## 3 Results and Discussion
### Biofilm Eradication  
Biofilms are a serious problem in the beverage industry, and can be up to 1000 times more resistant to disinfectants than planktonic cells [10]. Diffusion through biofilm structures is one of the main mechanisms explaining their low sensitivity to antimicrobial agents. An essential step to improve the disinfection process is therefore the loosening or eradication of biofilm structures. One common and useful method is the mechanical disruption of biofilm matrices [33]. However, this method is effective only on easily-accessible surfaces. An enzyme-based strategy is also commonly used to improve the cleaning process. However, its effectiveness depends on the EPS composition of the microbial biofilms [34]. In this study, we investigated the influence of 1% (\(w/v\)) saponin pretreatment on the eradication of mature bacterial biofilms. The effectiveness of QE was evaluated using three methods: (i) the plate count method, for detecting the number of living cells on the tested surface; (ii) luminometry, for measuring ATP levels in the biofilm structures; and (iii) light microscopy, for visualizing the bacterial cells in biofilm. Figure 2 shows the effect of QE solution (1% \(w/v\)) on the eradication of six-day bacterial biofilms treated with (Figure 2A) \(N\)-ethylo-\(N\),\(N\)-dimethylohexadecylioammonium bromide (QAC) or (Figure 2B) peracetic acid (PAA) at two different concentrations (0.125% and 0.250%). These concentrations were chosen on the basis of MIC results for less active PAA and planktonic cells. Biofilms are considered to be highly resistant to antimicrobial agents; therefore, the higher MIC values were used in these experiments. The results were obtained using the plate count method. The initial results for viable bacterial cells forming mature biofilms ranged from 5.0 \(\times\) 10\({}^{7}\) CFU/cm\({}^{2}\) for _As. bogorensis_ FFMW to 2.3 \(\times\) 10\({}^{8}\) CFU/cm\({}^{2}\) for _As. bogorensis_ ISD2. Eradication of _Asaia_ spp. biofilms led to a reduction in the number of living cells in the biofilms of around 4-5 logarithmic units. Generally, synergistic effects were observed between QE and QAC when the QAC concentration was lower; however, the final effect depended on the bacterial strain. The reason for this synergy is unclear. It may be due to changes in the permeability of bacterial cell walls and loosening of the EPS structure, which are known to occur as a result of surfactant action. However, the mechanisms of action of nonionic detergent QE and cationic surfactants such as QAC are generally different [35]. Due to obtained differences in the plate count method, luminometric measurement was used as the second method. Treatment with QE and PAA was more effective than eradication performed with QE and QAC. The reason for this was clearly related to the concentration of disinfectant used. It was thereby confirmed that cells forming biofilms are much less susceptible to biocidal than to planktonic action. The higher degree of eradication may have also depended on the type of action performed by the disinfectants. Both QE and QAC are surfactants with similar effects, whereas the action of PAA and QE is the combined effect of the detergent and the oxidizing agent. Figure 3 presents the results of luminescence testing (in RLU/cm\({}^{2}\)) for mature biofilms treated with disinfectants, QAA (Figure 3A) and PAA (Figure 3B), alone or in combination with 1% (\(w/v\)) QE. The initial results for six-day biofilms formed by _Asaia_ spp. strains varied, from 12,500 to 15,500 RLU/cm\({}^{2}\). Eradication of _Asaia_ spp. biofilms by antimicrobials led to as high as 10-fold reduction in RLU levels, and this effect was stronger in the case of combined treatment, especially with QE and PAA. In spite of general reduction of the obtained values of RLU/cm\({}^{2}\), for biofilms  
formed by _As. lantensis_ IFMW and _As. lantensis_ FMW1, the results were higher for pretreatment by 0.250% QAC in comparison to 0.125% QAC.