# Shedding Light on the Volatile Composition of _Broa_, a Traditional Portuguese Maize Bread
## 3 Results and Discussion
### Characterization of the Volatile Composition of Maize Flows  
The majority of the compounds identified in maize flours were aldehydes (hexanal, heptanal, octanal, 2-octenal, nonanal, 2-nonenal, decanal), alcohols (1-pentanol, 1-hexanol, 1-heptanol, 1-octen-3-ol), hydrocarbons and 2-pentylfuran, which have been described in cereal flours as secondary products of lipid oxidation of unsaturated fatty acids [15; 20; 37; 44]. Other abundantly detected volatile compounds were geranylacetone, ionone and 6-methyl-5-hepten-2-one, derived from carotenoids' oxidation [40; 59; 60; 61]. In order to evaluate the differences among maize flours and to study possible relations among the different volatiles, a cluster analysis was performed and the Spearman coefficients among the main volatiles were determined. Twenty-nine compounds, described in Supplementary Table S1, were considered for these analyses, since the remaining fifteen were only present in trace amounts in all maize samples and it was not possible to accurately measure their peak areas. A PCA was performed in order to reduce the number of variables, since it was higher (29) than the number of samples. Following the Kaiser criteria, four components were retained after PCA analysis, which explained 93% of the total variance. The optimal number of clusters obtained by the elbow point and silhouette methods was two, one corresponding to all the traditional maize varieties (F1 to F11) and the other to the commercial maize sample (F12). The commercial sample was distinguished from the traditional varieties due to the presence of benzaldehyde and higher amounts (\(p<0.05\)) of other aldehydes (2-octenal (\(E\)), 2-nonenal (\(E\)), hexanal, heptanal, octanal, 2-heptenal (\(Z\)), 2-undecenal, decanal and 6-nonenal), alcohols (1-hexanol, 1-heptanol, 1-octen-3-ol, 1-octanol, 1-nonanol and 1-pentanol) and 3-ethyl-2-methyl-1,3-hexadiene, 2-pentylfuran, limonene, trimethyldodecane, 2-heptanone and pantolactone. The mentioned aldehydes showed very strong and positive correlations among them (Supplementary Table S2). Since these volatiles are mainly derived from the oxidation of polyunsaturated fatty acids (Figure 2) [44; 62], these results suggest that more lipid oxidation reactions were occurring in the commercial flour. Very strong and positive correlations were also found between these aldehydes and 3-ethyl-2-methyl-1,3-hexadiene (Supplementary Table S2). Although the origin of this compound is not known, these high correlations suggest that it may also be a product of lipid oxidation. In fact, a recent study has shown that it was formed after black rice storage (>3 months) [63], and it has been recently described in purple sweet maize [64] and maize milk [65]. The highest concentration in lipid oxidation derivatives observed in F12 can be explained by its different genetic origin. In particular, the concentration of 2-octenal (\(E\)) and 2-nonenal (\(E\)), two by-products of linoleic acid oxidation, one of the most abundant lipids in maize kernel [66], was recently associated with a gene that codes for a linoleate 9S-lipoxygenase, an enzyme involved in linoleic acid metabolism [40]. Therefore, a higher enzymatic activity in F12 can explain its higher levels in 2-octenal (\(E\)) and 2-nonenal (\(E\)). Additionally, F12 was acquired already milled, and it may have been kept at room temperatures for a longer period than the traditional maize flours, contributing to its higher amounts in lipid oxidation products [67; 68]. Another possibility for these differences could be the different granulometry of the commercial sample, since it presented a higher mean diameter and a large particle distribution range compared to traditional maize flours [43]. However, research has shown the opposite: finely milled cereal flours tend to be more susceptible to lipid oxidation reactions than flours with higher particle sizes [67; 68], possibly due to the higher surface area that favors the contact with oxygen [67].