## 3 Results
### Analysis of Samples  
Microbiological analyses of seeds, leaves, and flowers revealed a good hygien quality of all the parts of SO plants since all the results presented values that are below the limit proposed by European Commission and International Commission on Microbiological Specifications for Foods [28] (Table 3). Model samples for the GC/MS analysis were prepared in order to mimic as far as possible the real food and drink preparation and consumption modalities. To minimise the loss of volatiles, autolysed samples were prepared by dissolving samples in water in a vial, which was rapidly sealed with a magnetic screw cap equipped with silicon/polytetrafluoroethylene septa (PTFE). The two main pungent ITCs, compounds **2** and **4**, were determined by triplicate analysis; the average value is reported in Table 2. Since other ITCs could be not determined in these experimental conditions, the total ITCs content was calculated as the sum of compounds **2** and **4**. Then, the total ITCs release has been calculated in a standard portion of each food or drink as in its common use, which is assumed to be 1 spoon for honey, 1 teacup = 125 mL for infuse and voice drink, 1 can = 330 mL for beer and 1 tablet. Samples of honey showed a low ITCs release; only sample S5, containing seeds and submitted to grinding before analysis, has an ITCs content much higher than the others, confirming that ITCs are well preserved in the plant material and their release is favoured by the activation of enzymatic hydrolysis. Since the precursor glucosinolates are most abundant in flowers than in seeds [13], the lower content of ITCs in honey with flowers can be related to a loss due to various enzymatic processes, which can involve flowers more than seeds. In fact, flowers' tissues have about 90% water, whereas seeds are mainly constituted by oil and are therefore protected by enzymatic hydrolysis until grinding occurs. Samples of honey have an ITCs release comparable or higher to that of the commercial tablets (S10). The analysis of infusion (S6) showed a release of active ITCs of 38.56 \(\upmu\)g/kg for compound **2** and 8.29 \(\upmu\)g/kg for compound **4**. \"Classic\" infusion with whole dried plant has a total ITCs content per portion higher than that of the voice drink (5.85 \(\upmu\)g/portion vs. 0.95 \(\upmu\)g/portion), indicating that the contribution of ITC released from dried leaves is relevant. On the other hand, the commercial infusion is made by the whole dried plant, including those parts containing low or no active principles, and it has an overall herbaceous, mild bitter, and astringent flavour and taste. Instead, the voice drink is made with seeds and flowers and results cleaner with a mild taste profile where the pungency of ITCs can be easily perceived. The peculiar taste and somatosensory notes of ITCs are not easily detected in the fermented drinks, where they are covered by a number of taste active compounds and volatile flavours. The ITCs content resulted quite high in beer (S8), with a value of 7.62 \(\upmu\)g/portion considering a can as a standard portion (330 mL); calculating the ITC concentration in the same volume, the content is about half that of infusion (17.84 vs. 31.03 \(\upmu\)g/L). Whereas the \"singers' beer\" could be a new and attractive product for consumers, it is clear that the presence of alcohol could not be recommended in a functional food or drink preparation. \begin{table}
\begin{tabular}{l c c c c} \hline  & **Leaves** & **Flowers** & **Seeds** & **Reference** \\ \hline Total bacterial count & 4.1 \(\times\) 10\({}^{3}\) CFU/g & 3.4 \(\times\) 10\({}^{3}\) CFU/g & 2.5 \(\times\) 10\({}^{3}\) CFU/g & [28] \\ Yeasts and moulds & \(<\)10\({}^{2}\) CFU/g & \(<\)10\({}^{2}\) CFU/g & \(<\)10\({}^{2}\) CFU/g & [28] \\ _Escherichia coli_ & \(<\)10 CFU/g & \(<\)10 CFU/g & \(<\)10 CFU/g & [29] \\ _Bacillus cereus_ & \(<\)10\({}^{2}\) CFU/g & \(<\)10\({}^{2}\) CFU/g & \(<\)10\({}^{2}\) CFU/g & \\ \hline \end{tabular}
\end{table} 
Table 3: Microbial counts of SO seeds, leaves, and flowers.