## 3 Results and Discussion
### Effect of the Stabilization Process
NonStab: non-stabilized brandies; ColdT: brandy stabilized at cold temperature; RoomT: brandy stabilized at room temperature. Differences in the ester profile of the brandies related to the stabilization process are shown in the scores and loadings plots of the ML-PLS-DA model (Figures 3A and 5). As was expected from the previous PCA, the brandies submitted to room temperature stabilization and filtration displayed the most distinct ester profile, being separated from the rest in component 1 (X-variate 1), which explained a high 54% of the variance. This stabilization process resulted in higher contents of many esters, such as isoamyl octanoate, ethyl isovalerate, ethyl isobutyrate, isobutyl hexaneoate, ethyl 2-methylbutyrate, propyl acetate, ethyl butyrate, isoamyl acetate, methyl lexanoate, ethyl valerate, methyl decanoate, and isobutyl acetate. The percentage of change related to the control  
Figure 4: Results from the permutation test (\(n=1000\)) performed on the multilevel partial least squares discriminant analysis (ML-PLS-DA). Histogram obtained from the permutation test for (**A**) the balanced error rate (BER), (**B**) area under the receiver operating characteristic curve (AUROC), and (**C**) the average number of misclassified (NMC). The real average performance of the model for the three statistics obtained from the double cross-validation is shown in each plot. brandy (before stabilization) is shown in Supplementary Table S1. Meanwhile, other esters such as isoamyl hexanoate, isoamyl butanoate, ethyl octanoate, hexyl acetate, phenylethyl acetate, ethyl cinnamate, ethyl decanoate, ethyl heptanoate, ethyl palmitate, ethyl dihydrate inconnamate, ethyl stearate, ethyl nonanoate, and ethyl myristate displayed a significant decrease in their concentration. The brandies submitted to cold stabilization were separated from those before stabilization in component 2 (X-variate 2), which explained 11% of the total variance (Figures 3A and 4B). This meant that the volatile profile of these brandies was less impacted by the stabilization process. This was mainly driven by a decrease in ethyl esters of long-chain fatty acids: ethyl palmitate, ethyl stearate, and ethyl myristate, with this method being effective for reducing these undesirable compounds, since turbidity of brandies is associated with ethyl esters of long-chain fatty acids due to decreases in their solubility, mainly related to low storage temperatures [7]. Therefore, a reduction of these compounds is beneficial in terms of ensuring the stability of brandies [33]. However, these ethyl esters displayed a greater decrease in brandies stabilized at room temperature, especially for ethyl stearate (decreasing below the detection limit in all the brandies, Supplementary Table S1). Meanwhile, both stabilization procedures yielded decreases of ethyl palmitate between 70-98% of the initial values and ethyl myristate between 11-87% (Supplementary Table S1). A previous study showed a similar reduction of ethyl myristate after brandy stabilization [7]. These results (lower content of ethyl esters of long-chain fatty acids and similar profile to the rest of the esters present in samples before stabilization) indicated a lower impact on the ester profile when cold stabilization was used, compared to room temperature stabilization. Figure 5: Loadings contribution barplot on component 1 (**A**) and component 2 (**B**) of the multilevel partial least squares discriminant analysis (ML-PLS-DA) carried out to discriminate between brandy samples before and after cold stabilization and filtration. Color indicates the class for which the compound had a maximal mean value. Bar length represents the multivariate regression coefficient, with either a positive or negative sign for that particular feature of each component, i.e., the importance of each variable in the model. NonStab: non-stabilized Brandies; ColdT: brandy stabilized at cold temperature; RoomT: brandy stabilized at room temperature.