## 3 Results and Discussion
### Anti-Quorum-Sensing Activity  
The inhibitory activity of the hawthorn extracts against quorum sensing was studied using the _C. violaceum_ 85 WT biosensor bacterium. The violacein pigment synthesis in the model organism is regulated by the quorum-sensing signaling system, and test extracts that can inhibit the pigment production are supposed to be samples that could affect the regulation process [29].  
Figure 5: Inhibitory capacity of hawthorn fruit extracts prepared by acetate buffer or via enzyme-assisted extraction against _W. anomalous_ and _D. hansenii_ yeast biofilms. C–acetate buffer extraction, S1–extraction with _R. miehei_ cellulase cocktail, S2–extraction with pectinase supplemented _R. miehei_ cellulase cocktail. Extracts with concentrations of 120.25 (C), 176.25 (S1) and 155.75 (S2) mg/mL were used in the tests. The control (Control) represents the biofilm formation in the absence of hawthorn residue extract. The values shown are averages of the data obtained from three measurements; error bars represent the standard deviation. The different letters above the columns indicate significant differences according to one-way ANOVA followed by [PERSON]’s multiple comparison test (\(p<0.05\)).  
The paper disk assay showed a quorum-sensing inhibitory effect for enzyme-free extracts made with acetate buffer extraction. The formation of a non-purple ring around the disk indicated bacterial growth with reduced violacein pigment production (Figure 6A). Both samples prepared via enzyme-assisted extraction did not result in any pigment-free ring around the disks. However, in the liquid culture assay, a significant (_p_ < 0.05) reduction in the violacein production was registered for the samples after carbohydrate treatments as well compared to control (Figure 6B). The violacein production was 90.3%, 87.2%, and 90.9% in the presence of acetate buffer extract and _R. miehei_ cellulase and _R. miehei_ cellulase-_A. niger_ pectinase-treated extracts, respectively. Treatment with _R. miehei_ cellulase significantly stimulated (_p_ < 0.05) the anti-quorum-sensing activity of the hawthorn residue compared to the enzyme-free extract.  
Anti-quorum-sensing activity has been demonstrated for other plant extracts as well, including those prepared from various medicinal plant and fruit residue samples [27; 71; 72; 73; 74; 75]. Natural antioxidative phenolic compounds could also show an inhibitory effect against bacterial quorum sensing [75; 76]. However, relatively few reports are available on the anti-quorum-sensing activity of hawthorn samples. Ethanolic extract of _C. monogyna_ Jacq. leaves, stems, and flowers could efficiently inhibit the \(\delta\)-toxin production of methicillin-resistant _S. aureus_ (MRSA) [77]. The resulted 57% inhibition of toxin production indicated a moderate anti-quorum-sensing activity in this pathogenic bacterium [77]. A 14.2 mm pigment-free halo was identified for the acetone-water extract of the _Crataegus cuneata_ fruit in the study of [PERSON] and [PERSON] [78], using a disk diffusion test based on a _C. violaceum_ CV026 biosensor. In fact, no study has reported to date that cellulase treatment increases the quorum-sensing inhibitory potential of hawthorn residue samples.