2 5 dimethylpyrazine

2-Methyl-1-propanol, 3-methyl-1-butanol, 3-methyl-butanal, 3-octanone, p-xylene, styrene, benzeneacetaldehyde, phenylethyl alcohol, 4-ethyl-phenol, 2,5-dimethyl-pyrazine, trimethyl-pyrazine, and 3-ethyl-2,5-dimethyl-pyrazine were purchased from Sigma-Aldrich China Inc. (Beijing, China). The 18 amino acid standards, including glycine (Gly), asparagine (Asn), alanine (Ala), glutamic acid (Glu), lysine (Lys), arginine (Arg), proline (Pro), valine (Val), tyrosine (Tyr), isoleucine (Ile), leucine (Leu), tryptophan (Try), serine (Ser), histidine (His), threonine (Thr), methionine (Met), cysteine (Cys) and phenylalanine (Phe), were all purchased from Biosharp.
High-performance liquid chromatography (HPLC)-grade acetonitrile was supplied by Tedia Company (Fairfield, OH, USA). Phenylisothiocyanate (PITC, lot code: 10113227) was purchased from Alfa Aesar Chemical Co. Ltd (Tianjin, China), with a purity higher than 97%. Triethanolamine (TEA, analytical grade) and n-hexane (analytical grade) were purchased from Sinopharm Chemical Reagent Co. Ltd. (Shanghai, China). Pure water was obtained using a Milli-Q purification system (Millipore, Bedford, MA, USA).

Standard 2-hexanone, 1H-1-ethyl-pyrrole, benzeneacetaldehyde, cis-linalool oxide, trans-linalool oxide, linalool, 3-octen-2-ol, phenylethyl alcohol, α-terpineol, safranal, camphene, geraniol, indole, 3-hexanol, hexanal, 1-octanol and 2,5-dimethylpyrazine were purchased from Sigma Co. Ltd. (St. Louis, MO, USA). Benzyl alcohol, trans-β-damascenone, trans-β-ionone, 2, 4-ditert-butylphenol, cedrol and caryophyllene oxide were purchased from Alfa Aesar Co. Ltd. (Heysham, Lancashire, UK). Standard chemical series of C₈-C₂₀ alkanes that were used to determine the liner retention index (RI) and the internal standard (Cyclohexanone) were obtained from Sigma Co. Ltd. (St. Louis, MO, USA). The other chemicals were purchased from Sinopharm Chemical Reagent Co. Ltd. (Shanghai, China).

Beef from the hind legs of the Qinchuan cow was purchased from Yichuan's Xinbai market (Ningxia, China). The following chemicals were purchased from Sigma-Aldrich (Shanghai, China):1,2-dichlorobenzene (internal standard) and n-alkanes (C₇-C₄₀, ≥ 97%), hexanal (95%), heptanal (97%), 1-heptanol (97%), octanal (99%), (E)-2-octenal (97%), non-anal (99.5%), (E)-2-non-enal (97%), benzaldehyde (99.5%), (E, E)-2,4-decadienal (94%), 2-pentylfuran (98%), 2,5-dimethyl pyrazine (98%) and 1-octen-3-ol (98%). (E)-2-undecenal (l (94%), 2-pentylfuran (98%), 2,5-dime. (E, E)-2,4-non-adienal (95.8%) was from TCI Development Co., Ltd. (Shanghai, China). (E)-2-hexenal (97%) and (E)-2-heptenal (97%) were purchased from TCI (Shanghai, China). Methanol (analytical grade) was purchased from Thermo Fisher Scientific Co., Ltd (Shanghai, China).

