## 5 Discussion  
Bacterial biofilms play an important role in different types of infections, including chronic lung infections, and it represent a serious public health problem. Thus, it is necessary to study targeted antibacterial therapies in synergism with antibiotics to counteract the resistance drug of this bacterium [30]. For these reasons, NAC and NACESOLTM can play an important role. The aim of the biofilm prevention assay was to separate planktonic cells from sessile cells, trapped in the exopolysaccharide matrix; therefore, this approach allows a concrete assessment of the presence of a biofilm and the effectiveness of active compounds in contrasting it. All the samples were treated according to an identical standardized protocol. The first washes are carried out in order to detach the planktonic cells, then the scraping has the role to detach the cell more adhered to the surface, and, at the end, the sonication was adopted to remove all the cells on the surface. Each step was made by transferring the surface in sterile water, so that the cells resulting from each step could be counted. The validity of this protocol was also confirmed by the homogeneity of the control colonies, that are the colonies deriving from the biofilm not treated with NAC (\(2.4\times 10^{6}\pm 3.6\times 10^{5}\) for NAC experiments, and \(2.6\times 10^{6}\pm 2.5\times 10^{5}\) for NACESOLTM experiments). N-Acetyl-t-cysteine, in a free form or in a multi-composite, was inserted into the cell culture after 24 h, not when the biofilm was fully formed, but during its formation. This protocol, applied to evaluate a preventive activity of NAC, involves the use of stressed condition of the environment in which the active must exert its activity. The number of bacterial cells detected was used as an indicator to evaluate the reduction of the biofilm. Evaluation of free NAC against _P. aeruginosa_ biofilm led to results showing its progressive reduction with the highest value (89.52%) at a concentration of 4 mg:mL-1. At 0.5 and 2 mg:mL-1 the action of the free NAC can be compared (19.42% \(\pm\) 4.28% and 20.87% \(\pm\) 4.34%), while it significantly increases to 4 mg:mL-1. As observed in literature _P. aeruginosa_ biofilms showed significant structural differences in the presence of the NAC regimen. The biomass, substratum coverage, average thickness, maximum thickness, and surface area of the biomass all decreased for biofilms grown in the presence of NAC [16; 17]. The introduction of the new multi-composite NACESOLTM derives from the problems related to free NAC molecule--its low bioavailability (below 5%), due to its N-deacetylation in the intestinal mucosa and first pass metabolism in the liver, its affinity to oxidation, and its acidity (pH = 2.2) when inhaled, which could produce caught and bronchospasm. \begin{table}
\begin{tabular}{c c c} \hline \hline  & **S Content (\%)** & **S.D.** \\ \hline CTR & 0.43 & 0.018 \\ ACTIVE & 0.24 & 0.095 \\ \hline \hline \end{tabular}
\end{table} 
Table 1: Atom % of sulfur on the control disk and on the disk with the active. The choice to use a compound such as NACESOLTM, and thus the use of \(\alpha\)-Cyclodextrin (\(\alpha\)-CD), is because it has several advantages over the problems of the NAC:  
* Stabilization to oxidation;
* Protection against degradation of substances or by microorganisms;
* Masking of the smell;
* Catalytic activity of CDs with guest molecule. The active molecule was inserted into the host creating the multi-composite (MComp), characterized by a rapid onset of action and a powerful release effect with additional functions that improve the characteristics of the NAC. The advantages are due to the molecular separation as the individual molecules, and not crystals, are released. Furthermore, there is a vector effect caused by the weak bonds between guest and host [31]. Components for each MComp are based on the properties of the bioactive and on the desired target; thus, each MComp is specifically designed to optimize the performance of a given ingredient.