## 3 Results and Discussion
### Influence of Hawthorn Extracts Against Biofilms  
In this assay, five common food-contaminating microorganisms, namely _E. coli_, _P. aeruginosa_, _B. subtilis_, _W. anomalous_, and _D. hansenii_ involved in the antimicrobial tests, were subjected to antibiotic film effect analysis. None of the hawthorn extracts tested showed an inhibitory effect against the formation of _E. coli_ and _P. aeruginosa_ biofilms (Table 3). In _B. subtilis_, extracts prepared by enzyme-assisted treatment resulted in a significant (_p_ < 0.05) reduction in biofilm mass compared to positive control; however, the effect varied depending on the type of enzyme treatment (Table 3). Namely, about 15.1% inhibition was registered against the growth of the _B. subtilis_ biofilm when the hawthorn extract treated with _R. miehei_ cellulase was added, while the biofilm formation was reduced by about 84.4% in the presence of the extract treated with _R. miehei_ cellulase-_A. niger_ pectinase, compared to the corresponding enzyme-free sample. Overall, the highest inhibitory effect was registered in _B. subtilis_. In this context, previous studies also identified phenolic-enriched plant extracts, e.g., solvent extracts of olive leaves [67], _Potentilla visianii_[68], and pomeramate peel [69], as efficient agents against the growth of _B. subtilis_ biofilms. \begin{table}
\begin{tabular}{c c c c} \hline \hline \multirow{2}{*}{**Extracts**} & \multicolumn{3}{c}{**Biofilm Formation (\%)**} \\ \cline{2-4}  & _E. coli_ & _P. aeruginosa_ & _B. subtilis_ \\ \hline Positive control & 100 \(\pm\) 0 a & 100 \(\pm\) 0 a & 100 \(\pm\) 0 a \\ C & 105.4 \(\pm\) 1.4 b & 98.6 \(\pm\) 1.7 a & 98.3 \(\pm\) 2.0 a \\ S1 & 101.6 \(\pm\) 2.2 a & 99.3 \(\pm\) 3.1 a & 84.9 \(\pm\) 3.2 b \\ S2 & 104.5 \(\pm\) 0.7 a & 98.8 \(\pm\) 0.3 a & 15.6 \(\pm\) 1.7 c \\ \hline \hline \end{tabular}
\end{table} 
Table 3: Effect of acetate buffer (C)-, and cellulase (S1)-, and cellulase–pectinase (S2)-treated hawthorn fruit residues on the biofilm formation (%) of food-contaminating bacteria. The biofilm formation measured in the absence of any extract was taken as the positive control (100%). The biofilm growth of yeasts was generally more sensitive to the extracts than that of bacteria. The biofilm growth of _W. anomalous_ was significantly (\(p<0.05\)) decreased with inhibitory effects of 36% to 43% in the presence of the extracts, irrespectively of the extraction method (Figure 5). In _D. hansenii_, the decreasing effects of the cellulase and cellulase-pectinase-treated extracts against biofilm formation were 61% and 52%, respectively. However, no significant inhibition towards biofilm growth was registered for the acetate buffer-extracted sample compared to the extract-free positive control (\(p>0.05\)) (Figure 5). As can be seen, the enzyme treatments increased the anti-biofilm formation effect of hawthorn fruit extracts in _D. hansenii_ compared to the inhibitory data from the acetate buffer-extracted sample (Figure 5). The _W. anomalous_ and _D. hansenii_ biofilms were sensitive to the presence of some individual phenolics, e.g., vanillin, vanillic acid, ferulic acid, or quercetin, in the study of [PERSON] et al. [66]. For comparison, a considerable increase was recorded in the concentration of most of these biofilm inhibitor phenolic compounds after the enzymatic treatment of the hawthorn residue (see Table 1). The biofilm inhibitory potential of hawthorn extracts has been less investigated. In the study of [PERSON] et al. [4], the upregulation of genes related to biofilm formation was identified in the presence of a phenolic-rich solvent extract of _Crataegus pinnatifida_ Bge. The increased expression of these stress response and oxidative stress genes may be related to cell oxidative and environmental stress caused by the hawthorn extract [4]. No IC\({}_{50}\) was identified against methicillin-resistant _S. aureus_ biofilms by [PERSON] et al. [70] even at 128 ug/mL concentration for ethanolic extracts from leaves, stems, and flowers of _Crataegus monogyna_ Jacq.