## 3 Results and Discussion
### Building a Formulation as RM for the Winey-Vinegary Defect
\begin{table}
\begin{tabular}{c c c c c c c c} \hline
**1** & **2** & **3** & **4** & **5** & **6** & **7** & **Sensory Descriptors** & **Code** \\ \hline
80 & 600 & 40 & - & 20 & - & 40 & Slightly vinegary, sweet, slightly rancid & AV1 \\
200 & 600 & 600 & 50 & 50 & 50 & 50 & Vinegary, glue, chemicals, wood & AV2 \\
400 & 1200 & 1200 & 100 & 100 & 100 & 100 & Vinegary, chemicals, glue, rancid, mushrooms & AV3 \\
200 & 600 & 600 & 10 & 10 & 10 & 10 & Vinegary, glue, no natural, slightly vinegary & AV4 \\
40 & 5000 & 600 & 10 & 10 & 10 & 10 & Slightly vinegary, vinegary & AV5 \\ - & 5000 & 600 & 1.5 & 1.5 & 1.5 & 1.5 & Less intense to vinegary & AV6 \\ - & 10,000 & 1200 & 3 & 3 & 3 & 3 & Vinegary at high and medium-high intensities & AV7 \\ - & 20,000 & 2000 & 5 & 5 & 5 & 5 & Vinegary, alcohol, artificial vinegary & AV8 \\ - & 32,000 & 4800 & 7.5 & 7.5 & 7.5 & 7.5 & Vinegary, high intensity of acetic acid and alcohol & AV9 \\ \hline \end{tabular}
\end{table} 
Table 3: Main formulations (volatile compounds and concentrations in mg/kg) emulating winey-vinegary aroma in VOO that resulted from modified simplex procedure and were evaluated by assessors in terms of suitability as possible RM. Note: **1**, ethyl acetate; **2**, ethanol; **3**, acetic acid; **4**, hexanal; **5**, 1-hexanol; **6**, _E_-2-hexenal; **7**, _E_-3-hexen-1-ol. evaluation and sensory notes that were not related with winey-vinegary defect (rancid, wood and mushrooms aromas) were identified by some assessors, while vinegary sensory note was only reported as \"slight intensity\" and \"non-natural\". Secondly, all assessors agreed in the characteristics of \"non-natural\" and low representativeness of the aromas, and the sensory notes of glue and chemicals were identified as the reason for this non-natural aroma. Individual dilutions of compounds identified ethyl acetate as being responsible for these sensory notes, particularly at concentrations higher than 80 mg/kg. It was thus decided to remove this compound from subsequent formulations. The third conclusion was that the increase of the concentration of acetic acid did not lead to a proportional increase of the intensity of vinegary aroma. On the contrary, the mixture of acetic acid and ethanol at different concentrations led to abrupt changes of the perception of the vinegary aroma, which explained the increase of the concentration of ethanol as aroma enhancer. The fourth conclusion was that the volatiles responsible for green aroma at a concentration of \(>\)50 mg/kg, although they contribute to the natural aroma similar to VOO, they also have a negative effect on the aroma. This negative effect consists in producing an \"analytical\" perception of the aroma, in which the individual effect of some compounds is perceived with rancid notes. This effect was considered as undesirable since the goal was to produce an aroma characterized with a \"holistic\" perception (i.e., no individual contribution of the compounds diluted in the formulation) of a winey-vinegary aroma as it occurs in natural VOOs. A programmed set of formulations based on modified simplex design using AV4 led to AV5 and AV6 (Table 3). In these two formulations, the addition of ethyl acetate was omitted because of its contribution to \"non-natural\" perception, even if it is an excellent marker of winey-vinegary defect and was initially identified as a \"primary compound\" to emulate the aroma of this defect. The new strategy for subsequent steps (AV7-AV9, Table 3) in modified simplex design was based on increasing the concentrations of all the volatiles as described in AV6. Table 3 shows the full agreement of the assessors in evaluation of these three formulations. In these formulations, the assessors clearly identified vinegary aroma and did not detect any masking effect or interferences from other compounds. From these formulations, AV7 and AV8 were considered by all assessors as satisfactory in terms of representativeness and aroma persistence, keeping the composition simple as in the previous formulations.