## 2 Materials and Methods
### Optimization of Extraction of Bioactive Compounds from R. typhimurium Fruits
#### 2.3.2 Bead-Beating Extraction (BBE)  
BBE was performed using a bead beater (Bead Genie, Scientific Industries, Inc., New York, NY, USA) set to a speed of 2640 strokes per minute (1320 rpm) without a pulse. The optimal values of solvent concentration (%) and solvent/solid ratio (\(v/w\)), according to the results of UAE, were applied for BBE using two different extraction times (5 min and 10 min) and different types of beads (1.4 mm ceramic beads, 2.8 mm ceramic beads, 2.4 mm metal beads). Content of Bioactive Compounds (Phenolics, Anthocyanins, Flavonoids, Tannins, Carotenoids) and HPLC Phenolic Profile  
The content of the investigated bioactive compounds was analyzed spectrophotometrically using the Spectord 200 Plus UV-Vis spectrophotometer (Analytik Jena, Jena, Germany). The results were expressed based on dry weight (DW). The total anthocyanin content (TAC), total phenolic content (TPC), total flavonoid content (TFC), total tannin content (TTC), and carotenoid content were determined according to studies in the literature [38; 39; 40; 41; 42] and expressed as mg cyanidin-3-O-glucoside/100 g DW, mg gallic acid equivalents (GAE)/100 g DW, mg quercetin equivalents (QE)/100 g DW, mg catechin equivalents (CE)/100 g DW, and mg \(\beta\)-carotene/100 g DW, respectively. The HPLC profile of phenolic compounds was investigated using a previously published method [43]. The identification of the phenolic compounds in the investigated extract was performed by comparing their retention times and UV spectral data with those of the authentic compounds (certified reference material) since polyphenols absorb light in the ultraviolet (UV) region. A 10 \(\upmu\)L sample of the BBE extract obtained using 61.51% ethanol, solvent/solid ratio of 20/1, extraction time of 5 min, and 2.8 mm ceramic beads was analyzed on the Shimadzu chromatographic instrument LC-20 AT with the following components: a quaternary pump, controller, solvent degasser, column oven, autosampler, and a diode array detector (DAD). The compounds were fully separated in a chromatographic column (150 X 4.6 mm, 5 \(\upmu\)m Kinetex C18 column, Agilent Technologies, Santa Clara, CA, USA) using mobile phases consisting of two components: component A (acidified water, pH 2.3) and component B (acetonitrile). A gradient elution mode was used by varying the percentage of component B (between 5% and 90%) for a total run of 45 min, keeping the column at 35 \({}^{\circ}\)C, with a flow rate of 0.8 mL/min. To avoid introducing air bubbles into the system, the mobile phase was degassed before daily runs and filtered by PVDF 0.20 \(\upmu\)m filters. Standard solutions were prepared for fifteen investigated phenolic acids and flavonoids, namely gallic acid, protocatechuric acid, caffe acid, chlorogenic acid, catechin, epicatechin, ellagic acid, kaempfer, myrietin, apigenin, quercetin, isoquercetin, naringenin, \(p\)-coumaric acid, and ferulic acid (all standards were purchased from Sigma-Aldrich, Merck, Darmstadt, Germany). The stock solutions were prepared by dissolving a certain amount of each standard in HPLC grade methanol (Chromasolv) to produce a solution of 1 mg/mL concentration; these were further used to prepare the calibration solutions. Following the analysis of the individual phenolic standards by UV-Vis spectra, four wavelengths were chosen for identification: 280 nm (gallic acid, catechin, epicatechin, ellagic acid, apigenin), 260 nm (protocatechuric acid), 320 nm (caffeic, chlorogenic, \(p\)-coumaric acid, ferulic acid, naringenin), and 360 nm (myricetin, kaemberol, quercetin, isoquercetin). The concentrations of the compounds were expressed in \(\upmu\)g/mL of extract. \begin{table}
\begin{tabular}{c c c c c c c c c c c c c c c} \hline \hline
**Sample** & **1** & **2** & **3** & **4** & **5** & **6** & **7** & **8** & **9** & **10** & **11** & **12** & **13** & **14** & **15** \\ \hline Final temperature (\({}^{\circ}\)C) & 45 & 48 & 58.6 & 57.2 & 43.5 & 43.7 & 50.9 & 51 & 43.2 & 53.9 & 40.9 & 50.8 & 48.8 & 55.7 & 49.6 \\ \hline \hline \end{tabular}
\end{table}