## 3 Results and Discussion
### Changes in Lipid Order Parameter in P. aeruginosa Cell Membrane  
Tannins can influence bacterial activity through the interaction with their cell membrane, which occurrs by interaction with polar parts of membrane or by incorporation deeply, into hydrophobic fatty-acid tails of phospholipids. Such a process results in changes in membrane physicochemical properties [25]. Therefore, the effects of C\({}_{55}\)H\({}_{40}\)O\({}_{34}\) on bacterial cell membrane structure were investigated using fluorescence techniques. Two labels, TMA-DPH and DPH, located in different parts of the lipid bilayer were used to analyze changes in _P. aeruginosa_ membrane fluidity. TMA-DPH fluorescent marker is located in the membrane's outer (hydrophilic) part, while DPH binds to the inner phospholipid's region (hydrophobic). Firstly, the fluorescence anisotropy changes in _P. aeruginosa_ without and in the presence of studied compound were monitored. It was noted that the bacterial membrane became more fluid with increasing concentration of C\({}_{55}\)H\({}_{40}\)O\({}_{34}\). Changes were observed in both the hydrophilic and hydrophobic parts. Based on the fluorescence anisotropy, the order parameter 'S' was calculated according to Equation (2), presented as the ratio (S/S\({}_{0}\)) and demonstrated below (Figure 2). The obtained results presents concentration-dependent changes in the ordering parameter (S). The polar region of the membrane experienced strong fluidization for the highest C\({}_{55}\)H\({}_{40}\)O\({}_{34}\) concentration (10 \(\mu\)M) order parameter lowered from 1 (control) up to 0.59 \(\pm\) 0.11. It can be the result from the interaction between C\({}_{55}\)H\({}_{40}\)O\({}_{34}\) and phospholipid leads on the lipid bilayer surface. Such effect was previously described for the tannins'-OH group's interaction with phospholipids' PO\({}^{-2}\) groups [26]. A lower effect was observed for non-polar membrane parts where, for the highest C\({}_{55}\)H\({}_{40}\)O\({}_{34}\) concentration, the order parameter decreased from 1 (control) up to 0.86 \(\pm\) 0.10. There were similar effects for PGG and dGVG interaction with _S. aureus_ (8325-4) [19]. The fluidization of the bacterial membrane may lead to modifications in membrane transport processes and increased permeability, resulting in a loss in cell integrity [30]. For example, resveratrol derivatives [31], catechins [32], and rutin [33] were capable of damaging bacterial cell membranes. Also, [PERSON] et al. [30] demonstrated that tannins isolated from three South American trees disrupted the membrane structure of _P. aeruginosa_, leading to a rapid loss of bacterial viability. Figure 1: (**A**) Survival of _P. aeruginosa_ under C\({}_{55}\)H\({}_{40}\)O\({}_{34}\) and (**B**) its logistic function. Experimental data represented by mean \(\pm\) SD values (\(n=6\)), *** \(p\) \(<\) 0.001. Analysis of P. aeruginosa Membrane Microdomains in the Presence of C\({}_{55}\)H\({}_{40}\)O\({}_{34}\)  
Bacterial microdomains are highly active cell membrane areas, rich in sterols (e.g., cholesterol) and sphingolipids. Their function is to segregate or concentrate specific membrane proteins and lipids. The above results clearly demonstrate that C\({}_{55}\)H\({}_{40}\)O\({}_{34}\) interacts with _P. aeruginosa_ membrane and changes its physicochemical properties. Such interaction can additionally trigger the formation of membrane microdomains. Therefore, it was also investigated if C\({}_{55}\)H\({}_{40}\)O\({}_{34}\) influences the level of membrane microdomains in _P. aeruginosa_ cells. It was observed (Figure 3) that C\({}_{55}\)H\({}_{40}\)O\({}_{34}\) caused stiffening and increased the level of membrane microdomains in _P. aeruginosa_ in a concentration-dependent manner. For the most significant amount of C\({}_{55}\)H\({}_{40}\)O\({}_{34}\), the microdomain level increased from 1 to 1.60 \(\pm\) 0.06. Figure 3: Changes in fluorescence intensity of the di-4-ANEPDHQ probe located within _P.