Vibramax 100

Sample matrices were prepared from store-bought raspberries, blueberries, and strawberries as follows. About 30 g of berries was weighed into plastic boxes to which 200 ml of 0.1 or 1% Alconox^TM (Alconox, Inc., NY, United States) was added. The boxes were then placed on an automatic shaker (Heidolph Vibramax 100); raspberry samples were shaken at 300 rpm for 10 min, whereas blueberry and strawberry samples were shaken at 600 rpm for 10 min.
The eluate was then transferred into four 50 ml tubes for concentration by centrifugation at 1,690 rcf for 10 min and the supernatant removed by vacuum suction, leaving 10 ml of the sediment. The pooled sediment was centrifuged at 3,803 rcf for 10 min with a deceleration break set to 6 (on a scale of 0–9) and about 1.5 ml of the sediment was further concentrated down to 250 μl by centrifugation at 13,000 rcf for 5 min.

After treatment with solutions, the thalli were gently rinsed in deionized water to remove unbounded ions (Yemets et al. 2015 (link)). Ca 100 mg of air-dried lichen material was weighed and intended for analysis. The initial electrical conductivity in μS cm⁻¹ (Ci) of the distilled water was measured using a pH/conductivity meter (Seven Go Duo SG23-FK5, Mettler Toledo). The samples were placed in 50 ml of distilled water in glass weight bottles, sealed with glass stoppers, and shaken on a vibrating shaker for 1 h (Vibramax 100, Heidolph). The conductivity of the samples was measured after soaking the thalli (Cv). The thalli were then boiled for 10 min at 100 °C to disrupt the cell membranes. The conductivity after the samples had cooled down to 20 °C was measured again (Cf). Relative electrical conductivity (EC), indicating the loss of cell membrane integrity, was calculated according to the formula: ((Cv–Ci)/Cf) × 100 (%). Five replicates for each experimental group were included. See Osyczka and Rola (2019 (link)) for details on the measurement procedure.

Fifty milligrams of ground cucumber samples were extracted with 1.5 mL of 0.4 M HCl for 3.5 h on the platform shaker at 600 rotations per minute (Heidolph Vibramax 100, Schwabach, Germany). Then, the extracts were centrifuged at 13,000× g for 30 min and filtered using syringe filters (nylon membrane, pore size 0.45 μm).
The phytic acid content in the extracts was determined by the method of Haug and Lantzsh [61 (link)] with minor modifications. Up to 0.5 mL of extract and 1 mL of ferric (III) solution (ammonium iron (III) sulphate · 12 H₂O in 2 M HCl) were added. The mixture was heated (100 °C) for 30 min in a dry heating block (myBlock Mini Dry Bath; Benchmark, Sayreville, NJ, USA). After cooling at 4 °C for 30 min to precipitate the iron–phytic acid complex, the samples were centrifuged for 30 min at 13,000 rpm, and the supernatant was collected. Then, 750 µL of 2,2′-bipyridine solution (1% 2,2′-bipyridine and 1% thioglycolic acid in water) was added to 500 µL of the supernatant. The absorbance was measured at 519 nm against distilled water. The phytic acid concentration was calculated based on the calibration curve (y = −0.0036x + 0.9983, R² = 0.9986) using sodium phytate as a reference. The amount of phytate in the tested sample was expressed as mg of phytate per gram of dry mass (mg g⁻¹ DW).