# Using Science and Technology to Unveil The Hidden Delicacy Terfezia arenaria, a Desert Truffle
## 2 Materials and Methods  
### Terfezia arenaria Samples  
Desert truffles naturally fruit in the spring from February to May. For three weeks of the 2019 spring season, sixty-three _T. arenaria_ truffles were harvested in Alentejo (south of Portugal) (see Figure S1). The samples were collected in four sampling sites--S1, S2, S3 and S4--with an area of 200 m\({}^{2}\) each; the sampling sites were separated by 1 to 2 km distance. In all the sampling sites, the Cistaceae host plants were abundant (_Tuberaria guttata_), but the forest-dominant species differed: _Quercus super_ in sites 1 and 2, _Pinus pinea_ in site 3 or mixed in site 4 (Figure S1). Specimens were freed from substrate debris at the site and further cleaned in the laboratory and used to analyse _T. arenaria_'s nutritional and chemical composition and volatile profile (using 2 techniques). _T. arenaria_ samples were (i) kept at \(-20\)\({}^{\circ}\)C until molecular analysis; (ii) dried for nutritional and chemical composition analyses; and (iii) kept at 4 \({}^{\circ}\)C until volatile profile analysis in the first 48 h post-harvest. The specimens were identified by molecular analysis. For the nutritional and chemical analyses, we used three dry samples of _T. arenaria_ truffles collected in three of the sampling sites (sites S1, S2, S3, S4) during the first week of April 2019. From each sampling site, one desert truffle with similar size and appearance was analysed. For the volatiles profile analysis, we used three fresh samples of _T. arenaria_ truffles collected in three of the sampling sites (sites S2, S3, S4) during the first week of April 2019. From each sampling site, one desert truffle with similar size and appearance was analysed. To validate _T. arenaria_'s identification using the e-nose, we used a total of five mushroom and truffle species: _T. arenaria_, _A. bisporus_, _L. edodes_, _P. ostreatus_ and _T. melanosporum_. Mature _T. arenaria_ truffles were harvested in Alentejo (south of Portugal) as described in Section 2.1. _A. bisporus_, _L. edodes_, and _P. ostreatus_ were purchased in a local supermarket, and _T. melanosporum_ was purchased at Espora Gourmet, SL. All fresh mushrooms and truffles samples were kept at 4 \({}^{\circ}\)C until analysis in the first 48 h post-harvest. Comparing T. arenaria's Nutritional Value with That of Other Edible Mushrooms and Truffles, and Meat  
To showcase its nutritional value, we collected and analysed _T. arenaria_'s samples for their nutritional and mineral composition. Furthermore, _T. arenaria_'s data was compared with that reported in the literature for other edible mushroom and truffle species (_A. bisporus_, _L. edodes_, _P. ostreatus_ and _T. melanosporum_), and with meat (cow, pig and chicken). The criteria for selecting the other edible mushroom and truffle species and the types of meat were wide consumption and easy to buy and find in the supermarkets. Twelve _T. arenaria_ composite samples, three for each sampling site, were prepared. The samples were dried at 40 \({}^{\circ}\)C for 72 h to determine their moisture content, and the dry material was powdered in a porcelain mortar and kept in brand-new sealed polyethylene bags under dry conditions at room temperature until analysis. Using the AOAC procedures [57], the dry samples were analysed for their (i) crude protein content (applying the conversion factor of N \(\times\) 4.38), which was estimated by the macro-Kjeldahl method, (ii) crude fat, which was determined by extracting a known weight of powdered sample with petroleum ether, using a Soxhlet apparatus, and (iii) ash concentration, which was determined by incineration at 600 \(\pm\) 15 \({}^{\circ}\)C. A bomb calorimeter (Parr 6200 Isoperibol Calorimeter) was used to estimate the energy of the samples.