# Traditional Grain-Based vs. Commercial Milk Refirs, How Different Are They?
## 2 Materials and Methods
### Assessment of VOCs by Headspace-Gas Chromatography-Ion Mobility Spectrometry (HS-GC-IMS)  
For analysis of VOCs, a prototypic ion mobility spectrometer (IMS, Gesellschaft fur Analytische Sensorsysteme mbH (G.A.S. mbH, Dortmund, Germany), was coupled to an Agilent 6890 N gas chromatograph (GC, Agilent Technologies, Palo Alto, CA, USA). The system was equipped with a CombiPal GC autosampler (CTC Analytics, Zwingen, Switzerland) with a headspace (HS) sampling unit and a 2.5 mL gas-tight heatable syringe [17].  
For the isolation of VOCs, 0.5 g of 47 \(\pm\) 1 h fermented traditional kefirs (Section 2.1), commercial kefirs, yogurts, and milk, which served as reference in this experiment, were transferred into a 20 mL HS vial, mixed with 0.5 mL saturated NaCl solution, and sealed with a 2 mm silicon cap. Subsequently, the samples were incubated for 20 min at 75 \({}^{\circ}\)C and 750 rpm. Following incubation, 1 mL of HS was acquired at a speed of 350 uL s\({}^{-1}\) using a syringe temperature of 80 \({}^{\circ}\)C to avoid condensation effects. Before each analysis, the syringe was automatically flushed with a stream of nitrogen for 5 min to avoid cross-contamination. Injection was performed into a split/splitless injector, operated at 150 \({}^{\circ}\)C in split mode (split 1:10). Chromatographic separation was performed on an HP-5 capillary column with a 30 m \(\times\) 0.32 mm \(\times\) 0.25 um 5% phenyl-methylpolysiloxane film (Agilent Technologies, Santa Clara, CA, USA, SN: USB345942H). Nitrogen of 99.99% purity was used as carrier gas at an initial column flow of 1.5 mL min\({}^{-1}\). The pressure was set constant to 6.7 psi. The GC-oven temperature was programmed as follows: the GC oven was preheated to 40 \({}^{\circ}\)C and ramped up to 140 \({}^{\circ}\)C at 10 \({}^{\circ}\)C min\({}^{-1}\), which corresponds to 10 min of GC runtime [17; 18].  
Following separation, the analytes were ionized in the IMS ionization chamber by a \({}^{3}\)H ionization source (300 MBq activity). The drift-tube length was 5.3 cm, operated at a constant voltage of 2.5 kV, with a nitrogen flow of 150 mL-min\({}^{-1}\). The gas flow was controlled by a mass-flow controller (Voegtlin Instruments, Aesch, Switzerland). The IMS cell was operated in positive-ion mode at a temperature of 120 \({}^{\circ}\)C. Each spectrum was the average of six scans obtained using injection pulse widths of 100 us, sampling frequencies of 228 kHz, and repetition rates of 21 ms. The data were collected using LAV software version 2.2.1 from G.A.S. mbH. The determination of the VOCs was performed in duplicate.  
Key compounds were identified through a literature search [19; 20] and subsequent confirmation through measurement of reference substances. Stock solutions of reference substances were prepared in LC-MS grade water (HiPerSolv Chromanorm, VWR International, Germany). For semi-quantitative evaluation of each substance detected via HS-GC-IMS measurements, the signals above the plateau of the local minimum were integrated using the 'volume above the minimal area' command from VOCal software version 0.1.0 from G.A.S. mbH, Germany. The peak volume was subsequently normalized to the preselected GC-IMS peak area. Measurements were conducted using consistent sample concentration and preparation, therefore the peak volume measurements in GC-IMS for a particular substance are comparable across dairy samples (further referred to as abundance). The absolute substance concentrations, however, were not quantified. For visualization of similarities between the peak volume of kefir and yogurt samples, a hierarchically clustered heatmap was generated. Python version 3.9.6 using the 'clustermap' command from the 'Seaborn' library was used. The distance metric applied was 'euclidean' and the linkage method which was used for calculating the clusters was 'ward'.