## 3 Results and Discussion
### Antibacterial Activity Is Also Dependent on the Length of Fermentation
#### 3.4.6 Microbial Markers  
1. Aerobic Microorganisms at 30 \({}^{\circ}\)C  
Enumeration of AM (aerobic microorganisms at 30 \({}^{\circ}\)C) is an indicator of quality and gives an estimate of total viable populations, both endogenous and contaminant microbiota, since microorganisms are inevitably introduced during manipulations [48]. However, in general, the initial contamination of vegetables reflects the microbiota environment in which they were grown [49; 50]. Before treatment (day 0), the initial AM was 6.10 log cfu-g\({}^{-1}\). Other works have found similar results in whole vegetables [28; 51; 52]. As can be observed in Figure 7a, AM counts sharply decreased after all treatments (\(p<0.05\)). Throughout the storage, samples treated with whey always presented a significantly lower AM count (\(p<0.001\)) when compared to water and chlorine, independent of time (\(p<0.001\)). This is a strong indication of the effectiveness of fermented whey as an alternative disinfectant.  
Figure 6: Overall acceptability of the lettuce treated with fermented whey and chlorine over 10 days in cold storage. Results represent the average of at least three replicate experiments (n = 3) \(\pm\) SD.  
Figure 7: _Cont._2. Psychrotrophic Microorganisms  
Similarly to AM, psychotrophic microorganisms (PM) counts were significantly reduced (\(p<0.05\)) in all treatments at day 1 (Figure 7b). Also, the highest count reduction (\(p<0.001\)) to 2.20 log cfu-g\({}^{-1}\) occurred in samples treated with whey. With chlorine, the count reduction found was 1.13 log cfu-g\({}^{-1}\). In the case of the PM, both for the lettuce treated with whey as well as with chlorine, after 5 days in cold storage, there were no significant differences between both treatments in respect to these counts.  
3. Lactic Acid Bacteria  
Because whey contains naturally high counts of LAB (in this case 10\({}^{7}\) cfu-g\({}^{-1}\)), which could induce a higher bacterial load into the product, we investigated the evolution of LAB counts in all treatments throughout cold storage time, and results are presented in Figure 7c. As can be seen, although on day 1 there was a significant difference between LAB counts in all treatments, from day 3 onward, the presence of live LAB from fermented whey did not confer a significant difference (\(p>0.05\)) when compared to chlorine and water treatments. This type of bacteria is normally present in MP vegetables in loads from 2 to 6 log cfu-g\({}^{-1}\) (Figure 7c; at day 0, our reference sample was around 3.5 log cfu-g\({}^{-1}\)), so LAB counts ended up being similar in all treatments tested, meaning that the introduction of LAB from whey would not pose any noticeable difference to the final product. Furthermore, it has been shown that the presence of these LAB could have a positive antimicrobial effect in lettuce and apple  
Figure 7: Packed shredded lettuce: effect of sanitation on shredded lettuce Aerobic Microorganisms (**a**), Psychotropic Microorganisms (**b**), and Lactic Acid Bacteria (**c**) over 10 days in cold storage. Results represent the average of at least three replicate experiments (n = 3) \(\pm\) SD.  
taste [35]. Additionally, the presence of lactic acid, antibacterial peptides, and low pH are contributive factors for the inhibitory effects of whey [53; 54; 55].