# Black Truffle Aroma Evaluation: SPME-GC-MS vs. Sensory Experts
## 3 Results and Discussion
### Volatile Organic Compounds Analysis via SPME-GC-MS
In January (time 2), the dominant VOCs were very similar but with different percentages: DMS (21.8%), 2-methyl-1-butanol (17.7%), butanal-2-methyl (13.2%), methylpropyl formate (10.3%), and 2-methyl-1-propanol (5.8%). At the end of the season, in February (time 3), the most abundant molecules were 2-methyl-1-butanol (29.25%), DMS (11.1%), butanal-2-methyl (8.8%), DMDS (6.3%), and 2-methyl-1-propanol (5.6%). Apart of them, some of the key truffle VOCs described for black truffle such as 3-methyl-1-butanol, 2-methyl-1-butanol, 2-butanone, and anisole [1, 2, 3] were also found in our samples. Some key compound contents changed their relative abundance throughout the season, i.e., butanal-3-methyl decreased from 17.7% to 4.5%, whereas 2-methyl-1-butanol increased from 3.1% to 21.3%. However, 2-propanone, butanal-2-methyl, and anisole maintained  
\begin{table}
\begin{tabular}{l l l l l l l l l} \hline \hline \multicolumn{1}{c}{**No**} & \multicolumn{1}{c}{**Name**} & \multicolumn{1}{c}{**CAS**} & \multicolumn{1}{c}{**RT**} & \multicolumn{1}{c}{**RIexp**} & \multicolumn{1}{c}{**RIlit**} & \multicolumn{1}{c}{**1**} & \multicolumn{1}{c}{**2**} & \multicolumn{1}{c}{**3**} \\ \hline C42 & 3-methyl-acid & 589-59-3 & 12.500 & 1007  
constant percentages in the aromatic profile. According to [PERSON] et al. (2020) [31], aromatic compounds can be used as ripening markers, and those previously described could be potential markers to establish VOCs profile for different truffle maturity stages. A PCA was used to explore the possible linkages between harvesting time and VOCs content detected via SPME-GC-MS (Figure 1). The PCA explained 47.1% of the data variability with the two first PCA components. The first component allowed us to clearly separate the first harvesting time from the others, whereas the second did not explain the maturation degree which might be due to other factors. An early harvesting time was positively associated with 2-butanone (C10), 2-methyl-1-propanol (C12), 2-methyl-1-butanol (C22), 3-octanol (C39), 2-ethyl-1-hexanol (C44), isoamyl-2 methylbutyrate (C50), 2-methyl-butanoic acid (C52), and benzeneethanol (C54). Some of these molecules have wine and onion aromas (2-methyl-1-butanol), a mushroom-like aroma (3-octanol), rose and green odors (2-ethyl-1-hexanol), and honey and rose odors (benzeneethanol) [32]. Other compounds such as butanal-3-methyl (C13), methional (C33), nonanal (C51), 2,5-dimethoxyytoluene (C63), or 2-undecanone (C65) were positively associated with truffles harvested in January and February. Some of them show malt (butanal-3-methyl), cooked potato (methional), fat and citrus (nonanal), and orange odors (2-undecanone) [32]. This difference might be due to some degree of aromatic immaturity in December truffles with respect to those harvested in January and February. Moreover, truffle aromas can vary depending on many factors, such as bacteria community, host tree, and geographical origin, among others [33; 34; 35]. For example, [PERSON] et al. (2016) identified some markers linked to the host tree: DMS was higher in hazel and holm oak truffles, whereas isoamyl alcohol was higher in Portuguese and kermes oak [35]. [PERSON] et al. (2020) reported a wide volatilome study including more than 450 truffle samples from 11 different countries. The study revealed an aromatic model able to differentiate between species with an overall classification rate of 97%. The article indicated that many factors, including genotypic variability, maturation, microbial community as well as geographical origin need to be considered for the understanding of the aromas [34]. Our results showed that SPME-GC-MS technique was able to differentiate fresh truffles depending on the harvest time during the season, even though they all showed optimal maturity according to spores and gleba color [25; 36]. Figure 1: _Cont._