Yeast Growth Inhibition by Pyrazines and Imidazoles

To determine if the detected pyrazines and imidazoles contributed toward the inhibition of yeast in the 2012 ASGH, we grew yeast in 2010 ASGH supplemented with similar levels of pyrazines and imidazoles found in the 2012 ASGH. 10 μL of exponentially growing Y128 S. cerevisiae [17 (link)] at a cell density of OD₆₀₀ = 1.0 was inoculated in 96-well microtiter plates containing 190 μL. We grew triplicate, 200 µL cultures of S. cerevisiae Y128 aerobically in each of the following hydrolysates: 2012 ASGH and 2010 ASGH supplemented with 0×, 1×, 10×, 25×, 37.5×, or 50× concentrations of the following pyrazines and imidazoles from 2012 ASGH dissolved in ddH₂O: 1 µM 1-methylimidazole, 140 µM 2-methylimidazole, 360 µM 4-methylimidazole, 6 µM 2,4-dimethylimidazole, 430 µM 2-methylpyrazine, 9 µM 2,5-dimethylpyrazine, and 38 µM 2,6-dimethylpyrazine (Sigma, USA). Cultures were incubated at 30 °C for 72 h and read every 11.3 min using a TECAN M1000 multimode plate reader housed within in an anaerobic chamber (Coy) maintained with 10% H₂, 10% CO₂, and 80% N₂ gases.