Wheat phytase

Nucleotides (ATP and UDP) and wheat phytase used in this study were purchased from Sigma-Aldrich (St. Louis, MO, USA). wheat phytase was reconstituted in endotoxin-free water (Sigma-Aldrich, USA) and residual inorganic phosphate was removed through Pi-bond resin (Innova Biosciences, Cambridge, UK). A malachite green-based Pi Color Lock gold phosphate detection kit was procured from Innova Biosciences (UK). L-phenylalanine and L-homoarginine as inhibitors of dephosphorylation, were procured from Sigma-Aldrich (USA). For cell viability assay, an EZ-CYTOX kit was purchased from DogenBio (Seoul, Korea). Cymax human interleukin-6 (IL-6) and IL-8 enzyme-linked immunosorbent assay (ELISA) kits used for IL-6 and IL-8 secretion assay were obtained from Ab FRONTIER (Seoul, Korea). A caspase-3/CPP32 colorimetric assay kit used to measure the activity of caspase-3, marker of programmed cell death, was sourced from BioVision (Milpitas, CA, USA). Human colorectal adenocarcinoma cell line, HT-29, was obtained from ATCC (Manassas, VA, USA). Cells were cultured in McCoy’s 5A medium purchased from Gibco Life technologies (Carlsbad, CA, USA). The medium was supplemented with 10% fetal bovine serum and 1% penicillin-streptomycin solution, both of which were purchased from Gibco Life technologies (USA). These cells were cultured at 37°C in a humidified air incubator with 5% CO₂.

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)].

Long-chain polyP, P700 with average 1,150 Pi residues was procured from Kerafast (Boston, MA, USA). Wheat phytase and Griess reagent were purchased from Sigma-Aldrich. Cymax mouse tumor necrosis factor α (TNF-α) enzyme-linked immunosorbent assay (ELISA) kit and Raw 264.7 cell (mouse macrophage cell line) were obtained from Ab Frontier and Korean Cell Line Bank, respectively. These cells were maintained in Dulbecco’s modified eagle medium (Gibco Life Technologies, Waltham, MA, USA) containing 10% fetal bovine serum and 1% penicillin-streptomycin (Gibco Life Technologies, USA), and were cultured in a humidified incubator at 37°C with 5% CO₂.