Ten compounds of interest were semi-quantified in the PPI, due to their involvement in the ‘beany’ off-flavor [[27] , [28] (link), [29] (link), [30] (link)]. The following standards were purchased from Sigma-Aldrich: hexanal (98 % purity, CAS 66-25-1), nonanal (>98 %, CAS 124-19-6), trans-2-nonenal (97 %, CAS 18829-56-6), 3-methylbutanal (97 %, CAS 590-86-3), 1-octen-3-ol (98 %, CAS 3391-86-4), 3-octen-2-one (98 %, CAS 1669-44-9), 2-pentylfuran (98 %, CAS 3777-69-3), benzaldehyde (99 %, CAS 100-52-7), 2,5-dimethylpyrazine (98 %, CAS 123-32-0) and 1-hexanol (98 %, CAS 111-27-3). An external calibration method, previously optimized for PPI [26 (link)] was used. The calibration curves of each of the ten compounds were obtained for concentrations ranging from 0.001 to 2.5 ppm, in distilled water. The amount of each compound in the sample was calculated as in the following example with hexanal for PEA1 before ionization. Semi-quantification steps were as following, with a and b from the calibration curve of hexanal (a = slope, b = intercept of the regression): $\text{Area}\text{<a class="product" href="/product/uSPhCZIBPBHhf-iFxrFA/" target="\_blank">hexanal</a>}=5303819\mathrm{A}.\mathrm{U}\text{.}$ ${\left[\text{<a class="product" href="/product/uSPhCZIBPBHhf-iFxrFA/" target="\_blank">hexanal</a>}\right]}_{\text{in}\text{the}\text{assay}}\left(\mathrm{\mu }\mathrm{g}/\text{mL}\right)=\left(\text{Area <a class="product" href="/product/uSPhCZIBPBHhf-iFxrFA/" target="\_blank">hexanal</a>}-\mathrm{b}\right)/\mathrm{a}=\left(5303819-157037\right)/1\times {10}^7=0.51\mathrm{\mu }\mathrm{g}/\text{mL}$ ${\left[\text{<a class="product" href="/product/uSPhCZIBPBHhf-iFxrFA/" target="\_blank">hexanal</a>}\right]}_{\text{in}\text{the}\text{sample}}\left(\mathrm{\mu }\mathrm{g}/\mathrm{g}\right)={\left[\text{<a class="product" href="/product/uSPhCZIBPBHhf-iFxrFA/" target="\_blank">hexanal</a>}\right]}_{\text{in}\text{the}\text{assay}}\left(\mathrm{\mu g}/\text{mL}\right)\times {\text{V}}_{\text{solution}}\left(\text{mL}\right))/{\text{ m}}_{\text{sample}}\left(\mathrm{g}\right)$ $=\left(0.51\times 2\right)/0.2568=4.01\mathrm{\mu }\mathrm{g}/\mathrm{g}$ $\mathrm{m}{\left[\text{<a class="product" href="/product/uSPhCZIBPBHhf-iFxrFA/" target="\_blank">hexanal</a>}\right]}_{\text{in}\text{the}\text{sample}}\left(\mathrm{n}=3\right)=4.1\pm 0.2\mathrm{\mu }\mathrm{g}\text{of}\text{<a class="product" href="/product/uSPhCZIBPBHhf-iFxrFA/" target="\_blank">hexanal</a>}/\mathrm{g}\text{of}\text{PPI}$

The following reference compounds: acetic acid, acetoin, 2-methylbutanal, 2-methylpyrazine, 2,5-dimethylpyrazine, 2-furanmethanethiol, 2-acetyl-1-pyrroline, benzaldehyde, 1-octen-3-one, octanal, linalool, 2-acetylthiazole, 2-acetylpyridine, 4-hydroxy-2,5-dimethyl-3(2H)-furanone, 2-methoxyphenol, 3-hydroxy-4,5-dimethyl-2(5H)-furanone, hexadecanol, octadecanal, 2.3-pentanedione, butanoic acid, β-iso-methyl ionone, β-ionone, were from Sigma-Aldrich (St. Louis MO). Stock standard solutions 10³ or 10⁴ µg mL⁻¹ of each compound was prepared as described earlier [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.