## 2 Results and Discussion
### Potential Medium Chain Fatty Acid (MCFA)-Derived Precursors of Ethyl Esters
#### 2.1.3 Acyl-Carnitine Conjugates, Putative Transport Forms of MCFAs  
No significant difference was observed in the ethyl nonanoate concentrations of the control wines and those produced after the addition of 1 and 10 \(\upmu\)M nonanoyl carnitine. However, the addition of 100 \(\upmu\)M nonanoyl carnitine to MGJM significantly increased the ethyl nonanoate concentration in the resulting wine (Figure 1C). Increasing the concentration of nonanoyl carnitine from 100 \(\upmu\)M to 1 mM also caused significant increases in ethyl nonanoate production in the subsequent fermentations (Figure 1C). The addition of 1 and 10 \(\upmu\)M dodecanoyl carnitine to the MGJM caused a significant difference in ethyl dodecanoate content of the model wines, and this was further enhanced when the model must was supplemented with 100 \(\upmu\)M dodecanoyl carnitine (Figure 1C). When 1 mM dodecanoyl carnitine was added to the MGJM the yeast appeared to lose viability and fermentation of the MGJM did not occur, denoted by no loss of weight and no flocculence in the must (data not shown).  
The apparent toxicity of the high concentration of dodecanoyl carnitine compared to the free fatty acid was unexpected. Fatty acids appear to be imported into yeast cells in a process that requires activation of the molecule with CoA [34]. The carnitine conjugate may be transported more readily as it is more soluble than the free fatty acid and because it is already activated. The increased transportation of the carnitine conjugate into the cell, followed by liberation of the carnitine, may lead to toxic intracellular levels of the free fatty acid that is not seen when the free fatty acid alone is added to the model must. The mechanism for the transport of extracellular carnitine or conjugates into the cell in _Saccharomyces cerevisiae_ is still unclear.  
Paradoxically, the nonanoyl carnitine conjugate did not inhibit fermentation when added to the must at a concentration of 1 mM, whereas the free fatty acid did. This may reflect the difference in the fattyacid chain length which may allow more diffusion of the C\({}_{9}\) fatty acid into the cell compared to both nonanoyl carnitine and free dodecanoic acid. Certainly the stimulation of ethyl nonanoate production was 10-fold lower after the addition of 10 or 100 mM nonanoyl carnitine compared to the equivalent amounts of nonanoic acid (Figure 1). Alternatively, the odd number of carbons in the nonanoyl carnitine conjugate may inhibit any further metabolism of this compound into something that may be toxic to the yeast (for example, the free acid) compared to the even-numbered dodecanoyl carnitine.  
The concentration of acyl carnitine conjugates in grape berries is unknown. A study that quantified free carnitine and carnitine esters in many different foods found that fleshy fruits such as apples, guavas and oranges contain concentrations of total carnitine esters of between 0.05-1.45 mg\(\cdot\)kg\({}^{-1}\)[35]. The grape seed, being a source of lipids, may contain more carnitine esters than the rest of the berry, but further work is required to determine if these conjugates from grapes can alter ester composition in wines.