## 3 Results and Discussion
### Antibacterial Activity Is Also Dependent on the Length of Fermentation  
Although several works have already shown that fermented whey can be used as an antibacterial agent in minimally processed salads, being able to reduce overall microorganism growth [8; 40], most studies also use fermentation periods around 24 to 72 h [33; 39]. Since lactic acid production was higher in longer fermentation periods, we set out to search the best timing along fermentation for higher antibacterial activity. Hence, whey samples were collected daily throughout the 120 h, and firstly tested against reference Gram-positive _Listeria monocytogenes_ 4b and Gram-negative _Escherichia coli_ O157:H7, and MIC were determined for each day (Table 1). Results show that our fermented whey reduced the growth of both bacteria, from day 1 in _Escherichia coli_ O157:H7 and from day 4 in _L. monocytogenes_ 4b, concomitantly to the increase in lactic acid produced (Figure 1), respectively, of approximately 5 and 15 g-L\({}^{-1}\). However, more importantly, activities were significantly higher on the final day of fermentation, where MICs were more significantly reduced. Thus, our results indicate that fermentation periods longer than 72 h should be preferred in this type of study to obtain the best antibacterial activities. \begin{table}
\begin{tabular}{c c c} \hline \hline \multirow{2}{*}{**Days of Fermentation**} & \multicolumn{2}{c}{**Strains**} \\ \cline{2-3}  & _Listeria monocytogenes_ **4b** (MIC) & _Escherichia coli_ **O157:H7 (MIC)** \\ \hline
1 & NI & 25.0 \\
2 & NI & 25.0 \\
3 & NI & 25.0 \\
4 & 25.0 & 25.0 \\
5 & 1.56 & 3.13 \\ \hline \hline \end{tabular} Note. \({}^{\star}\) NI = No inhibition observed. Figures are averages of three independent experiments, expressed in % (r/r) of whey diluted in growth media.  
\end{table} 
Table 1: Minimum inhibitory concentrations (MIC) of fermented whey to control two selected bacterial strains \({}^{\star}\). Figure 1: Metabolite profile of whey during fermentation by mesophilic starter bacteria. Results reflect the average of three independent trials \(\pm\) standard deviation. Five Days-Fermented Whey Presents Dose-Dependent Antibacterial Activities and Is Effective Against Several Bacterial Species  
Based on the results in Figure 1 and Table 1, whey fermented for 120 h was selected to further analyze antibacterial activities towards thirteen pathogenic and spoilage strains, and results are presented in Figure 2. Results show that growth inhibition was induced in all strains, either being Gram-negative or Gram-positive species, corroborating a broad range of antibacterial activity. Subsequently, 120 h whey samples were evaluated against _L. monocytogenes_ and assessed at three different dilutions (Table 2) and compared to milk and non-fermented whey. Results show there was a dose-dependent effect in 120 h fermented whey, whereas neither milk nor unfermented whey exhibited an antibacterial effect (\(p<0.05\)) (Table 2). Industrial Whey Is an Effective Disinfecting Agent When Applied to Lettere, with Better or Similar Results When Compared Chlorine  
Because we aimed to test our 120 h fermented whey in a more realistic manner, we set out to determine its efficacy as a lettuce disinfect, testing quality indices such as texture and color, including its evaluation by a sensory panel, and also assessing its microbiological quality.