# How Different Fermentation Type Affects Volatile Composition of Plum Jerkums
## 2 Materials and Methods
### Chemical Composition of Fresh and Fermented Plum Mashes  
The concentration of ethanol, reducing sugars, sugar-free extract, total extract, and sucrose concentrations and tittratable acidity were determined using officially approved methods described by the International Organization of Vine and Wine (OIV) [11]. Tittrable acidity (TA) was calculated from the results obtained with Mettler DL 25 titrator (Mettler Toledo, Greifensee, Switzerland). We used 0.1 M NaOH as a titrant. Samples were titrated to pH 7.0. Results were shown as grams of malic acid per liter. For the calculation of fermentation efficiency, we applied theoretical correlations which were as follows--1 g of reducing sugars or sucrose could be transformed to 0.511 g or 0.538 g of ethanol, respectively. Ninhydrin method was used for the determination of free amino nitrogen (FAN). Then, 2 mL of diluted (\(\times\)50) plum musts or jerkums (1 mL) were transferred to caped glass tubes. Then, we added 1 mL of minhydrin reagent and heated tubes in the boiling water bath for 16 min. After cooling samples, we added 5 mL of dilution reagent. Finally, the absorbance was measured at 575 nm against distilled water with minhydrin as a blank [12]. Determination of Sugar and Main Organic Acids Content by High-Performance Liquid Chromatography (HPLC)  
Centrifuged (MPW-6 SR, MPW Med. Instruments, Warszawa, Poland--14,000\(\times\)\(g/5\) min) musts or jerkums were diluted (\(\times\)5) with deionized water. Then we used syringe filters (0.45 \(\upmu\)m pore density, Sartorius AG, Getinge, Germany) to filtrate obtained dilutions. We analyzed organic acids on a Shimadzu NEXERA XR chromatograph (Kyoto, Japan). That piece of equipment has a pump system, and a UV/Vis detector (monitored at 210 nm). Malic, succininic, acetic, tartaric, lactic, and citric acids (Sigma-Aldrich, St. Louis, MO, USA) were analyzed using Rezex ROA-Organic Acid Aminex HPX-87H column (300 \(\times\) 7.8 mm) (Rezex, Torrance, CA, USA). Isocrating elutions of samples was carried out at 40 \({}^{\circ}\)C with 0.005 M H\({}_{2}\)SO\({}_{4}\)) at a flow rate of 0.4 mL/min. We analyzed sugar profile with the same piece of equipment, however, we replaced the UV/Vis detector with a refracometer detector RF-20A. Sugars were separated on an Asahipak NH2P-50, 4.6 \(\times\) 250 mm Shodex column (Showa Denko America, Munich, Germany) at 30 \({}^{\circ}\)C. The isocratic elution program (0.8 mL/min) lasted 16 min. We used an aqueous solution of acetonitrile (70%) for elution. We prepared standard curves for each tested compound: fructose, glucose, sucrose, and glycerol. Volatile Composition of Fresh and Fermented Plum Mashes Lsing Solid Phase Microextraction-Gas Chromatography-Mass Spectrometry (SPME-GC-MS) and Gas Chromatography-Flame Ionization Detector (GC-FID) Methods  
Analysis of volatile compounds was performed using gas chromatography with mass spectrometry [1; 6]. We suspended 0.05 mg of 4-methyl-2-pentanol/L and 0.5 \(\upmu\)g of ethyl nonanoate, (Sigma-Aldrich, St. Louis, MO, USA) in 1 mL saturated saline and that solution was used as an internal standard. We transferred 1 mL of juice or jerkums to a 10 mL vial. We conditioned the SPME device (Supelco Inc., Bellefonte, PA, USA, 100 \(\upmu\)m polydimethylsiloxane fiber) in the GC injector port at 250 \({}^{\circ}\)C for 1 h. After inserting the fiber in the glass vials, samples were stirred (300 rpm/40 \({}^{\circ}\)C/30 min) and then fibers were transferred to the injector port Agilent Technologies 7890B chromatograph system (Agilent Technologies, Santa Clara, CA, USA). The system is equipped with an LECO Pegasus HT, High Throughput time-of-flight mass spectrometry (TOFMS) detector. Fibers were held for 3 min in the inlet. Transfers of the fiber were automated with GERSTEL MultiPurpose Sampler (MPS). Volatile compounds were separated on the Rtx-1 ms capillary column (Crossbond 100% dimethyl polysiloxane, 30 m \(\times\) 0.53 mm \(\times\) 0.5 \(\upmu\)m).