Phytic acid, mineral cations and
phytase interactions
Roselina Angel – University of Maryland
Todd Applegate – Purdue University
Phytic Acid
Fe
Zn
Protein
Ca
Starch
Protein
Cu
P
C
H
O
Cation “Impairment” of P
hydrolysis
vs.
Cation “release” capacity of
phytase
Background
• Mineral inhibition of phytase
– Maenz et al. (1999)
• Zn2+ > > Fe2+ >Mn2+ >Fe3+ >Ca2+ >Mg2+
• Soluble Ca - chelate at 1 mM Ca : 10 mM phytate
• Soluble Zn - chelate at equal concentrations Zn : phytate
• Mineral complex stability
– Vohra et al. (1965)
• Cu2+ >Zn2+ >Ni2+ >Co2+ >Mn2+ >Fe3+ >Ca2+
– Maddaiah et al. (1964)
• Zn2+ >Cu2+ >Co2+ >Mn2+ >Ca2+
• At the same pH
Cation Inhibition of Phytate Hydrolysis*
Zn++
1.6
Ca++
120
Zn (mM)
1.2
80
0.8
40
0.4
0
0
4
5
6
6.5
7
pH
Maenz et al., 1999
* mM mineral conc. for 50% inhibition
Cation Inhibition of Phytate Hydrolysis*
Zn++
1.6
Ca++
120
80
0.8
Ca (mM)
Zn (mM)
1.2
40
0.4
0
0
4
5
6
6.5
7
pH
Maenz et al., 1999
* mM mineral conc. for 50% inhibition
2 mmol Phytic Acid + 30 mmol Calcium
[Corn/SBM dietary phytic acid + 0.9% Calcium (2:1; H20:feed)]
Angel and Applegate, 2000
2 mmol Phytic Acid + 30 mmol Calcium
[Corn/SBM dietary phytic acid + 0.9% Calcium (2:1; H20:feed)]
pH = 2.5
Angel and Applegate, 2000
2 mmol Phytic Acid + 30 mmol Calcium
[Corn/SBM dietary phytic acid + 0.9% Calcium (2:1; H20:feed)]
pH = 2.5
pH = 6.5
Angel and Applegate, 2000
2 mmol Phytic Acid + 30 mmol Calcium
[Corn/SBM dietary phytic acid + 0.9% Calcium (2:1; H20:feed)]
pH = 2.5
pH = 6.5
Angel and Applegate, 2000
2 mmol phytic acid+ 30 mmol Calcium
[corn/SBM St diet phytic acid content+ 0.9% Ca (2:1; H20:diet)]
100
100
100
97.9
84.7
62.1 51.7
26.4
11.1
7.9
1.5
% of the phytic acid P in solution
Angel, 2007
2 mmol Phytic Acid+ 30 mmol Calcium
[Corn/SBM St diet phytic acid content + 0.9% Ca (2:1; H20:diet)]
100
100
100
97.9
84.7
62.1 51.7
26.4
11.1
7.9
1.5
% of the phytic acid P in solution
Proventriculus/ Gizard
Proximal
duodenum
Distal duodenum to Ileum
Angel, 2007
% Phytate P Hydrolysis
Ca & Ileal Phytate-P Hydrolysis: Chick
80
70
60
50
40
30
20
10
0
0.4 % Ca
0.9 % Ca
*
*
Exp. 2
Exp. 3
Applegate et al., 2003
Ca & Ileal Phytate-P Hydrolysis: Chick
75
0.2% Ca
0.4% Ca
0.7% Ca
65
0.9% Ca
%
55
45
35
25
15
Exp 1
Exp 2
Exp 3
Tamim et al 2003, 2004, unpublished
Exp 2
Exp 3
Applegate et al., 2003
75
0.2% Ca
0.4% Ca
0.7% Ca
65
0.9% Ca
Ca & Ileal Phytate-P Hydrolysis
55
45
35
25
15
Exp 1
Exp 1
Exp 2
Exp 3
Exp2
Exp 2
Exp 3
Exp 2
Exp 3
Exp3
--------------------------- (% PP Hydrolysis)-------------------
0.2% Ca
67.1
69.2
68.9
0.4% Ca
0.7% Ca
18.9
25.4
74.1
35.2
49.7
23.1
28.5
0.9% Ca
------------------------------- (PP, % of diet) ------------------------
Δ PP hydrolysis
- 0.13
- 0.12
- 0.11
- 0.063 -0.031
Tamim et al., 2003, 2004, unpublished; Applegate et al., 2003
Phytic Acid + 250 ppm Cu (CuSO4)
pH = 2.5
pH = 6.5
Copper Sulfate addition to
Broiler Diets
% phosphorus retention
65
no copper sulfate
2 lb/ton copper sulfate
0
600
60
55
50
45
40
35
Phytase U/kg
Banks & Applegate, 2003
Copper Sulfate addition to
Broiler Diets
% phosphorus retention
65
no copper sulfate
2 lb/ton copper sulfate
a
60
a,b
55
b
50
45
c
40
35
0
Phytase U/kg
600
Banks & Applegate, 2003
Copper Sulfate addition to
Broiler Diets
% phosphorus retention
65
no copper sulfate
2 lb/ton copper sulfate
a
60
-0.055
(% of diet)
a,b
55
b
-0.066
(% of diet)
50
45
c
40
35
0
Phytase U/kg
600
Banks & Applegate, 2003
Solubility of different Cu sources at different
concentrations at pH6.5
120
Solubility(%)
100
62.5
80
125
60
250
40
500
20
0
Cu Sul
Cu CL
TBCC
Cu Lys
Cu CIT
Cu sources
Pang & Applegate, 2004
Cu source & Phytase activity – pH6.5
Relative P release (%)
0
62.5
125
250
500
120
100
80
60
40
20
0
0
CuSO
1 4
CuCl
2
TBCC
3
Cu-Lys
Cu-Citrate
4
5
6
Pang & Applegate, 2004
Apparent Phosphorus Retention
(% of diet) as affected by Cu source
% 40
a
P
30
c
0
Chloride
0
Lysinate
nPP nPP
c
Citrate
0.20 0.40
b
Sulfate
R
e
t 20
e
n
t 10
i
o
n
0
b
b
250
250
250
250
ppm Cu
Banks & Applegate, 2004
a-c: P ≤ 0.05
Ca & Micro-mineral source – in vivo
Phytate – P hydrolysis
Ca:
Micro mineral:
Ca*Micro:
------ 0 % Calcium ------
P < 0.0001
P=0.39
P = 0.63
------ 0.5 % Calcium -----Tamim & Angel, 2003
Conclusions
(& Unresolved questions…….)
a) Calcium has a huge impact on PP hydrolysis. This effect is
heavily affected by pH --- but even at acidic pHs it can
influence PP hydrolysis by phytase
b) In laying hens vs chicks – lower impact of phytase on PP
release. Possibly because of better PP hydrolysis in the
absence of phytase
c) Cu (up to 250 ppm) & Zn (up to 160 ppm) have
inconsistent impacts on PP hydrolysis & no effect on
phytase efficacy (i.e. no interaction of phytase * Cu –orphytase * Zn)