## 4 Discussion
Furthermore, in the literature there is a scarce number of studies related to the optimization of protein and polyphenol extraction parameters from green leaves, while at the same time, there are no available studies on the optimization of ultrasonic extraction for proteins from sugar beet leaves. In addition to changes in protein and phenol yield, changes in physical parameters were also observed. During ultrasound treatment, the structure of the cell membrane was disturbed, and a certain number of cells were destroyed, which resulted in the release of electrolytes into the solution, as a result of which the pH value, after the treatment, changed compared to the pH value of the untreated sample. Disruption of biological membranes occurs as a result of the combined action of cavitation and resulting shearing of heating, heating of the medium, and the formation of free radicals. When it comes to aqueous media, ultrasound treatment results in the formation of H\(\bullet\) and \(\bullet\)OH radicals (H\({}_{2}\)O \(\rightarrow\) H\(\bullet\) + \(\bullet\)OH), which additionally affects changes in pH value [49]. Considering the significantly higher proportion of dry matter, a higher value of electrical conductivity was observed in extracts obtained by extraction of dried sugar beet leaves compared to extracts obtained by extraction of fresh leaves. Furthermore, compared to ultrasonically treated samples, an increase in electrical conductivity of thermally treated samples was observed. As the temperature increases, the mobility of ions in the solution increases and their number also increases due to the dissociation of molecules, which consequently leads to an increase in the value of electrical conductivity [50]. Considering the emphasis of this research on sustainability, it is very important to pay attention to the power and energy consumption of thermal and ultrasonic extraction treatments. In this research, lower energy consumption was evaluated in ultrasonically treated samples compared to thermally treated samples. Ultrasound, as a non-thermal extraction technique, along with the use of lower temperatures, reduces the required extraction time, and therefore significantly affects the reduction of energy consumption [25]. With the increase in energy consumption, the emission of CO\({}_{2}\) and other pollutants also increases [51]. Consequently, in addition to lower energy consumption, CO\({}_{2}\) emissions were reduced in samples treated with ultrasound compared to thermally treated samples. Considering the benefits of ultrasonic extraction in the extraction of specialized plant metabolites from the by-products of the sugar industry as well as others, and the growing awareness of the population regarding sustainable development, the use of ultrasound in the processing and utilization of by-products is increasingly certain and inevitable in the near future. Furthermore, in later experiments, for the purpose of extracting a certain enzyme, water as a green solvent shows promising results in terms of sustainability. The excellent solubility of the protein in water and the distance from its isoelectric point, pl (4-6), will potentially allow higher yields of the target enzyme. Furthermore, the mechanismsunderlying the antioxidant power of bioactive compounds will be considered. In addition to ultrasound, for the same purpose, the authors will investigate the influence of other non-thermal extraction techniques, such as high voltage electric discharge (HVED) plasma. Conceptualization, A.R.J. and J.D. ; methodology, J.D., A.R.J. and M.N. ; software, A.R.J. ; validation, A.R.J. ; formal analysis, J.D. ; investigation, J.D. and M.H. ; resources, A.R.J. ; data curation, A.R.J. and J.D. ; writing--original draft preparation, J.D. ; writing--review and editing, A.R.J. ; visualization, J.D. and M.N. ; supervision, A.R.J. ; project administration, A.R.J. ; funding acquisition, A.R.J. and J.D. All authors have read and agreed to the published version of the manuscript.