## 2 Results
### Effect of Plasma Water on the Lipolytic Activity of the Y. lipolytica Yeast
However, it should be taken into account that the ricinoleic acid released in the hydrolysis process is partially involved in the \(\beta\)-oxidation cycle and converted into GDL or other lactones. The determined  
Figure 4: Acid value in media based on low-pressure glow plasma water saturated with air (PWTA), nitrogen (PWTN) or molecular oxygen (Nantes plasma water) with castor oil as a hydrophobic substrate. Mean values followed by the same letter are not significantly different according to HSD Tuk-ey (\(p\leq\) 0.05). Figure 3: Kinetics of extracellular lipolytic activity (dashed lines) and production of gamma-decalactone (solid lines) in media based on low-pressure glow plasma water saturated with air (PWTA), nitrogen (PWTN) or molecular oxygen (Nantes plasma water). acid value is reduced by the amounts of ricinoleic acid, which is involved in the yeast catabolic cycle. Analysis of the kinetics of extracellular lipolytic activity (Figure 3) in combination with the determined acid value (Figure 4) resulted in the new hypothesis that plasma treatment under varying conditions of the water used in microbial cultures affects lipase production/secretion, or more meaningfully, affects the activity of the enzyme itself. To unequivocally answer this question, the lipolytic activity of the native enzyme YLIip2, isolated from the culture medium and purified by chromatography, was determined. In addition, tests were carried out with four substrates varying in carbon chain length \(p\)-nitrophenyl esters, using \(p\)-nitrophenyl octanoate, laurate, stearate and oleate. The results of this experiment are presented in Figure 5.  
The data collected showed that there was no clear correlation between the plasma treatment of water and the activity of the native extracellular enzyme YLLip2. However, such a correlation was visible within the type of a substrate used. The length of the carbon chain of the \(p\)-nitrophenyl ester and the chain saturation had a significant impact on the catalytic activity of the enzyme. Lip2p showed the highest activity, irrespective of the type of water used in the culture medium, toward \(p\)-nitropenol oleate (C18 monounsaturated acid). In the case of media based on commercial Nantes plasma water or PWTN, the activity toward this substrate was approximately 0.048 \(\pm\) 0.002 U/mL and 0.042 \(\pm\) 0.003 U/mL, respectively, while in the presence of \(p\)-nitrophenyl stearate (C18 saturated acid), the activity decreased to 0.018 \(\pm\) 0.001 U/mL and 0.025 \(\pm\) 0.003 U/mL, respectively. Therefore, it is clear that unsaturated fatty acids enhanced the catalytic activity of lipases. The effect of carbon chain length becomes significantly apparent when comparing YLLip2 activity toward stearate (C18) and \(p\)-nitrophenyl octanoate (C8). In relation to octanoate, the activity was about 0.027 U/mL \(\pm\) 0.001 U/mL for the reaction carried out in the presence of commercial Nantes plasma water, then 0.034 U/mL \(\pm\) 0.001 U/mL using WPTN, and 0.032 \(\pm\) 0.002 U/mL for the control substrate. When \(p\)-nitrophenyl stearate was used, the activities were 0.009 to 0.012 U/mL lower for each water variant. Hence, it can be concluded that the shorter carbon chain of the hydrolysis substrate affected the level of determined YLLip2 catalytic activity. It also confirmed that the studied enzyme was a \"true\" lipase. Figure 5: Lipolytic activity of extracellular native lipase YLLip2 verified in the context of the type of plasma water used in culture medium and the substrate used. Mean values followed by the same letter are not significantly different according to HSD Tukey (\(p\leq 0.05\)).