1. Phytic acid, mineral cations and phytase interactions Roselina Angel – University of Maryland Todd Applegate – Purdue University

2. Phytic Acid P C H O Ca Protein Cu Starch Zn Fe Protein

3. Cation “Impairment” of P hydrolysis vs. Cation “release” capacity of phytase

4. Phytase Phosphate Inositol 5 Phosphate

5. Background Mineral inhibition of phytase – Maenz et al. (1999) Zn 2+ > > Fe 2+ >Mn 2+ >Fe 3+ >Ca 2+ >Mg 2+ 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) Cu 2+ >Zn 2+ >Ni 2+ >Co 2+ >Mn 2+ >Fe 3+ >Ca 2+ – Maddaiah et al. (1964) Zn 2+ >Cu 2+ >Co 2+ >Mn 2+ >Ca 2+ At the same pH

6. Cation Inhibition of Phytate Hydrolysis* Maenz et al., 1999 * mM mineral conc. for 50% inhibition

7. Cation Inhibition of Phytate Hydrolysis* * mM mineral conc. for 50% inhibition Maenz et al., 1999

8. 2 mmol Phytic Acid + 30 mmol Calcium [Corn/SBM dietary phytic acid + 0.9% Calcium (2:1; H 2 0:feed)] Angel and Applegate, 2000

9. pH = 2.5 2 mmol Phytic Acid + 30 mmol Calcium [Corn/SBM dietary phytic acid + 0.9% Calcium (2:1; H 2 0:feed)] Angel and Applegate, 2000

10. pH = 2.5 pH = 6.5 2 mmol Phytic Acid + 30 mmol Calcium [Corn/SBM dietary phytic acid + 0.9% Calcium (2:1; H 2 0:feed)] Angel and Applegate, 2000

11. pH = 2.5 pH = 6.5 2 mmol Phytic Acid + 30 mmol Calcium [Corn/SBM dietary phytic acid + 0.9% Calcium (2:1; H 2 0:feed)] Angel and Applegate, 2000

12. 2 mmol phytic acid+ 30 mmol Calcium [corn/SBM St diet phytic acid content+ 0.9% Ca (2:1; H 2 0: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

13. 2 mmol Phytic Acid+ 30 mmol Calcium [Corn/SBM St diet phytic acid content + 0.9% Ca (2:1; H 2 0: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 Proventriculus/ Gizard Proximal duodenum Distal duodenum to Ileum

14. Ca & Ileal Phytate-P Hydrolysis: Chick * * Applegate et al., 2003

15. Ca & Ileal Phytate-P Hydrolysis: Chick Tamim et al 2003, 2004, unpublished Applegate et al., 2003 %

16. Ca & Ileal Phytate-P Hydrolysis Tamim et al., 2003, 2004, unpublished; Applegate et al., 2003 Exp 1Exp 2Exp 3Exp2Exp3 --------------------------- (% PP Hydrolysis)------------------- 0.2% Ca67.169.268.9 0.4% Ca74.135.2 0.7% Ca18.925.428.5 0.9% Ca49.723.1 ------------------------------- (PP, % of diet) ------------------------ Δ PP hydrolysis- 0.13- 0.12- 0.11 - 0.063-0.031

17. pH = 2.5 pH = 6.5 Phytic Acid + 250 ppm Cu (CuSO 4 )

18. Banks & Applegate, 2003 Phytase U/kg Copper Sulfate addition to Broiler Diets

19. a,b c a b Banks & Applegate, 2003 Phytase U/kg

20. Copper Sulfate addition to Broiler Diets a,b c a b Banks & Applegate, 2003 Phytase U/kg -0.066 (% of diet) -0.055 (% of diet)

21. Pang & Applegate, 2004

22. CuSO 4 CuCl TBCC Cu-Lys Cu-Citrate Cu source & Phytase activity – pH6.5 Pang & Applegate, 2004

23. Apparent Phosphorus Retention (% of diet) as affected by Cu source a c c b b 0 0 250 250 250 250 ppm Cu b 0.20 nPP 0.40 nPP a-c: P ≤ 0.05 Sulfate Citrate Lysinate Chloride Banks & Applegate, 2004

24. Ca & Micro-mineral source – in vivo Phytate – P hydrolysis ------ 0 % Calcium ------ ------ 0.5 % Calcium ------ Tamim & Angel, 2003 Ca: P < 0.0001 Micro mineral: P=0.39 Ca*Micro: P = 0.63

25. 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 –or- phytase * Zn)