# Does Organic Farming Increase Raspberry Quality, Aroma and Beneficial Bacterial Biodiversity?
## 2 Materials and Methods
### Fruit Volatile Analysis  
Raspberry fruit volatiles were analysed by gas chromatography-mass spectrometry (GC-MS). Each replicate (n = 3) consisted of a pool of three berries. Berries were put in a glass vial, closed with a lid equipped with a pierceable silicon cap and stored at \(-\)80 \({}^{\circ}\)C. Samples were taken out from \(-\)80 \({}^{\circ}\)C and put at \(-\)20 \({}^{\circ}\)C for 20 min before equilibration. After adding 1-octanol 0.05% (\(v/v\) in H\({}_{2}\)O) as internal standard, equilibration was performed for 20 min at 40 \({}^{\circ}\)C. Samples were then exposed to a 50/30-\(\upmu\)m divinylbenzene/carboxen/polydimethyl siloxane (DVB Carboxen PDMS) Stable Flex 2 cm solid phase microextraction (SPME) fibre (Supelco, Bellefonte, PA, USA) for 40-min exposure. Afterwards, the SPME fibre was desorbed in a Shimadzu GC-MS-QP2010 Plus (Shimadzu, Tokyo, Japan) at 250 \({}^{\circ}\)C for 10 min in the split mode. The chromatographic separation of volatile compounds was performed on an RTX-WAX fused-silica capillary column (30 m \(\times\) 0.25 mm i.d. \(\times\) 0.25 \(\upmu\)m) coated with polyethylene glycol (PEG) (Restek, Bellefonte, PA, USA). A three-step heating oven program was set: (1) 45 \({}^{\circ}\)C, 10 min; (2) 4 \({}^{\circ}\)C min\({}^{-1}\) temperature increase to 200 \({}^{\circ}\)C; (3) 200 \({}^{\circ}\)C, 8 min. Helium was used as the carrier gas at a constant flow rate of 1 mL/min. A mass range from 33 to 400 \(m/z\) was scanned at a rate of 769 amu s\({}^{-1}\). Mass spectra and linear retention indices (calculated according to the retention times of linear C8-C20 standard alkanes) were used for the identification of volatile compounds, based on the NIST/EPA/NIH Mass Spectral Database (NIST 08, National Institute of Standards and Technology, Gaithersburg, MD, USA) and the ChemSpider information resource ([[http://www.chemspider.com](http://www.chemspider.com)]([http://www.chemspider.com](http://www.chemspider.com)), accessed on October 2020). Bacterial Volatile Analysis by Proton-Transfer Reaction\(-\)Mass Spectrometry (PTR-MS) and in Silico Fruit Validime Assembly  
10 mL commercial raspberry juice (Bocon, Italy) diluted with distilled water to 75%, adjusted to pH 7, were inoculated with 100 \(\mu\)L of freshly grown bacterial culture and incubated for 24 h. The experimental set foresaw three replicates for each bacterial isolate. Headspace volatiles were measured as described in [49] by using a proton-transfer reaction time of flight-mass spectrometer (PTR-ToF-MS) 8000 (Ionicon Analytik GmbH, Innsbruck, Austria). The drift tube conditions were as follows: 110 \({}^{\circ}\)C drift tube temperature, 2.8 mbar drift pressure, 428 V drift voltage, ion funnel (18 V). This leads to an E/N ratio of about 130 Townsend (Td), with E corresponding to the electric field strength and N to the gas number density (1 Td = 10\(-\)17 Vcm\({}^{-2}\)). The sampling time per channel of ToF acquisition was 0.1 ns, amounting to 350,000 channels for a mass spectrum ranging up to \(m/z=400\). The sample headspace was withdrawn through PTR-MS inlet with 40 sccm flow for 60 cycles resulting in an analysis time of 60 s/sample. Pure nitrogen was flushed continuously through the vial to prevent pressure drop. Each measurement was conducted automatically after 25 min of sample incubation at 40 \({}^{\circ}\)C and 5 min between each measurement was applied in order to prevent memory effect. All steps of measurements were automated by an adapted GC autosampler (MPS Multipurpose Sampler, GERSTEL) coupled to PTR-ToF-MS. The analysis of PTR-ToF-MS spectra proceeded as described in [49]. The array of masses detected with PTR-ToF-MS was reduced by applying noise and correlation coefficient thresholds. The first removed peaks that were not significantly different from blank samples; the latter excluded peaks with over 99% correlation, which mostly correspond to isotopes of monoisotopic masses. PTR-ToF-MS outputs of the isolates were employed to build the in silico fruit volatileome.