Table 1.

Preparation of enzymatic dephosphorylation reactions for calculating free and total phosphorus (g P 100 g.⁻¹)

Stepwise additions for the reactions	Free P (mL)	Total P (mL)
Distilled water	0.62	0.6
Solution 1 (buffer provided by Megazyme)	0.2	0.2
Neutralized sample extract	0.05	0.05
Suspension 2 (phytase prepared as per Megazyme kit instructions)	–	0.02
Mixed well and incubated for 10 min at 40 °C
Distilled water	0.02	–
Solution 3 (buffer provided by Megazyme)	0.02	0.02
Suspension 4 (alkaline phosphatase provided by Megazyme)	–	0.02
Solutions were mixed well on a vortex mixer and incubated in a water bath for 15 min at 40 °C
Trichloroacetic acid (50% w/v)	0.30	0.30

Solutions were centrifuged at 13,000 rpm for 10 min and 1.0 mL of supernatant was transferred to a new tube for spectrophotometric estimation