Solid phase microextraction fiber

To detect whether there was an interaction between plant and mVOCs while co-culturing, part of the co-culture systems (yeast-plant co-culture tubes) that incubated at 20 °C were involved in this experiment. The VOCs present in the co-culture system of plant and yeasts were collected and tested by microextraction and GC-MS, respectively. First, a small hole was drilled in the lid of the co-culture tube (without touch of the suspended Eppendorf tube and A. thaliana) and a pre-cleaned solid-phase microextraction fiber (75 µm, 1 cm, carboxen/polydimethylsiloxane; Supelco, Bellafonte, PA, USA) was inserted into it. The tube was placed vertically at room temperature (about 22°C) for 1.5 days, and then the fibers were taken to the GC-MS for analyte desorption, separation and detection, immediately. The adsorption conditions and operation program of GC-MS were performed the same as described in 2.2. All treatments were performed with three biological replicates and blank control (without yeast inoculation) were also involved in this experiment (n=3). Then, the information of detected VOCs (including the kind, retention time, peak area) would be acquired. Excluding the VOCs detected in blank control or yeast culture, the finial VOCs would be considered as new VOCs for they only be produced while and yeast interacting.

Volatile flavor compounds were extracted according to Vasta et al. [17 (link)]. Briefly, a muscle sample (5 g) was placed in a 15 mL vial, and a solid-phase microextraction fiber (Supelco, Bellefonte, USA) was exposed over the sample and extracted at 60 °C for 40 min. Then, it was desorbed for 3 min. The gas chromatograph (TRACE 1300, Thermo Fisher Scientific, Waltham, USA) settings were as follows: the oven temperature was held at 40 °C for 5 min, increased to 200 °C (5 °C/min), and then ramped at a rate of 20 °C/min to 250 °C. The mass spectra were acquired at 70 eV with a scan range of 30 to 400 m/z. The flavor substances were identified using the NIST MS Search 2.0 database. The relative contents of volatiles were analyzed using the area normalization method.

Fresh samples (0.3 g) were ground in 2 mL potassium phosphate buffer (50 m mol/L Tris and 20 m mol/L NaCl, pH = 8.0) on ice, and then extracted using solid-phase microextraction fiber (Supelco, Bellefonte, PA, USA) coated with an absorbent phase made of polydimethysiloxane/carboxen/divinylbenzene under headspace mode at 40 °C for 50 min with stirring. Vanillin was used as a reference standard. After extraction, the extraction device was inserted into the injection port and maintained for 5 min at 250 °C. Analysis was performed using a Shimadzu QP2010 GC-MS equipped with a vocol column (60 m × 0.32 mm × 0.18 μm, Supelco, Bellefonte, PA, USA). Helium was used as the carrier gas at a constant flow rate of 0.81 mL/min. The program was set as 35 °C for 3 min, then raised to 40 °C at 3 °C/min and held for 1 min, before finally being raised to 210 °C at 5 °C/min and held for 25 min. The electron ionization system at an ionization energy of 70 eV was used, and the mass spectra scan ranged from m/z 45 to 1000. Identification was conducted based on comparison of molecular weights and mass spectra fragmentation patterns with those recorded in the Nist 147 and Wiley 7 Spectrometry Library data (GC/MS), utilizing previous analyses of pure references that are commercially available.

We weighed 5 g of essential oil and microcapsules containing the same amount of essential oil into a headspace extraction bottle, placed it in a thermo-static oscillator with a 150 g oscillation frequency, a used solid-phase microextraction fiber (Supelco, Bellefonte, PA, USA) to perform extraction at 60 °C for 40 min, collected the volatile compounds in the LCEO, and analyzed them with GC-MS (QP2010 Shimadzu, Kyoto, Japan) and a DB-5ms capillary column (Agilent Technologies Inc., Palo Alto, CA, USA).The carrier gas was nitrogen, and the flow rate was 1 mL/min. The temperature increase program moved from 40 °C to 180 °C at 5 °C/min, the temperature was maintained at 10 °C/min to 230 °C for 5 min, and the temperature at the injection port was 230 °C. The compounds were identified in the preliminary stage by comparing the mass spectra to real samples from the NIST MS collection [18 (link)]. Normalized quantification was carried out with the peak area of the chromatographic peak of the total ion flow of GC-MS, and the identified components accounted for 99.42% of the total area of the chromatographic peak.