2 6 dimethylpyrazine

Green coffee beans (Coffea arabica, from Brazil) were bought from a commercial market in Seoul, Korea. The origin of green coffee beans was verified by the importers and experts. 2-Methylpyrazine, 2,5-dimethylpyrazine, 2,6-dimethylpyrazine, 2-ethyl-3-methylpyrazine, 2-ethyl-5-methylpyrazine, 3-ethyl-2,5-dimethylpyrazine, furfural, furfuryl acetate, furfuryl propionate, furfuryl alcohol, 1-furfurylpyrrole, 4-ethylguaiacol, 2-methoxy-4-vinylphenol, 5-methylfurfural, quinoxaline, and C7–C30 alkane standard were obtained from Sigma–Aldrich Chemical Co. (St. Louis, MO, USA). 2-Ethylpyrazine, 2,3-dimethylpyrazine, 1-methylpyrrole, guaiacol, and 2-acetylfuran were bought from Tokyo Chemical Industry Co., Ltd. (Tokyo, Japan). Divinylbenzene/carboxen/polydimethylsiloxane (DVB/CAR/PDMS, 50-μm film thickness) solid-phase microextraction (SPME) fiber was purchased from Supelco, Inc. (Bellefonte, PA, USA).

Soybeans, raw wheat flour and edible salt (NaCl) were purchased from a local supermarket (Zhenjiang, China, 2021). 3-Methylbutanal, ethanol, 3-methyl-1-butanol, ethyl propanoate, ethyl acetate, ethyl isovalerate, 2-phenylethanol, benzeneacetaldehyde, 3-(methylthio) propyl acetate, ethyl 3-(methylthio) propionate, methyl benzoate, (E)-β-damascenone, 2-phenylethyl acetate, 2-furanmethanol, 3-methylthio-1-propanol, 3-methylthio propanal, 2,6-dimethyl pyrazine, trimethyl pyrazine, 2-acetyl pyrroline, 4-hydroxy-2,5-dimethyl-3(2H)-furanone (HDMF) and 4-hydroxy-2(or 5)-ethyl-5(or 2)-methyl-3(2H)-furanone (HEMF) were all ordered from Sigma-Aldrich (Shanghai, China, 2021). 3-Furaldehyde, 4-ethylphenol, 4-ethylguaiacol, 4-vinylguaiacol, 3-methylbutanoic acid, acetic acid, 2-methylbutanoic acid, dimethyl trisulfide, 1-octen-3-ol and C6-C33 n-alkanes were provided by Aladdin Holdings Group (Shanghai, China, 2021). Other analytically pure reagents were purchased from Sinopharm Chemical Reagent Co., Ltd. (Shanghai, China, 2021). A. oryzae 3.042 were obtained from the Guangdong Institute of Microbiology (Guangzhou, China, 2021).

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.