# Enlarging Knowledge on Lager Beer Volatile Metabolites Using Multidimensional Gas Chromatography
## 3 Results and Discussion
### Profiling the Lager Beer Volatile Metabolites  
The quality and trait of fermented foods, such as beer, can be monitored by metabolomics, e.g., evaluation of the changes of the metabolic profiles during fermentation and prediction of fermented foods quality, among others [33]. The comprehensive characterization of the beer volatile metabolites, a metabolite profiling strategy, was selected taking into account two potential sources of volatile metabolites in beer: raw materials (cereals and hops) and yeasts (Figure 1). In fact, monoterenic and sesquiterpene compounds may arise from raw materials, once they are reported to be synthetized through the mevalonate (MVA) and methylerythnitol 4-phosphate (MEP, only expressed in plants metabolism) metabolic pathways [9]. Cereal and hops may also contribute to the presence of norisoprenoids in beer volatile composition, through the carotenoids' degradation in plants [10]. Yeasts metabolism is the main responsible for the unique aroma profiles of beer, through the production of a wide range of volatile metabolites, particularly the target chemical families that were selected for this study: acids, alcohols, esters, monoterenic compounds, norisoprenoids, sesquiterpene compounds, and sulfur compounds. Indeed, they may arise from metabolism of carbohydrates and amino acids [5; 10; 34; 35; 36; 37]; from biosynthesis of monoterenic compounds, sesquiterpene compounds, and fatty acids [38; 39; 40; 41]; and also from de novo synthesis of sulfur compounds [34]. The 329 detected volatile metabolites were systematized in Figure 4, in which the detected volatile metabolites (target nodes) were displayed and organized according to the respective chemical families (nodes). The chemical families with higher number of detected metabolites present in Figure 4 were esters and monoterenic compounds (ca. 90 metabolites each), followed by alcohols (60), sesquiterpene compounds (42), acids (17), norisoprenoids (16), sulfur compounds (13), and volatile phenols (3). Moreover, the amount of each metabolite (median GC chromatographic peak area in all lager beer under study) is reflected in the edge' thickness, and also in the size of the volatile metabolites' name (Figure 4), i.e., the higher GC chromatographic peak area is proportional to the highest volatile metabolites' names and edge thickness (e.g., octanoic acid and phenylethanol were the volatile metabolites with the highest GC chromatographic peak area, considering all the lager beers under study). The variety and diversity of the detected chemical structures from the 8 targeted families, even within each chemical family and also the wide range of detected amount (variation of GC chromatographic peak areas from \(10^{4}\) to \(10^{9}\)) was only possible due to the methodology used. In fact, the orthogonal mechanism and ToF analyzer of GC\(\times\)GC-ToFMS increases the chromatographic and spectral resolution and also the sensitivity, allowing the simultaneous analysis of major and trace analytes of beer within a single analysis. From the detected volatile metabolites, it was possible to observe that 168 were present in all lager beers under study being called as common volatile metabolites. They were listed in Table 2 and represent ca. 51% of the total detected metabolites. An in-depth analysis of these 168 common metabolites allowed to construct Table 3, in which it is possible to compare the number and percentage of the common volatile metabolites within each chemical family. Table 3 shows that the number of common volatile metabolites varied between 22% and 100% from the total number of detected volatile metabolites depending on the chemical family. In fact, the chemical families can be ordered according to the increase of the percentage of common volatile metabolites: monoterenic compounds \(<\) sesquiterpene compounds \(<\) norisoprenoids \(<\) acids \(<\) alcohols \(<\) esters \(<\) sulfur compounds \(<\) volatile phenols.