Enzymatic Phytic Acid Hydrolysis

Enzymatic hydrolysis of phytic acid (Sigma, Poznań, Poland) was performed as described elsewhere [14 (link)] with modifications. The reaction was conducted in a total volume of 5 mL containing 100 mg of phytate and 50 mg of wheat phytase (Sigma, Poznań, Poland) at a pH of 5.15 and a temperature of 55 °C. The reaction was stopped after two hours by heating (100 °C for 5 min). Next, the obtained hydrolysate of phytic acid (hPA120) was cooled down on ice and precipitated enzymatic proteins were centrifuged (12,000× g, 10 °C, 10 min). The supernatant was collected, filter sterilized (filter pore 0.22 µm), aliquoted, and stored at −20 °C. Directly before use, the hydrolysate was defrosted and diluted twice with McCoy 5A medium without antibiotics. Before applying onto colonocytes, the hPA120 was 10-fold diluted in a medium appropriate for investigated cell lines.
The composition of inositol phosphates in phytate hydrolysate was determined by applying HPLC-MS (LC-20, Shimadzu, Kyoto, Japan; QTRAP 5500 mass spectrometer, AB SCIEX, Vaughan, Canada) and identified using real standards comprised the retention time and the presence of the respective parent and daughter ion (negative) pairs (Table S1) [15 (link)].