## 3 Results and Discussion
### Biotin and Thiamine Impact on the Wine Metabolome
#### 3.4.2 Impact of Thiamine on the Wine Metabolome  
In order to determine the extent of the influence of thiamine on the yeast exometabolome, an ANOVA was performed on the 3870 extracted features, and resulted in the isolation of 515 that were associated with significant differences (\(p<0.01\)) in regard to the initial concentration of the vitamin, therefore accounting for nearly 15% of the extracted features. However, only 378 of those specific features were assigned possible chemical formulas through putative annotations in online databases, in a similar fashion to what was performed on biotin-associated features, and these were the sole ones to be considered for their significant influence in the _S. cerevisiae_ thiamine-dependent metabolome. It is also relevant to note that the number of features associated with thiamine here was significantly higher than those associated with biotin, since it more than doubled its amount, also suggesting a greater influence of thiamine on the yeast metabolism. Similar to what was observed in the assessment of the influence of biotin on the yeast exometabolome, a high proportion of those features were significantly more intense in high thiamine, amounting to 378, while only 69 features appeared significantly more intense (\(p<0.01\)) in low thiamine wines. Such a differentiation also appeared quite clearly through hierarchical clustering of these 515 total biomarkers, since it resulted in a distinct discrimination between high and low thiamine wines (Figure 3D), strongly testifying on the influence exerted by the vitamin on the wine exometabolome. An investigation of the chemical composition of the thiamine-affected features was led, in order to find the precise nature of the effect exercised by the vitamin on the yeast metabolism during fermentation. As such, Van Krevelen diagrams were drawn based on the O/C and H/C atomic ratios of the specific features for both the low and high thiamine conditions, and their elemental composition was investigated in an effort to define their chemical identities. Surprisingly, as opposed to what was observed for biotin, there was no clear difference between the elemental composition proportions of the high and low thiamine features (Figure 5), although there was a slight increase in the percentage of CHO markers in high biotin, amounting to 11.7% of the features, against 7.2% in low thiamine, as well as a slight increase in phosphorus-containing ones in low thiamine, reaching 39.1% of the features, against 33.0% in high thiamine. Interestingly, however, these CHO features in high thiamine conditions appeared to be amongst the most intense ones, found mostly where the amino sugars (O/C \(>0.6\) and H/C \(>1.5\)) and carbohydrates (O/C \(>0.8\), \(1.6<\) H/C \(<2.7\)) were expected on the Van Krevelen diagrams. Plus, it appears relevant to note that the strongly represented CHON biomarkers, not significantly varying in proportion between both thiamine conditions, also appeared to display similar behaviors in regard to their intensities and nature, whether obtained from low or high thiamine wines. They are, indeed, associated with a number of high intensities, as observed on Van Krevelen diagrams, and both were found in the area in which polyphenols were expected (O/C \(<1.2\) and H/C \(<1.3\)), consistent with the high proportion of polyphenol compounds that were predicted [58]. Thiamine did not appear, consequently, to interfere in any way with the frequency of formation of neither polyphenols nor lipids; however, the high thiamine exometabolome did display increases in the proportion of carbohydrates and amino sugars formed during alcoholic fermentation, while low thiamine appeared to induce noticeable increases in the proportion of synthetized proteins. A limited number of biomarkers for both thiamine conditions was annotated at level 3 [57] in online databases, since less than 5% of those were matched with an identification.