## 3 Results and Discussion
### Wine Flavour Profiling  
After the optimization of the HS-SPME/GC-MS method, the best conditions of the analysis were applied to the untargeted flavour profiling of the wines which had been obtained by fermentation with mixed cultures of yeasts and which were composed of the _S. cerevisiae_ strain in association with the ND1 strain. This was tested both in free and immobilized cells, in comparison to the control, which was the experimental wine that had been obtained using single fermentation with the EC1118 strain. The SPME-GC/MS analysis allowed the identification of the 21 volatile organic compounds in the wine sample obtained by the mixed starters of _S. cerevisiae_ and the free cells of _H. osmophila_ (sample W1), which was a considerably greater number compared to the 8 compounds identified in the W2 wine sample that had been fermented with immobilized cells of the ND1 strain in co-culture with EC1118, and of the 6 compounds identified in the control (C), i.e., the wine sample obtained through the fermentation with _S. cerevisiae_ alone. Such a difference in the number of VOCs detected in W1 and W2 wines could be due to a remarkable reduction in the enzymatic activity of yeasts, potentially caused by the diffusion limitations or the breakage of the microcapsules [33]. Figure 2 shows the total ion current chromatograms, obtained at optimized conditions, of W1 and W2 wine samples and of the control C.  
All the volatile organic compounds detected in the three samples were identified by comparing their mass spectra with those available in the literature [34; 35; 36] and in the NIST electronic Mass Spectral Database. They belonged to five classes, namely higher alcohols, esters, volatile acids, terpenes, aldehydes and volatile phenols. The retention times, the quality match values with the NIST data and the main chemical-physical characteristics of the identified compounds are reported in Table 3, along with the occurrence or not of each VOC in the two wine samples and in the control. The wine obtained by co-inoculation of the free cells of _H. osmophila_ and _S. cerevisiae_ had a high number of higher alcohols, representing about the 44.9% of the total volatile organic compounds (Figure 3) and includ ethyl acetate  
Figure 1: Response surface plot for the total sum of peaks areas of the standard mixture used as model for the HS-SPME optimization according to the DoE face-centred composite design (95% of the explained variance). (compound **2**), 2-butanol-3-methyl (compound **3**), 1-butanol, 3-methyl- (compound **4**), 1-heptanol (compound **5**), 2,3-butanediol (compound **8**), phenylethyl alcohol (compound **9**) and 1-dodecanol (compound **11**). Higher alcohols are also referred to as fused alcohols and are alcohols which have more than two carbons in their chain; thus, they have higher molecular weights and higher boiling points than ethanol. The discussion regarding the role that these compounds play in improving the quality of enoological products remains controversial. Higher alcohols impart a range of organoleptic attributes, ranging from solvent-like to floral. which could positively influence wine flavour [37]. However, several authors attributed to higher alcohols the pungent and unpleasant character of some wines, especially when present at high levels [38; 39]. This is the case of phenylethyl alcohol and 1-butanol, 3-methyl-, known also as isoamyl alcohol, both among the major fused alcohols occurring in the W1 Aglianico wine analyzed in this study. Figure 3: Pie chart reporting the percentage composition of higher alcohols, esters, volatile acids, terpenes, aldehydes and volatile phenols detected in the headspace of W1 wine sample, based on average chromatographic peaks areas. Figure 2: Total ion current chromatograms obtained by the HS-SPME/GC-MS analysis of the three wine samples, i.e., Aglianico wine obtained by fermentation with the mixed starters _S. cerevisiae_ and free cells of _H. osmophila_ (W1); Aglianico wine obtained by fermentation with the mixed starters _S.