# _Torusspora delbrueckii_ Strain Behaviour within Different
## 3 Results and Discussion
### Volatile Organic Compounds  
Volatile compounds can be found in raw materials or may be formed during fermentation and maturation. The influence of _T. delbrueckii_ on the volatile organic compound (VOC) production in elder has been scarcely investigated, while more information is available for wine and beer [19; 46], where _T. delbrueckiii_ has been associated with an enhanced fusel alcohol and ester production. Esters, alcohols, and acids are the main constituents of the fermentation bouquet [21]. Twenty-six VOCs were detected and quantified (examples of the chromatograms are reported in Figure S3a,b), whereby 17 were identified as esters (11 ethyl esters), five as alcohols, and four as acids. Most of the VOCs appear to be produced during fermentation as previously observed [47] and only the 2-methylbutyric acid methyl ester was exclusively found in the juice sample. To evaluate the correlations among the VOCs, strains, and production method, a principal component analysis (PCA) was carried out. The PCA-score plot and biplot (Figure 2) are showing the separation of ciders fermented with strain A from samples fermented with strain B (Figure 2A) according to their metabolite profile. PC1 explained 32.3% of the total variance and allows us to identify the compounds correlated to yeast strains, while PC2, explaining 28.2% of the total variance, allows a separation of the samples based on the production methods (Figure 2B). The ciders obtained with strain A clustered in the right side of the score plot, correlating with alcohols and most of the detected esters. The ciders fermented by strain B clustered on the left side of the plot correlating with acids and some esters (Figure 2B). For both strains, the base ciders were placed on the upper side of the plot, while the sparkling ciders were also influenced by negative loadings and partially or totally projected on the lower part of the plot. Figure 1: Dimethyl sulphide (DMS) amount found in apple juice (Juice), in base cider (Cider), Pét-Nat, pressured-tank Charmat method (Charmat), and traditional bottle-fermented cider (Champenoise) inoculated with _Toruslospora delbrueckii_ strain A or strain B. Data are reported as average and error bars indicate the standard deviation. Different letters identify significant differences (\(p<0.05\)). Figure 2: Principal component analysis of volatile organic compounds: (**A**) Score plot of ciders produced with different _T. delbrueckii_ strains; (**B**) score plot of ciders obtained with different methods; (**C**) biplot based on VOC composition of samples. C: base cider; PN: Pét-Nat; TM: Campenoise method, CM: Charmat method. A and B indicate ciders fermented by T. delbrueckii strain A and strain B, respectively. (1) Acetic acid ethylester; (2) i-butanol; (3) propionic acid ethylester; (4) 3-methylbutanol; (5) 2-methyl-butanol; (6) i-butyric acid ethylester; (7) 2-methylbutyric acid methylester; (8) butyric acid ethylester; (9) lactic acid ethylester; (10) i-valeric acid; (11) 2-methylbutyric acid ethylester; (12) hexanol; (13) acetic acid 3-methylbutylester; (14) acetic acid 2-methylbutylester; (15) hexanoic acid; (16) hexanoic acid ethylester; (17) acetic acid hexylester; (18) 2-hydroxy-4 methylvaleric acid ethylester; (19) 2-phenyl-ethanol; (20) octanoic acid; (21) succinic acid diethylester; (22) octanoic acid ethylester; (23) benzeneacetic acid ethylester; (24) acetic acid phenylethylester; (25) decanoic acid; and (26) decanoic acid ethylester. The final products obtained with strain B have been separated in a cluster showing the least diverse volatile composition. These results are better highlighted in the heatmap (Figure 3) where the distance between the links is smaller among the strain B cider clusters than strain A cider clusters. The quantification of the VOCs is reported in Table S2a,b. The ANOVA analysis shows significant differences among the strains for all the metabolites, while the applied production method generated significant differences for 20 compounds.