**3. Results**

The two-way ANOVA results (Table 1) show that the ageing technology and the kind of wood had a highly significant effect on the colour and total phenolic content acquired by the wine spirits during the ageing process (after 6, 12, and 18 months). Among these factors, greater influence was exerted by the ageing technology (higher percentage of the variance explained) on the chromatic characteristics, while a similar weight of both factors was observed in the phenolic content. Regardless of the sampling time, the wine spirits aged with micro-oxygenation and chestnut wood staves (AC) exhibited a significantly lower value of lightness (L \*) and significantly higher values of saturation (C \*) and chromaticity coordinates (a \* and b \*) than the others. This set of chromatic characteristics reflects a more evolved colour, since lower L \* and higher C \* correspond to a more intense/darker colour, and the combination of higher a \* and b \* (yellow and red hues, respectively) is associated with a greater intensity of amber or orange hue, which made these spirits look older than the others. The colour of wine spirits from Limousin wooden barrels (TL) was on the opposite side, while the wine spirits aged with micro-oxygenation and Limousin wood staves (AL) and those aged in chestnut wooden barrels (TC) presented intermediate characteristics.


**Table 1.** Effect of the ageing technology and kind of wood on the chromatic characteristics and total phenolic index acquired by the wine spirits after 6, 12, and 18 months of ageing. AC: Alternative Chestnut, AL: Alternative Oak, TC: Traditional Chestnut, TL: Traditional Oak.

L \*—lightness; a \*, b \*—chromaticity coordinates; C \*—saturation; TPI—total phenolic index. For each sampling time (6, 12 and 18 months), mean values with the same letter in a column are not statistically different. NS, *p* > 0.05; \* 0.01 < *p* < 0.05; \*\* 0.001 < *p* < 0.01; \*\*\* *p* < 0.001.

In a previous work, this research team have already identified furfural, ellagic acid, vanillin and coniferaldehyde as markers of the ageing technology and the kind of wood used [8]. The results obtained for these compounds are shown in Table 2. It should be stressed that most of the low molecular weight compounds contents were mainly dependent on the ageing technology (higher percentage of the variance explained), as aforementioned for the chromatic characteristics and TPI, but ellagic acid content was closely related to the kind of wood. Indeed, significantly higher levels of furfural, vanillin, and coniferaldehyde were found in the wine spirits aged by the alternative technology; the first two were higher in the modality comprising chestnut wood staves (AC), whereas the latter was higher in the modality comprising Limousin wood staves (AL). Slight differences were found between the wine spirits from chestnut barrels (TC) and Limousin oak barrels (TL). Regarding ellagic acid, higher content was promoted by the chestnut wood (AC and TC), especially in the alternative technology. The contact with Limousin oak together with micro-oxygenation (AL) induced a slightly lower content of this phenolic acid, and a weak performance was showed by the Limousin oak barrels (TC).

Besides the differences between the two ageing technologies and the two kinds of wood, the results also reveal a remarkable role of the ageing time, as indicated by the percentage of variance observed for each factor in ANOVA (Table 2). Regardless of the ageing modality, there was a gradual decrease of lightness and a gradual increase of saturation and chromatic coordinates that correspond to the colour development over the ageing process.


**Table 2.** Effect of the ageing technology and kind of wood on the contents of low molecular weight compounds (mg/L absolute ethanol) of the wine spirits after 6, 12, and 18 months of ageing.

Furf—furfural; Ellag—ellagic acid; Vanil—vanillin; Cofde—coniferaldehyde; sumHPLC—total content of low molecular weight compounds determined by HPLC. For each sampling time (6, 12, and 18 months), mean values with the same letter in a column are not. NS, *p* >0.05; \* 0.01 < *p* <0.05; \*\* 0.001 < *p* <0.01; \*\*\* *p* <0.001.

The levels of significance shown in Table 3 reveal that the variation of chromatic characteristics, phenolic content (TPI and sumHPLC), and most of individual compounds contents were mostly significant between 6 months and 12 months. Significant differences between the three sampling times marked the wine spirits aged by the alternative technology with Limousin staves. In general, the L \* parameter decreased over the ageing time, while the other parameters (a \*, b \*, and C \*) increased. Furfural, ellagic acid, vanillin, coniferaldehyde and total content of low molecular weight compounds increased. Similar results were observed in the first months of ageing time of this kind of wine spirit [8].


