Rex W. Newkirk

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

Abstract Minerals can readily bind to phytic acid and thus have the potential to form mineral–phytate complexes that may be resistant to hydrolysis by phytase activity of animal, plant and microbial origin. In simple solution, at pH 7.0, mineral concentrations from 0.053mM for Zn 2+ up to 4.87mM for Mg 2+ caused a 50% inhibition of phytate-P hydrolysis by microbial phytase. The rank order of mineral potency as inhibitors of phytate hydrolysis was Zn 2+ ⪢Fe 2+ >Mn 2+ >Fe 3+ >Ca 2+ >Mg 2+ at neutral pH. Acidification of the media to pH 4.0 decreased the inhibitory potency of all of the divalent cations tested. The inhibitory potency of Fe 3+ showed a moderate increase with declining pH. Inclusion of 25mM ethylenediamine-tetraacetic acid (EDTA) completely blocked Ca 2+ inhibition of phytate hydrolysis at pH 7. Inorganic P comprised 0.20–0.25 of the total P in a slurry of canola meal. Incubation with microbial phytase increased inorganic P up to 0.50 of total P levels. Supplementation with chelators such as EDTA, citrate and phthalate increased the efficacy of microbial phytase in hydrolyzing phytic acid. Incubation of canola meal with 100mM phthalic acid plus microbial phytase resulted in complete hydrolysis of phytate-P. Competitive chelation by compounds such as EDTA, citric acid or phthalic acid has the potential to decrease enzyme-resistant forms of phytic acid and thereby improve the efficacy of microbial phytase in hydrolyzing phytic acid.

In vitro hydrolysis of phytate in canola meal with purified and crude sources of phytase

Pre-treatment conditions required to hydrolyze phytate in canola meal with crude and purified phytases were investigated. Phytase (10,000, 100,000, 340,000 U/kg Natuphos®) and the same phytase (10,000, 100,000 U/kg) that had been purified by gel filtration were added to canola meal without pH adjustment (pH 5.8, 2:1 H2O: meal). Phytate hydrolysis was incomplete after 30 and 60 min (50°C) indicating that a portion of the phytate was resistant to hydrolysis. Purified and crude phytases (10,000 U/kg) were used to hydrolyze canola meal at pH 2.0, 2.5, 3.0, 3.5, 4.0, 4.5, 5.0 and 5.8. Phytase was more effective between pH 3 and 5. Crude phytase was significantly more effective (P<0.0001) than purified enzyme, suggesting that non phytase enzymes facilitated the action of phytase. The levels of lower inositol phosphates in response to incubation pH were similar to phytate, suggesting that they too are susceptible to the formation of complexes which prevent hydrolysis. Complete hydrolysis with crude phytase (pH 5.0, 5000 U/kg, 50°C, 2:1 H2O: meal) in 250 g batches was accomplished within 23 h. In conclusion, a portion of the phytate in canola meal is resistant to hydrolysis by phytase. Therefore, meal pretreatment, which includes pH modification, temperature and moisture control, and the addition of phytase and other enzymes, is required for the effective hydrolysis of phytate.