Dry bath system

DNA was extracted from the bone powder received from UoS using a bead-beating approach. Initially, 700 μl bacterial lysis buffer (Roche UK- Cat. No. 4659180001) was added to the powder and vortexed before being transferred to a bead beating tube (Lysis Matrix B, 2ml tube (116911050, MP Biomedicals). The sample was bead-beaten for 2 minutes at full speed on a Tissue Lyser bead beater (Qiagen- Cat. No. 69980) and then pulse centrifuged (Eppendorf UK centrifuge model 5424). An aliquot (420 μl) was then transferred to a fresh Eppendorf tube and 20 μl of proteinase K (>600mAu/ml) (Qiagen, Cat. No. 19133) was added. The sample was then incubated at 65°C on a dry bath system (StarLab UK) for 10 minutes. Finally, DNA was purified on the MagNA Pure Compact machine (Roche UK) using the Roche MagnaPure Compact DNA_bacteria_V3_2 protocol (MagNA pure compact NA isolation kit I, Roche UK- product 03730964001).

Fresh, non-clarified pomegranate juice was pipetted into 1 mL sterile centrifuge tubes, closed, and heated in a Dry Bath System (Star Lab, Hamburg, Germany) for 5, 10, and 20 min at 80 °C. After thermal treatment, samples were put on ice and centrifuged for 5 min at 15,000× g. The clear supernatants were filtered through a PTFE filter (0.45 µm) and analyzed with HPLC-DAD as described in Section 2.12. The same thermal treatment and analysis were applied for (i) an unheated control of fresh, non-clarified pomegranate juice, (ii) as well as a mixture of individual anthocyanin standards that was adjusted to the pH of the pomegranate juice.
On a bigger scale, thermal treatment of 0.5 L of fresh, non-clarified pomegranate juice was performed for 10 min at 80 °C, with induction heating (Electrolux, Stockholm, Sweden). The treated juice was stored in sterile 50 mL centrifuge tubes, either overnight at 6 °C or for 3 to 5 months at 18 °C, which were followed up by centrifugation and HPLC analysis.