## 2 Materials and Methods
### Analysis of the Fruit Volatiles
#### 2.3.3 Analysis of the Apple Volatiles by HS-SPME-GC-MS  
For the analysis of primary aroma compounds, aliquots of the homogenized samples (250 mg puree) were transferred into 20 mL headspace vials. 2-Octanol (purity \(>\) 99%, Sigma Aldrich, Darmstadt, Germany) was used as internal standard (30 ng absolute in 10 \(\mu\)L methanol corresponding to 120 \(\mu\)g/kg puree, purity \(>\) 99%, Fisher Scientific, Schwerte, Germany). For the analysis of secondary aroma compounds, 100 \(\mu\)L of apple fruit juice was used with the addition of 100 mg NaCl and 2-octanol (30 ng absolute in 10 \(\mu\)L methanol corresponding to 300 \(\mu\)g/L juice, purity \(>\) 99%, Fisher Scientific, Schwerte, Germany) in 20 mL headspace vials [33]. All samples were analyzed in duplicate in randomized order. The extraction/enrichment of the volatiles was performed by headspace solid-phase micro extraction (HS-SPME) using a CTC Combi PAL sampler (CTC Analytics, Zwingen, Switzerland). A 50/30 \(\mu\)m DVB/Car/PDMS 2 cm stable flex SPME fibre (Supelco, Bellefonte, PA, USA) was used for the enrichment of the volatiles. Prior to the extraction of the volatiles, the samples were equilibrated in the oven of the autosampler at 40 \({}^{\circ}\)C for 5 min and the fibre was exposed in the headspace to the equilibrated sample at 40 \({}^{\circ}\)C for 20 min. Samples were stirred thoroughly during the equilibration and enrichment process in the oven of the CTC Combi PAL sampler adding a glass-coated stirrer into the headspace vial. Immediately after exposure, the fibre was transferred to the injector of the GC system for thermo-desorption. The SPME fibre was left in the injection port for re-conditioning (20 min) before it was exposed into the headspace of the next sample. Analysis of the volatiles was performed with 1-dimensional gas chromatography-mass spectrometry (GC-MS) (Shimadzu GCMS-QP 2010 Plus, Shimadzu Europa GmbH) on a semi-polar column (Rxi(r) 5 ms, 30 m \(\times\) 0.25 mm \(\times\) 1 \(\mu\)m, Restek Corporation, Bellefonte, PA, USA). The following conditions were used: temperature program starting at \(-10\)\({}^{\circ}\)C for 1 min with a temperature ramp of \(8\)\({}^{\circ}\)C/min up to 270 \({}^{\circ}\)C (1 min holding time), splitless injection with a sampling time of 1 min and an injection temperature of 270 \({}^{\circ}\)C was applied. SPME liners with a constant inner diameter of 0.75 mm were used in the GC injection system and cryo-focusing was performed by blowing liquid nitrogen into the GC oven to reach the start temperature of \(-10\)\({}^{\circ}\)C. Helium was used as carrier gas with a linear velocity of 35.0 cm/s. Mass-selective detection was performed in the scan mode (35-350 \(m/z\), EI (70 eV), interface temperature 280 \({}^{\circ}\)C, ion source temperature 200 \({}^{\circ}\)C). The obtained chromatograms were analyzed with the free deconvolution software PARADISe [36], which is based on the PARAFAC2 model [37]. The identification of the compounds was performed on probability-based matching of the mass spectra to those from MS libraries (NIST14, Adams Essential Oils library, FFNSC 4.0) or authentic reference compounds as well as on linear-temperature programmed retention indices (RI) [38]. RI was calculated using the homologous series of n-alkanes (C5-C26) and was compared to RI from authentic reference compounds or data from the literature. The relative concentrations (semi-quantification) were calculated by comparison of the respective peak areas obtained from the total ion chromatograms deconvolution to the internal standard 2-octanol assuming a response factor of 1 for all compounds as described by [PERSON] (2015) [39]. Relative concentrations of primary aroma compounds were calculated from the sample quantity taking the relative fruit content of the puree into account and are given in mg/kg. Relative concentrations of secondary aroma compounds were calculated directly from the juice and are given in mg/L juice. To follow changes within compound class (alcohols, esters, etc.