## 2 Materials and Methods
### Labeling of Samples and Extraction
#### 2.3.2 Ultrasonic Extraction  
Experimental design and optimization of ultrasonic extraction parameters with maximum output values were performed in the STATGRAPHICS Centurion program (Statgraphics Technologies Inc., The Plains, VA, USA). The experiment includes 4 series of 9 samples (LUDI1-9, LUDW1-9, LUWII-9, and LUWWI-9). Multilevel factorial design was used to determine the potential impact of input (independent) variables on output (dependent) variables. The input parameters of the experiment are amplitude (50, 75, and 100%) and treatment time (3, 6, and 9 min) as shown in Table 1, while the output variables include total proteins [mg(g\({}_{\text{d.m.}}\))\({}^{-1}\)], total phenols [mg(g\({}_{\text{d.m.}}\))\({}^{-1}\)], pH, electrical conductivity [mSm\({}^{-1}\)], and total energy change [W]. In the first part of the experiment, ultrasound-assisted extraction of proteins and specialized plant metabolites from sugar beet leaves was performed using the Q700 CA Sonicator ultrasonic apparatus (Qsonica, Newtown, CT, USA), with deionized water as a green extraction solvent. The diameter of the probe of the ultrasound apparatus was 12 mm. The treatment was performed at different values of amplitude (50, 75, and 100%) and duration (3, 6, and 9 min) for 4 series of samples (LUDI, LUDW, LUWI, and LUWW) as shown in Table 1. During the individual sample extraction, the temperature did not exceed 40 \({}^{\circ}\)C in order to avoid denaturation of thermolabile proteins present in the sugar beet leaf.  
A total of 2 \(\pm\) 0.1000 g of weighed crushed sugar beet leaf samples (dry/fresh) were transferred into 250 mL laboratory beakers and 100 mL of deionized room temperature (22 \({}^{\circ}\)C) or cold (4 \({}^{\circ}\)C) water was added into them. Ice cubes and a certain amount of water were placed in a plastic container for better heat transfer. The samples prepared in this way were placed in the housing of the device. The housing of the device is used for sound isolation because of the noise produced by the device due to sonication. An ultrasonic probe was placed in the laboratory beaker with the sample, which was located in the  
\begin{table}
\begin{tabular}{c c c c c c} \hline \hline  & **Sample Name** & \multicolumn{2}{c}{**Amplitude**} & \multicolumn{2}{c}{**Treatment Time**} \\  & & & **[\%]** & **[min]** \\ \hline LUDI1 & LUDW1 & LUWI1 & LUWW1 & 75 & 6 \\ \hline LUDI2 & LUDW2 & LUWI2 & LUWW2 & 75 & 3 \\ \hline LUDI3 & LUDW3 & LUWI3 & LUWW3 & 50 & 6 \\ \hline LUDI4 & LUDW4 & LUWI4 & LUWW4 & 50 & 9 \\ \hline LUDI5 & LUDW5 & LUWI5 & LUWW5 & 75 & 9 \\ \hline LUDI6 & LUDW6 & LUWI6 & LUWW6 & 100 & 9 \\ \hline LUDI7 & LUDW7 & LUWI7 & LUWW7 & 50 & 3 \\ \hline LUDI8 & LUDW8 & LUWI8 & LUWW8 & 100 & 6 \\ \hline LUDI9 & LUDW9 & LUWI9 & LUWW9 & 100 & 3 \\ \hline \hline \end{tabular}
\end{table}
Table 1: Variations of parameters (amplitude and time) of ultrasound-assisted extraction for individual samples.  
\begin{table}
\begin{tabular}{c c c} \hline \hline \multicolumn{1}{c}{**Sample Name**} & \multicolumn{1}{c}{**Temperature**} & \multicolumn{1}{c}{**Treatment Time**} \\  & & [\({}^{\circ}\)C] & **[min]** \\ \hline LD0/3 & LW0/3 & 60 & 3 \\ \hline LD0/6 & LW0/6 & 60 & 6 \\ \hline LD0/9 & LW0/9 & 60 & 9 \\ \hline \hline \end{tabular}
\end{table}
Table 2: Variations of thermal extraction duration for individual samples.  
center of the beaker, immersed in the liquid about 2.4 cm, and sufficiently spaced from the bottom. A thermocouple was also placed in the sample beaker to measure the temperature of the system, in such a way that it does not touch the walls of the beaker or the ultrasonic probe. Prior to treatment, it is necessary to close the sound-insulated housing and enter the appropriate ultrasonic extraction parameters on the control LCD screen of the device in accordance with the data in Table 1. Extracts obtained by high-power ultrasonic treatment were filtered using a Buchner funnel and analyzed.