**Table 3.** Differences observed for each samples group (technology vs. wood) during the ageing time.

The spectra obtained for wine spirit are similar to those reported by others authors [28].

The representative absorbance spectra of wine spirit samples studied is plotted in Figure 2; their spectral information is in accordance with previous reports of other authors [48]. The IR region from 2990 to 3626 cm<sup>−</sup><sup>1</sup> has a very strong influence due to water present in the samples [49]. Nevertheless, for these analyses, the background was measured with water.

**Figure 2.** Fourier transform infrared spectroscopy–Attenuated Total Reflection (FTIR-ATR) absorbance spectra of wine spirit samples.

There is IR information in the regions from 3000 to 2900 cm<sup>−</sup><sup>1</sup> due to the O–H stretching of alcohols and C–H stretching of CH3 and CH2, and consequently related to the presence of ethanol and methanol in the alcoholic beverages [49].

Regarding the region from 1500 to 860 cm<sup>−</sup>1, it corresponds to C–C and C–O vibrations in volatile compounds [12,14].

The small peak at 1450 cm<sup>−</sup><sup>1</sup> was assigned to C–OH bending deformation, and the peak at 1275 was assigned to C–O stretching in the acid molecules [11,50].

According to Stuart (2004), the region from 1300 to 840 cm<sup>−</sup><sup>1</sup> shows other absorption bands assigned to the C=O and C=C groups present in furanic compounds. The highest peaks at 1086 and 1044 cm<sup>−</sup><sup>1</sup> are ascribed to the C–O stretch absorption bands, which are important regions for ethanol and methanol identification and quantification respectively, and C–C absorption bands, which are related to ethanol and some organic compounds such as sugars, phenols, alcohols, and esters [14,49,51].

The peak at 879 cm<sup>−</sup><sup>1</sup> could be ascribed to out-of-plane C–H bending of aromatic compounds [10], and to CH–OH, C–C, C–O, and C–H bond stretching due to water, sugars, and phenolic compounds [51].

According to these regions of the FTIR-ATR absorption spectra (with baseline correction) of the wine spirit, a mathematical analysis has been performed to determine the differences between the groups studied. On the one hand, a functional ANOVA (FANOVA) model has been considered using two different tests: FP, the permutation test based on a representation of the base function (Equation (9)); and FB, the F test with the reduced bias estimation method (Equation (11)). On the other hand, a vector analysis based on the classical Analysis of Variance and the non-parametric Kruskal test have been carried out. Although all the areas of the curves have been analysed following the same process, only two were plotted each time to show the differences between the samples (space problem).

First, the different groups based on the ageing technology and kind of wood (AC, AL, TC, and TL) were tested. The contrast is different for each ageing time (18 months, 12 months, and 6 months). With an 18-month period, the hypothesis of similarity between all groups is rejected in all areas of the whole curve and from both points of view (Table 4). The samples of wine spirits aged by the alternative technology, on average, have higher absorbance units than the aged by the traditional one. Within the alternative technology, the wine spirit aged in oak wood always showed higher absorbance units

than the one aged in chestnut wood (Figure 3). With an ageing period of 12 months, the similarity hypothesis is also rejected in all areas by the two analyses (Table 4). Even so, in this case, the di fferences between alternative and traditional ageing technologies are small. The spectrometric curves can hardly be di fferentiated in the functional part of Figure 4, and the *p*-values obtained are higher (Table 4). With an ageing time of 6 months, significant reasons were found to reject the similarity hypothesis in all areas through functional and vector analysis (Table 4). With this sample, the di fference is more significant than with the 12-month sample but less than with the 18-month sample. Moreover, it can be seen that the wine spirit that gets higher absorbance units in the alternative sample is the one aged in oak, but with the traditional sample, it is the one aged in chestnut (Figure 5). These results are in accordance with those previously observed for the colour and analytical parameters (Tables 1 and 2).

**Table 4.** Numerical results of the similarity contrast between the groups AC, AL, TC, and TL, depending on the ageing time of the wine spirit samples. Functional results (FDA) are based on FANOVA using two di fferent tests (FP: permutation test based on a representation of the base function, FB: F test with a reduced bias estimation method). In addition, the results of the ANOVA and Kruskal's test representing the vectorial results (VA) are shown. All the results are *p*-values based on a 5% significance level.


