## 4 Discussion  
To gain deep understanding of the mechanism of UFAs regulating chemical and aromatic compounds, we investigate the effect of UFAs addition on intra-metabolites, major fermentation products, and aromatic compounds profiles of Cabernet Sauvignon wine fermented with two _S. cerevisiae_ strains (EC1118 and BDX), respectively. The results shows that pre-fermentative addition of UFAs mixture significantly alters the physiological and energetic state of the cell, and affects the metabolism of carbon and nitrogen source, which consequently modify the chemical and aromatic component of the final wines. Consistent with previous results [23], UFAs supplementation promoted yeast growth and biomass of both strains (Figure 1), but unexpectedly, the promotion did not correspondingly increased fermentation activity. Conversely, decreased sugar consumption rate and prolonged fermentation duration time, especially in high-UFAs added fermentation, was noticed. This is inconsistent with the previous result of [PERSON], [PERSON], and [PERSON] [24] who proposed that viable yeast cell concentration is positive with the fermentation rates in wine fermentation. We ascribed this inconsistency to the changes of metabolism patterns of yeast in response to excess UFAs in the environment. Under anaerobic fermentation condition, the absence of oxygen suppresses the cells de novo synthesis of UFAs, and yeast cells have to directly uptake UFAs from grape juice in order to maintain the cell membrane integrity and resist various fermentative stresses, while the uptake of UFAs is ATP-dependent [25]. Moreover, once in the cytoplasm, the cells need to compartmental  
Figure 4: The comparison of aroma gene expressions in EC1118 and BDX strains fermented in high-UFAs addition vs. the control juice. T1, T2, T3, and T4 represented the mid-exponential phase, the early-stationary phase, the mid-stationary phase, and the end of fermentation, respectively. ize UFAs into the peroxisome and to use ATP again [26]. The requirement for abundant ATP would activate the flux of glycolysis pathway because it is the only way for yeast to produce ATP under anaerobic condition [25], and consequently alter the physiological and energetic state of cells. The decreased levels of glycolysis intermediates and ATP in cells support this conclusion. The activation of ATP production leads to two consequences. Firstly, intracellular NADH levels are increased and cause redox imbalance (Figure 3). Secondly, the NADH-consume reactions would be activated and the NAD\({}^{+}\) dependent reactions are restrained because cells need to re-oxidize excess NADH to NAD\({}^{+}\)[27]. This explains the increased production of glycerol and decreased acetic acid synthesis observed (Table 1). Glycerol is the main metabolite produced by yeasts to maintain the intracellular redox balance (NADH/NAD\({}^{+}\) turnover) [28]. Under anaerobic conditions at high glucose concentration, such as wine fermentation condition, the cytosolic redox balance is mainly restored by glycerol formation [27], which consequently lead to decreased acetic acid production [29]. The link of decreasing acetic acid and increased glycerol production was observed in previous work [30]. We also observed the slight increment of ethanol content in response to UFAs addition. This finding implied that under the fermentation condition with more exogenous UFAs, glycerol and ethanol syntheses is the strategy for cells to re-oxidize excess NADH, in which, glycerol plays the major function. The large production of ATP is usually accompanied with increased cell growth, which is in agreement with the results of biomass concentration in Figure 1. Certainly, the increased resistance of cells to stress conditions, such as ethanol toxicity due to incorporating more UFAs, is possibly another reason for increased biomass. Under anaerobic condition, the metabolism of TCA cycle is limited. The decrement of TCA cycle intermediates might be drawn for producing biomass and amino acids.