## 4 Discussion  
Our study hypothesis was rejected, as the results of the present research show that the pre-disinfection of PMMA discs by soaking in a disinfecting solution for 10 min prior to exposing them to incubation with whole saliva significantly reduces the presence of malodor-producing bacteria in the biofilm formed on these discs concomitant with the reduction in proteolytic activity and resulting VSC and malodor production. Salivary incubation assays are routinely used in oral malodor investigations and were shown to be a useful tool in screening for anti-malodor agents [7].  
The disinfecting solutions used in this study are commercially available mouthwash formulations with claims of oral malodor reduction. The chlorhexiidine formulation was the most effective pre-disinfecting solution in reducing malodor-producing bacteria in  
Figure 5: Confocal Laser Scanning Microscopy (CLSM) images showing VSC-producing bacteria (stained red) within the biofilm (black) formed on the PMMA discs (green) following pre-disinfection using: (**a**) Saline (negative) control, (**b**) essential oil formulation, (**c**) herbal extract formulation, and (**d**) chlorhexiidine formulation.  
Figure 6: The mean results (\(\pm\)SD) of the quantification of VSC-producing bacteria within the biofilm formed on the PMMA discs measured using confocal microscopy for the various tested solutions: Saline, essential oils (EO), herbal extracts (HE), and chlorhexiidine (CHX) formulations as indicated.  
the biofilm and resulting malodor-related parameters. This is in agreement with other malodor studies [8; 9]. However, the use of 0.2% chlorhexidine is not without the risk of side effects, the most common of which is staining and discoloration [10] that may pose serious aesthetic challenges in temporary crowns. Fortunately, in this study, a 10 min duration exposure was used, which is the equivalent of a 5-day rinsing in routine life. Such an extent of exposure is unlikely to yield a significant color change [11]. Furthermore, the results of the present study imply that natural-based products may also offer an esthetically safe and effective alternative.  
Although routine removal of temporary crowns is often accompanied by the emission of malodor it was not clear to what extent this contributes to the overall problem of oral malodor in everyday life. A recent study comparing VSC levels in the oral cavities of patients with or without fixed partial dentures (i.e., dental crowns and bridges) found that crowns and bridges, especially faulty ones, were a significant risk factor for elevated VSC levels [5]. Temporary crowns and bridges are more than likely to exacerbate this problem, mainly due to the porosity of PMMA [3].  
Taken together, the results of the present study suggest that pre-disinfection of temporary crowns may be beneficial in reducing the crowns' contribution to oral malodor production. However, as this is an in vitro study, further clinical trials are warranted. The mechanism responsible for such results remains to be also investigated under in vitro conditions. It may well be discovered that the porous PMMA harbored a substantial reservoir of chlorhexidine, which influenced the malodor production. The experiment should be repeated with the samples thoroughly washed after exposure to the disinfection solutions. This will reveal the effect of each solution due to the actual 10 min exposure with less impact of the remnant disinfect material.  
Conceptualization, O.R. and G.M.; methodology, N.S.; software, E.D.; validation, S.L.; U.J. and H.H.; formal analysis, G.B.I.; writing--original draft preparation, N.S.; writing--review and editing, U.J., O.R., G.M., E.D. and H.H. visualization, S.L.; supervision, O.R. and G.B.I.; project administration, N.S. All authors have read and agreed to the published version of the manuscript.  
This research received no external funding.  
The Research protocol was approved by Tel Aviv university ethics committee.  
Not applicable.  
Author (NS) declare ownership of patent EP2066304.