# Construction of IsoVoc Database for the Authentication of Natural Flavours
## 3 Results and Discussion
### Selection of Authentic Reference Sample  
To address the usefulness of fresh fruits instead of distillates for database creation, we compared the \(\delta^{13}\)C values of specific VOCs present in distillates and fresh fruit samples of apple and strawberry. Only aroma compounds that occur in at least five samples per group (fruit samples and distillates), wherein the compound must be present in both groups,were accepted for further data analysis. In this way, we identified 15 characteristic aroma compounds for apple and 11 aroma compounds for strawberry. We included 49 recovery aromas and 99 samples of fresh apple juice (Figure 1). For the 15 aroma compounds, \(\delta^{13}\)C values ranged between \(-43.7\%\) (acetone in fruit) and \(-24.1\%\) (methyl hexanoate in fruit). A Mann-Whitney U test revealed statistically significant differences in median isotope values for (E)-2-hexanal (\(-42.2\%\); \(-35.1\%\)), amyl acetate (\(-36.5\%\); \(-31.4\%\)), butyl acetate (\(-36.2\%\); \(-33.6\%\)), butyl propionate (\(-36.8\%\); \(-30.5\%\)), hexanal (\(-40.1\%\); \(-35.9\%\)), hexyl 2-methyl butyrate (\(-33.9\%\); \(-37.6\%\)) and hexyl acetate (\(-36.7\%\); \(-32.3\%\)) between fruit samples and distillates respectively. For strawberry, 17 distillates and 17 fresh strawberry samples were analysed (Figure 2). For the selected 11 aroma compounds, \(\delta^{13}\)C values ranged between \(-47.0\%\) ((E)-2-hexanal in fruit) and \(-26.0\%\) (2-methylbutyl acetate in distillate). In this case, the Mann-Whitney U test revealed statistically significant differences for (E)-2-hexanal, (\(-30.5\%\); \(-33.6\%\)), acetone (\(-42.1\%\); \(-37.7\%\)) and ethyl butyrate (\(-31.4\%\); \(-29.7\%\)) between fruit samples and distillates, respectively. Data analysis also revealed several outliers in apple and strawberry that were not a result of analytical problems or random errors but are likely due to differences in variety, stage of maturity, and geographical origin, although further investigation is required. The outliers were not removed from the database. It is also necessary to consider natural isotopic variation caused by, e.g., geographical location, variety, temporal and seasonal variation, and processing to make the database more robust. Further, before data analysis it is important to understand if various technological processes such as distillation cause any isotope fractionation in isotopic values of aroma compounds in specific sample type. When comparing distillates with the freshly prepared fruit juice, we observed a difference in \(\delta^{13}\)C values for seven investigated VOCs in apple and three in strawberry. Although [PERSON] et al. [28] did not find a significant isotopic effect caused by technological treatment for (E)-2-hexanal, (E)-2-hexanol, and hexanal, in this study, we found that for (E)-2-hexanal and hexanal, preparing the fresh juice can modify the isotopic data. In apple, the isotopic values for (E)-2-hexanal are lower for fruit juice than distillates. In the case of strawberry aroma, the opposite is true. Except for hexyl-2-methyl butyrate, the median isotope values  
Figure 1: Comparison of \(\delta^{13}\)C values of volatile organic compounds (VOCs) present in apple fruit and apple distillates. \({}^{*}p<0.05\) (Mann-Whitney test, statistically significant). are lower in the fruit samples. Isotope fractionation that we observed in many volatile compounds is most likely compound dependent. For volatiles that are produced as a result of tissue disruption (secondary compounds, like aldehydes (E)-2-hexanal and hexanal), it seems that different types of fruit processing can cause isotope fractionation in different ways resulting in the difference in isotopic ratios between distillates and fruit juice samples. Beside fruit processing, different fractionation of compounds could be a consequence of flavour changes occurring through maturation, harvest, and subsequent storage.