## 3 Results and Discussion
### Changes in Lipid Order Parameter in P. aeruginosa Cell Membrane
aeruginosa_ membrane microdomains under the influence of C\({}_{55}\)H\({}_{40}\)O\({}_{34}\). Experimental data represented by mean \(\pm\) SD values, (\(n=4\)), *** \(p<0.001\). Figure 2: Changes in the ordering parameter (S) of TMA-DPH and DPH probes in _P. aeruginosa_ membranes under the influence of C\({}_{55}\)H\({}_{40}\)O\({}_{34}\). Experimental data represented by mean \(\pm\) SD values (\(n=6\)), *** \(p<0.01\); *** \(p<0.001\). (S and S0 means ordering parameter in sample and control, respectively). The explanation of microdomain formation in bacteria is complex. There are only a few studies on bacterial microdomains and the most extensively studied Gram-negative bacteria so far include _Helicobacter pylori_, _Camyylobacter jejuni_, and _Borrelia burgdorferi_[34; 35]. We assume that _P. aeruginosa_, under stress caused by the antimicrobial action of the C\({}_{55}\)H\({}_{40}\)O\({}_{34}\), began synthesizing microdomains as a defense mechanism. According to [PERSON], tannins can impact membrane integrity by interacting with proteins and lipids or forming membrane pores [36]. Additionally, the compound may affect sterol levels or embedding itself in the membrane, as confirmed by studies using TMA-DPH and DPH markers, contributing to local phospholipid aggregation, acting as a binding molecule, and leading to an increase in microdomain levels compared to the control. Fluorescent Analysis of C\({}_{55}\)H\({}_{40}\)O\({}_{34}\) Binding to Membrane Proteins of P. aeruginosa  
The biological activity of tannins is connected with their strong interaction with lipids and proteins, including proteins anchored in the membrane structure. Tannin-protein interactions can be also responsible for the tannins' antimicrobial activity [37]. Therefore, it was investigated if C\({}_{55}\)H\({}_{40}\)O\({}_{34}\) interacts with membrane proteins of _P. aeruginosa_. In studies, the changes in fluorescence intensity descended from tryptophan in proteins were monitored. Tryptophan (Trp) fluorescence is highly sensitive to changes in their microenvironment. It allows for analyzing structural changes in the protein molecules [19]. As can be observed in Figure 4, C\({}_{55}\)H\({}_{40}\)O\({}_{34}\) was responsible for a concentration-dependent decrease in Trp fluorescence intensity at all three temperatures. Based on the obtained results, it was concluded that the studied tannin has a strong affinity to the membrane proteins of _P. aeruginosa_. C\({}_{55}\)H\({}_{40}\)O\({}_{34}\) interaction with proteins can trigger the changes in the protein's secondary and tertiary structure, as well as, disturb protein functions, which, in the case of membrane proteins like efflux pumps (one from _P. aeruginosa_ virulence factor responsible for MDR) can partially or entirely inhibit their activity leading to \"turn off\" one of the MDR crucial factors. Similar observations were described for other polyphenols whose activity led to the inactivation of several virulence factors in _S. aureus_[14; 38]. The above results showed that C\({}_{55}\)H\({}_{40}\)O\({}_{34}\) interacts with _P. aeruginosa_ membrane proteins. Some physicochemical parameters were calculated using Stern-Volmer plots to describe these interactions and better explain their mechanisms (Figure 5A,C,E). It allowed us to obtain the K\({}_{\text{SV}}\) constants, which informed us about the quencher's affinity to the quenched molecules. Received Stern-Volmer constants (Table 1) indicate a strong affinity of C\({}_{55}\)H\({}_{40}\)O\({}_{34}\) to the membrane proteins of _P. aeruginosa_. Figure 4: Relative quenching of Trp fluorescence of _P. aeruginosa_ membrane proteins under the influence of C\({}_{55}\)H\({}_{40}\)O\({}_{34}\). Experimental data represented by mean \(\pm\) SD values (\(n=5\)), *** \(p<0.001\). To determine whether C\({}_{55}\)H\({}_{40}\)O\({}_{34}\) causes static quenching (result in tannin-protein complex formation) or dynamic quenching (tannin-protein collisional encounters), the quenching constant (k\({}_{\rm q}\)) was calculated using Equation (4).