Table7ComparisonofmineralsourcesdifferencesforspecificconcentrationsforA.nigerphytaseactivity
FeZnCuMn
0.57.5250.57.5250.51551025
GLY78.7±0.2B47.8±0.9B8.9±0.5B96.8±0.4B59.8±0.9B42.7±0.5B66.7±0.9B54.3±1.3B34.0±0.7B92.0±1.1B73.1±0.6B59.8±0.9B
ACH69.3±0.6C20.9±1.1C14.2±0.7C92.2±1.4C57.1±0.8C41.5±0.8B70.0±1.2A52.7±0.8B32.6±0.3BN/AN/AN/A
PSC85.9±1.2D62.8±1.5D29.3±0.7D98.4±1.4B76.2±0.6A64.4±0.9C62.8±0.5C53.1±1.3B35.8±0.2C95.8±0.2C82.7±1.1C76.2±0.6C
SO489.1±0.4E55.3±1.3E24.0±1.2E98.8±0.3B78.6±0.3D66.0±0.7C61.6±0.3C44.6±0.8C19.1±0.9D94.6±1.5AC81.8±0.2C78.6±0.3D
DataarethemeansSD(n=3)thatcorrespondtoE.coliphytaserelativeactivity(%)
Foreachconcentrationofmineral(columns),sourcemeansmarkedbydifferentitalicizedletterssignificantlydiffer(p<0.05;Tukey’sHSDposthoctests)
PROproteinate,GLYglycinate,ACHaminoacidchelate,PSCpolysaccharidecomplex,SO4,sulphate
Santos et al.

oilcakes in solid-state fermentation by Mucor racemosus. Bioresour
Technol 97(3):506–511
12. Adeola O, Cowieson AJ (2011) Board-invited review: opportunities
and challenges in using exogenous enzymes to improve nonruminant
animal production. J Anim Sci 89(10):3189–3218
13. Ullah AH, Dischinger HC Jr (1993) Aspergillus ficuum phytase:
complete primary structure elucidation by chemical sequencing.
Biochem Biophys Res Commun 192(2):747–753
14. Ramachandran S, Roopesh K, Nampoothiri KM, Szakacs G, Pandey
A (2005) Mixed substrate fermentation for the production of phytase
by Rhizopus spp. using oilcakes as substrates. Process Biochem
40(5):1749–1754
15. Casey A, Walsh G (2004) Identification and characterization of a
phytase of potential commercial interest. J Biotechnol 110(3):313–322
16. Paripatananont T, Lovell RT (1997) Comparative net absorption of
chelated and inorganic trace minerals in channel catfish Ictalurus
punctatus diets. J World Aquacult Soc 28(1):62–67
17. Veum TL, Carlson MS, Wu CW, Bollinger DW, Ellersieck MR
(2004) Copper proteinate in weanling pig diets for enhancing growth
performance and reducing fecal copper excretion compared with
copper sulfate. J Anim Sci 82(4):1062–1070
18. Mondal MK, Das TK, Biswas P, Samanta CC, Bairagi B
(2007) Influence of dietary inorganic and organic copper salt
and level of soybean oil on plasma lipids, metabolites and
mineral balance of broiler chickens. Anim Feed Sci Technol
139(3–4):212–233
19. Garg AK, Mudgal V, Dass RS (2008) Effect of organic zinc supple-
mentation on growth, nutrient utilization and mineral profile in
lambs. Anim Feed Sci Technol 144(1–2):82–96
20. AAFCO (1998) Official Publication of the Association of American
Feed Control Officials Incorporated. In: Bachman PM (ed). pp 237–238.
21. Shah BG (1981) Chelating agents and bioavailability of minerals.
Nutr Res 1(6):617–622
22. Byrne LA, Hynes MJ, Connolly CD, Murphy RA (2011) Analytical
determination of apparent stability constants using a copper ion
selective electrode. J Inorg Biochem 105(12):1656–1661
23. Radcliffe JS, Aldridge BE, Saddoris KL (2007) Understanding or-
ganic mineral uptake mechanisms: experiments with Bioplex® Cu.
(14/02/14).
24. Guo R, Henry PR, Holwerda RA, Cao J, Littell RC, Miles RD,
Ammerman CB (2001) Chemical characteristics and relative bio-
availability of supplemental organic copper sources for poultry. J
Anim Sci 79(5):1132–1141
25. Näsi JM, Helander EH, Partanen KH (1995) Availability for growing
pigs of minerals and protein of a high phytate barley-rapeseed meal
diet treated with Aspergillus niger phytase or soaked with whey.
