# Insights into Native Fermentation Process of Apples (_Malus domestica_) in Low Sodium Conditions
## 2 Materials and Methods
### Physical and Chemical Analysis
#### 2.3.6 Concentration of Cations  
The cations from the samples (apple peels and brine) were determined by analyzing the potassium (K), sodium (Na), and magnesium (Mg), respectively, through the analytical method of high-resolution continuous source flame atomic absorption spectrometry (HR-CS-FAAS) and by using the ContrAAA 700 instrument (AnalytikJena, Jena, Germany). The continuum source and the high-resolution monochromator provide the spectrum for the analysis of multiple elements. The instrument was calibrated before the working session using the ICP multi-element standard solution IV 23 elements (Merck, Darmstadt, Germany, Certipur(r)). The cations were extracted from the calculated peel samples in an acidic solution. The extraction was performed according to the method described by [PERSON] ([PERSON]) et al. [17]. Approximately 0.5 g of the calcinated peel was weighed and placed into polytetrafluoroethylene containers in order to disaggregate the cations in an acidic aqueous solution by microwave-assisted digestion. Digestion was achieved using an extraction matrix consisting of nitric acid (HNO\({}_{3}\) 65% Suprapur) and hydrogen peroxide (H\({}_{2}\)O\({}_{2}\) 30% Emsure). The treated samples were placed into the TopWave (AnalytikJena, Germany) microwave digestion system, using a program of 200 \({}^{\circ}\)C temperature for 45 min. After digestion, the samples were diluted with ultrapure water (Evoqua (Pittsburgh, PA, USA), Siemens (Seoul, Republic of Korea)) (volume = 50 mL) in previously rinsed Falcon tubes. The brine samples were filtered in Falcon tubes, rinsed beforehand, and mineralized with HNO\({}_{3}\) 65% Suprapur. The results are expressed as mg/g dry weight in the case of the peel samples and mg/L in the case of the brine samples. 2.3.7 Phytochemical Analysis of Fresh and Fermented Apple Total Phenolic Content (TPC) Determination  
The Folin-Ciocalteu method was used to estimate the total phenolic content using gallic acid as standard as presented by [PERSON] et al. [18]. Up to 0.1 mL of the extract (1 mg/mL), 1 mL Folin-Ciocalteu reagent (diluted 1:10) was added, and the mixture was left out for 5 min, and then 1 mL (75 g/L) of sodium carbonate, Na\({}_{2}\)CO\({}_{3}\), was added. The resulting solutions were incubated at room temperature for 90 min. The absorbance of the samples was read at 765 nm. The total phenolic content was estimated from the gallic acid calibration curve of 0, 25, 50, 75, 100, 125, and 150 \(\mu\)g/mL concentrations, prepared in methanol 95%; the results were expressed as mg gallic acid equivalent per gram of extract (mg GAE/g). Total Flavonoid Content (TFC)  
The TFC was estimated by means of the method of [PERSON] et al. [11]. The extract (1 mL, 1 mg/mL) was diluted with 1.25 mL distilled water, and 75 \(\mu\)L 5% NaNO\({}_{2}\) was added to the mixture. After 6 min, 150 \(\mu\)L 10% AlCl\({}_{3}\) was added; afterwards, another 5 min, 1 mL 1 M NaOH was added to the mixture. The absorbance was assessed at 510 nm versus water blank. A standard curve of catechin was prepared using 5-1000 \(\mu\)g/mL of catechin, and the results were expressed as milligrams of catechin equivalents per gram of sample (mg CE/g). Determination of Total Carotenoids and Chlorophyll  
In both cases of determining the total carotenoids and chlorophyll, 1 g of finely ground sample was mixed with organic solvents mixture, 10 mL of hexane-acetone 3:1 in a Falcon tube of 50 mL, as [PERSON] et al. [19] described. Then, it was placed in the ultrasonication water bath (ARGO LAB Digital Ultrasonic Cleaner, ARGO LAB, Carpi, Italy) at 30-40 \({}^{\circ}\)C for 30 min to accelerate the reaction, because heat will increase the kinetic energy of the molecules, thus improving the extraction of carotenoids and chlorophylls. After the ultrasonication treatment, the samples were centrifuged with Hettich 320 R at 9000 rpm, 4 \({}^{\circ}\)C, 5-10 min. The filtrate was used to read the absorbances of the samples.