Spherisorb column

Freeze-dried sauerkraut powder (2 g) was extracted with 20 mL of water/perichloric acid (95:5, v:v). Mixture was sonicated (amplitude 60) using a macro-probe (Vibra-Cell sonicator; Sonic and Materials Inc., Danbury, CT) for 1 min (2 cycles, 30 s/cycle, 5-min interval between cycles) in an ice bath. The suspension was held under stirring conditions at room temperature for 1 h, kept at 4°C overnight, and centrifuged at 10,000 rpm for 10 min. Water-soluble extracts (WSE) were filtered and stored at −20°C until further use. Concentrations of glucose, fructose, and mannitol were determined through a HPLC system Ultimate 3000 (Dionex, Germering, Germany) equipped with a Spherisorb column (Waters, Millford, USA) and a Perkin Elmer 200a refractive index detector (55 ). Lactic, acetic, and citric acids were determined by a HPLC system Ultimate 3000 (Dionex, Germering, Germany) equipped with an Aminex HPX-87H column (ion exclusion, Bio-Rad) and a UV detector operating at 210 nm (56 (link)). Organic acids and sugars standards were purchased from Sigma-Aldrich (Steinheim, Germany).

Organic acids and sugars were determined every four hours throughout the growth kinetics with a Dionex UltiMate 3000 (Thermo Fisher, Waltham, MA, USA) HPLC apparatus. Organic acids were analyzed by using an Aminex HPX-87H column (Biorad, Hercules, CA, USA) according to the method described by Zeppa et al. [19 (link)]. Sugars were determined by using a Spherisorb column (Waters, Millford, MA, USA) as described by Rizzello et al. [20 (link)].
The results were expressed as means of three biological replicates analyzed in triplicate ± standard deviations.

Equal volumes of perchloric acid (5%, vol/vol) were added to fermented and unfermented CP aliquots as precipitating agent. The suspension was kept at 4°C overnight, centrifuged at 10,000 × g, 10 min, and filtered through a Millex-HA 0.22-mm pore size filter (Millipore Co., Bedford, MA). The concentration of glucose, fructose and sucrose was determined through HPLC analysis, using an ÄKTA Purifier system (GE Healthcare) equipped with a Spherisorb column (Waters, Millford, USA) and a Perkin Elmer 200a refractive index detector. Elution was at 32°C with a flow rate of 1 ml/min, using acetonitrile 80% as the mobile phase [25 ]. Organic acids were determined by HPLC, using an ÄKTA Purifier system (GE Healthcare) equipped with an Aminex HPX-87H column (ion exclusion, Biorad) and a UV detector operating at 210 nm. Elution was at 60°C with a flow rate of 0.6 ml/min, using 10 mM H₂SO₄ as the mobile phase [26 (link)]. Peaks were identified by comparing elution times and spiking samples with known quantities of standard solutions of acetic and lactic acid. Total and individual free amino acids were analyzed by a Biochrom 30 series Amino Acid Analyzer (Biochrom Ltd., Cambridge Science Park, England), with a Na-cation-exchange column (20 by 0.46 cm inner diameter) as described by Rizzello et al. [27 ].

Total titratable acidity (TTA) was determined on 10 g of samples homogenized with 90 mL of distilled water using a Stomacher apparatus (Seward, London, UK), and expressed as the amount (mL) of 0.1 M NaOH to reach a pH of 8.3. The value of pH was measured by a Foodtrode electrode (Hamilton, Bonaduz, Switzerland). Chemical analyses concerning total proteins, fat content, and dry matter were determined by MilkoScan™ FT6000 (Foss Electric A/S, Hillerod, Denmark), based on Fourier-transformed infrared technology [26 (link)].
Carbohydrates were determined in water-soluble extract (W-SE) by HPLC analysis. Two grams of freeze-dried fruit powder were extracted with 20 mL of water/perchloric acid (95:5, v:v). In an ice bath, the mixture was sonicated (amplitude 60) with a macro-probe (Vibra-Cell sonicator; Sonic and Materials Inc., Danbury, CT, USA) for 1 min (two cycles, 30 s/cycle, 5 min interval between cycles). The suspension was stirred at room temperature for 1 h, kept at 4 °C overnight, and centrifuged for 10 min at 10,000 rpm. W-SE was filtered and stored at −20 °C until further use. A Spherisorb column (Waters, Milford, CT, USA) and a Perkin Elmer 200a refractive index detector were used to determine the concentrations of glucose, fructose, mannitol, and sucrose [25 (link)]. Carbohydrates standards were purchased from Sigma-Aldrich (Steinheim, Germany).

Soluble reducing sugars determination followed the method described in Reference [26 (link)]. Defatted sample (0.5 g) was weighted in centrifuge tube. Soluble reducing sugars were extracted with 10 mL 70% ethanol under ultrasonic condition for 20 min. The supernatant was collected after 2000 r/min centrifugation for 10 min. The ethanol extraction and centrifuge procedure were repeated with the residue. Two parts of supernatant were filtered and vacuum rotary evaporated under 50 °C. The volume was made constant at 1 mL with 70% ethanol for analysis. The detection was performed on HPLC (Agilent 1260 Infinity, Agilent Technologies, Santa Clara, CA, USA) with diode array detector (G4212B). Spherisorb column (4.6 mm × 250 mm, 5 μm, Waters, Milford, MA, USA) was used. The mobile phase was 70% acetonitrile at a flow rate of 1 mL/min. The results were expressed as gram sugar per kilogram samples.

Tocopherol compounds in oil samples were determined using a high‐performance liquid chromatography (HPLC) system (Waters ACQUITY UPLC^® System) with a Spherisorb column (25 cm × 4 mm i.d., WATERS) packed with silica (5 μm particle size), and a fluorescence detector operating at an excitation wavelength of 290 nm and an emission wavelength of 330 nm was utilized. The used mobile phase consisted of acetonitrile and water (90:10, v/v) at a flow rate of 0.5 ml/min. Tocopherols in the test samples were verified by the comparison of the retention time with the available reference standards (Tavakoli, Hashemi, et al. (2018 (link))).

The pigments profile was determined according to Wright et al. [56 (link)] using a Waters Alliance 2695 HPLC and Waters 2996 photodiode array detector (PAD) coupled to a Waters Spherisorb column (5 µm, 4.6 × 250 mm). Briefly, samples were extracted with methanol, filtered through 0.2-µm syringe filters, and injected in the HPLC. The standards of alloxanthin, diatoxanthin, lutein, neoxanthin, violaxanthin, and zeaxanthin were obtained from DHI Lab Products (Hørsholm, Denmark), while β-carotene was supplied by Sigma-Aldrich.