In general terms, complete alcoholic fermentation of Tempranillo and Cabernet could be divided into two different stages: tumultuous and slow. The duration of tumultuous fermentation varied according to the composition of the must and the temperature at which it was carried out. Grapes were stored at 21ºC in a cooler before crushing. Cellar temperature was set at 22ºC and temperature in the tank was held at 28ºC at the tumultuous stage to ensure good extraction of polyphenols. This step should be carefully considered to avoid uncontrolled fermentations and make this methodology reliable. The yeasts developed comfortably, thus ensuring the total transformation of all sugar into alcohol in both performances. Density rapidly decreased after the second day of fermentation for each variety and vessel (Figure 1).
Approximately 5 and 9 days after the start of fermentation, for Tempranillo and Cabernet respectively, a small quantity of sugar corresponding to a density of ρ=1010kg/m3 remained in the must. At this point, the second stage of fermentation (slow fermentation process) began, which over the following 3 days transformed the remaining final grams of sugar into alcohol. Tempranillo showed a rapid decrease until the 5th day of fermentation when it reached ρ=997.8, 1007.0, 1006.5 and 1005.0 kg/m3 respectively for each increasing small scale volume (25, 50, 75 and 100L). Fermentation kinetics in Tempranillo required 8 days to ferment all the reducing sugars, showing a slow decrease for the last 3 days. The Cabernet required 12 days to complete the fermentation process. Temperatures never reach more than 28ºC for both kinetics under the same conditions of controlled room temperature and size vessel. After fermentation, the temperature decreased to 22ºC in both.
Modelling data using linear functions proved easier to predict the kinetics of the fermentation processes of both varieties/volume studies. As tumultuous fermentation occurred with a different duration for each variety than that of the slow stage, two regression curves were calculated for each combination variety/volume. As expected, tumultuous phase (when maximal fermentation activity occurs) and slow fermentation stage (happening after tumultuous fermentation) clearly show two different slopes (equations are given on Table 3b).
According to Table 3b, two clear slopes were distinguished on the fermentation curves for both varieties, Tempranillo and Cabernet Sauvignon. Linear regression slopes of tumultuous stage ranged between -21.933 and -24.850 for Tempranillo and -12.286 and -17.321 for Cabernet Sauvignon, indicating a faster kinetic for Tempranillo in the tumultuous fermentation stage. The coefficient of determination is higher in the tumultuous stage. Considering now all volume vessels, Tempranillo slopes from the tumultuous stage would not indicate much different kinetics between volumes, even if 10L capacity vessel seems to decrease faster, with a curve done by y = -24.8x + 1121, than 25, 50 and 100L, which have y = -22.4x + 1124, y = -22.1x + 1122, and y = -21.9x + 1120, respectively, showing very similar slopes. However, slow stage reveals a similar tendency, having the lowest slope for 10L. Cabernet sauvignon showed a proportional relation between slopes and volume. The 10L volume tank has the higher slope in the tumultuous stage (y = -17.3x + 1119) and so the lower slope on the slow stage (y = -1.9x + 1016), indicating that the tumultuous part of fermentation proceeds faster in the 10L than any other vessel evaluated.
Effect of small-scale tank volume on wine composition
With regards to wine composition basic parameters, tank size was observed not to influence ABV, pH and TA neither in CS nor in TE (Table 4), but it affected phenolic composition (TAnt, and tannins). The highest TAnt values were observed in the intermediate sizes (25 and 50 L), whereas for tannins content the highest values were found in the greater tanks (50 and 100 L) in both varieties. One of the most relevant effects that tank size could have from the research point of view is increasing or decreasing the variability of the composition of the wine obtained from replicates. When the coefficients of variation (CV) obtained for each variable, tank size and variety are compared (Fig 2a and 2b), all values were low, especially for ABV, pH and TA (CV<4%), but also for TAnt and tannin content (CV<8%). Taking into account that the observed CVs were satisfactory for all tank sizes and varieties (less than 5%), there is a slightly greater variability in intermediate sizes (25 and 50 L) in TE. This fact supports the repeatability of the wines made at any of the tank sizes considered with regards to the major wine composition parameters.
Total anthocyanin composition (Table 5a and 5b) in the medium size-tanks (25 and 50 L) was higher than any other volumes (10 and 100 L) in TE. Malvidin glucosides (G) proved much more extracted (up to one third) than the acetyl glucosides (AG). Furthermore, the latter showed almost the same concentration of coumarylglucosides (GC). In CS, the greatest anthocyanin contents were found in the biggest volumes (Table 9), CS tanks of 10 and 25 L showed delayed extraction of anthocyanins (table 9), giving 117.7mg/L of total anthocyanins in 10 L and 128.5mg/L in 25 L and 361.9mg/L in 100 L and 384.4mg/L in 50 L. Thus, in the case of CS it appears that, the larger the tank, the greater the extraction (50, 100). In CS, the difference between glycosides (G) and acetyl glucosides (AG) total concentration was not remarkable, with lower extractions observed in the smaller volumes for them all. Reproducibility in terms of anthocyanin content can be said to be satisfactory, since CVs for all anthocyanin families were below 20%, the median CV being 13 % for CS and 10% for TE (Figure 2c and 2d). Tank size appeared to affect reproducibility, although the observed effect was different for each variety. In TE, the lowest CVs were found for 100 and 25 L tanks, whereas in CS this occurred in 10 and 50 L tanks.
Variability in procyanidins content (Figure 2e and 2f) was relatively similar to that observed for anthocyanins; median value was just 9% except for CS-25 and TE-50 (12-22%, respectively). CV lower and upper values ranged between 10-22% in CS and 5-17% in TE, dimers showing the higher CV, which indicates that reproducibility was in general terms very satisfactory, particularly in TE, where almost always was below 10%. In both CS and TE, the lower CVs were associated to 10 L and 100 L volumes. In general, the effect of tank size on procyanidins content repeatability was less relevant than it was for anthocyanins.
Comparison with commercial volume
Principal component analyses allowed condensing the information provided by all the analysis variables included in the study into a reduced number of components with a minimum loss of information in both varieties. Thus, in CS, the first component accounted for 44.6 % of variability, the second one for 36.6 % and the third one for 5.9 % (Figure 3a), whereas in TE they were 44.0, 27.3 and 7.2% respectively (Figure 3b). In both varieties, the first component included mainly anthocyanin-content variables, the second one procyanidins-content variables, and the third one was linked mainly to acidity (pH in CS, and titratable acidity in TE).
PCA scores for all small-scale tanks, average scores for each small-scale volume and commercial scale tank scores are represented in Figure 3a and 3b for CS and TE, respectively. For both varieties, the composition of the wine obtained in 100 L tanks was clearly more similar to commercial scale wine for the main (first) component, related to anthocyanin content. For the second component, related to procyanidins, wines obtained in 10 and 100 L volumes were the most similar to commercial scale in CS, whereas in TE differences were smaller in this axis, 10 L, 25 L and 100 L showing similar scores for this component to that in the commercial scale wine (Figure 3c and 3d).