**Figure 3.** Plots of two of the four significant areas of the curves with an ageing time of 18 months. In the first row, vectorial analysis by means of boxplots. In the second row, functional data analysis (FDA) through curves in the studied interval. The wine spirit sample is divided into four groups (AC, AL, TC, and TL).

**Figure 4.** Plots of two of the four significant areas of the curves with an ageing time of 12 months. In the first row, vectorial analysis by means of boxplots. In the second row, FDA through curves in the studied interval. The wine spirit sample is divided into four groups (AC, AL, TC, and TL).

**Figure 5.** Plots of two of the four significant areas of the curves with an ageing time of 6 months. In the first row, vectorial analysis by means of boxplots. In the second row, FDA through curves in the studied interval. The wine spirit sample is divided into four groups (AC, AL, TC, and TL).

Secondly, the similarity between the three different ageing times was contrasted. This contrast is different depending on the ageing technology and kind of wood used (AC, AL, TC, and TL). Figure 6 shows the boxplots and curves of the sample resulting from alternative technology with chestnut wood. It can be seen that there is little difference between the three ageing times. Especially at the spectral region of 1150–960 cm<sup>−</sup><sup>1</sup> (second d column of Figure 6), in which the similarity hypothesis is rejected in the vector analysis. Instead, FDA is able to detect these differences between the curve samples (Table 5). This region is characteristic of the absorption bands assigned to C=O and C=C groups existing in furanic compounds, C–O stretch absorption bands related to ethanol and methanol, and C–C absorption bands also related to ethanol and some organic compounds such as sugars, phenols, alcohols, and esters previously reported, and all of them are important to differentiate the samples in this study. They are mainly identified at the peaks of the 1044 cm<sup>−</sup><sup>1</sup> and 1086 cm<sup>−</sup>1, which are chiefly related to the presence of ethanol and methanol but also related to some organic compounds such as sugars, phenols, alcohols, and esters existing in minor concentration in the beverages. In addition, similarity in the other areas of the entire curve within the sample resulting from alternative technology with chestnut wood is rejected. In the case of the alternative technology, the wine spirits aged with Limousin oak wood are very similar to those aged with chestnut wood but with more distance between the three ageing times (Figure 7), as observed in the chemical analysis. The similarity hypothesis is rejected in all areas and from both points of view (Table 5). Figure 8 shows the differentiation within the samples resulting from chestnut barrels. In this case, the two areas drawn from the whole curve are closer, but the differences between the three ageing times are greater. The hypothesis of similarity between the samples is rejected in all areas and from the vectorial and functional approach (Table 5). Finally, regarding the wine spirits aged in Limousin oak barrels, the 18 and 12-month samples show higher absorbance units than the 6-month sample (Figure 9). The spectrometric curves of the functional graph can be easily distinguished. There are significant reasons to reject the similarity between the three samples in all areas of the full curve from the two analyses (Table 5).

**Figure 6.** Plots of two of the four significant areas of the Alternative Chestnut (AC) curves. In the first row, vectorial analysis by means of boxplots. In the second row, FDA through curves in the studied interval. The wine spirit sample is divided into three groups depending on the ageing time (18, 12, and 6 months of ageing).

**Figure 7.** Plots of two of the four significant areas of the Alternative Oak (AL) curves. In the first row, vectorial analysis by means of boxplots. In the second row, FDA through curves in the studied interval. The wine spirit sample is divided into three groups depending on the ageing time (18, 12, and 6 months of ageing).

**Table 5.** Results of the similarity contrast between the three ageing times (18 months, 12 months, and 6 months), depending on the ageing technology. Functional results (FDA) are based on functional ANOVA (FANOVA) using two different tests (FP: permutation test based on a representation of the base function, FB: F test with a reduced bias estimation method). In addition, the results of the ANOVA and Kruskal's test representing the vectorial results (VA) are shown. All the results are *p*-values based on a 5% significance level.


**Figure 8.** Plots of two of the four significant areas of the Traditional Chestnut (TC) curves. In the first row, vectorial analysis by means of boxplots. In the second row, FDA through curves in the studied interval. The wine spirit sample is divided into three groups depending on the ageing time (18, 12, and 6 months of ageing).

**Figure 9.** Plots of two of the four significant areas of the Traditional Oak (TL) curves. In the first row, vectorial analysis by means of boxplots. In the second row, FDA through curves in the studied interval. The wine spirit sample is divided into three groups depending on the ageing time (18, 12, and 6 months of ageing).
