## 3 Results and Discussion
### Evaluation of the Fermentation and Drying Effects on Coffee Metabolomic Profile  
To evaluate the effect of the two times of fermentation (12 h and 24 h), the RF approach was applied on green and roasted coffees, using either the matrices of bucketed spectra or the matrices of metabolites (Supplementary Materials Figure S8a-d). Considering all four models created, the fermentation time mostly affected the profile of green coffee, while the effect was not remarkable in roasted coffee. The RF model built on green metabolites resulted to be the most effective in discriminating the fermentation times (pred. acc%. 72.2, Supplementary Materials Figure S8b). A total of 24 h fermented coffee beans were  
Figure 4: **Log\({}_{2}\)(FC) of metabolites’ concentrations characteristic of distinct farms. ** Positive Log\({}_{2}\)(FC) values represent higher level of green coffee (**a**) and roasted coffee (**b**) metabolites in farm 1 compared to farm 3 (negative Log\({}_{2}\)(FC) values) considering catuai rojo batches; positive values represent higher levels of green coffee (**c**) and roasted coffee (**d**) metabolites in farm 1 compared to farm 2 (negative Log\({}_{2}\)(FC) values) considering bourbon batches. Dark gray bars represent the metabolites which are statistically significant after the FDR _p_-value correction (FDR < 0.05), gray bars for those metabolites that show a _p_-value < 0.05 but lose significance after the False Discovery Rate correction. Asterisks represent the Cliff’s delta effect-size, were “***” means large effect, “***” medium effect. characterized by higher levels of acids (in particular: malic acid, acetic acid, chlorogenic acids, 5-CQA, citric acid, fumaric acid, GABA, quinic acid, as reported in Supplementary Materials Figure S8b1). Univariate analysis corroborates the fact that malic acid levels are statistically different in the two groups. Among all the fifteen quantified metabolites in green coffee beans, only malic acid remained statistically significant after the false discovery rate (FDR) correction (malic acid: \(p\)-value = 0.0002, FDR = 0.003, cliff's delta = small). Based on the good performance of the model reported as b in Supplementary Materials Figure S8, to check if distinct varieties reacted differently to 12 h or 24 h of fermentations, the effect was evaluated on green coffee metabolites considering each variety separately. As can be seen in Figure 5, each variety reacts differently to the time of fermentation. In particular, the profiles of fermentation times can be distinguished with a predictive accuracy \(\sim\)100% in maracaturra, pacamara and bourbon rojo (Figure 5b,e,f), suggesting a remarkable change induced by the time of fermentation. As previously reported, the coffee batches longer fermented are characterized by higher levels of acids. Figure 5: _Cont._  
The drying effect was also evaluated considering coffee batches processed \"under shade\" and those at \"direct sun\" exposure. Four RF models were created using available data (Supplementary Materials Figure S9a-d): also, in this case the effect of the different drying procedure is more remarkable in green coffee (Supplementary Materials Figure S9a,b), and in particular the RF model built on metabolites provided a better discrimination of green coffee batches processed in the two different manners (overall predictive accuracy: 71%, Supplementary Materials Figure S9b). Among the quantified metabolites, amino acids (valine and alanine) seem to be the most affected by these procedures (Supplementary Materials Figure S9b1). Univariate analysis confirms valine as the only metabolite which remained significantly altered after the FDR correction (valine: \(p\)-value = 0.0006, FDR = 0.008, cliff's delta = small). The effect of the drying procedure is detectable in each variety Figure 6. In Figure 6 it emerges that valine levels are higher in all coffee beans batches dried at direct sun.