# Enlarging Knowledge on Lager Beer Volatile Metabolites Using Multidimensional Gas Chromatography
## 3 Results and Discussion
### Profiling the Lager Beer Volatile Metabolites
\begin{table}
\begin{tabular}{c c c c c c c c} \hline \hline \({}^{1}\)Ra (s) & \({}^{2}\)Ra (s) & \({}^{3}\) & **Compound** & **CAS Number** & **MSI Level \({}^{4}\)** & **RI Calc.\({}^{5}\)** & **RI Lum \({}^{6}\)** & **Previously Reported on Lager Beer** \\ \hline
78 & 0.33 & Dimethyl/sulfide & 75-18.3 & 2 & 580 & 526 & [53] \\
162 & 0.58 & Dimethyl/sulfide & 642-0.2 & 2 & 727 & 731 & [53] \\
300 & 1.02 & 13.5-Ovalaine & 646-12.8 & 2 & 509 & 913 & [53] \\
408 & 0.88 & Dimethyl/sulfide & 3068-08.8 & 2 & 963 & 969 & [53] \\
438 & 2.86 & 3-Methyl/sulfide & 505-10.2 & 2 & 965 & 989 & [43,46,50–23] \\
456 & 0.74 & 5.5-Methyl/s-formaldehyde & 5903-05.8 & 2 & 996 & 995 & – \\
606 & 0.99 & Emyl 3-(methyl/s) fluorophore & 13327-65.5 & 2 & 1101 & 1098 & – \\
642 & 1.09 & 3-Methyl/spropyl acetate & 16605-0.0 & 2 & 1126 & 1125 & – \\
780 & 2.03 & Benchmarke & 95-16.9 & 2 & 1224 & 1230 & – \\
816 & 0.67 & 2-(1,1-Dimethyl/s)-thiophene & 1669-78.7 & 2 & 1251 & 1251 & – \\ Volatile phenols & 90-05.1 & 1 & 1090 & 1095 & [49] \\
988 & 2.01 & 4.5-Displaced & 278-59.9 & 2 & 1283 & 1290 & [59] \\
500 & 2.99 & 4-Vinyl/suaised & 7786-61.0 & 2 & 1316 & 1333 & [43,46,57,70,51] \\ \hline \hline \end{tabular}  
* Retention times for first (\({}^{1}\)Ra) and second (\({}^{7}\)Ra) dimensions in seconds, \({}^{5}\) Level of metabolite identification according to [PERSON] et al. [29]: (1) Identified compounds; (2) Putatively annotated compounds; (3) Putatively characterized compound classes; (4) Unknown compounds. \({}^{6}\) RI Retention Index obtained through the modulated chromatogram. \({}^{4}\) RI, Retention Index reported in the literature for Equity-5 CC column or equivalents.  
\end{table} 
Table 2: _Cont._A set of 12 volatile metabolites were the major ones in lager beers that corresponded to ca. 81% of the total area of the targeted chemical families, namely, 3 acids--ctanoic acid, decanoic acid, and hexanoic acid; 3 alcohols--phenylethanol, 2-methyl-1-butanol, and 3-methyl-1-butanol; and 6 esters--2-phenylethyl acetate, 2/3-methylbutyl acetate, ethyl octanoate, ethyl decanoate, ethyl hexanoate, and ethyl acetate. These 12 volatile metabolites were highlighted in bold in Table 2, which contributed highly to the highest total amount of the acids, alcohols, and esters. Indeed, all of these previously mentioned metabolites are frequently detected in beer volatile composition [46; 50], being their main source related with yeast metabolism [55; 56]. Moreover, these results are in agreement with those reported by [PERSON] et al. [49], which showed a similar set of metabolites that had the highest concentration in beers, namely, 3-methyl-1-butanol, ethyl octanoate, ethyl hexanoate, 3-methylbutyl acetate, 2-phenylethyl acetate, and octanoic acid. The beer profiling can be helpful for the simultaneous screenings and/or follow-up of different aspects, such as the monitoring of the beer aroma (reported in Beer Flavor Wheel [57]) once these targeted chemical families have a huge impact on beer aroma properties, e.g., alcohol (n-propanol, 2-methyl-1-propanol), sour apple (ethyl hexanoate, ethyl ocanoate), rose (phenylethanol, phenylethyl acetate), cheese (butanoic, hexanoic, octanoic acids), or fruity (\(\beta\)-damascenone, ethyl decanoate) aromas, among others. Furthermore, the detected volatile metabolites may be used to monitor the yeast metabolism [4; 5; 23; 35], for instance the yeasts' aminoacids uptake [35], e.g., uptake of leucine promotes the formation of 3-methyl butanol (one of the major volatile metabolites present in all lager beers under study). It also allows the detection of different off-flavors that may be monitored in the final product or along brewing process, namely, dimethyl sulfide (vegetables aroma, formed from malt during wort boiling) [58]; acids (cheese aromas) and sulfur-containing metabolites (putrid aromas), which can be associated to be produced by bacteria spoilage (e.g., _Pediococcus_ spp., _Lactobacillus_ spp., _Megasphaera_ spp., _Pectinatus_ spp.)