Trimethylsilyl propionate

The polar non-volatile compounds in fermented grain were assayed using the proton nuclear magnetic resonance (¹H NMR). Metabolites were extracted by suspending 300 mg of fermented grain in 1.5 mL of cold ultra-pure water (Milli-Q; Millipore, Bedford, MA, USA), followed by grinding using a Mini-Beadbeater for 60 s and cooling on ice for 10 min. After centrifugation at 13,000× g for 10 min, 1.0 mL of supernatant was mixed with 1.0 mL of phosphate buffer (0.1 M sodium phosphate consisting of 10% deuterium oxide (D₂O) (v/v), 100 mM of imidazole, 0.2% (w/v) sodium azide, and 1 mM of trimethylsilylpropionate ([Sigma-Aldrich, St. Louis, MO, USA] as an internal standard)), which was then centrifuged at 16,060× g and 4 °C for 5 min. Then, 600 μL of the supernatant was transferred to 5 mm NMR tubes. The NMR spectra were recorded using an Avance III 600 FT-NMR spectrometer (Bruker, Billerica, MA, USA) at 14.1 T (600.13 MHz proton frequency). The experimental NMR spectra were compared with those of known metabolites using the Chenomx NMR Suite software (version 6.0; Chenomx, Edmonton, AB, Canada) to identify the metabolites.