3.2. Fruit Processing and Ellagitannin Extraction

Ellagitannins were extracted from raspberry press cake, following juice pressing from fruit pulp. Prior to processing, 30 kg of fruits was defrosted and ground using a fruit grinder (Model 886.9, Zelmer, Rzeszów, Poland). The pulp was heated to 50 °C and treated with the enzymatic preparation Rohapect 10 L (AB Enzymes, Darmstadt, Germany) at a dose of 0.2 mL per 1 kg of pulp. Enzyme treatment lasted for 1 h at 50 °C, with the pulp stirred every 10 min. Subsequently, the pulp was pressed using a laboratory basket press (self-manufactured, Lodz University of Technology, Lodz, Poland) to separate juice from the press cake. The latter was extracted with water (1/2 of press cake weight) for 15 min at 50 °C to remove water-soluble anthocyanins. Next, the press cake was pressed again to obtain secondary juice and press cake (3.4 kg).

The press cake was extracted in two steps using 60% acetone at ambient temperature. The mass ratio of the press cake to the extractant was 1:5 and extraction time was 8 h per one step. The extraction process was augmented with shaking using an orbital shaker (Elmi DOS-10 L, Aizkraukles, Riga, Latvia) at 150 rpm. After the first step, raw extract was filtered through cotton filter cloth in order to separate solids, while in the second step, the post-extraction residue was extracted according to the same procedure. The extracts obtained from the two steps were mixed and filtered through a 3.6 mm thick Hobrafilt S40N cellulose filter with 5 µm nominal retention (Hobra-Školnik S.R.O., Broumov, Czech). Acetone was removed from the raw extract using a Basis Hei-VAP HL rotary vacuum evaporator (Heidolph, Schwabach, Germany) at 60 °C and under reduced pressure of 450–72 mbar. The extract without acetone was again passed through a Hobrafilt S40N filter, and then purified on a 90 cm × 1.6 cm column packed with Amberlite XAD 1600 N resin (DOW, Midland, MI, USA). The extract was injected at a rate of approx. 15 mL/min. Elution was carried out at 10 mL/min using water/ethanol solutions with ethanol concentrations of 10%, 20%, 30%, 40%, 50%, and 60%, consecutively. The volume of solutions with each ethanol concentration equaled the volume of the column. The eluate obtained using the 40% ethanol solution contained the highest concentrations of lambertianin C and sanguiin H-6. Subsequently, ethanol was removed and the eluate was concentrated to approx. 5° Brix using a Basis HEI-VAP HL rotary vacuum evaporator at 60 °C under pressure reduced from 135 to 72 mbar. The concentrated extract was then freeze-dried (−32 °C, 48 h; Christ, Alpha 1–2 LDplus, Osterode am Harz, Germany). In the process, 30.8 g of raspberry ellagitannin preparation (REP) was obtained in the form of a red powder.