# _Torusspora delbrueckii_ Strain Behaviour within Different
## 3 Results and Discussion
### Volatile Organic Compounds
#### 3.7.3 Esters  
Esters are fermentative metabolites produced by yeasts via the enzymatic reaction involving a carboxylic acid and an alcohol (Figure 4). They represent key flavour compounds in fermented beverages, contributing to a pleasant fruity aroma. _T. delbrueckii_ has a deep influence on the development and production of esters in wine [20]. In our study, esters accounted for the 33% of the quantified compounds. A PCA analysis of the esters (Figure 5) allowed a separation of the samples according to PC1 and PC2, explaining the 37% and the 22.3% of variance, respectively. PC1 contributes to separate samples in relation to the yeast strain employed for the fermentation. The ciders obtained with strain B formed a small cluster on the left side of the graph, correlating with the lactic acid ethyl ester, acetic acid ethyl ester, and 2-hydroxy-4-valeric acid ethyl ester. Among the strain A ciders, CA is the only product located on the left and lower side of the graphic together with PNB, and their position is correlated with hexanoic acid ethyl esters and with benzene acetic acid ethyl ester as negative loadings. Sparkling ciders produced with strain A are located on the right side of the graphic with a separation along PC2 among PNA, on the lower side of the graphic, and samples obtained with the Charmat and Champenoise methods that are positioned in the upper right quadrant. PNA is closer to CA than CMA and TMA, and correlated with the esters of butyric acid, succinic acid diethyl ester, and propionic acid diethyl ester. The CMA and TMA ciders correlated with acetic acid 2-methylbutylester, acetic acid 3-methylbutylester, acetic acid phenyl ethyl ester, decanoic acid ethyl ester, and acetic acid ethyl ester. Figure 5: _Cont._Figure 5: (**a**). PCA analysis of esters. (**A**): Score plot of ciders produced with different _T. delbrueckii_ strains. PC1 accounts for 37% of the variability; PC2 accounts for 22.3% of the variability. (**B**): Score plot of ciders obtained with different methods: base cider (C), Pét-Nat (PN), Champenoise method (TM), Charmat method (CM). In sample label, letter A and B indicate ciders fermented by _T. delbrueckii_ strain (A,B), respectively. (**b**). PCA analysis: biplot based on chemical analysis of esters: base cider (C), Pét-Nat (PN), Champenoise method (TM), Charmat method (CM). In sample label, letter A and B indicate ciders fermented by _T. delbrueckii_ strain A and B, respectively. (1) Acetic acid ethylester; (2) propionic acid ethylester; (3) i-butyric acid ethylester; (4) 2-methylbutyric acid methylester; (5) butyric acid ethylester; (6) lactic acid ethylester; (7) 2-methylbutyric acid ethylester; (8) acetic acid 3-methylbutylester; (9) acetic acid 2-methylbutylester; (10) hexanoic acid ethylester; (11) acetic acid hexylester; (12) 2-hydroxy-4-methylvaleric acid ethylester; (13) succinic acid diethylester; (14) octanoic acid ethylester; (15) benzeneacetic acid ethylester; (16) acetic acid phenylethylester; and (17) decanoic acid ethylester. For all the identified esters, an ANOVA analysis was performed and significant differences in the compound concentration due to the strain, method, and method-strain interactions were highlighted (Table S2a,b). Acetic acid ethyl ester is the most common ester produced during alcoholic fermentation. It can be produced by both fermentative yeasts and acetic acid bacteria in low-oxygen conditions. In all the analysed samples, acetic acid ethyl ester was the most abundant ester, ranging from 11.36 mg/L to 31.11 mg/L in PNA and TMB, respectively. Acetic acid ethyl ester, when in a low concentration, is associated with a fruity aroma, while, when its concentration exceeds the human perception threshold (100-200 mg/L), it is associated with an unpleasant aroma of solvent [50].