*2.3. Volatile Analysis*

Volatiles were analyzed from the vial headspace using solid-phase micro-extraction, and gas chromatography–mass spectrometry (HS-SPME-GC-MS), as described by Wang et al. [25] with modifications. The volatile substances were extracted using a 2 cm SPME fiber (50/30 μm DVB/Carboxen/PDMS; Supelco, Bellefonte, PA, USA). The homogenate mixture was incubated for 30 min at 40 ◦C and another 30 min was needed for exposure to the fiber.

The volatile compounds were desorbed in a GC-MS (Model 7890 GC coupled with a 5975 MS detector; Agilent, Santa Clara, CA, USA) by using the HP-5 column (50 m × 0.32 mm × 1.05 μm, J&W Scientific, Agilent, Santa Clara, CA, USA). The GC oven temperature was programmed to increase from 40 to 230 ◦C at a rate of 4 ◦C min−<sup>1</sup> and the holding time was 11.70 min. Helium (37 kPa) was used as the carrier gas at a constant flow of 1.5 mL min−1. For the MS system, the temperatures of the inlet, ionizing source, and transfer line were 250, 230, and 280 ◦C, respectively; electron impact mass units were recorded at 70 eV ionization voltages.

Volatile compounds were tentatively identified by matching their mass spectra to entries in the NIST 14 library (National Institute of Standards and Technology, Gaithersburg, MA, USA) and the retention indexes were compared with the standard volatile compounds. A calibration curve (peak area vs. concentration of reference standards) was prepared from the serial dilutions of the standard and used for sample quantification. Standard aroma compounds (all volatiles identified in this study) were purchased from Sigma-Aldrich (St. Louis, MO, USA) and Fluka Chemical Corporation (Buchs, Switzerland).
