# The Effect of Apple Juice Concentration on Cider Fermentation and Properties of the Final Product
## 3 Results and Discussion
### Production of Volatile Esters  
The presence of 8 esters was monitored across the samples. PLS-DA was employed as a statistical approach to observe the differences in ester production (Figure 4). PLS-DA method identifies the differences between the sample groups, which are shown as ellipses on the biplots with overlap indicating similarity. The characteristic differences between the groups are evaluated on an axis that goes through the origin point at (0,0) towards the position of a volatile ester label. The best representation of the results was achieved when viewing the samples according to the yeast species--_S. bayanus_ yeasts (Figure 4A) were compared to _S. cerevisiae_ yeasts (Figure 4B). According to the biplots, all ciders fermented with _S. cerevisiae_ yeasts clustered closely together showing more consistency across the samples in volatile ester composition than _S. bayanus_ yeasts.  
Figure 3: Residual malic acid concentration at the end of fermentation (_p_ < 0.05). The color green represents control samples, orange—supplemented with DAP, purple—supplemented with Fermaid O. (**A**) represents the samples fermented at 18 \({}^{\circ}\)C; (**B**)—at 30 \({}^{\circ}\)C. Red lines represent initial malic acid content either in juice or reconstituted concentrate.  
The cider samples made with the concentrate correlated with the highest production of isoamyl acetate (up to 6700 \(\upmu\)g L\({}^{-1}\) in the concentrate; up to 5100 \(\upmu\)g L\({}^{-1}\) in the juice). Isoamyl acetate can be synthesized by yeast either from amino acids leucine and valine or from isoamyl alcohol [34; 35]. As was noted previously, the diluted concentrate had elevated amounts of most of the identified amino acids, including leucine. Higher content of leucine might have been successfully utilized by the strains used in this study for isoamyl acetate production. _S. cerevisiae_ yeasts used in the study accounted for the highest amount of isoamyl acetate produced (up to 6700 \(\upmu\)g L\({}^{-1}\)) as a result of additional leucine utilization than _S. bayanus_ yeasts (up to 5300 \(\upmu\)g L\({}^{-1}\)).  
According to the biplots, the use of juice concentrate in cider fermentation has also resulted in the production of selected long-chain fatty acid esters (ethyl decanoate, ethyl dodecanoate). Long-chain fatty acid esters possess a characteristic \"fatty\" or \"soapy\" odor description; their overproduction when using juice concentrate in fermentations might increase the risk of the off-flavor formation [36; 37]. On the other hand, the same esters have also been reported to increase the perceived fruitiness in wine [38].  
It should be noted that long-chain fatty acids esters have been previously tied to the onset of cell death [39; 40; 41]. Due to their low permeability through the cell membrane, the long-chain fatty acid esters are released along with other compounds (e.g., amino acids, fatty acids, lipids, glycoproteins, mannoproteins) into the environment during cell autolysis [39]. The correlation of long-chain fatty acids esters with the cider samples produced using concentrate could signify a higher cell death rate in comparison to the samples prepared with apple juice.  
The nutritional supplementation approaches used in this study did not possess any significant influence on the production of volatile esters in _S. cerevisiae_ strains. On the other hand, organic supplementation has increased the production of medium-chain fatty acid ethyl esters (ethyl butyrate, ethyl hexanoate, ethyl octanoate) in _S. bayanus_ strains and reduced the formation of long-chain fatty acid esters. Supplementation of nitrogen with organic sources has proven to be efficient at stimulating ethyl ester production in multiple previous studies [7; 38; 42]. The supplementation requirements for the visible increase in ester production, however, have shown in this study to be strain-specific.