Anim Feed Sci Technol 56(1–2):83–98
26. Revy PS, Jondreville C, Dourmad JY, Nys Y (2004) Effect of zinc
supplemented as either an organic or an inorganic source and of
microbial phytase on zinc and other minerals utilisation by weanling
pigs. Anim Feed Sci Technol 116(1–2):93–112
27. Jondreville C, Lescoat P, Magnin M, Feuerstein D, Gruenberg B, Nys
Y (2007) Sparing effect of microbial phytase on zinc supplementa-
tion in maize–soya-bean meal diets for chickens. Anim: Int J Anim
Biosci 1(6):804–811
28. Schlegel P, Nys Y, Jondreville C (2010) Zinc availability and diges-
tive zinc solubility in piglets and broilers fed diets varying in their
phytate contents, phytase activity and supplemented zinc source.
Anim: Int J Anim Biosci 4(2):200–209
29. Engelen AJ, van der Heeft FC, Randsdorp PH, Smit EL (1994)
Simple and rapid determination of phytase activity. J AOAC Int
77(3):760–764
30. Rao D, Rao KV, Reddy TP, Reddy VD (2009) Molecular character-
ization, physicochemical properties, known and potential applica-
tions of phytases: an overview. Crit Rev Biotechnol 29(2):182–198
31. Wyss M, Brugger R, Kroenberger A, Remy R, Fimbel R, Osterhelt G,
Lehmann M, van Loon A (1999) Biochemical characterization of
f u n g a l p h y t a s e s ( m y o- i n o s i t o l he x a k i s p h o s p h a t e
phosphohydrolases): catalytic properties. Appl Environ Microbiol
65(2):367–373
32. Mullaney EJ, Ullah AHJ (2003) The term phytase comprises several
different classes of enzymes. Biochem Biophys Res Commun
312(1):179–184
33. George TS, Simpson RJ, Gregory PJ, Richardson AE (2007)
Differential interaction of Aspergillus niger and Peniophora lycii
phytases with soil particles affects the hydrolysis of inositol phos-
phates. Soil Biol Biochem 39(3):793–803
34. Greiner R, Konietzny U, Jany KD (1993) Purification and character-
ization of two phytases from Escherichia coli. Arch Biochem
Biophys 303(1):107–113
35. Greiner R, Haller E, Konietzny U, Jany K-D (1997) Purification and
characterization of a phytase from Klebsiella terrigena. Arch
Biochem Biophys 341(2):201–206
36. Dvorakova J, Volfova O, Kopecky J (1997) Characterization of
phytase produced by Aspergillus niger. Folia Microbiologica 42(4):
349–352
37. Greiner R, da Silva LG, Couri S (2009) Purification and
characterisation of an extracellular phytase from Aspergillus
niger 11T53A9. Braz J Microbiol: [Publ Braz Soc Microbiol]
40(4):795–807
38. Greiner R, Farouk A-E (2007) Purification and characterization of a
bacterial phytase whose properties make it exceptionally useful as a
feed supplement. Protein J 26(7):467–474
39. Ejechi B (2003) Immobilization of Cu(II) and Cr(IV) in
basidiomycete-colonized sawdust. World J Microbiol Biotechnol
19(2):135–137
40. Javaid A (2012) Biosorption of electroplating heavy metals by some
basidiomycetes. Mycopath 6(1 & 2)
41. Li S, Luo X, Liu B, Crenshaw TD, Kuang X, Shao G, Yu S (2004)
Use of chemical characteristics to predict the relative bioavailability
of supplemental organic manganese sources for broilers. J Anim Sci
82(8):2352–2363
42. Cao J, Henry PR, Guo R, Holwerda RA, Toth JP, Littell RC,
Miles RD, Ammerman CB (2000) Chemical characteristics
and relative bioavailability of supplemental organic zinc
sources for poultry and ruminants. J Anim Sci 78(8):2039–
2054
43. Pang Y, Applegate TJ (2006) Effects of copper source and concen-
tration on in vitro phytate phosphorus hydrolysis by phytase. J Agric
Food Chem 54(5):1792–1796
44. Nutrient requirements of poultry: ninth revised edition, 1994 (1994).
The National Academies Press.
45. Nutrient requirements of swine: 10th revised edition (1998). The
National Academies Press.

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