radual decline at higher pH levels were observed for the enzyme
(Fig. 4). This narrow pH range was previously shown to be
desirable for phytase applied to fermented calcium-fortified
soymilk (Tang et al., 2010) and the diet for broiler chickens
(Elkhalil et al., 2007). As expected, the change in the free
calcium concentration in the soymilk samples followed the same
trend as the enzyme. Therefore, the release of calcium ions
resulting from the action of phytase was pH-dependent. Such a
phytase activity–calcium release relationship was similar to that
observed at different temperatures (Fig. 3). Namely, the free
calcium concentration in soymilk was strongly correlated with the
phytase activity (r at 0.992 for temperature dependency and r at
0.982 for pH dependency). The outlier point of 65 8C was not
included in the correlation plot because the enzyme appeared to
have lost considerable amount of activity at this high temperature
probably due to thermal denaturation (Fig. 5). Therefore, whether
the release of Ca2+ by phytase was achieved under varying pH,
hydrolysis temperatures, and possibly other conditions, the ISE
method was sensitive and reliable for the detection of phytase
activity.