Chromafil ao 20 25

The phenolic compounds were extracted in an ice-cold ultrasonic bath for 60 min with 70% EtOH using an equivalent of 30 mg/mL dry weight. The homogenates were centrifuged for 8 min at 9500 rpm. The supernatant was filtered through a Chromafil AO-20/25 polyamide ester filter produced by Macherey-Nagel (Düren, Germany).

The samples were prepared according to Mikulic-Petkovsek et al.^{22 (link)}. The extract for sugars and acids was prepared from 5 g of fruit pericarp without the peel of A. chinensis and 5 g of whole fruit of A. arguta, as the fruits of A. arguta are eaten without peeling and the fruits of A. chinensis must be peeled before consumption. Plant material was cut into small pieces and 25 ml of double distilled water was added to the test tubes of each sample. Samples were placed on a shaker for 30 min on room temperature. After they were put in a centrifuge (Eppendorf Centrifuge 5810 R) for 5 min at 5000 rpm at 4 °C. Samples were filtered through a cellulose filter Chromafil A-20/25 produced by Macherey–Nagel (Düren, Germany), transferred to a vial and stored at − 20 °C until analysed by high-performance liquid chromatography (HPLC).
Extract for phenols was prepared according to Mikulic-Petkovsek et al.^{23 (link)} in test tubes from 5 g of fruit pericarp without peel of A. chinensis and 5 g of whole fruit of A. arguta, and 10 ml of methanol with 3% formic acid was added. Test tubes were places in a cooled ultrasonic bath for 60 min. After the sample was centrifuged for 8 min at 8000 rpm, supernatant was filtered through a polyamide filter Chromafil AO-20/25 produced by Macherey–Nagel (Düren, Germany), transferred to a vial and stored at − 20 °C until the start of the analysis.

The phenolic compounds and dicarboxylic acid derivatives were extracted according to the protocol described by Medic et al. [6 (link)]. Twenty chestnuts per cultivar were used, with five chestnuts per replicate. The chestnuts were weighed; then, the seed coat was removed, and the pellicles were peeled. The peeled raw chestnuts were then ground using a mechanical mill (A10 basic; IKA Works GmbH & Co. KG, Staufen, Germany). Briefly, 1 g of the sample was then extracted with 80% methanol in bi-distilled water. The extraction ratio was 1:2.5 (w/v). The samples were then vortexed (TOP-MIX 94500 vortex mixer; Heidolph, Schwabach, Germany), sonicated in iced water for 60 min (Sonis 4 ultrasonic bath; Iskra pio, Sentjernej, Slovenia), and centrifuged at 10,000× g for 10 min at 4 °C. Samples were then filtered with a 0.2 µm polyamide filter (Chromafil AO −20/25; Macherey-Nagel, Düren, Germany), transferred to vials, and stored at −20 °C for further analysis.

For the analysis of the sugars, organic acids, and phenolic compounds, the method described in previous protocols was used [16 (link),17 (link),18 (link)]. All the treatments, i.e., the various juices, were filtered using 0.2 µm cellulose filters (Chromafil Xtra MV-20/25; Macherey-Nagel, Düren, Germany) into vials for the subsequent analysis of the sugars and organic acids. For the analysis of the phenolic compounds, 0.2 µm polyamide filters (Chromafil AO-20/25; Macherey-Nagel, Düren, Germany) were used for filtration. The vials containing the filtered samples were stored at −20 °C until further analysis of the organic acids, sugars, and phenolic compounds. The ascorbic acid was extracted in a 1:1 ratio, whereby 1 mL of apple juice was mixed with 1 mL of 2% metaphosphoric acid.