## 3 Results
### Effects of Pre-Ferrantative Addition of UFAs Mixture on Intra-Metabolite Profile  
Samples for intracellular metabolite analysis were taken at four stages in fermentation, which corresponded to around 20%, 50%, 80% sugar consumption, and the end of the fermentation, respectively. A total of fifty-five intracellular metabolites were identified,  
Figure 2: Principle components analysis using thirty-five volatile compounds and major fermentation products (including ethanol, glycerol, acetic acid, malic acid, citric acid, and succinic acid) in wines treated by different levels of UFAs (**(A)** Scatter plot; **(B)** loading plot). E_Control, E_L, and E_H represented the control, low-UFAs, and high-UFAs added wines fermented by EC1118 strain, respectively; B_Control, B_L, and B_H represented the control, low-UFAs, and high-UFAs added wines fermented by BDX strain, respectively. including twenty-three glycolysis and TCA cycle intermediates, twenty-two amino acids, ten fatty acids, and energy pool, which are presented by heat map in Figure 3. UFA addition resulted in significant variation of metabolite levels in central carbon and nitrogen metabolisms, which are largely associated with the strain and fermentation stages. In general, the relative abundance of intermediates in glycolysis and TCA cycle, amino acids, ATP, and saturated fatty acid were decreased, especially before around 50% sugar consumption, while the levels of NADH, NADH/NAD+, NADPH, NADPH/NADP+, and unsaturated fatty acids were increased compared to the control wine samples. In the glycolysis pathway, the decreased intermediates included glucose-6-phosphate, 3-phosphoglyceric acid, glycerol 3-phosphate, fructose-1, 6-biophosphate, phosphoenolpyruvate acid, and pyruvate acid, which were profound in BDX strain. In comparison, the levels of TCA cycle intermediates are dependent on fermentation stage. In the early stage (corresponding to around 50% sugar consumption), the abundance of ketoglutaric acid, fumaric acid, malic acid, and succinic acid decreased in both strains, but were improved after cells entered the late stage (corresponding to around 80% sugar consumption), especially in the strain of BDX. Noticeably, we found that the ATP level in cells showed decrement in UFAs added fermentation, suggesting that energy is largely consumed by cells in response to more UFAs. In addition, acetyl coenzyme A (Acetyl CoA) was accumulated in both strains with UFAs addition. Acetyl CoA is the key intermediate in the central metabolism of yeast, and it is used to synthesize a vast amount of functional products, however, its level is usually kept in equilibrium in normal conditions [22]. The accumulation of acetyl CoA confirmed again that yeast physiology is disturbed by exogenous UFAs. A global reduction of amino acid contents was observed in both strains, especially in the early fermentation, including phenylalanine, methionine, leucine, isoleucine, valine, lysine, asparagine, aspartate, and serine, suggesting that the most amino acids are used for cells proliferation. The intracellular contents of LA, OA, and ALA are correspondingly increased due to the incorporation of exogenous UFAs, which results in decreased production of saturated fatty acids in cells (except for tetradecanoic acid in BDX strain). This result is in agreement with the data of the contents of medium-chain fatty acids (MCFAs) in the wines (Table S7). Effects of Pre-Fementative Addition of UFAs Mixture Genes Expression Related to Aroma Compounds Formation  
To reveal the mechanism of UFAs regulating aroma at molecular level, the relative expression levels of twenty genes associated with aroma syntheses were quantified at different stages of fermentation by real-time PCR (Figure 4). These genes involved in amino acid transportation (_GAP1_, _BAP2_) and higher alcohol biosynthesis (_BAT1_, _BAT2_, _PDC1_, _PDC5_, _PDC6_, _ARO10_, and _ADH1_), fatty acid biosynthesis (_ACC1_, _FAS1_, _FAS2_, _FAT3_, _FAA1_, _OLE1_, and _ELO1_) and ester biosynthesis (_ATF1_, _IAH1_, _EEB1_, and _EHT1_).