4 methylimidazole

4-methylimidazole (purity > 98%), 1-methylimidazole (1-MeI) and glycerol (GLY) were purchased from Sigma-Aldrich Chemie GmbH (Steinheim, Germany). Ethylene glycol (EG), sodium chloride (NaCl), sodium hydroxide (NaOH), hydrochloric acid (HCl) and all other reagents (analytical grade) were purchased from Merck. Nitrogen gas (Sabalan Co., Tehran, Iran) with 99.999% purity was used for gas flow of GF-HS-SDME system. Stock solution of 4-MeI and 1-MeI were prepared by dissolving 1.0 g of each compound separately in 1 L of ethanol to obtain a concentration of 1000.0 mg L⁻¹. All solutions were kept at 4 °C in a dark place when not in use, being stable for 3 months [21 (link)].

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.